MULTIFUNCTIONAL MOLECULES THAT BIND TO CALRETICULIN AND USES THEREOF

REFERENCE TO A SEQUENCE LISTING XML

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Feb. 16, 2023, is named 53676-740_301_SL.xml and is 2,710,544 bytes in size.

BACKGROUND

Myeloproliferative neoplasms (MPNs) are a group of conditions that cause blood cells to grow abnormally in the bone marrow. Common myeloproliferative neoplasms include primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), and chronic myelogenous leukemia (CML). Primary myelofibrosis is a chronic blood cancer in which excessive scar tissue forms in the bone marrow and impairs its ability to produce normal blood cells. Given the ongoing need for improved treatment of myeloproliferative neoplasms such as myelofibrosis, new compositions and treatments targeting myeloproliferative neoplasms are highly desirable.

SUMMARY OF THE INVENTION

Provided herein, inter alia, in an aspect, is a composition comprising a polypeptide molecule comprising: (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), e.g., a calreticulin-targeting antigen binding domain disclosed in any one of Table 4, Table 5, Table 6, Table 24, Table 25, Table 16, Table 17, Table 18, or Table 19, and (ii) a second antigen binding domain that binds to TCRβV, e.g., an anti-TCRβV antigen binding domain disclosed in any one of Table 30, Table 31, Table 32, Table 10, Table 11, Table 12, or Table 13, or a second antigen binding domain that binds to NKp30, e.g., an anti-NKp30 antigen binding domain disclosed in Table 7, Table 8, Table 35, Table 36, Table 9, Table 10, or Table 34.

In some embodiments, the polypeptide molecule is a multifunctional polypeptide molecule.

In some embodiments, the polypeptide molecule is a multispecific polypeptide molecule.

In some embodiments, the second antigen binding domain binds to TCRβV.

In some embodiments, the second antigen binding domain activates a T cell or the second antigen binding domain does not activate a T cell.

In some embodiments, the second antigen binding domain binds to TCRβV12 or TCRβV6 (e.g., comprising the amino acid sequence of SEQ ID NO: 1044).

In some embodiments, the second antigen binding domain comprises one or more amino acid sequences as listed in Table 30, Table 31, Table 32, Table 10, Table 11, Table 12, or Table 13.

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 3 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 4 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 5 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 8 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 45 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 46 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 47 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 51 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 52 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 53 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or (d) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 48 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 49 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 50 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 54 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 55 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 56 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of a VH in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of a VL in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto) (iii) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (iv) the VL comprises the amino acid sequence of SEQ ID NO: 10 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of SEQ ID NO: 11 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO: 1312 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of SEQ ID NO: 1314 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 63 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 66 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 67 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 68 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO: 15 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of SEQ ID NO: 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises: the amino acid sequence of SEQ ID NO: 23 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 24 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 25 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or (ii) the VL comprises: the amino acid sequence of SEQ ID NO: 26 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 27 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 28 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 30 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the composition as describe herein comprises: a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to TCR (e.g., TCRVβ) (e.g., a first scFv that binds to TCR (e.g., TCRVβ), a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to TCR (e.g., TCRVβ) (e.g., a second scFv that binds to TCR (e.g., TCRVβ), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein: the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH form a third antigen binding domain that binds to a second calreticulin protein, optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In some embodiments, the second antigen binding domain binds to NKp30.

In some embodiments, the second antigen binding domain is chosen from an antibody molecule, e.g., an antigen binding domain, or ligand that binds to (e.g., activates) NKp30, e.g., the second antigen binding domain is an antibody molecule or ligand that binds to (e.g., activates) NKp30.

In some embodiments, the second antigen binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 7313 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions; and/or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 7326 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (i) a VH comprising the amino acid sequence of any of SEQ ID NOs: 7298 or 7300-7304 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of SEQ ID NOs: 7298 or 7300-7304); and/or (ii) a VL comprising the amino acid sequence of any of SEQ ID NOs: 7299 or 7305-7309 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to any of SEQ ID NOs: 7299 or 7305-7309).

In some embodiments, the second antigen binding domain comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7305 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7305); or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7309 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7309).

In some embodiments, the second antigen binding domain comprises: (i) an amino acid sequence of SEQ ID NO: 7310 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7310); or (ii) an amino acid sequence of SEQ ID NO: 7311 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7311).

In some embodiments, the second antigen binding domain comprises: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

In some embodiments, the second antigen binding domain comprises: (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) having an amino acid sequence of a VHFWR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 having an amino acid sequence of a VHFWR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 having an amino acid sequence of a VHFWR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 having an amino acid sequence of a VHFWR4 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), and/or (2) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) having an amino acid sequence of a VLFWR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 having an amino acid sequence of a VLFWR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 having an amino acid sequence of a VLFWR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 having an amino acid sequence of a VLFWR4 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the second antigen binding domain comprises: (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, a VHFWR2 amino acid sequence of SEQ ID NO: 6004, a VHFWR3 amino acid sequence of SEQ ID NO: 6005, or a VHFWR4 amino acid sequence of SEQ ID NO: 6006, and (3) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292, or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the second antigen binding domain comprises: (i) a VH comprising the amino acid sequence of a VH of Table 7, Table 35, Table 9, Table 10, or Table 34 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) a VL comprising the amino acid sequence of a VL of Table 8, Table 36, Table 9, Table 10, or Table 34 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto).

In some embodiments, the second antigen binding domain comprises a light chain comprising the amino acid sequence of a light chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the second antigen binding domain comprises a heavy chain comprising the amino acid sequence of a heavy chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and a light chain comprising the amino acid sequence of a light chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the composition as described herein comprises: a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30), a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein: the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH from a third antigen binding domain that binds to a second calreticulin protein, optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In some embodiments, the calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6285-6312 or 1001, optionally wherein the calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6313-6346 or 1002-1003.

In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

In some embodiments, the first antigen binding domain binds to an epitope located within the C-terminus of the calreticulin protein, optionally wherein the first antigen binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286.

In some embodiments, the composition as described herein further comprises a third antigen binding domain that binds to a second calreticulin protein, e.g., wherein the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein: (i) the third antigen binding domain is different from the first antigen binding domain, or (ii) the third antigen binding domain is the same as the first antigen binding domain.

In some embodiments, the second calreticulin molecule is the same as the calreticulin molecule bound by the first antigen binding domain.

In some embodiments, the second calreticulin molecule is different from the calreticulin molecule bound by the first antigen binding domain.

In some embodiments, the second calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6285-6312 or 1001, optionally wherein the second calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6313-6346 or 1002-1003.

In some embodiments, the calreticulin protein bound by the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 6285 or 1001, and the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

In some embodiments, the third antigen binding domain binds to an epitope located within the C-terminus of the second calreticulin protein, optionally wherein the third antigen binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286.

In some embodiments, the first antigen binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); (iii) a VH comprising the amino acid sequence of a VH in Table 24, Table 25, or Table 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); (iv) a VL comprising the amino acid sequence of a VL in Table 24, Table 25, or Table 16 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto); (v) a VH comprising a heavy chain framework region 1 (VHFWR1) having an amino acid sequence of a VHFWR1 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 having an amino acid sequence of a VHFWR2 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 having an amino acid sequence of a VHFWR3 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 having an amino acid sequence of a VHFWR4 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or (vi) a VL comprising a light chain framework region 1 (VLFWR1) having an amino acid sequence of a VLFWR1 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 having an amino acid sequence of a VLFWR2 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VLFWR3 having an amino acid sequence of a VLFWR3 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or a VLFWR4 having an amino acid sequence of a VLFWR4 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the multifunctional molecule further comprises a tumor-targeting moiety.

In some embodiments, the tumor-targeting moiety binds to a tumor antigen.

In some embodiments, the tumor antigen is selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In some embodiments, the tumor-targeting moiety comprises an antibody molecule, e.g., that binds to a tumor antigen selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In some embodiments, the tumor-targeting moiety comprises a VH and/or VL sequence, e.g., as listed in Table 38 or Table 20.

In some embodiments, the multifunctional molecule preferentially binds to a myeloproliferative neoplasm cell over a non-tumor cell, optionally wherein the binding between the multifunctional molecule and the myeloproliferative neoplasm cell is more than 10, 20, 30, 40, 50-fold greater than the binding between the multifunctional molecule and a non-tumor cell.

In some embodiments, the myeloproliferative neoplasm cell is chosen from a myelofibrosis cell, an essential thrombocythemia cell, a polycythemia vera cell, or a chronic myeloid cancer cell, optionally wherein: the myeloproliferative neoplasm cell does not comprise a JAK2 V617F mutation, or the myeloproliferative neoplasm cell does not comprise a MPL mutation.

In some embodiments, the composition as described herein further comprises a linker, e.g., a linker between the first antigen binding domain and the second antigen binding domain.

In some embodiments, the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.

In some embodiments, the linker is a peptide linker.

In some embodiments, the peptide linker comprises Gly and Ser.

In some embodiments, the peptide linker comprises an amino acid sequence chosen from SEQ ID NOs: 6214-6217 or 6220-6221 and 77-78.

In another aspect, provides herein is a multifunctional molecule comprising: (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), e.g., a calreticulin-targeting antigen binding domain disclosed in any one of Table 4, Table 5, Table 6, Table 24, Table 25, Table 16, Table 17, Table 18, or Table 19, and (ii) a second antigen binding domain that binds to TCRβV, e.g., an anti-TCRβV antigen binding domain disclosed in any one of Table 30, Table 31, Table 32, Table 10, Table 11, Table 12, or Table 13, or a second antigen binding domain that binds to NKp30, e.g., an anti-NKp30 antigen binding domain disclosed in Table 7, Table 8, Table 35, Table 36, Table 9, Table 10, or Table 34.

In some embodiments, the second antigen binding domain binds to TCRβV.

In some embodiments, the second antigen binding domain activates a T cell or the second antigen binding domain does not activate a T cell.

In some embodiments, the second antigen binding domain binds to TCRβV12 or TCRβV6 (e.g., comprising the amino acid sequence of SEQ ID NO: 1044).

In some embodiments, the second antigen binding domain comprises one or more amino acid sequences as listed in Table 30, Table 31, Table 32, Table 10, Table 11, Table 12, or Table 13.

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of a VH in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of a VL in Table 30, Table 31, Table 10, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto) (iii) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (iv) the VL comprises the amino acid sequence of SEQ ID NO: 10 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of SEQ ID NO: 11 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO: 1312 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of SEQ ID NO: 1314 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 63 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 66 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 67 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 68 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises the amino acid sequence of SEQ ID NO: 15 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or (ii) the VL comprises the amino acid sequence of SEQ ID NO: 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein: (i) the VH comprises: the amino acid sequence of SEQ ID NO: 23 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 24 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 25 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or (ii) the VL comprises: the amino acid sequence of SEQ ID NO: 26 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 27 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 28 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 30 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the multifunctional molecule as described herein comprises: a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to TCR (e.g., TCRVβ) (e.g., a first scFv that binds to TCR (e.g., TCRVβ), a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to TCR (e.g., TCRVβ) (e.g., a second scFv that binds to TCR (e.g., TCRV 0), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein: the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH form a third antigen binding domain that binds to a second calreticulin protein, optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In some embodiments, the second antigen binding domain binds to NKp30.

In some embodiments, the second antigen binding domain comprises: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 7313 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions; and/or (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 7326 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises: (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

In some embodiments, the multifunctional molecule as described herein comprises: a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30), a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein: the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH from a third antigen binding domain that binds to a second calreticulin protein, optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

In some embodiments, the multifunctional molecule as described herein further comprises a third antigen binding domain that binds to a second calreticulin protein, e.g., wherein the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein: (i) the third antigen binding domain is different from the first antigen binding domain, or (ii) the third antigen binding domain is the same as the first antigen binding domain.

In some embodiments, the second calreticulin molecule is the same as the calreticulin molecule bound by the first antigen binding domain.

In some embodiments, the second calreticulin molecule is different from the calreticulin molecule bound by the first antigen binding domain.

In some embodiments, the multifunctional molecule further comprises a tumor-targeting moiety.

In some embodiments, the tumor-targeting moiety binds to a tumor antigen.

In some embodiments, the tumor antigen is selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In some embodiments, the tumor-targeting moiety comprises a VH and/or VL sequence, e.g., as listed in Table 38 or Table 20.

In some embodiments, the multifunctional molecule as described herein further comprises a linker, e.g., a linker between the first antigen binding domain and the second antigen binding domain.

In some embodiments, the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.

In some embodiments, the linker is a peptide linker.

In some embodiments, the peptide linker comprises Gly and Ser.

In some embodiments, the peptide linker comprises an amino acid sequence chosen from SEQ ID NOs: 6214-6217 or 6220-6221 and 77-78.

In another aspect, provides herein is a nucleic acid molecule encoding the multifunctional molecule as described herein.

In another aspect, provides herein is a vector, e.g., an expression vector, comprising the nucleic acid molecule as described herein.

In another aspect, provides herein is a cell comprising the nucleic acid molecule as described herein or the vector as described herein.

In another aspect, provides herein is a method of making, e.g., producing, the multifunctional molecule as described herein, comprising culturing the cell as described herein, under suitable conditions, e.g., conditions suitable for gene expression and/or homo- or heterodimerization.

In another aspect, provides herein is a pharmaceutical composition comprising the composition as described herein, the multifunctional molecule as described herein, the nucleic acid molecule as described herein, the vector as described herein, or the cell as described herein, and a pharmaceutically acceptable carrier, excipient, diluent, or stabilizer.

In another aspect, provides herein is a method of treating a cancer, comprising administering to a subject in need thereof the composition as described herein, the multifunctional molecule as described herein, the nucleic acid molecule as described herein, the vector as described herein, the cell as described herein, or the pharmaceutical composition as described herein, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

In another aspect, provides herein is a use of the composition as described herein, the multifunctional molecule as described herein, the nucleic acid molecule as described herein, the vector as described herein, or the cell as described herein for the manufacture of a medicament for treating a cancer.

In some embodiments, the subject has cancer cells that express the first and/or second calreticulin protein.

In some embodiments, the subject has the JAK2 V617F mutation.

In some embodiments, the subject does not have the JAK2 V617F mutation.

In some embodiments, the subject has a MPL mutation.

In some embodiments, the subject does not have a MPL mutation.

In some embodiments, the cancer is a hematological cancer, optionally wherein the cancer is a myeloproliferative neoplasm, e.g., primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML), optionally wherein the cancer is myelofibrosis.

In some embodiments, the cancer is a solid tumor cancer.

In some embodiments, the method as described herein or the use as described herein further comprises administering a second therapeutic treatment.

In some embodiments, the second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormonal therapy), radiation, or surgery.

In some embodiments, the therapeutic agent is selected from: a chemotherapeutic agent, or a biologic agent.

In another aspect, provides herein is a method of detecting calreticulin (e.g., wild-type and/or mutant calreticulin) in a sample or subject, comprising: contacting the sample or subject with an anti-calreticulin (e.g., wild-type and/or mutant calreticulin) antibody molecule described herein; and detecting formation of a complex between the antibody molecule and the sample or subject, thereby detecting calreticulin (e.g., wild-type and/or mutant calreticulin).

In some embodiments, calreticulin (e.g., wild-type and/or mutant calreticulin) is detected in vitro or in vivo.

In some embodiments, the method as described herein further comprises contacting a reference sample or subject with the antibody molecule; and detecting formation of a complex between the antibody molecule and the reference sample or subject, wherein a change, e.g., a statistically significant change, in the formation of the complex in the sample or subject, relative to the reference sample or subject is indicative of the presence of calreticulin (e.g., wild-type and/or mutant calreticulin) in the sample or subject.

In some embodiments, the method as described herein further comprises obtaining a sample from a subject.

In some embodiments, sample comprises one or more of plasma, tissue (e.g., cancerous tissue), biopsy, blood (e.g., whole blood), PBMCs, bone marrow, and/or lymphatic tissue, e.g., lymph node.

In some embodiments, the sample has not been frozen and/or fixed.

In some embodiments, the sample has been frozen (e.g., snap frozen) and/or fixed (e.g., formalin-fixed paraffin-embedded (FFPE)).

In some embodiments, the subject has, or is at risk of having, a disease or disorder described herein (e.g., cancer, e.g., myelofibrosis).

In some embodiments, the method as described herein further comprises performing a flow analysis, e.g., using a multi-panel method.

In some embodiments, the method as described herein further comprises assessing T-cell clonality, e.g., to determine the presence and/or level of T cell malignancy.

In some embodiments, the method as described herein further comprises measuring the level of calreticulin+(e.g., wild-type calreticulin+ and/or mutant calreticulin+) cells from the biological sample (e.g., determining if the calreticulin+ cells are depleted, e.g., relative to a reference sample or subject).

In some embodiments, the method as described herein further comprises measuring the intracellular level of calreticulin (e.g., wild-type and/or mutant calreticulin).

In some embodiments, the method as described herein further comprises measuring the membrane level of calreticulin (e.g., wild-type and/or mutant calreticulin).

In some embodiments, the method as described herein further comprises evaluating the subject for a change in prognosis, severity, or presence or absence of a disease or disorder (e.g., cancer, e.g., myelofibrosis), e.g., after treatment (e.g., with an antibody molecule described herein).

In some embodiments, the antibody molecule is detectably labeled.

In another aspect, provides herein is a method of evaluating a subject, comprising: contacting a sample (e.g., a sample described herein) from the subject with an anti-calreticulin (e.g., wild-type and/or mutant calreticulin) antibody molecule described herein; and detecting formation of a complex between the antibody molecule and the sample, thereby evaluating the subject.

In some embodiments, the subject has, or is at risk of having, a disease or disorder described herein (e.g., cancer, e.g., myelofibrosis).

In some embodiments, the subject has not been treated with an antibody molecule described herein.

In some embodiments, the subject has been treated with an antibody molecule described herein.

In another aspect, provides herein is a kit comprising an anti-calreticulin (e.g., wild-type and/or mutant calreticulin) antibody molecule described herein and instructions for use in a method of detecting calreticulin (e.g., wild-type and/or mutant calreticulin) in a sample or subject.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1B shows the alignment of the Antibody A source mouse VH and VL framework 1, CDR 1, framework 2, CDR 2, framework 3, CDR3, and framework 4 regions with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs are shown in italics, and combined CDRs are shown in boxes. The framework positions that were back mutated are double underlined. FIG. 1A shows VH sequences for murine Antibody A (SEQ ID NO: 1) and humanized Antibody A-H (SEQ ID NO: 9). FIG. 1B shows VL sequences for murine Antibody A (SEQ ID NO: 2) and humanized Antibody A-H (SEQ ID NO: 10 and SEQ ID NO: 11).

FIGS. 2A-2B shows the alignment of the Antibody B source mouse VH and VL framework 1, CDR 1, framework 2, CDR 2, framework 3, CDR3, and framework 4 regions with their respective humanized sequences. Kabat CDRs are shown in bold, Chothia CDRs are shown in italics, and combined CDRs are shown in boxes. The framework positions that were back mutated are double underlined. FIG. 2A shows the VH sequence for murine Antibody B (SEQ ID NO: 15) and humanized VH sequences B-H. 1A to B-H.1C (SEQ ID NOs: 23-25). FIG. 2B shows the VL sequence for murine Antibody B (SEQ ID NO: 16) and humanized VL sequences B-H.1D to B-H.1H (SEQ ID NOs: 26-30).

FIG. 3 depicts the phylogenetic tree of TCRBV gene family and subfamilies with corresponding antibodies mapped. Subfamily identities are as follows: Subfamily A: TCRβ V6; Subfamily B: TCRβ V10; Subfamily C: TCRβ V12; Subfamily D: TCRβ V5; Subfamily E: TCRβ V7; Subfamily F: TCRβ V11; Subfamily G: TCRβ V14; Subfamily H: TCRβ V16; Subfamily I: TCRβ V18; Subfamily J: TCRβ V9; Subfamily K: TCRβ V13; Subfamily L: TCRβ V4; Subfamily M: TCRβ V3; Subfamily N: TCRβ V2; Subfamily O: TCRβ V15; Subfamily P: TCRβ V30; Subfamily Q: TCRβ V19; Subfamily R: TCRβ V27; Subfamily S: TCRβ V28; Subfamily T: TCRβ V24; Subfamily U: TCRβ V20; Subfamily V: TCRβ V25; and Subfamily W: TCRβ V29 subfamily. Subfamily members are described in detail herein in the Section titled “TCR beta V (TCRβV)”.

FIGS. 4A-4C show human CD3+ T cells activated by anti-TCR Vβ13.1 antibody (A-H.1) for 6-days. Human CD3+ T cells were isolated using magnetic-bead separation (negative selection) and activated with immobilized (plate-coated) anti-TCR Vβ13.1 (A-H.1) or anti-CD3 E (OKT3) antibodies at 100 nM for 6 days. FIG. 4A shows two scatter plots (left: activated with OKT3; and right: activated with A-H.1) of expanded T cells assessed for TCR Vβ13.1 surface expression using anti-TCR Vβ13.1 (A-H.1) followed by a secondary fluorochrome-conjugated antibody for flow cytometry analysis. FIG. 4B shows percentage (%) of TCR Vβ13.1 positive T cells activated by anti-TCR Vβ13.1 (A-H.1) or anti-CD3e (OKT3) plotted against total T cells (CD3+). FIG. 4C shows relative cell count acquired by counting the number of events in each T cell subset gate (CD3 or TCR Vβ13.1) for 20 seconds at a constant rate of 60 μl/min. Data shown as mean value from 3 donors.

FIGS. 5A-5B show cytolytic activity of human CD3+ T cells activated by anti-TCR Vβ13.1 antibody (A-H.1) against transformed cell line RPMI 8226. FIG. 5A depicts target cell lysis of human CD3+ T cells activated with A-H.1 or OKT3. Human CD3+ T cells were isolated using magnetic-bead separation (negative selection) and activated with immobilized (plate-coated) A-H.1 or OKT3 at the indicated concentrations for 4 days prior to co-culture with RPMI 8226 cells at a (E:T) ratio of 5:1 for 2 days. Samples were next analyzed for cell lysis of RPMI 8226 cells by FACS staining for CFSE/CD138-labeled, and membrane-impermeable DNA dyes (DRAQ7) using flow cytometry analysis. FIG. 5B shows target cell lysis of human CD3+ T cells activated with A-H.1 or OKT3 incubated with RPMI-8226 at a (E:T) ratio of 5:1 for 6 days followed by cell lysis analysis of RPMI 8226 cells as described above. Percentage (%) target cell lysis was determined by normalizing to basal target cell lysis (i.e. without antibody treatment) using the following formula, [(x−basal)/(100%−basal), where x is cell lysis of sample]. Data shown is a representative of n=1 donor.

FIGS. 6A-6B show IFNg production by human PBMCs activated with the indicated antibodies. Human PBMCs were isolated from whole blood from the indicated number of donors, followed by solid-phase (plate-coated) stimulation with the indicated antibodies at 100 Nm. Supernatant was collected on Days 1, 2, 3, 5, or 6. FIG. 6A is a graph comparing the production of IFNg in human PBMCs activated with the antibodies indicated activated with anti-TCR Vβ13.1 antibodies (A-H.1 or A-H.2) or anti-CD3e antibodies (OKT3 or SP34-2) on Day 1, 2, 3, 5, or 6 post-activation. FIG. 6B shows IFNg production in human PBMCs activated with the antibodies indicated activated with the indicated anti-TCR Vβ13.1 antibodies or anti-CD3e antibody (OKT3) on Day 1, 2, 3, 5, or 6 post-activation.

FIGS. 7A-7B show IL-2 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described for FIGS. 6A-6B was used.

FIGS. 8A-8B show IL-6 production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described for FIGS. 6A-6B was used.

FIGS. 9A-9B show TNF-alpha production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described for FIGS. 6A-6B was used.

FIGS. 10A-10B show IL-1beta production by human PBMCs activated with the indicated antibodies. A similar experimental setup as described for FIGS. 6A-6B was used.

FIGS. 11A-11B are graphs showing delayed kinetics of IFNg secretion in human PMBCs activated by anti-TCR Vβ13.1 antibody A-H.1 when compared to PBMCs activated by anti-CD3e antibody OKT3. FIG. 11A shows IFNg secretion data from 4 donors. FIG. 11B shows IFNg secretion data from 4 additional donors. Data shown is representative of n=8 donors.

FIG. 12 depicts increased CD8+ TSCM and Temra T cell subsets in human PBMCs activated by anti-TCR Vβ13.1 antibodies (A-H.1 or A-H.2) compared to PBMCs activated by anti-CD3e antibodies (OKT3 or SP34-2).

FIGS. 13A-13F show characterization of an anti-TCRVb antibody. FIG. 13A is a graph depicting proliferation of T cells activated with anti-CD3 (OKT3) antibody or anti-TCRVb antibody. FIG. 13B shows selective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) cells with anti-TCRVb antibodies. Tn=naïve T cell; Tscm=stem cell memory T cell; Tcm=central memory T cell; Tem=effector memory T cell; Temra=effector memory CD45RA+ T cell. FIG. 13C is a graph showing IFN-g secretion by PBMCs stimulated with an anti-TCRVb antibody, or anti-CD3 antibodies. FIG. 13D shows target cell lysis by T cells stimulated with an anti-TCRVb antibody, or anti-CD3 antibodies. Cells were stimulated for 4 days followed by 2 days incubation with multiple myeloma target cells for assessment of cell killing. FIG. 13E is a graph showing perforin secretion by T cells stimulated with an anti-TCRVb antibody, or an anti-CD3 antibody. Perforin was analyzed by FACS staining in TCRVB-positive and TCRVB-negative T cells in PBMCs after 5 days of stimulation with 100 ng/ml plate-bound antibody. FIG. 13F is a graph showing Granzyme B by T cells stimulated with an anti-TCRVb antibody, or an anti-CD3 antibody. Granzyme B was analyzed by FACS staining in TCRVB-positive and TCRVB-negative T cells in PBMCs after 5 days of stimulation with 100 ng/ml plate-bound antibody.

FIGS. 14A-14B show production of IL-2 and IL-15 and expansion of human NK cells by stimulation of PBMCs with anti-TCRVb antibody for 6 days at a dose of 100 nM. FIG. 14A shows secretion of IL-2 or IL-15 in T cells stimulated with an anti-TCRVb antibody, or anti-CD3 antibodies. FIG. 14B depicts flow cytometry dot plots showing NKp46 staining vs CD56 antibody staining in cells stimulated with an anti-TCRVb antibody or an anti-CD3 antibody or a control sample.

FIGS. 15A-15C show secretion of cytokines in PBMCs stimulated with an anti-TCRVb antibody, or anti-CD3 antibodies.

FIGS. 16A-16B show killing of MM cells by dual targeting BCMA-TCRvb antibody molecules. FIG. 16A shows in vitro killing by one of the following dual-targeting antibody molecules: BCMA-TCRVb, BCMA-CD3, or Control-TCRVb; or an isotype control. FIG. 16B shows in vivo killing of MM cells by a dual-targeting BCM-TCRVb antibody.

FIG. 17 shows lysis of MM target cells with a dual targeting antibody which recognized FcRH5 on one arm and TCRVb on the other arm.

FIGS. 18A-18C are schematic representations of exemplary formats and configurations of functional moieties attached to a dimerization module, e.g., an immunoglobulin constant domain. FIG. 18A depicts moieties A, B, C and D, covalently linked to a heterodimeric Fc domain. FIG. 18B depicts moieties A, B, C and D, covalently linked to a homodimeric Fc domain. FIG. 18C depicts moieties A, B, C and D, covalently linked to heterodimeric heavy and light constant domains (e.g., a Fab CH₁and a Fab CL). In some embodiments, the functional moiety is an antigen binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein). In some embodiments, the functional moiety is an antigen binding domain that binds to a wild-type calreticulin protein and a calreticulin mutant protein with approximately the same affinity. In some embodiments, the functional moiety is an antigen binding domain that preferentially binds to a calreticulin mutant protein over a wild type calreticulin protein, e.g., wherein the first calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286 and the wild type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001. In some embodiments, the functional moiety is an immune cell engager chosen from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager. In some embodiments, the functional moiety is a cytokine molecule. In some embodiments, the functional moiety is a stromal modifying moiety.

FIGS. 19A and 19B are schematic representations of exemplary formats and configurations of a multifunctional molecule comprising a first antigen binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), a second antigen binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), and one or more moieties that bind to CD3 (e.g., an scFv that binds to CD3). In one embodiment, the first antigen binding domain (e.g., the first Fab) binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein) disclosed herein, e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., Type 1 or Type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6113 or 6314. In one embodiment, the second antigen binding domain (e.g., the second Fab) binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein) disclosed herein, e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., Type 1 or Type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314.

FIGS. 20A and 20B are schematic representations of exemplary formats and configurations of a multifunctional molecule comprising a first antigen binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), a second antigen binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), and one or more moieties that bind to TCR (e.g., TCR p) (e.g., an scFv that binds to TCR (e.g., TCR P)). In one embodiment, the first antigen binding domain (e.g., the first Fab) binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein) disclosed herein, e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., Type 1 or Type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. In one embodiment, the second antigen binding domain (e.g., the second Fab) binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein) disclosed herein, e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., Type 1 or Type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314.

FIGS. 21A and 21B are schematic representations of exemplary formats and configurations of a multifunctional molecule comprising a first antigen binding domain (e.g., a first Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), a second antigen binding domain (e.g., a second Fab) that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), and one or more moieties that bind to NKp30 (e.g., an antibody molecule or ligand that binds to NKp30). In one embodiment, the first antigen binding domain (e.g., the first Fab) binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein) disclosed herein, e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., Type 1 or Type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314. In one embodiment, the second antigen binding domain (e.g., the second Fab) binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein) disclosed herein, e.g., a calreticulin mutant protein disclosed in Table 2 or 3, e.g., Type 1 or Type 2 calreticulin mutant protein disclosed in Table 2 or 3, e.g., a calreticulin mutant protein comprising the amino acid sequence of SEQ ID NO: 6313 or 6314.

FIG. 22 is a graph showing binding of NKp30 antibodies to NK92 cells. Data was calculated as the percent-AF747 positive population.

FIG. 23 is a graph showing activation of NK92 cells by NKp30 antibodies. Data were generated using hamster anti-NKp30 mAbs.

FIGS. 24A-24D are schematics showing exemplary multispecific molecules comprising a TGFβ inhibitor. In some embodiments, the TGFβ inhibitor comprises a TGF-beta receptor ECD homodimer. In some embodiments, the TGFβ inhibitor comprises a TGFBR2 ECD heterodimer. In FIGS. 24A and 24B, the two TGFBR ECD domains are linked to the C-terminus of two Fc regions. In some embodiments, the CH1-Fc-TGFBR ECD region shown in FIG. 24A or 24B comprises the amino acid sequence of SEQ ID NO: 6405 or 3193. In some embodiments, the Fc-TGFBR ECD region shown in FIG. 24A or 24B comprises the amino acid sequence of SEQ ID NO: 6407 or 6408. In FIGS. 24C and 24D, the two TGFBR ECD domains are linked to CH1 and CL, respectively. In some embodiments, the TGFBR ECD-CH1-Fc region shown in FIG. 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6409 or 6410. In some embodiments, the TGFBR ECD-CL region shown in FIG. 24C or 24D comprises the amino acid sequence of SEQ ID NO: 6411 or 6412. In some embodiments, the multispecific molecule comprises a binding moiety A and a binding moiety B. In some embodiments, the binding moiety A or binding moiety B is a calreticulin-targeting antigen binding domain disclosed herein.

FIGS. 25A-25B are a series of graphs showing enzyme-linked immunosorbent assay (ELISA) results showing the level of binding of the parental IgG form of antibody 6C10 (BKM0106) to wild-type calreticulin (CALR WT) and two calreticulin mutants (CALR ins and CALR del, as described herein). FIG. 25A shows ELISA results when the indicated antigen (CALR WT, CALR ins, or CALR del) was coated on the plate. FIG. 25B shows ELISA results when the BKM0106 antibody was coated on the plate.

FIGS. 26A-26B are a series of graphs showing binding of the parental IgG form of antibody 6C10 (BKM0106) to cells expressing one of two calreticulin mutants (CALR ins and CALR del, as described herein), as assessed by FACS.

FIG. 27 is a graph showing therapeutic efficacy of various antibody molecules in an in vivo murine model of myelofibrosis. Antibody molecules tested included ADCC-enabled antibody molecules against mutant calreticulin (mtCalR), bispecific antibodies comprising a mtCalR-binding domain and a second binding domain specific to another target (i.e., TCRvβ or CD3) and an LALAPG variant Fc region. Naïve mouse spleen and vehicle were used as controls.

FIG. 28 is a table showing in vitro binding of exemplary anti-CD3 antibody molecules BKM0020, BKM0025, BKM0028, BKM0038, as described herein, to human CD3e (huCD3e) and cynomolgus CD3e (cCD3e).

FIG. 29 is a graph showing binding of exemplary anti-CD3 antibody molecule BKM0020, as described herein, to Jurkat cells expressing human CD3e (huCD3e).

FIGS. 30A and 30B are schematics showing the alignments of affinity matured humanized Antibody A-H sequences. FIG. 30A shows the alignment of affinity matured humanized Antibody A-H VL sequences (SEQ ID NOs: 3377-3389, respectively, in order of appearance). FIG. 30B shows the alignment of affinity matured humanized Antibody A-H VH sequences (SEQ ID NOS 3390-3436, respectively, in order of appearance).

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are multifunctional molecules (also referred to herein as “multispecific molecules”) that include a plurality of (e.g., two or more) functionalities (or binding specificities), comprising (i) an antigen binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), e.g., wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, and (ii) one, two, or all of: (a) an immune cell engager chosen from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (b) a cytokine molecule; (c) a stromal modifying moiety, and (d) a tumor-targeting moiety (e.g., which binds to a tumor antigen chosen from: G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1). In some embodiments, the antigen binding domain binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a mutant calreticulin protein, e.g., as described herein). In some embodiments, the antigen binding domain binds to a calreticulin mutant protein disclosed in Table 2 or Table 3. In some embodiments, the antigen binding domain binds to Type 1 calreticulin mutant protein disclosed in Table 2 or Table 3. In some embodiments, the antigen binding domain binds to Type 2 calreticulin mutant protein disclosed in Table 2 or Table 3. In some embodiments, the antigen binding domain binds to both Type 1 and Type 2 calreticulin mutant proteins disclosed in Table 2 or Table 3. In some embodiments, the T cell engager comprises an additional antigen binding domain that binds to the variable chain of the beta subunit of TCR (TCR PV), e.g., a TCR p V6 or TCR p V12.

In an embodiment, the multispecific or multifunctional molecule is a bispecific (or bifunctional) molecule, a trispecific (or trifunctional) molecule, or a tetraspecific (or tetrafunctional) molecule. In an embodiment, the multispecific or multifunctional molecule is a bispecific molecule.

Without being bound by theory, the multispecific or multifunctional molecules disclosed herein are expected to localize (e.g., bridge) and/or activate an immune cell (e.g., an immune effector cell chosen from a T cell, an NK cell, a B cell, a dendritic cell or a macrophage), in the presence of a cell expressing the calreticulin protein, e.g., on the surface. Increasing the proximity and/or activity of the immune cell, in the presence of the cell expressing the calreticulin protein, using the multispecific or multifunctional molecules described herein is expected to enhance an immune response against the target cell, thereby providing a more effective therapy.

Novel multifunctional, e.g., multispecific, molecules that include (i) a stromal modifying moiety and (ii) an antigen binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein and/or a calreticulin mutant protein), e.g., wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286 are disclosed. Without being bound by theory, the multifunctional molecules disclosed herein are believed to inter alia target (e.g., localize to) a cancer site, and alter the tumor stroma, e.g., alter the tumor microenvironment near the cancer site. The multifunctional molecules can further include one or both of: an immune cell engager (e.g., chosen from one, two, three, or all of a T cell engager, NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); and/or a cytokine molecule. Accordingly, provided herein are, inter alia, multifunctional, e.g., multispecific molecules, that include the aforesaid moieties, nucleic acids encoding the same, methods of producing the aforesaid molecules, and methods of treating a cancer using the aforesaid molecules.

Accordingly, provided herein are, inter alia, multispecific or multifunctional molecules (e.g., multispecific or multifunctional antibody molecules) that include the aforesaid moieties, nucleic acids encoding the same, methods of producing the aforesaid molecules, and methods of treating a disease or disorder, e.g., cancer, using the aforesaid molecules.

Definitions

In some embodiments, the multifunctional molecule includes an immune cell engager. “An immune cell engager” refers to one or more binding specificities that bind and/or activate an immune cell, e.g., a cell involved in an immune response. In some embodiments, the immune cell is chosen from a T cell, an NK cell, a B cell, a dendritic cell, and/or the macrophage cell. The immune cell engager can be an antibody molecule, a receptor molecule (e.g., a full length receptor, receptor fragment, or fusion thereof (e.g., a receptor-Fc fusion)), or a ligand molecule (e.g., a full length ligand, ligand fragment, or fusion thereof (e.g., a ligand-Fc fusion)) that binds to the immune cell antigen (e.g., the T cell, the NK cell antigen, the B cell antigen, the dendritic cell antigen, and/or the macrophage cell antigen). In some embodiments, the immune cell engager specifically binds to the target immune cell, e.g., binds preferentially to the target immune cell. For example, when the immune cell engager is an antibody molecule, it binds to an immune cell antigen (e.g., a T cell antigen, an NK cell antigen, a B cell antigen, a dendritic cell antigen, and/or a macrophage cell antigen) with a dissociation constant of less than about 10 nM.

As used herein, the terms “T cell receptor beta variable chain,” “TCRVβ,” “TCRVb,” and “TCRβV” are used interchangeably to refer to an extracellular region of the T cell receptor beta chain which comprises the antigen recognition domain of the T cell receptor. The term TCRVβ or TCRβV includes isoforms, mammalian, e.g., human TCRBV, species homologs of human and analogs comprising at least one common epitope with TCRBV. Human TCRβV comprises a gene family comprising subfamilies including, but not limited to: a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, or a TCRβ V29 subfamily. In some embodiments, the TCRβ V6 subfamily comprises: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01. In some embodiments, TCRβV comprises TCRβ V6-5*01. TCRβ V6-5*01 is also known as TRBV65; TCRBV6S5; TCRBV13S1, or TCRβ V13.1. The amino acid sequence of TCRβ V6-5*01, e.g., human TCRβ V6-5*01, is known in that art, e.g., as provided by IMGT ID L36092. In some embodiments, TCRβ V6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO: 1043, or a sequence having 85%, 90%, 95%, 99% or more identity thereof. In some embodiments, TCRβ V6-5*01 comprises the amino acid sequence of SEQ ID NO: 1044, or a sequence having 85%, 90%, 95%, 99% or more identity thereof.

In some embodiments, the multifunctional molecule includes a cytokine molecule. As used herein, a “cytokine molecule” refers to full length, a fragment or a variant of a cytokine; a cytokine further comprising a receptor domain, e.g., a cytokine receptor dimerizing domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor, that elicits at least one activity of a naturally-occurring cytokine. In some embodiments the cytokine molecule is chosen from interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or a fragment or variant thereof, or a combination of any of the aforesaid cytokines. The cytokine molecule can be a monomer or a dimer. In some embodiments, the cytokine molecule can further include a cytokine receptor dimerizing domain. In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor chosen from an IL-15Ra or IL-21R.

As used herein, the term “molecule” as used in, e.g., antibody molecule, cytokine molecule, receptor molecule, includes full-length, naturally-occurring molecules, as well as variants, e.g., functional variants (e.g., truncations, fragments, mutated (e.g., substantially similar sequences) or derivatized form thereof), so long as at least one function and/or activity of the unmodified (e.g., naturally-occurring) molecule remains.

In some embodiments, the multifunctional molecule includes a stromal modifying moiety. A “stromal modifying moiety,” as used herein refers to an agent, e.g., a protein (e.g., an enzyme), that is capable of altering, e.g., degrading a component of, the stroma. In some embodiments, the component of the stroma is chosen from, e.g., an ECM component, e.g., a glycosaminoglycan, e.g., hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan and keratin sulfate; or an extracellular protein, e.g., collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

Certain terms are defined below.

As used herein, the articles “a” and “an” refer to one or more than one, e.g., to at least one, of the grammatical object of the article. The use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

As used herein, “about” and “approximately” generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.

“Antibody molecule” as used herein refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. An antibody molecule encompasses antibodies (e.g., full-length antibodies) and antibody fragments. In an embodiment, an antibody molecule comprises an antigen binding or functional fragment of a full-length antibody, or a full-length immunoglobulin chain. For example, a full-length antibody is an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that is naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes). In some embodiments, an antibody molecule refers to an immunologically active, antigen-binding portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment, e.g., functional fragment, is a portion of an antibody, e.g., Fab, Fab′, F(ab′)₂, F(ab)₂, variable fragment (Fv), domain antibody (dAb), or single chain variable fragment (scFv). A functional antibody fragment binds to the same antigen as that recognized by the intact (e.g., full-length) antibody. The terms “antibody fragment” or “functional fragment” also include isolated fragments consisting of the variable regions, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). In some embodiments, an antibody fragment does not include portions of antibodies without antigen binding activity, such as Fc fragments or single amino acid residues. Exemplary antibody molecules include full length antibodies and antibody fragments, e.g., dAb (domain antibody), single chain, Fab, Fab′, and F(ab′)₂fragments, and single chain variable fragments (scFvs).

As used herein, an “immunoglobulin variable domain sequence” refers to an amino acid sequence which can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally-occurring variable domain. For example, the sequence may or may not include one, two, or more N- or C-terminal amino acids, or may include other alterations that are compatible with formation of the protein structure.

In some embodiments, an antibody molecule is monospecific, e.g., it comprises binding specificity for a single epitope. In some embodiments, an antibody molecule is multispecific, e.g., it comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence has binding specificity for a first epitope and a second immunoglobulin variable domain sequence has binding specificity for a second epitope. In some embodiments, an antibody molecule is a bispecific antibody molecule. “Bispecific antibody molecule” as used herein refers to an antibody molecule that has specificity for more than one (e.g., two, three, four, or more) epitope and/or antigen.

“Antigen” (Ag) as used herein refers to a molecule that can provoke an immune response, e.g., involving activation of certain immune cells and/or antibody generation. Any macromolecule, including almost all proteins or peptides, can be an antigen. Antigens can also be derived from genomic recombinant or DNA. For example, any DNA comprising a nucleotide sequence or a partial nucleotide sequence that encodes a protein capable of eliciting an immune response encodes an “antigen.” In some embodiments, an antigen does not need to be encoded solely by a full-length nucleotide sequence of a gene, nor does an antigen need to be encoded by a gene at all. In some embodiments, an antigen can be synthesized or can be derived from a biological sample, e.g., a tissue sample, a tumor sample, a cell, or a fluid with other biological components. As used, herein a “tumor antigen” or interchangeably, a “cancer antigen” includes any molecule present on, or associated with, a cancer, e.g., a cancer cell or a tumor microenvironment that can provoke an immune response. As used, herein an “immune cell antigen” includes any molecule present on, or associated with, an immune cell that can provoke an immune response.

The “antigen-binding site,” or “binding portion” of an antibody molecule refers to the part of an antibody molecule, e.g., an immunoglobulin (Ig) molecule, that participates in antigen binding. In some embodiments, the antigen binding site is formed by amino acid residues of the variable (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches within the variable regions of the heavy and light chains, referred to as hypervariable regions, are disposed between more conserved flanking stretches called “framework regions,” (FRs). FRs are amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In some embodiments, in an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface, which is complementary to the three-dimensional surface of a bound antigen. The three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” The framework region and CDRs have been defined and described, e.g., in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917. Each variable chain (e.g., variable heavy chain and variable light chain) is typically made up of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the amino acid order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

“Cancer” as used herein can encompass all types of oncogenic processes and/or cancerous growths. In some embodiments, cancer includes primary tumors as well as metastatic tissues or malignantly transformed cells, tissues, or organs. In some embodiments, cancer encompasses all histopathologies and stages, e.g., stages of invasiveness/severity, of a cancer. In some embodiments, cancer includes relapsed and/or resistant cancer. The terms “cancer” and “tumor” can be used interchangeably. For example, both terms encompass solid and liquid tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

As used herein, an “immune cell” refers to any of various cells that function in the immune system, e.g., to protect against agents of infection and foreign matter. In some embodiments, this term includes leukocytes, e.g., neutrophils, eosinophils, basophils, lymphocytes, and monocytes. Innate leukocytes include phagocytes (e.g., macrophages, neutrophils, and dendritic cells), mast cells, eosinophils, basophils, and natural killer cells. Innate leukocytes identify and eliminate pathogens, either by attacking larger pathogens through contact or by engulfing and then killing microorganisms, and are mediators in the activation of an adaptive immune response. The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are important types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow. B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response. The term “immune cell” includes immune effector cells.

“Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include, but are not limited to, T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NK T) cells, and mast cells.

The term “effector function” or “effector response” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.

The compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence provided herein.

The term “variant” refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

The term “functional variant” refers to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and is capable of having one or more activities of the reference amino acid sequence.

Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

It is understood that the molecules of the present invention may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.

The term “amino acid” is intended to embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Exemplary amino acids include naturally-occurring amino acids; analogs, derivatives and congeners thereof, amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” includes both the D- or L-optical isomers and peptidomimetics.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms “polypeptide”, “peptide” and “protein” (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. The polypeptide can be isolated from natural sources, can be a produced by recombinant techniques from a eukaryotic or prokaryotic host, or can be a product of synthetic procedures.

The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence,” and “polynucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be either single-stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

The term “isolated,” as used herein, refers to material that is removed from its original or native environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated by human intervention from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of the environment in which it is found in nature.

As used herein, the term “transforming growth factor beta-1 (TGF-beta 1)” refers to a protein that in humans is encoded by the gene TGFB1, or its orthologs. Swiss-Prot accession number P01137 provides exemplary human TGF-beta 1 amino acid sequences. An exemplary immature human TGF-beta 1 amino acid sequence is provided in SEQ ID NO: 6378. An exemplary mature human TGF-beta 1 amino acid sequence is provided in SEQ ID NO: 6395.

As used herein, the term “transforming growth factor beta-2 (TGF-beta 2)” refers to a protein that in humans is encoded by the gene TGFB2, or its orthologs. Swiss-Prot accession number P61812 provides exemplary human TGF-beta 2 amino acid sequences. An exemplary immature human TGF-beta 2 amino acid sequence is provided in SEQ ID NO: 6379. An exemplary mature human TGF-beta 2 amino acid sequence is provided in SEQ ID NO: 6396.

As used herein, the term “transforming growth factor beta-3 (TGF-beta 3)” refers to a protein that in humans is encoded by the gene TGFB3, or its orthologs. Swiss-Prot accession number P10600 provides exemplary human TGF-beta 3 amino acid sequences. An exemplary immature human TGF-beta 3 amino acid sequence is provided in SEQ ID NO: 6380. An exemplary mature human TGF-beta 3 amino acid sequence is provided in SEQ ID NO: 6397.

As used herein, a “TGF-beta receptor polypeptide” refers to a TGF-beta receptor (e.g., TGFBR1, TGFBR2, or TGFBR3) or its fragment, or variant thereof.

As used herein, the term “transforming growth factor beta receptor type 1 (TGFBR1)” (also known as ALK-5 or SKR4) refers to a protein that in humans is encoded by the gene TGFBR1, or its orthologs. Swiss-Prot accession number P36897 provides exemplary human TGFBR1 amino acid sequences. Exemplary immature human TGFBR1 amino acid sequences are provided in SEQ ID NOs: 6381, 6382, and 6383. Exemplary mature human TGFBR1 amino acid sequences are provided in SEQ ID NOs: 6398, 6399, and 6400. As used herein, a “TGFBR1 polypeptide” refers to a TGFBR1 or its fragment, or variant thereof.

As used herein, the term “transforming growth factor beta receptor type 2 (TGFBR2)” refers to a protein that in humans is encoded by the gene TGFBR2, or its orthologs. Swiss-Prot accession number P37173 provides exemplary human TGFBR2 amino acid sequences. Exemplary immature human TGFBR2 amino acid sequences are provided in SEQ ID NOs: 6384 and 6385. Exemplary mature human TGFBR2 amino acid sequences are provided in SEQ ID NOs: 6401 and 6402. As used herein, a “TGFBR2 polypeptide” refers to a TGFBR2 or its fragment, or variant thereof.

As used herein, the term “transforming growth factor beta receptor type 3 (TGFBR3)” refers to a protein that in humans is encoded by the gene TGFBR3, or its orthologs. Swiss-Prot accession number Q03167 provides exemplary human TGFBR3 amino acid sequences. Exemplary immature human TGFBR3 amino acid sequences are provided in SEQ ID NOs: 6392 and 6393. Exemplary mature human TGFBR3 amino acid sequences are provided in SEQ ID NOs: 6403 and 6404. As used herein, a “TGFBR3 polypeptide” refers to a TGFBR3 or its fragment, or variant thereof.

Various aspects of the invention are described in further detail below. Additional definitions are set out throughout the specification.

Antibody Molecules

In some embodiments, a multifunctional molecule, multispecific molecule, and/or an antigen binding domain as described herein comprises an antibody molecule. In one embodiment, the antibody molecule binds to a cancer antigen, e.g., a tumor antigen or a stromal antigen. In some embodiments, the cancer antigen is, e.g., a mammalian, e.g., a human, cancer antigen. In other embodiments, the antibody molecule binds to an immune cell antigen, e.g., a mammalian, e.g., a human, immune cell antigen. For example, the antibody molecule binds specifically to an epitope, e.g., linear or conformational epitope, on the cancer antigen or the immune cell antigen.

In an embodiment, an antibody molecule is a monospecific antibody molecule and binds a single epitope. E.g., a monospecific antibody molecule having a plurality of immunoglobulin variable domain sequences, each of which binds the same epitope.

In an embodiment an antibody molecule is a multispecific or multifunctional antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domains sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a multispecific antibody molecule comprises a third, fourth or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

In an embodiment a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a scFv or a Fab, or fragment thereof, have binding specificity for a first epitope and a scFv or a Fab, or fragment thereof, have binding specificity for a second epitope.

In an embodiment, an antibody molecule comprises a diabody, and a single-chain molecule, as well as an antigen-binding fragment of an antibody (e.g., Fab, F(ab′)₂, and Fv). For example, an antibody molecule can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In an embodiment an antibody molecule comprises or consists of a heavy chain and a light chain (referred to herein as a half antibody. In another example, an antibody molecule includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab′, F(ab′)₂, Fc, Fd, Fd′, Fv, single chain antibodies (scFv for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen or receptor. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4) of antibodies. The a preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule can also be a human, humanized, CDR-grafted, or in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from, e.g., kappa or lambda. The term “immunoglobulin” (Ig) is used interchangeably with the term “antibody” herein.

Examples of antigen-binding fragments of an antibody molecule include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv), see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

Antibody molecules include intact molecules as well as functional fragments thereof. Constant regions of the antibody molecules can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).

Antibody molecules can also be single domain antibodies. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine. According to another aspect of the invention, a single domain antibody is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example. For clarity reasons, this variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; such VHHs are within the scope of the invention.

The VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR or FW).

The extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).

The terms “complementarity determining region,” and “CDR,” as used herein refer to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. In general, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme). As used herein, the CDRs defined according the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”

For example, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).

Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The antibody molecule can be a polyclonal or a monoclonal antibody.

The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody can be made by hybridoma technology or by methods that do not use hybridoma technology (e.g., recombinant methods).

The antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods.

Phage display and combinatorial methods for generating antibodies are known in the art (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) JMol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated by reference herein).

In one embodiment, the antibody is a fully human antibody (e.g., an antibody made in a mouse which has been genetically engineered to produce an antibody from a human immunoglobulin sequence), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat antibody). Methods of producing rodent antibodies are known in the art.

Human monoclonal antibodies can be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. International Application WO 92/03918; Kay et al. International Application 92/03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, L. L. et al. 1994 Nature Genet. 7:13-21; Morrison, S. L. et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326).

An antibody molecule can be one in which the variable region, or a portion thereof, e.g., the CDRs, are generated in a non-human organism, e.g., a rat or mouse. Chimeric, CDR-grafted, and humanized antibodies are within the invention. Antibody molecules generated in a non-human organism, e.g., a rat or mouse, and then modified, e.g., in the variable framework or constant region, to decrease antigenicity in a human are within the invention.

An “effectively human” protein is a protein that does substantially not evoke a neutralizing antibody response, e.g., the human anti-murine antibody (HAMA) response. HAMA can be problematic in a number of circumstances, e.g., if the antibody molecule is administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A HAMA response can make repeated antibody administration potentially ineffective because of an increased antibody clearance from the serum (see, e.g., Saleh et al., Cancer Immunol. Immunother., 32:180-190 (1990)) and also because of potential allergic reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).

Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J Natl Cancer Inst. 80:1553-1559).

A humanized or CDR-grafted antibody will have at least one or two but generally all three recipient CDRs (of heavy and or light immuoglobulin chains) replaced with a donor CDR. The antibody may be replaced with at least a portion of a non-human CDR or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding to the antigen. Preferably, the donor will be a rodent antibody, e.g., a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, the immunoglobulin providing the CDRs is called the “donor” and the immunoglobulin providing the framework is called the “acceptor.” In one embodiment, the donor immunoglobulin is a non-human (e.g., rodent). The acceptor framework is a naturally-occurring (e.g., a human) framework or a consensus framework, or a sequence about 85% or higher, preferably 90%, 95%, 99% or higher identical thereto.

As used herein, the term “consensus sequence” refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A “consensus framework” refers to the framework region in the consensus immunoglobulin sequence.

An antibody molecule can be humanized by methods known in the art (see e.g., Morrison, S. L., 1985, Science 229:1202-1207, by Oi et al., 1986, BioTechniques 4:214, and by Queen et al. U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, the contents of all of which are hereby incorporated by reference).

Humanized or CDR-grafted antibody molecules can be produced by CDR-grafting or CDR substitution, wherein one, two, or all CDRs of an immunoglobulin chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science 239:1534; Beidler et al. 1988 J. Immunol. 141:4053-4060; Winter U.S. Pat. No. 5,225,539, the contents of all of which are hereby expressly incorporated by reference. Winter describes a CDR-grafting method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987; Winter U.S. Pat. No. 5,225,539), the contents of which is expressly incorporated by reference.

Also within the scope of the invention are humanized antibody molecules in which specific amino acids have been substituted, deleted or added. Criteria for selecting amino acids from the donor are described in U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, e.g., columns 12-16 of U.S. Pat. No. 5,585,089, the contents of which are hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al. EP 519596 A1, published on Dec. 23, 1992.

The antibody molecule can be a single chain antibody. A single-chain antibody (scFv) may be engineered (see, for example, Colcher, D. et al. (1999) Ann N YAcad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). The single chain antibody can be dimerized or multimerized to generate multivalent antibodies having specificities for different epitopes of the same target protein.

In yet other embodiments, the antibody molecule has a heavy chain constant region chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly, chosen from, e.g., the (e.g., human) heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In another embodiment, the antibody molecule has a light chain constant region chosen from, e.g., the (e.g., human) light chain constant regions of kappa or lambda. The constant region can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and/or complement function). In one embodiment the antibody has: effector function; and can fix complement. In other embodiments the antibody does not; recruit effector cells; or fix complement. In another embodiment, the antibody has reduced or no ability to bind an Fc receptor. For example, it is a isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region.

Methods for altering an antibody constant region are known in the art. Antibodies with altered function, e.g. altered affinity for an effector ligand, such as FcR on a cell, or the C1 component of complement can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see e.g., EP 388,151 Al, U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of all of which are hereby incorporated by reference). Similar type of alterations could be described which if applied to the murine, or other species immunoglobulin would reduce or eliminate these functions.

An antibody molecule can be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a “derivatized” antibody molecule is one that has been modified. Methods of derivatization include but are not limited to the addition of a fluorescent moiety, a radionucleotide, a toxin, an enzyme or an affinity ligand such as biotin. Accordingly, the antibody molecules of the invention are intended to include derivatized and otherwise modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody molecule is produced by crosslinking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

Multispecific or Multifunctional Antibody Molecules

Exemplary structures of multispecific and multifunctional molecules defined herein are described throughout. Exemplary structures are further described in: Weidle U et al. (2013) The Intriguing Options of Multispecific Antibody Formats for Treatment of Cancer. Cancer Genomics & Proteomics 10: 1-18 (2013); and Spiess C et al. (2015) Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular Immunology 67: 95-106; the full contents of each of which is incorporated by reference herein).

In some embodiments, multispecific antibody molecules can comprise more than one antigen-binding site, where different sites are specific for different antigens. In some embodiments, multispecific antibody molecules can bind more than one (e.g., two or more) epitopes on the same antigen. In some embodiments, multispecific antibody molecules comprise an antigen-binding site specific for a target cell (e.g., cancer cell) and a different antigen-binding site specific for an immune effector cell. In some embodiments, the multispecific antibody molecule is a bispecific, trispecific, or tetraspecific antibody molecule. In one embodiment, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG appended with an additional antigen-binding moiety; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates.

BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include but are not limited to crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-holes assembly, charge pair, Fab-arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm; and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises heavy chains that are engineered for heterodimerization. For example, heavy chains can be engineered for heterodimerization using a “knobs-into-holes” strategy, a SEED platform, a common heavy chain (e.g., in κλ-bodies), and use of heterodimeric Fc regions. See Spiess et al. Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knobs-in-holes, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See Id. BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification/assembly into a BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, e.g., using protein A and sequential pH elution.

IgG appended with an additional antigen-binding moiety is another format of bispecific antibody molecules. For example, monospecific IgG can be engineered to have bispecificity by appending an additional antigen-binding unit onto the monospecific IgG, e.g., at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy chain or variable light chain), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). See Id. Examples of appended IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds IL-1α and IL-1β; and ABT-122 (AbbVie), which binds TNF and IL-17A.

Bispecific antibody fragments (BsAb) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAb lack an Fc region. In some embodiments, bispecific antibody fragments include heavy and light chain regions that are connected by a peptide linker that permits efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include but are not limited to nanobody, nanobody-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab′)₂, F(ab′)₂-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, and intrabody. See Id. For example, the BiTE format comprises tandem scFvs, where the component scFvs bind to CD3 on T cells and a surface antigen on cancer cells

Bispecific fusion proteins include antibody fragments linked to other proteins, e.g., to add additional specificity and/or functionality. An example of a bispecific fusion protein is an immTAC, which comprises an anti-CD3 scFv linked to an affinity-matured T-cell receptor that recognizes HLA-presented peptides. In some embodiments, the dock-and-lock (DNL) method can be used to generate bispecific antibody molecules with higher valency. Also, fusions to albumin binding proteins or human serum albumin can be extend the serum half-life of antibody fragments. See Id.

In some embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and/or antibody fragments, can be used to create BsAb molecules. See Id. An exemplary bispecific antibody conjugate includes the CovX-body format, in which a low molecular weight drug is conjugated site-specifically to a single reactive lysine in each Fab arm or an antibody or fragment thereof. In some embodiments, the conjugation improves the serum half-life of the low molecular weight drug. An exemplary CovX-body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides inhibiting either VEGF or Ang2. See Id.

The antibody molecules can be produced by recombinant expression, e.g., of at least one or more component, in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification/assembly. Alternatively, the antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods such as affinity chromatography, e.g., using protein A and sequential pH elution. In other embodiments, affinity tags can be used for purification, e.g., histidine-containing tag, myc tag, or streptavidin tag.

CDR-Grafted Scaffolds

In some embodiments, the antibody molecule is a CDR-grafted scaffold domain. In some embodiments, the scaffold domain is based on a fibronectin domain, e.g., fibronectin type III domain. The overall fold of the fibronectin type III (Fn3) domain is closely related to that of the smallest functional antibody fragment, the variable domain of the antibody heavy chain. There are three loops at the end of Fn3; the positions of BC, DE and FG loops approximately correspond to those of CDR1, 2 and 3 of the VH domain of an antibody. Fn3 does not have disulfide bonds; and therefore Fn3 is stable under reducing conditions, unlike antibodies and their fragments (see, e.g., WO 98/56915; WO 01/64942; WO 00/34784). An Fn3 domain can be modified (e.g., using CDRs or hypervariable loops described herein) or varied, e.g., to select domains that bind to an antigen/marker/cell described herein.

In some embodiments, a scaffold domain, e.g., a folded domain, is based on an antibody, e.g., a “minibody” scaffold created by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). The “minibody” can be used to present two hypervariable loops. In some embodiments, the scaffold domain is a V-like domain (see, e.g., Coia et al. WO 99/45110) or a domain derived from tendamistatin, which is a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460). For example, the loops of tendamistatin can be modified (e.g., using CDRs or hypervariable loops) or varied, e.g., to select domains that bind to a marker/antigen/cell described herein. Another exemplary scaffold domain is a beta-sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00/60070).

Other exemplary scaffold domains include but are not limited to T-cell receptors; MHC proteins; extracellular domains (e.g., fibronectin Type III repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zinc finger domains; DNA-binding proteins; particularly monomeric DNA binding proteins; RNA binding proteins; enzymes, e.g., proteases (particularly inactivated proteases), RNase; chaperones, e.g., thioredoxin, and heat shock proteins; and intracellular signaling domains (such as SH2 and SH3 domains). See, e.g., US 20040009530 and U.S. Pat. No. 7,501,121, incorporated herein by reference.

In some embodiments, a scaffold domain is evaluated and chosen, e.g., by one or more of the following criteria: (1) amino acid sequence, (2) sequences of several homologous domains, (3) 3-dimensional structure, and/or (4) stability data over a range of pH, temperature, salinity, organic solvent, oxidant concentration. In some embodiments, the scaffold domain is a small, stable protein domain, e.g., a protein of less than 100, 70, 50, 40 or 30 amino acids. The domain may include one or more disulfide bonds or may chelate a metal, e.g., zinc.

Antibody-Based Fusions

A variety of formats can be generated which contain additional binding entities attached to the N or C terminus of antibodies. These fusions with single chain or disulfide stabilized Fvs or Fabs result in the generation of tetravalent molecules with bivalent binding specificity for each antigen. Combinations of scFvs and scFabs with IgGs enable the production of molecules which can recognize three or more different antigens.

Antibody-Fab Fusion

Antibody-Fab fusions are bispecific antibodies comprising a traditional antibody to a first target and a Fab to a second target fused to the C terminus of the antibody heavy chain. Commonly the antibody and the Fab will have a common light chain. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. It seems like the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15:159.

Antibody-scFv Fusion

Antibody-scFv Fusions are bispecific antibodies comprising a traditional antibody and a scFv of unique specificity fused to the C terminus of the antibody heavy chain. The scFv can be fused to the C terminus through the Heavy Chain of the scFv either directly or through a linker peptide. Antibody fusions can be produced by (1) engineering the DNA sequence of the target fusion, and (2) transfecting the target DNA into a suitable host cell to express the fusion protein. It seems like the antibody-scFv fusion may be linked by a (Gly)-Ser linker between the C-terminus of the CH3 domain and the N-terminus of the scFv, as described by Coloma, J. et al. (1997) Nature Biotech 15:159.

Variable Domain Immunoglobulin DVD

A related format is the dual variable domain immunoglobulin (DVD), which are composed of VH and VL domains of a second specificity place upon the N termini of the V domains by shorter linker sequences.

Other exemplary multispecific antibody formats include, e.g., those described in the following US20160114057A1, US20130243775A1, US20140051833, US20130022601, US20150017187A1, US20120201746A1, US20150133638A1, US20130266568A1, US20160145340A1, WO2015127158A1, US20150203591A1, US20140322221A1, US20130303396A1, US20110293613, US20130017200A1, US20160102135A1, WO2015197598A2, WO2015197582A1, U.S. Pat. No. 9,359,437, US20150018529, WO2016115274A1, WO2016087416A1, US20080069820A1, U.S. Pat. Nos. 9,145,588B, 7,919,257, and US20150232560A1. Exemplary multispecific molecules utilizing a full antibody-Fab/scFab format include those described in the following, U.S. Pat. No. 9,382,323B2, US20140072581A1, US20140308285A1, US20130165638A1, US20130267686A1, US20140377269A1, U.S. Pat. No. 7,741,446B2, and WO1995009917A1. Exemplary multispecific molecules utilizing a domain exchange format include those described in the following, US20150315296A1, WO2016087650A1, US20160075785A1, WO2016016299A1, US20160130347A1, US20150166670, U.S. Pat. No. 8,703,132B2, US20100316645, U.S. Pat. No. 8,227,577B2, US20130078249.

Fc-Containing Entities (Mini-Antibodies)

Fc-containing entities, also known as mini-antibodies, can be generated by fusing scFv to the C-termini of constant heavy region domain 3 (CH3-scFv) and/or to the hinge region (scFv-hinge-Fc) of an antibody with a different specificity. Trivalent entities can also be made which have disulfide stabilized variable domains (without peptide linker) fused to the C-terminus of CH3 domains of IgGs.

Fc-Containing Multispecific Molecules

In some embodiments, the multispecific molecules disclosed herein includes an immunoglobulin constant region (e.g., an Fc region). Exemplary Fc regions can be chosen from the heavy chain constant regions of IgG1, IgG2, IgG3 or IgG4; more particularly, the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.

In some embodiments, the immunoglobulin chain constant region (e.g., the Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function.

In other embodiments, an interface of a first and second immunoglobulin chain constant regions (e.g., a first and a second Fc region) is altered, e.g., mutated, to increase or decrease dimerization, e.g., relative to a non-engineered interface, e.g., a naturally-occurring interface. For example, dimerization of the immunoglobulin chain constant region (e.g., the Fc region) can be enhanced by providing an Fc interface of a first and a second Fc region with one or more of: a paired protuberance-cavity (“knob-in-a hole”), an electrostatic interaction, or a strand-exchange, such that a greater ratio of heteromultimer to homomultimer forms, e.g., relative to a non-engineered interface.

In some embodiments, the multispecific molecules include a paired amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, e.g., of the Fc region of human IgG1 For example, the immunoglobulin chain constant region (e.g., Fc region) can include a paired an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), and T366W (e.g., corresponding to a protuberance or knob).

In other embodiments, the multifunctional molecule includes a half-life extender, e.g., a human serum albumin or an antibody molecule to human serum albumin.

Heterodimerized Antibody Molecules & Methods of Making

Various methods of producing multispecific antibodies have been disclosed to address the problem of incorrect heavy chain pairing. Exemplary methods are described below. Exemplary multispecific antibody formats and methods of making said multispecific antibodies are also disclosed in e.g., Speiss et al. Molecular Immunology 67 (2015) 95-106; and Klein et al mAbs 4:6, 653-663; November/December 2012; the entire contents of each of which are incorporated by reference herein.

Heterodimerized bispecific antibodies are based on the natural IgG structure, wherein the two binding arms recognize different antigens. IgG derived formats that enable defined monovalent (and simultaneous) antigen binding are generated by forced heavy chain heterodimerization, combined with technologies that minimize light chain mispairing (e.g., common light chain). Forced heavy chain heterodimerization can be obtained using, e.g., knob-in-hole OR strand exchange engineered domains (SEED).

Knob-In-Hole

Knob-in-Hole as described in U.S. Pat. Nos. 5,731,116, 7,476,724 and Ridgway, J. et al. (1996) Prot. Engineering 9(7): 617-621, broadly involves: (1) mutating the CH3 domain of one or both antibodies to promote heterodimerization; and (2) combining the mutated antibodies under conditions that promote heterodimerization. “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); “Holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and/or Y407V).

For bispecific antibodies including an Fc domain, introduction of specific mutations into the constant region of the heavy chains to promote the correct heterodimerization of the Fc portion can be utilized. Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety. These techniques include the “knobs-into-holes” (KiH) approach which involves the introduction of a bulky residue into one of the CH3 domains of one of the antibody heavy chains. This bulky residue fits into a complementary “hole” in the other CH3 domain of the paired heavy chain so as to promote correct pairing of heavy chains (see e.g., U.S. Pat. No. 7,642,228).

Exemplary KiH mutations include S354C, T366W in the “knob” heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain. Other exemplary KiH mutations are provided in Table 1, with additional optional stabilizing Fc cysteine mutations.

TABLE 1

Exemplary Fc KiH mutations and optional Cysteine mutations

Position
Knob Mutation
Hole Mutation

T366
T366W
T366S

L368
—
L368A

Y407
—
Y407V

Additional Cysteine Mutations to form a stabilizing disulfide bridge

Position
Knob CH3
Hole CH3

S354
S354C
—

Y349
—
Y349C

Other Fe mutations are provided by Igawa and Tsunoda who identified 3 negatively charged residues in the CH3 domain of one chain that pair with three positively charged residues in the CH3 domain of the other chain. These specific charged residue pairs are: E356-K439, E357-K370, D399-K409 and vice versa. By introducing at least two of the following three mutations in chain A: E356K, E357K and D399K, as well as K370E, K409D, K439E in chain B, alone or in combination with newly identified disulfide bridges, they were able to favor very efficient heterodimerization while suppressing homodimerization at the same time (Martens T et al. A novel one-armed antic-Met antibody inhibits glioblastoma growth in vivo. Clin Cancer Res 2006; 12:6144-52; PMID: 17062691). Xencor defined 41 variant pairs based on combining structural calculations and sequence information that were subsequently screened for maximal heterodimerization, defining the combination of S364H, F405A (HA) on chain A and Y349T, T394F on chain B (TF) (Moore G L et al. A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens. MAbs 2011; 3:546-57; PMID: 22123055).

Other exemplary Fc mutations to promote heterodimerization of multispecific antibodies include those described in the following references, the contents of each of which is incorporated by reference herein, WO2016071377A1, US20140079689A1, US20160194389A1, US20160257763, WO2016071376A2, WO2015107026A1, WO2015107025A1, WO2015107015A1, US20150353636A1, US20140199294A1, U.S. Pat. No. 7,750,128B2, US20160229915A1, US20150344570A1, U.S. Pat. No. 8,003,774A1, US20150337049A1, US20150175707A1, US20140242075A1, US20130195849A1, US20120149876A1, US20140200331A1, U.S. Pat. No. 9,309,311B2, U.S. Pat. No. 8,586,713, US20140037621A1, US20130178605A1, US20140363426A1, US20140051835A1 and US20110054151A1.

Stabilizing cysteine mutations have also been used in combination with KiH and other Fc heterodimerization promoting variants, see e.g., U.S. Pat. No. 7,183,076. Other exemplary cysteine modifications include, e.g., those disclosed in US20140348839A1, U.S. Pat. No. 7,855,275B2, and U.S. Pat. No. 9,000,130B2.

Strand Exchange Engineered Domains (SEED)

Heterodimeric Fc platform that support the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) C(H)3 heterodimers are known. These derivatives of human IgG and IgA C(H)3 domains create complementary human SEED C(H)3 heterodimers that are composed of alternating segments of human IgA and IgG C(H)3 sequences. The resulting pair of SEED C(H)3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-C(H)2-[SEED C(H)3], that may be genetically linked to one or more fusion partners (see e.g., Davis J H et al. SEEDbodies: fusion proteins based on strand exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng Des Sel 2010; 23:195-202; PMID:20299542 and U.S. Pat. No. 8,871,912. The contents of each of which are incorporated by reference herein).

Duobody

“Duobody” technology to produce bispecific antibodies with correct heavy chain pairing are known. The DuoBody technology involves three basic steps to generate stable bispecific human IgG1 antibodies in a post-production exchange reaction. In a first step, two IgG1s, each containing single matched mutations in the third constant (CH3) domain, are produced separately using standard mammalian recombinant cell lines. Subsequently, these IgG1 antibodies are purified according to standard processes for recovery and purification. After production and purification (post-production), the two antibodies are recombined under tailored laboratory conditions resulting in a bispecific antibody product with a very high yield (typically >95%) (see e.g., Labrijn et al, PNAS 2013; 110(13):5145-5150 and Labrijn et al. Nature Protocols 2014; 9(10):2450-63, the contents of each of which are incorporated by reference herein).

Electrostatic Interactions

Methods of making multispecific antibodies using CH3 amino acid changes with charged amino acids such that homodimer formation is electrostatically unfavorable are disclosed. EP1870459 and WO 2009089004 describe other strategies for favoring heterodimer formation upon co-expression of different antibody domains in a host cell. In these methods, one or more residues that make up the heavy chain constant domain 3 (CH3), CH3-CH3 interfaces in both CH3 domains are replaced with a charged amino acid such that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favorable. Additional methods of making multispecific molecules using electrostatic interactions are described in the following references, the contents of each of which is incorporated by reference herein, include US20100015133, U.S. Pat. No. 8,592,562B2, U.S. Pat. No. 9,200,060B2, US20140154254A1, and U.S. Pat. No. 9,358,286A1.

Common Light Chain

Light chain mispairing needs to be avoided to generate homogenous preparations of bispecific IgGs. One way to achieve this is through the use of the common light chain principle, i.e. combining two binders that share one light chain but still have separate specificities. An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable light chain to interact with each of the heteromeric variable heavy chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common light chain as disclosed in, e.g., U.S. Pat. No. 7,183,076B2, US20110177073A1, EP2847231A1, WO2016079081A1, and EP3055329A1, the contents of each of which is incorporated by reference herein.

CrossMab

Another option to reduce light chain mispairing is the CrossMab technology which avoids non-specific L chain mispairing by exchanging CH1 and CL domains in the Fab of one half of the bispecific antibody. Such crossover variants retain binding specificity and affinity, but make the two arms so different that L chain mispairing is prevented. The CrossMab technology (as reviewed in Klein et al. Supra) involves domain swapping between heavy and light chains so as to promote the formation of the correct pairings. Briefly, to construct a bispecific IgG-like CrossMab antibody that could bind to two antigens by using two distinct light chain-heavy chain pairs, a two-step modification process is applied. First, a dimerization interface is engineered into the C-terminus of each heavy chain using a heterodimerization approach, e.g., Knob-into-hole (KiH) technology, to ensure that only a heterodimer of two distinct heavy chains from one antibody (e.g., Antibody A) and a second antibody (e.g., Antibody B) is efficiently formed. Next, the constant heavy 1 (CH1) and constant light (CL) domains of one antibody are exchanged (Antibody A), keeping the variable heavy (VH) and variable light (VL) domains consistent. The exchange of the CH1 and CL domains ensured that the modified antibody (Antibody A) light chain would only efficiently dimerize with the modified antibody (antibody A) heavy chain, while the unmodified antibody (Antibody B) light chain would only efficiently dimerize with the unmodified antibody (Antibody B) heavy chain; and thus only the desired bispecific CrossMab would be efficiently formed (see e.g., Cain, C. SciBX 4(28); doi:10.1038/scibx.2011.783, the contents of which are incorporated by reference herein).

Common Heavy Chain

An exemplary method of enhancing the formation of a desired bispecific antibody from a mixture of monomers is by providing a common variable heavy chain to interact with each of the heteromeric variable light chain regions of the bispecific antibody. Compositions and methods of producing bispecific antibodies with a common heavy chain are disclosed in, e.g., US20120184716, US20130317200, and US20160264685A1, the contents of each of which is incorporated by reference herein.

Amino Acid Modifications

Alternative compositions and methods of producing multispecific antibodies with correct light chain pairing include various amino acid modifications. For example, Zymeworks describes heterodimers with one or more amino acid modifications in the CH1 and/or CL domains, one or more amino acid modifications in the VH and/or VL domains, or a combination thereof, which are part of the interface between the light chain and heavy chain and create preferential pairing between each heavy chain and a desired light chain such that when the two heavy chains and two light chains of the heterodimer pair are co-expressed in a cell, the heavy chain of the first heterodimer preferentially pairs with one of the light chains rather than the other (see e.g., WO2015181805). Other exemplary methods are described in WO2016026943 (Argen-X), US20150211001, US20140072581A1, US20160039947A1, and US20150368352.

Lambda Kappa Formats

Multispecific molecules (e.g., multispecific antibody molecules) that include the lambda light chain polypeptide and a kappa light chain polypeptides, can be used to allow for heterodimerization. Methods for generating bispecific antibody molecules comprising the lambda light chain polypeptide and a kappa light chain polypeptides are disclosed in PCT Publication No. WO2018057955 (corresponding to PCT/US17/53053, filed on Sep. 22, 2017), incorporated herein by reference in its entirety.

In some embodiments, the multispecific molecules includes a multispecific antibody molecule, e.g., an antibody molecule comprising two binding specificities, e.g., a bispecific antibody molecule. The multispecific antibody molecule includes:

- a lambda light chain polypeptide 1 (LLCP1) specific for a first epitope;
  - a heavy chain polypeptide 1 (HCP1) specific for the first epitope;
  - a kappa light chain polypeptide 2 (KLCP2) specific for a second epitope; and
  - a heavy chain polypeptide 2 (HCP2) specific for the second epitope.

“Lambda light chain polypeptide 1 (LLCP1)”, as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence, such that when combined with a cognate heavy chain variable region, can mediate specific binding to its epitope and complex with an HCP1. In an embodiment it comprises all or a fragment of a CH1 region. In an embodiment, an LLCP1 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sufficient sequence therefrom to mediate specific binding of its epitope and complex with an HCP1. LLCP1, together with its HCP1, provide specificity for a first epitope (while KLCP2, together with its HCP2, provide specificity for a second epitope). As described elsewhere herein, LLCP1 has a higher affinity for HCP1 than for HCP2.

“Kappa light chain polypeptide 2 (KLCP2)”, as that term is used herein, refers to a polypeptide comprising sufficient light chain (LC) sequence, such that when combined with a cognate heavy chain variable region, can mediate specific binding to its epitope and complex with an HCP2. In an embodiments it comprises all or a fragment of a CH1 region. In an embodiment, a KLCP2 comprises LC-CDR1, LC-CDR2, LC-CDR3, FR1, FR2, FR3, FR4, and CH1, or sufficient sequence therefrom to mediate specific binding of its epitope and complex with an HCP2. KLCP2, together with its HCP2, provide specificity for a second epitope (while LLCP1, together with its HCP1, provide specificity for a first epitope).

“Heavy chain polypeptide 1 (HCP1)”, as that term is used herein, refers to a polypeptide comprising sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with a cognate LLCP1, can mediate specific binding to its epitope and complex with an HCP1. In an embodiments it comprises all or a fragment of a CH1 region. In an embodiment, it comprises all or a fragment of a CH2 and/or CH3 region. In an embodiment an HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sufficient sequence therefrom to: (i) mediate specific binding of its epitope and complex with an LLCP1, (ii) to complex preferentially, as described herein to LLCP1 as opposed to KLCP2; and (iii) to complex preferentially, as described herein, to an HCP2, as opposed to another molecule of HCP1. HCP1, together with its LLCP1, provide specificity for a first epitope (while KLCP2, together with its HCP2, provide specificity for a second epitope).

“Heavy chain polypeptide 2 (HCP2)”, as that term is used herein, refers to a polypeptide comprising sufficient heavy chain (HC) sequence, e.g., HC variable region sequence, such that when combined with a cognate LLCP1, can mediate specific binding to its epitope and complex with an HCP1. In an embodiments it comprises all or a fragment of a CH1 region. In an embodiments it comprises all or a fragment of a CH2 and/or CH3 region. In an embodiment an HCP1 comprises HC-CDR1, HC-CDR2, HC-CDR3, FR1, FR2, FR3, FR4, CH1, CH2, and CH3, or sufficient sequence therefrom to: (i) mediate specific binding of its epitope and complex with an KLCP2, (ii) to complex preferentially, as described herein to KLCP2 as opposed to LLCP1; and (iii) to complex preferentially, as described herein, to an HCP1, as opposed to another molecule of HCP2. HCP2, together with its KLCP2, provide specificity for a second epitope (while LLCP1, together with its HCP1, provide specificity for a first epitope).

In some embodiments of the multispecific antibody molecule disclosed herein:

- LLCP1 has a higher affinity for HCP1 than for HCP2; and/or
- KLCP2 has a higher affinity for HCP2 than for HCP1.

In some embodiments, the affinity of LLCP1 for HCP1 is sufficiently greater than its affinity for HCP2, such that under preselected conditions, e.g., in aqueous buffer, e.g., at pH 7, in saline, e.g., at pH 7, or under physiological conditions, at least 75, 80, 90, 95, 98, 99, 99.5, or 99.9% of the multispecific antibody molecule molecules have a LLCP1 complexed, or interfaced with, a HCP1.

In some embodiments of the multispecific antibody molecule disclosed herein: the HCP1 has a greater affinity for HCP2, than for a second molecule of HCP1; and/or

- the HCP2 has a greater affinity for HCP1, than for a second molecule of HCP2.

In some embodiments, the affinity of HCP1 for HCP2 is sufficiently greater than its affinity for a second molecule of HCP1, such that under preselected conditions, e.g., in aqueous buffer, e.g., at pH 7, in saline, e.g., at pH 7, or under physiological conditions, at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9% of the multispecific antibody molecule molecules have a HCP1 complexed, or interfaced with, a HCP2.

In another aspect, disclosed herein is a method for making, or producing, a multispecific antibody molecule. The method includes:

- (i) providing a first heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both));
- (ii) providing a second heavy chain polypeptide (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both));
- (iii) providing a lambda chain polypeptide (e.g., a lambda light variable region (VL□), a lambda light constant chain (VL□), or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH); and
- (iv) providing a kappa chain polypeptide (e.g., a lambda light variable region (VL□), a lambda light constant chain (VL□), or both) that preferentially associates with the second heavy chain polypeptide (e.g., the second VH), under conditions where (i)-(iv) associate.

In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization.

In some embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) are introduced in a single cell, e.g., a single mammalian cell, e.g., a CHO cell. In some embodiments, (i)-(iv) are expressed in the cell.

In some embodiments, (i)-(iv) (e.g., nucleic acid encoding (i)-(iv)) are introduced in different cells, e.g., different mammalian cells, e.g., two or more CHO cell. In some embodiments, (i)-(iv) are expressed in the cells.

In one embodiment, the method further comprises purifying a cell-expressed antibody molecule, e.g., using a lambda- and/or kappa-specific purification, e.g., affinity chromatography.

In some embodiments, the method further comprises evaluating the cell-expressed multispecific antibody molecule. For example, the purified cell-expressed multispecific antibody molecule can be analyzed by techniques known in the art, include mass spectrometry. In one embodiment, the purified cell-expressed antibody molecule is cleaved, e.g., digested with papain to yield the Fab moieties and evaluated using mass spectrometry.

In some embodiments, the method produces correctly paired kappa/lambda multispecific, e.g., bispecific, antibody molecules in a high yield, e.g., at least 75%, 80, 90, 95, 98, 99 99.5 or 99.9%.

In other embodiments, the multispecific, e.g., a bispecific, antibody molecule that includes:

- (i) a first heavy chain polypeptide (HCP1) (e.g., a heavy chain polypeptide comprising one, two, three or all of a first heavy chain variable region (first VH), a first CH1, a first heavy chain constant region (e.g., a first CH2, a first CH3, or both)), e.g., wherein the HCP1 binds to a first epitope;
- (ii) a second heavy chain polypeptide (HCP2) (e.g., a heavy chain polypeptide comprising one, two, three or all of a second heavy chain variable region (second VH), a second CH1, a second heavy chain constant region (e.g., a second CH2, a second CH3, or both)), e.g., wherein the HCP2 binds to a second epitope;
- (iii) a lambda light chain polypeptide (LLCP1) (e.g., a lambda light variable region (VL1), a lambda light constant chain (VL1), or both) that preferentially associates with the first heavy chain polypeptide (e.g., the first VH), e.g., wherein the LLCP1 binds to a first epitope; and
- (iv) a kappa light chain polypeptide (KLCP2) (e.g., a lambda light variable region (VLk), a lambda light constant chain (VLk), or both) that preferentially associates with the second heavy chain polypeptide (e.g., the second VH), e.g., wherein the KLCP2 binds to a second epitope.

In some embodiments, the first and second heavy chain polypeptides form an Fc interface that enhances heterodimerization. In some embodiments, the multispecific antibody molecule has a first binding specificity that includes a hybrid VL1-CL1 heterodimerized to a first heavy chain variable region connected to the Fc constant, CH2-CH3 domain (having a knob modification) and a second binding specificity that includes a hybrid VLk-CLk heterodimerized to a second heavy chain variable region connected to the Fc constant, CH2-CH3 domain (having a hole modification).

Calreticulin-Targeting Antigen Binding Domains

The present disclosure provides, inter alia, multispecific (e.g., bi-, tri-, tetra-specific) or multifunctional molecules, that include, e.g., are engineered to contain, one or more antigen binding domains that bind to calreticulin, e.g., a wild-type calreticulin protein or a calreticulin mutant protein. In some embodiments, the multifunctional molecule binds to a wild-type calreticulin protein and a calreticulin mutant protein with similar affinity. In some embodiments, the multifunctional molecule preferentially binds to a calreticulin mutant protein over a wild type calreticulin protein.

An exemplary wild type human calreticulin is shown as SEQ ID NO: 6285.

(SEQ ID NO: 6285)

EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQ

TSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSL

DQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDE

FTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPE

DWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQ

NPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLD

LWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRL

KEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL

An additional exemplary wild type human calreticulin is shown as SEQ ID NO: 1001:

(SEQ ID NO: 1001)

MLLSVPLLLGLLGLAVAHHHHHHHHGGGGSEPAVYFKEQFLDGDGWTSRW

IESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFSNK

GQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIMFGPDIC

GPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYEVKID

NSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKPEDWD

KPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQIDNPDYKG

TWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLITNDEA

YAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEEEEAEDKED

DEDKDEDEEDEEDKEEDEEEDVPGQA

Calreticulin mutant proteins have been identified and found to be associated with myeloid cancers, e.g., see Nangalia et al., N Engl J Med. 2013 Dec. 19; 369(25):2391-2405, Klampfl et al., N Engl J Med. 2013 Dec. 19; 369(25):2379-90, and US20170269092, herein incorporated by reference in their entirety. Mutant calreticulin has a frameshift in exon 9 of the coding sequence of wild type calreticulin, resulting in the replacement of the C-terminal negatively charged amino acids of wild type calreticulin by a predominantly positively charged polypeptide. Table 2 discloses full-length amino acid sequences of 38 calreticulin mutant proteins. Table 3 discloses the C-terminal amino acid sequences of the 36 calreticulin mutant proteins. All 38 calreticulin mutant proteins comprise the amino acid sequence of RRKMSPARPRTSCREACLQGWTEA (SEQ ID NO: 6286).

The predominant mutations of calreticulin are Type 1 and Type 2 mutations (see Tables 2 and 3). Type 1 mutation is a 52-bp deletion (c.1092_1143del) whereas Type 2 mutation is a 5-bp insertion (c.1154_1155insTTGTC).

TABLE 2

Full-length amino acid sequences of calreticulin mutants

SEQ

ID NO
Type
Full length sequences of insertion/deletion frameshift mutations of calreticulin

SEQ
Type 1
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6313

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

WTEA

SEQ
Type 2
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6314

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDNCRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
Type 3
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6315

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

GWTEA

SEQ
Type 4
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6316

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type 5
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6317

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEGQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

WTEA

SEQ
Type 6
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6318

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEERRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

GWTEA

SEQ
Type 7
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6319

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

WTEA

SEQ
Type 8
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6320

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type 9
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:

QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6321

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEERQRTRRMMRTKMRMRRMRRTRRKMRRK

MSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
10
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6322

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDMCRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
11
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6323

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEDQRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

GWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
12
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6324

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
13
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6325

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRQRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
14
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6316

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
15
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6326

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLRRRERTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
16
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6327

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLQRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
17
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6328

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKRRQWTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

ACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
18
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6329

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

WTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
19
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6330

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEERQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCR

EACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
20
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6331

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEGRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPR

TSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
21
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6332

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEAFKRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRT

SCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
22
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6333

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDNAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKM

SPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
23
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6334

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDCVRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSP

ARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
24
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6335

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPR

TSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
25
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6336

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPR

TSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
26
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6337

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKNAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRK

MSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
27
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6338

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKCFAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRK

MSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
28
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6339

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKRRMMRTKMRMRRMRRTRRKMRRKMSPARPRT

SCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
29
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6340

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEPPLCLRRMMRTKMRMRRMRRTRRKMRRKMS

PARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
30
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6341

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEDHPCRRMMRTKMRMRRMRRTRRKMRRKMSP

ARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
31
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6342

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEGNCRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
32
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6343

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDCRRMMRTKMRMRRMRRTRRKMRRK

MSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
33
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6344

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDKCRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
34
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6345

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDTCRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
35
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6346

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDICRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
Type
EPAVYFKEQFLDGDGWTSRWIESKHKSDFGKFVLSSGKFYGDEEKDKGL

ID NO:
36
QTSQDARFYALSASFEPFSNKGQTLVVQFTVKHEQNIDCGGGYVKLFPNS

6344

LDQTDMHGDSEYNIMFGPDICGPGTKKVHVIFNYKGKNVLINKDIRCKD

DEFTHLYTLIVRPDNTYEVKIDNSQVESGSLEDDWDFLPPKKIKDPDASK

PEDWDERAKIDDPTDSKPEDWDKPEHIPDPDAKKPEDWDEEMDGEWEP

PVIQNPEYKGEWKPRQIDNPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGV

LGLDLWQVKSGTIFDNFLITNDEAYAEEFGNETWGVTKAAEKQMKDKQ

DEEQRLKEEEEDKKRKEEEEAEDKCRRMMRTKMRMRRMRRTRRKMRR

KMSPARPRTSCREACLQGWTEA

SEQ
mutCal
MLLSVPLLLGLLGLAVAHHHHHHHHGGGGSEPAVYFKEQFLDGDGWTS

ID NO:
R ins
RWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFS

1002

NKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIMFG

PDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYE

VKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKP

EDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQID

NPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLI

TNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRLKEEEEDKKRKEE

EEAEDNCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

WTEA

SEQ
mutCal
MLLSVPLLLGLLGLAVAHHHHHHHHGGGGSEPAVYFKEQFLDGDGWTS

ID NO:
R del
RWIESKHKSDFGKFVLSSGKFYGDEEKDKGLQTSQDARFYALSASFEPFS

1003

NKGQTLVVQFTVKHEQNIDCGGGYVKLFPNSLDQTDMHGDSEYNIMFG

PDICGPGTKKVHVIFNYKGKNVLINKDIRCKDDEFTHLYTLIVRPDNTYE

VKIDNSQVESGSLEDDWDFLPPKKIKDPDASKPEDWDERAKIDDPTDSKP

EDWDKPEHIPDPDAKKPEDWDEEMDGEWEPPVIQNPEYKGEWKPRQID

NPDYKGTWIHPEIDNPEYSPDPSIYAYDNFGVLGLDLWQVKSGTIFDNFLI

TNDEAYAEEFGNETWGVTKAAEKQMKDKQDEEQRTRRMMRTKMRMR

RMRRTRRKMRRKMSPARPRTSCREACLQGWTEA

TABLE 3

The C-terminal amino acid sequences of calreticulin mutants

SEQ ID

C-terminal sequences of insertion/deletion frameshift mutations of

NO
Type
calreticulin

SEQ ID
Type 1
TRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE

NO: 6287

A

SEQ ID
Type 2
NCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWT

NO: 6288

EA

SEQ ID
Type 3
QRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGW

NO: 6289

TEA

SEQ ID
Type 4
RRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACL

NO: 6290

QGWTEA

SEQ ID
Type 5
GQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

NO: 6291

WTEA

SEQ ID
Type 6
RRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

NO: 6292

GWTEA

SEQ ID
Type 7
RRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA

NO: 6293

SEQ ID
Type 8
RRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

NO: 6292

GWTEA

SEQ ID
Type 9
RQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

NO: 6294

WTEA

SEQ ID
Type 10
MCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWT

NO: 6295

EA

SEQ ID
Type 11
DQRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACL

NO: 6296

QGWTEA

SEQ ID
Type 12
RRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACL

NO: 152

QGWTEA

SEQ ID
Type 13
QRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREAC

NO: 6297

LQGWTEA

SEQ ID
Type 14
RRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACL

NO: 6290

QGWTEA

SEQ ID
Type 15
RRRERTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

NO: 6298

GWTEA

SEQ ID
Type 16
QRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACL

NO: 6299

QGWTEA

SEQ ID
Type 17
RRQWTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

NO: 6300

GWTEA

SEQ ID
Type 18
RMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA

NO: 6301

SEQ ID
Type 19
RQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

NO: 6294

WTEA

SEQ ID
Type 20
GRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACL

NO: 6302

QGWTEA

SEQ ID
Type 21
AFKRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

NO: 6303

GWTEA

SEQ ID
Type 22
NAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCR

NO: 6304

EACLQGWTEA

SEQ ID
Type 23
CVRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCRE

NO: 6305

ACLQGWTEA

SEQ ID
Type 24
RRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQ

NO: 6292

GWTEA

SEQ ID
Type 25
RQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

NO: 6294

WTEA

SEQ ID
Type 26
NAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCR

NO: 6304

EACLQGWTEA

SEQ ID
Type 27
CFAKRRRRQRTRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSC

NO: 6306

REACLQGWTEA

SEQ ID
Type 28
RRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA

NO: 6293

SEQ ID
Type 29
PPLCLRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

NO: 6307

WTEA

SEQ ID
Type 30
DHPCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQG

NO: 6308

WTEA

SEQ ID
Type 31
GNCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGW

NO: 6309

TEA

SEQ ID
Type 32
CRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE

NO: 6310

A

SEQ ID
Type 33
CRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE

NO: 6310

A

SEQ ID
Type 34
TCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWT

NO: 6311

EA

SEQ ID
Type 35
ICRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE

NO: 6312

A

SEQ ID
Type 36
CRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTE

NO: 6310

A

In some embodiments, the calreticulin-targeting antigen binding domain comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Tables 4-7, Table 24, and Table 25.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising one, two, three CDRs from murine 16B11.1 antibody, e.g., as described in Table 4. For example, in some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6358 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6360 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 227 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

Alternatively, or in combination with the calreticulin-targeting antigen binding domain comprising the VH comprising one, two, three CDRs from murine 16B11.1 antibody, the calreticulin-targeting antigen binding domain comprises a VL comprising one, two or three CDRs derived from murine 16B11.1 antibody, e.g., as described in Table 4. For example, in some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 251 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 246 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 248 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 251, a VLCDR2 amino acid sequence of SEQ ID NO: 253, and a VLCDR3 amino acid sequence of SEQ ID NO: 255. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 258, a VLCDR2 amino acid sequence of SEQ ID NO: 260, and a VLCDR3 amino acid sequence of SEQ ID NO: 262. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 265, a VLCDR2 amino acid sequence of SEQ ID NO: 267, and a VLCDR3 amino acid sequence of SEQ ID NO: 269. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 272, a VLCDR2 amino acid sequence of SEQ ID NO: 274, and a VLCDR3 amino acid sequence of SEQ ID NO: 276. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 279, a VLCDR2 amino acid sequence of SEQ ID NO: 281, and a VLCDR3 amino acid sequence of SEQ ID NO: 283.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6253 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6254 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6253, a VHCDR2 amino acid sequence of SEQ ID NO: 6254, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6255.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6259, a VLCDR2 amino acid sequence of SEQ ID NO: 6260, and a VLCDR3 amino acid sequence of SEQ ID NO: 6261.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising one, two, three, or four framework regions from humanized 16B11.1 antibody, e.g., as described in Table 4. For example, in some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6357, a VHFWR2 amino acid sequence of SEQ ID NO: 6359, a VHFWR3 amino acid sequence of SEQ ID NO: 6361, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6273. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6362, a VHFWR2 amino acid sequence of SEQ ID NO: 6363, a VHFWR3 amino acid sequence of SEQ ID NO: 226, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 229, a VHFWR2 amino acid sequence of SEQ ID NO: 6369, a VHFWR3 amino acid sequence of SEQ ID NO: 6371, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6373, a VHFWR2 amino acid sequence of SEQ ID NO: 6369, a VHFWR3 amino acid sequence of SEQ ID NO: 6371, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6374, a VLFWR2 amino acid sequence of SEQ ID NO: 6375, a VLFWR3 amino acid sequence of SEQ ID NO: 247, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 249. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 250, a VLFWR2 amino acid sequence of SEQ ID NO: 252, a VLFWR3 amino acid sequence of SEQ ID NO: 254, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 256. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 257, a VLFWR2 amino acid sequence of SEQ ID NO: 259, a VLFWR3 amino acid sequence of SEQ ID NO: 261, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 263. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 264, a VLFWR2 amino acid sequence of SEQ ID NO: 266, a VLFWR3 amino acid sequence of SEQ ID NO: 268, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 270. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 271, a VLFWR2 amino acid sequence of SEQ ID NO: 273, a VLFWR3 amino acid sequence of SEQ ID NO: 275, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 277. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 278, a VLFWR2 amino acid sequence of SEQ ID NO: 280, a VLFWR3 amino acid sequence of SEQ ID NO: 282, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 284.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, a VHFWR2 amino acid sequence of SEQ ID NO: 6226, a VHFWR3 amino acid sequence of SEQ ID NO: 6228, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, a VLFWR2 amino acid sequence of SEQ ID NO: 6240, a VLFWR3 amino acid sequence of SEQ ID NO: 6242, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6244. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6263, a VHFWR2 amino acid sequence of SEQ ID NO: 6264, a VHFWR3 amino acid sequence of SEQ ID NO: 6265, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277, a VLFWR2 amino acid sequence of SEQ ID NO: 6278, a VLFWR3 amino acid sequence of SEQ ID NO: 6279, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6347 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6347). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6348 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6348). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6349 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6349). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6350 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6350). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6351 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6351). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6352 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6352). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6353 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6353). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6354 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6354). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6355 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6355). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6356 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6356).

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6247). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247, and a VL comprising the amino acid sequence of SEQ ID NO: 6249.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising one, two, or all three CDR sequence as listed in a single row of Table 4, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (e.g., to one, two, or all three of the CDR sequences). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising one, two, or all three CDR sequence as listed in a single row of Table 5, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (e.g., to one, two, or all three of the CDR sequences). In some embodiments, the calreticulin-targeting antigen binding domain comprises: (i) a VH comprising one, two, or all three CDR sequence as listed in a single row of Table 4, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (e.g., to one, two, or all three of the CDR sequences); and (ii) a VL comprising one, two, or all three CDR sequence as listed in a single row of Table 5, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto (e.g., to one, two, or all three of the CDR sequences).

TABLE 4

Exemplary heavy chain CDRs and FWRs of calreticulin-targeting antigen binding domains

Ab ID
VHFWR1
VHCDR1
VHFWR2
VHCDR2
VHFWR3
VHCDR3
VHFWR4

AbH-1H
QVQLVQ
YSFTG
WVRQAP
YISCYN
RVTMTVDT
SSMDY
WGQG

SGAEVK
YYIH
GQELGW
GASSY
SISTAYTEL
(SEQ
TLVTV

KPGASVK
(SEQ
MG (SEQ
NQKFK
SSLRSEDT
ID NO:
SS (SEQ

VSCKASG
ID NO:
ID NO:
G (SEQ
ATYYCA
6255)
ID NO:

(SEQ ID
6253)
6264)
ID NO:
(SEQ ID NO:

228)

NO: 6263)

6254)
6265)

AbH-2H
QVTLKES
YSITSD
WIRQPP
YISYSG
RLSITKDTS
DPPYY
WGQG

GPVLVKP
YAWN
GKALEW
STSYNP
KSQVVLTM
YGS
TTVTV

TETLTLT
(SEQ
LA (SEQ
SLKS
TNMDPVDT
(SEQ
SS (SEQ

CTVSG
ID NO:
ID NO:
(SEQ ID
ATYYCAR
ID NO:
ID NO:

(SEQ ID
6256)
6267)
NO:
(SEQ ID NO:
6258)
6269)

NO: 6266)

6257)
6268)

AbM-1H
EVQLEQS
YSFTG
WVKQS
YISCYN
KATFTVDT
SSMDY
WGQG

GPELVKT
YYIH
HGKSLE
GASSY
SSSTAYMQ
(SEQ
TSVTV

GASVKIS
(SEQ
WIG
NQKFK
FNSLTSGD
ID NO:
SS (SEQ

CKASG
ID NO:
(SEQ ID
G (SEQ
SAVYYCA
6255)
ID NO:

(SEQ ID
6253)
NO: 6271)
ID NO:
(SEQ ID NO:

6273)

NO: 6270)

6254)
6272)

AbM-2H
DVQLQES
YSITSD
WIRQFP
YISYSG
RISITRDTS
DPPYY
WGQG

GPGLVK
YAWN
GNKLEW
STSYNP
KNQFFLQL
YGSNG
TSVTV

NSQSLSL
(SEQ
MG (SEQ
SLKS
NSVTPEDT
T (SEQ
SS (SEQ

TCTVTG
ID NO:
ID NO:
(SEQ ID
ATYYCAR
ID NO:
ID NO:

(SEQ ID
6256)
6275)
NO:
(SEQ ID NO:
6262)
6273)

NO: 6274)

6257)
6276)

Murine
EVKLVES
FSRYD
WVRQTP
TISSGG
RFTISRDNA
HSAYY
WGQG

anti-
GGGLVK
MS
EKRLEW
SYTYYP
RNTLYLQM
VNYEN
TSVTV

calreticulin
PGGSLKL
(SEQ
VA (SEQ
DSVKG
SSLRSEDT
AMDY
SS (SEQ

antibody
SCAASGF
ID NO:
ID NO:
(SEQ ID
ALYYCAR
(SEQ
ID NO:

16B11.1
A
6358)
6359)
NO:
(SEQ ID NO:
ID NO:
6273)

heavy chain
(SEQ ID

6360)
6361)
227)

variable
NO: 6357)

region

Humanized
QVQLVES
FSRYD
WIRQAP
TISSGG
RFTISRDNA
HSAYY
WGQG

anti-
GGGLVK
MS
GKGLEW
SYTYYP
KNSLYLQM
VNYEN
TLVTV

calreticulin
PGGSLRL
(SEQ
VA (SEQ
DSVKG
NSLRAEDT
AMDY
SS

heavy chain
SCAASGF
ID NO:
ID NO:
(SEQ ID
AVYYCAR
(SEQ
(SEQ ID

variable
A (SEQ ID
6358)
6363)
NO:
(SEQ ID NO:
ID NO:
NO:

region
NO: 6362)

6360)
226)
227)
228)

variant 1

Humanized
QVQLVES
FSRYD
WVRQAP
TISSGG
RFTISRDNS
HSAYY
WGQG

anti-
GGGVVQ
MS
GKGLEW
SYTYYP
KNTLYLQ
VNYEN
TLVTV

calreticulin
PGRSLRL
(SEQ
VA (SEQ
DSVKG
MNSLRAED
AMDY
SS (SEQ

heavy chain
SCAASGF
ID NO:
ID NO:
(SEQ ID
TAVYYCAR
(SEQ
ID NO:

variable
A
6358)
6369)
NO:
(SEQ ID NO:
ID NO:
228)

region
(SEQ ID

6360)
6371)
227)

variant 2
NO: 229)

Humanized
EVQLVES
FSRYD
WVRQAP
TISSGG
RFTISRDNS
HSAYY
WGQG

anti-
GGGLVQ
MS
GKGLEW
SYTYYP
KNTLYLQ
VNYEN
TLVTV

calreticulin
PGGSLRL
(SEQ
VA (SEQ
DSVKG
MNSLRAED
AMDY
SS (SEQ

heavy chain
SCAASGF
ID NO:
ID NO:
(SEQ ID
TAVYYCAR
(SEQ
ID NO:

variable
A (SEQ ID
6358)
6369)
NO:
(SEQ ID NO:
ID NO:
228)

region
NO: 6373)

6360)
6371)
227)

variant 3

6C10
EVQLVEK
FSEYW
WLRQAP
VIKYK
RFTISRDDS
GRDV
WGQG

Parental
GGGLVQ
MN
GKGLEW
YSNYA
KSSVYLQM
QDY
TMVTV

VH BK051
PGKSLKL
(SEQ
VG (SEQ
TEFAES
TNLRAEDT
(SEQ
SS (SEQ

SCTASGF
ID NO:
ID NO:
VKG
AIYYCAR
ID NO:
ID NO:

T (SEQ ID
7396)
7398)
(SEQ ID
(SEQ ID NO:
7403)
6006)

NO: 7394)

NO:
7402)

7400)

6C10
EVQLVES
FSEYW
WLRQAP
VIKYK
RFTISRDDS
GRDV
WGQG

humanized
GGGLVQ
MN
GKGLEW
YSNYA
KSIVYLQM
QDY
TMVTV

heavy chain
PGPSLRL
(SEQ
VG (SEQ
TEFAES
NSLKTEDT
(SEQ
SS (SEQ

variable
SCTASGF
ID NO:
ID NO:
VKG
AVYYCAR
ID NO:
ID NO:

region
T (SEQ ID
7396)
7398)
(SEQ ID
(SEQ ID NO:
7403)
6006)

variant 1
NO: 7423)

NO:
7424)

(BK197)

7400)

6C10_hum1_
EVQLVES
FSEYW
WLRQAP
VIKYK
RFTISRDDS
GRDV
WGQG

scFv VH
GGGLVQ
MN
GKGLEW
YSNYA
KSIVYLQM
QDY
TMVTV

(BKM0161)
PGRSLRL
(SEQ
VG (SEQ
TEFAES
NSLKTEDT
(SEQ
SS (SEQ

SCTASGF
ID NO:
ID NO:
VKG
AVYYCAR
ID NO:
ID NO:

T (SEQ ID
7396)
7398)
(SEQ ID
(SEQ ID NO:
7403)
6006)

NO: 7436)

NO:
7424)

7400)

6C10_hum5_
EVQLVES
SEYW
WLRQAP
VIKYK
RFTISRDDS
GRDV
WGQG

scFv VH
GGGLVQ
MN
GKGLEW
YSNYA
KSSVYLQM
QDY
TMVTV

(BKM0165)
PGGSLRL
(SEQ
VG (SEQ
TEFAES
NSLKTEDT
(SEQ
SS (SEQ

SCAASGF
ID NO:
ID NO:
VKG
AVYYCAR
ID NO:
ID NO:

TF (SEQ
7444)
7398)
(SEQ ID
(SEQ ID NO:
7403)
6006)

ID NO:

NO:
7445)

7443)

7400)

TABLE 5

Exemplary light chain CDRs and FWRs of calreticulin-targeting antigen binding domains

Ab ID
FWR1
CDR1
FWR2
CDR2
FWR3
CDR3
FWR4

AbH-1L /
DVVMTQ
KSSQSLL
WLQQRP
LVSKL
GVPDRFSG
WQGTH
FGGG

AbH-2L
SPLSLPV
DSDGKT
GQSPRR
DS
SGSGTDFT
FPYT
TKVEI

TLGQPAS
YLN
LIY (SEQ
(SEQ
LKISRVEA
(SEQ ID
K (SEQ

ISC (SEQ
(SEQ ID
ID NO:
ID NO:
EDVGVYH
NO:
ID NO:

ID NO:
NO: 6259)
6278)
6260)
C (SEQ ID
6261)
6280)

6277)

NO: 6279)

AbM-1L /
DVVMTQ
KSSQSLL
WLLQRP
LVSKL
GVPDRFTG
WQGTH
FGGG

AbM-2L
TPLTLSV
DSDGKT
GQSPKR
DS
SGSGTDFT
FPYT
TKLEI

TIGQPASI
YLN
LIY (SEQ
(SEQ
LKISRVEA
(SEQ ID
K (SEQ

SC (SEQ
(SEQ ID
ID NO:
ID NO:
EDLGVYH
NO:
ID NO:

ID NO:
NO: 6259)
6282)
6260)
C (SEQ ID
6261)
6284)

6281)

NO: 6283)

Murine
NIVLTQS
RASESV
WYQQRP
LASNL
GVPARFSG
QQNNE
FGAG

anti-
PASLAVS
DSFGISF
GQPPKL
ES
SGSRTDFT
DPLT
TKLEL

calreticulin
LGQRATI
MH (SEQ
LIY (SEQ
(SEQ
LTIDPVEA
(SEQ ID
K (SEQ

antibody
SC (SEQ
ID NO:
ID NO:
ID NO:
DDAATYY
NO: 248)
ID NO:

16B11.1
ID NO:
251)
6375)
246)
C (SEQ ID

249)

light chain
6374)

NO: 247)

variable

region

Humanized
DIVLTQT
RASESV
WYLQKP
LASNL
GVPDRFSG
QQNNE
FGQG

anti-
PLSLSVT
DSFGISF
GQSPQL
ES
SGSRTDFT
DPLT
TKLEI

calreticulin
PGQPASIS
MH (SEQ
LIY (SEQ
(SEQ
LKISRVEA
(SEQ ID
K (SEQ

light chain
C (SEQ ID
ID NO:
ID NO:
ID NO:
EDVGVYY
NO: 255)
ID NO:

variable
NO: 250)
251)
252)
253)
C (SEQ ID

256)

region

NO: 254)

variant 1

Humanized
DIVLTQS
RASESV
WYQQRP
LASNL
GVPDRFSG
QQNNE
FGQG

anti-
PLSLPVT
DSFGISF
GQSPRL
ES
SGSRTDFT
DPLT
TKLEI

calreticulin
LGQPASI
MH (SEQ
LIY (SEQ
(SEQ
LKISRVEA
(SEQ ID
K (SEQ

light chain
SC (SEQ
ID NO:
ID NO:
ID NO:
EDVGVYY
NO: 262)
ID NO:

variable
ID NO:
258)
259)
260)
C (SEQ ID

263)

region
257)

NO: 261)

variant 2

Humanized
DIVLTQT
RASESV
WYLQKP
LASNL
GVPDRFSG
QQNNE
FGQG

anti-
PLSLPVT
DSFGISF
GQSPQL
ES
SGSRTDFT
DPLT
TKLEI

calreticulin
PGEPASIS
MH (SEQ
LIY (SEQ
(SEQ
LKISRVEA
(SEQ ID
K (SEQ

light chain
C (SEQ ID
ID NO:
ID NO:
ID NO:
EDVGVYY
NO: 269)
ID NO:

variable
NO: 264)
265)
266)
267)
C (SEQ ID

270)

region

NO: 268)

variant 3

Humanized
EIVLTQSP
RASESV
WYQQK
LASNL
GIPARFSG
QQNNE
FGQG

anti-
ATLSLSP
DSFGISF
PGQAPR
ES
SGSRTDFT
DPLT
TKLEI

calreticulin
GERATLS
MH (SEQ
LLIY
(SEQ
LTISSLEPE
(SEQ ID
K (SEQ

light chain
C (SEQ ID
ID NO:
(SEQ ID
ID NO:
DFAVYYC
NO: 276)
ID NO:

variable
NO: 271)
272)
NO: 273)
274)
(SEQ ID

277)

region

NO: 275)

variant 4

Humanized
DIQLTQS
RASESV
WYQQK
LASNL
GVPSRFSG
QQNNE
FGQG

anti-
PSSLSAS
DSFGISF
PGKAPK
ES
SGSRTDFT
DPLT
TKLEI

calreticulin
VGDRVTI
MH (SEQ
LLIY
(SEQ
FTISSLQPE
(SEQ ID
K (SEQ

light chain
TC (SEQ
ID NO:
(SEQ ID
ID NO:
DIATYYC
NO: 283)
ID NO:

variable
ID NO:
279)
NO: 280)
281)
(SEQ ID

284)

region
278)

NO: 282)

variant 5

6C10
EIVLTQSP
STSSSVT
WYQQK
STSNL
GVPTRFSG
QQCLSS
FGAG

Parental
ASKAASQ
TNYLH
PDTPPKL
AS
SGSGTSYS
PCT
TKLEI

VL BK052
GEEVTIT
(SEQ ID
LIY (SEQ
(SEQ
LTISNMQG
(SEQ ID
K (SEQ

C (SEQ ID
NO: 7387)
ID NO:
ID NO:
EDVATYY
NO:
ID NO:

NO: 7386)

7388)
7389)
C (SEQ ID
7392)
7393)

NO: 7391)

6C10 VL
EIVLTQSP
STSSSVT
WYQQK
STSNL
GVPTRFSG
QQSLSS
FGAG

BK210 -
ASKAASQ
TNYLH
PDTPPKL
AS
SGSGTSYS
PST
TKLEI

C91S/C96S
GEEVTIT
(SEQ ID
LIY (SEQ
(SEQ
LTISNMQG
(SEQ ID
K (SEQ

mutant
C (SEQ ID
NO: 7387)
ID NO:
ID NO:
EDVATYY
NO:
ID NO:

NO: 7386)

7388)
7389)
C (SEQ ID
7410)
7393)

NO: 7391)

6C10 VL
EIVLTQSP
STSSSVT
WYQQK
STSNL
GVPTRFSG
QQSLSS
FGAG

BK210 -
ASKAASQ
TNYLH
PDTPPKL
AS
SGSGTSYS
PCT
TKLEI

C91S
GEEVTIT
(SEQ ID
LIY (SEQ
(SEQ
LTISNMQG
(SEQ ID
K (SEQ

mutant
C (SEQ ID
NO: 7387)
ID NO:
ID NO
EDVATYY
NO
ID NO:

NO: 7386)

7388)
7389)
C (SEQ ID
7415)
7393)

NO: 7391)

6C10 VL
EIVLTQSP
STSSSVT
WYQQK
STSNL
GVPTRFSG
QQCLSS
FGAG

BK210 -
ASKAASQ
TNYLH
PDTPPKL
AS
SGSGTSYS
PST
TKLEI

C96S
GEEVTIT
(SEQ ID
LIY (SEQ
(SEQ
LTISNMQG
SEQ ID
K (SEQ

mutant
C (SEQ ID
NO: 7387)
ID NO:
ID NO:
EDVATYY
NO:
ID NO:

NO: 7386)

7388)
7389)
C (SEQ ID
7417)
7393)

NO: 7391)

6C10
DIQLTQS
STSSSVT
WYQQK
STSNL
GVPSRFSG
QQCLSS
FGQG

humanized
PSFLSAS
TNYLH
PGKAPK
AS
SGSGTEYT
PCT
TKLEI

light chain
VGDRVTI
(SEQ ID
LLIY
(SEQ
LTISSLQPE
(SEQ ID
K (SEQ

variable
TC (SEQ
NO: 7387)
(SEQ ID
ID NO:
DFATYYC
NO:
ID NO:

region
ID NO:

NO: 280)
7389)
(SEQ ID
7392)
256)

variant 1
7426)

NO: 7427)

(BK198)

6C10
DIVLTQS
STSSSVT
WYQQK
STSNL
GVPDRFSG
QQCLSS
FGQG

humanized
PDSLAVS
TNYLH
PGQPPK
AS
SGSGTDYT
PCT
TKLEI

light chain
LGERATI
(SEQ ID
LLIY
(SEQ
LTISSLQAE
(SEQ ID
K (SEQ

variable
NC (SEQ
NO: 7387)
(SEQ ID
ID NO:
DVAVYYC
NO:
ID NO:

region
ID NO:

NO: 7430)
7389)
(SEQ ID
7392)
256)

variant 2
7429)

NO: 7431)

(BK199)

6C10
EIVLTQSP
STSSSVT
WYQQK
STSNL
GIPDRFSG
QQCLSS
FGQG

humanized
ATLSLSP
TNYLH
PGQAPK
AS
SGSGTDYT
PCT
TKLEI

light chain
GERATLS
(SEQ ID
LLIY
(SEQ
LTISRLEPE
(SEQ ID
K (SEQ

variable
C (SEQ ID
NO: 7387)
(SEQ ID
ID NO:
DFAVYYC
NO:
ID NO:

region
NO: 271)

NO: 7433)
7389)
(SEQ ID
7392)
256)

variant 3

NO: 7434)

(BK200)

6C10_
EIVLTQSP
STSSSVT
WYQQK
STSNL
GIPARFSG
QQCLSS
FGQG

hum2_scFv
ATLSLSP
TNYLH
PGQAPK
AS
SGSGTDYT
PCT
TKLEI

VL
GERATLS
(SEQ ID
LLIY
(SEQ
LTISSLQPE
(SEQ ID
K (SEQ

(BKM0162)
C (SEQ ID
NO: 7387)
(SEQ ID
ID NO:
DFAVYYC
NO:
ID NO:

NO: 271)

NO: 7433)
7389)
(SEQ ID
7392)
256)

NO: 7439)

6C10_hum3_
DIQLTQS
STSSSVT
WYQQK
STSNL
GVPSRFSG
QQSLSS
FGOG

scFv VL
PSFLSAS
TNYLH
PGKAPK
AS
SGSGTEYT
PST
TKLEI

(BKM0163)
VGDRVTI
(SEQ ID
LLIY
(SEQ
LTISSLQPE
(SEQ ID
K (SEQ

TC (SEQ
NO: 7387)
(SEQ ID
ID NO:
DFATYYC
NO:
ID NO:

ID NO:

NO: 280)
7389)
(SEQ ID
7410)
256)

7426)

NO: 7427)

6C10_hum4_
EIVLTQSP
STSSSVT
WYQQK
STSNL
GIPARFSG
QQSLSS
FGQG

scFv VL
ATLSLSP
TNYLH
PGQAPK
AS
SGSGTDYT
PST
TKLEI

(BKM0164)
GERATLS
(SEQ ID
LLIY
(SEQ
LTISSLQPE
(SEQ ID
K (SEQ

C (SEQ ID
NO: 7387)
(SEQ ID
ID NO:
DFAVYYC
NO:
ID NO:

NO: 271)

NO: 7433)
7389)
(SEQ ID
7410)
256)

NO: 7439)

TABLE 6

Exemplary FWRs of calreticulin-targeting antigen binding

SEQ ID NO
Description
Sequence

SEQ ID NO:
Ab-1
X₁VQLX₂QSGX₃EX₄X_5KX₆GASVKX₇SCKASG, wherein:

6224
VHFWR1
X₁ is not E,

X₂ is not E,

X₃ is not P,

X₄ is not L,

X₅ is not V,

X₆ is not T, or

X₇ is not I

SEQ ID NO:
Ab-1
WVX₁QX₂X₃GX₄X₅LX₆WX₇G, wherein:

6226
VHFWR2
X₁ is not K,

X₂ is not S,

X₃ is not H,

X₄ is not K,

X₅ is not S,

X₆ is not E, or

X₇ is not I

SEQ ID NO:
Ab-1
X₁X₂TX₃TVDTSX₄STAYX₅X₆X₇X₈SLX₉SX₁₀DX₁₁AX₁₂YYCA,

6228
VHFWR3
wherein:

X₁ is not K,

X₂ is not A,

X₃ is not F,

X₄ is not S,

X₅ is not M,

X₆ is not Q,

X₇ is not F,

X₈ is not N,

X₉ is not T,

X₁₀ is not G,

X₁₁ is not S, or

X₁₂ is not V

SEQ ID NO:
Ab-1
WGQGTX₁VTVSS, wherein:

6230
VHFWR4
X₁ is not S

SEQ ID NO:
Ab-2
X₁VX₂LX₃ESGPX₄LVKX₅X₆X₇X₈LX₉LTCTVX₁₀G, wherein:

6232
VHFWR1
X₁ is not D,

X₂ is not Q,

X₃ is not Q,

X₄ is not G,

X₅ is not N,

X₆ is not S,

X₇ is not Q,

X₈ is not S,

X₉ is not S, or

X₁₀ is not T

SEQ ID NO:
Ab-2
WIRQX₁PGX₂X₃LEWX₄X₅, wherein:

6234
VHFWR2
X₁ is not F,

X₂ is not N,

X₃ is not K,

X₄ is not M, or

X₅ is not G

SEQ ID NO:
Ab-2
RX₁SITX₂DTSKX₃QX₄X₅LX₆X₇X₈X₉X₁₀X₁₁PX₁₂DTATYYCAR,

6236
VHFWR3
wherein:

X₁ is not I,

X₂ is not R,

X₃ is not N,

X₄ is not F,

X₅ is not F,

X₆ is not Q,

X₇ is not L,

X₈ is not N,

X₉ is not S,

X₁₀ is not V,

X₁₁ is not T, or

X₁₂ is not E

SEQ ID NO:
Ab-2
WGQGTX₁VTVSS, wherein:

6230
VHFWR4
X₁ is not S

SEQ ID NO:
Ab-1/2
DVVMTQX₁PLX₂LX₃VTX₄GQPASISC, wherein:

6238
VLFWR1
X₁ is not T,

X₂ is not T,

X₃ is not S, or

X₄ is not I

SEQ ID NO:
Ab-1/2
WLX₁QRPGQSPX₂RLIY, wherein:

6240
VLFWR2
X₁ is not L, or

X₂ is not K

SEQ ID NO:
Ab-1/2
GVPDRFX₁GSGSGTDFTLKISRVEAEDX₂GVYHC, wherein:

6242
VLFWR3
X₁ is not T, or

X₂ is not L

SEQ ID NO:
Ab-1/2
FGGGTKX₁EIK, wherein:

6244
VLFWR4
X₁ is not L

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a VH amino acid sequence as listed in Table 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VL amino acid sequence as listed in Table 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 9800, or 9900 sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises: (i) a VH comprising a VH amino acid sequence as listed in Table 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and (ii) a VL comprising a VL amino acid sequence as listed in Table 24, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

TABLE 24

Exemplary variable regions of calreticulin-targeting antigen binding (underlining

indicates CDR sequences)

SEQ

ID NO
Description
Sequence

SEQ
AbH-1 heavy
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPG

ID NO:
chain variable
QELGWMGYISCYNGASSYNQKFKGRVTMTVDTSISTAYTELSSL

6247
region
RSEDTATYYCA SSMDYWGQGTLVTVSS

SEQ
AbH-2 heavy
QVTLKESGPVLVKPTETLTLTCTVSGYSITSDYAWNWIRQPPGK

ID NO:
chain variable
ALEWLAYISYSGSTSYNPSLKSRLSITKDTSKSQVVLTMTNMDP

6248
region
VDTATYYCARDPPYYYGSWGQGTTVTVSS

SEQ
AbH-1 / AbH-2
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQR

ID NO:
light chain
PGQSPRRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVG

6249
variable region
VYHCWQGTHFPYTFGGGTKVEIK

SEQ
AbM-1 heavy
EVQLEQSGPELVKTGASVKISCKASGYSFTGYYIHWVKQSHGKS

ID NO:
chain variable
LEWIGYISCYNGASSYNQKFKGKATFTVDTSSSTAYMQFNSLTS

6250
region
GDSAVYYCA SSMDYWGQGTSVTVSS

SEQ
AbM-2 heavy
DVQLQESGPGLVKNSQSLSLTCTVTGYSITSDYAWNWIRQFPGN

ID NO:
chain variable
KLEWMGYISYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTPED

6251
region
TATYYCARDPPYYYGSNGTWGQGTSVTVSS

SEQ
AbM-1 / AbM-2
DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRP

ID NO:
light chain
GQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGV

6252
variable region
YHCWQGTHFPYTFGGGTKLEIK

SEQ
Murine anti-
EVKLVESGGGLVKPGGSLKLSCAASGFAFSRYDMSWVRQTPEK

ID NO:
calreticulin
RLEWVATISSGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLR

6347
antibody
SEDTALYYCARHSAYYVNYENAMDYWGQGTSVTVSS

16B11.1 heavy

chain variable

region

SEQ
6C10 Parental
EVQLVEKGGGLVQPGKSLKLSCTASGFTFSEYWMNWLRQAPG

ID NO:
VH BK051
KGLEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMT

7418

NLRAEDTAIYYCARGRDVQDYWGQGTMVTVSS

SEQ
6C10
EVQLVESGGGLVQPGPSLRLSCTASGFTFSEYWMNWLRQAPGK

ID NO:
humanized
GLEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSIVYLQMNS

7425
heavy chain
LKTEDTAVYYCARGRDVQDYWGQGTMVTVSS

variable region

variant 1

(BK197)

SEQ
6C10_hum1_sc
EVQLVESGGGLVQPGRSLRLSCTASGFTFSEYWMNWLRQAPGK

ID NO:
Fv VH
GLEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSIVYLQMNS

7438
(BKM0161)
LKTEDTAVYYCARGRDVQDYWGQGTMVTVSS

SEQ
6C10_hum5_sc
EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYWMNWLRQAPGK

ID NO:
Fv VH
GLEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMNS

7446
(BKM0165)
LKTEDTAVYYCARGRDVQDYWGQGTMVTVSS

SEQ
Murine anti-
NIVLTQSPASLAVSLGQRATISCRASESVDSFGISFMHWYQQRPG

ID NO:
calreticulin
QPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATY

6348
antibody
YCQQNNEDPLTFGAGTKLELK

16B11.1 light

chain variable

region

SEQ
Humanized anti-
QVQLVESGGGLVKPGGSLRLSCAASGFAFSRYDMSWIRQAPGK

ID NO:
calreticulin
GLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLR

6349
heavy chain
AEDTAVYYCARHSAYYVNYENAMDYWGQGTLVTVSS

variable region

variant 1

SEQ
Humanized anti-
QVQLVESGGGVVQPGRSLRLSCAASGFAFSRYDMSWVRQAPGK

ID NO:
calreticulin
GLEWVATISSGGSYTYYPDSVKGRFTISRDNSKNTLYLQMNSLR

6350
heavy chain
AEDTAVYYCARHSAYYVNYENAMDYWGQGTLVTVSS

variable region

variant 2

SEQ
Humanized anti-
EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPGK

ID NO:
calreticulin
GLEWVATISSGGSYTYYPDSVKGRFTISRDNSKNTLYLQMNSLR

6351
heavy chain
AEDTAVYYCARHSAYYVNYENAMDYWGQGTLVTVSS

variable region

variant 3

SEQ
Humanized anti-
DIVLTQTPLSLSVTPGQPASISCRASESVDSFGISFMHWYLQKPG

ID NO:
calreticulin light
QSPQLLIYLASNLESGVPDRFSGSGSRTDFTLKISRVEAEDVGVY

6352
chain variable
YCQQNNEDPLTFGQGTKLEIK

region variant 1

SEQ
Humanized anti-
DIVLTQSPLSLPVTLGQPASISCRASESVDSFGISFMHWYQQRPG

ID NO:
calreticulin light
QSPRLLIYLASNLESGVPDRFSGSGSRTDFTLKISRVEAEDVGVY

6353
chain variable
YCQQNNEDPLTFGQGTKLEIK

region variant 2

SEQ
Humanized anti-
DIVLTQTPLSLPVTPGEPASISCRASESVDSFGISFMHWYLQKPGQ

ID NO:
calreticulin light
SPQLLIYLASNLESGVPDRFSGSGSRTDFTLKISRVEAEDVGVYY

6354
chain variable
CQQNNEDPLTFGQGTKLEIK

region variant 3

SEQ
Humanized anti-
EIVLTQSPATLSLSPGERATLSCRASESVDSFGISFMHWYQQKPG

ID NO:
calreticulin light
QAPRLLIYLASNLESGIPARFSGSGSRTDFTLTISSLEPEDFAVYYC

6355
chain variable

QQNNEDPLTFGQGTKLEIK

region variant 4

SEQ
Humanized anti-
DIQLTQSPSSLSASVGDRVTITCRASESVDSFGISFMHWYQQKPG

ID NO:
calreticulin light
KAPKLLIYLASNLESGVPSRFSGSGSRTDFTFTISSLQPEDIATYYC

6356
chain variable

QQNNEDPLTFGQGTKLEIK

region variant 5

SEQ
6C10 Parental
EIVLTQSPASKAASQGEEVTITCSTSSSVTTNYLHWYQQKPDTPP

ID NO:
VL BK052
KLLIYSTSNLASGVPTRFSGSGSGTSYSLTISNMQGEDVATYYCQ

7419

QCLSSPCTFGAGTKLEIK

SEQ
6C10 VL
EIVLTQSPASKAASQGEEVTITCSTSSSVTTNYLHWYQQKPDTPP

ID NO:
BK210 -
KLLIYSTSNLASGVPTRFSGSGSGTSYSLTISNMQGEDVATYYCQ

7420
C91S/C96S

QSLSSPSTFGAGTKLEIK

mutant

SEQ
6C10 VL
EIVLTQSPASKAASQGEEVTITCSTSSSVTTNYLHWYQQKPDTPP

ID NO:
BK210 - C91S
KLLIYSTSNLASGVPTRFSGSGSGTSYSLTISNMQGEDVATYYCQ

7421
mutant

QSLSSPCTFGAGTKLEIK

SEQ
6C10 VL
EIVLTQSPASKAASQGEEVTITCSTSSSVTTNYLHWYQQKPDTPP

ID NO:
BK210 - C96S
KLLIYSTSNLASGVPTRFSGSGSGTSYSLTISNMQGEDVATYYCQ

7422
mutant

QCLSSPSTFGAGTKLEIK

SEQ
6C10
DIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQKPGKAP

ID NO:
humanized light
KLLIYSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQ

7428
chain variable

CLSSPCTFGQGTKLEIK

region variant 1

(BK198)

SEQ
6C10
DIVLTQSPDSLAVSLGERATINCSTSSSVTTNYLHWYQQKPGQPP

ID NO:
humanized light
KLLIYSTSNLASGVPDRFSGSGSGTDYTLTISSLQAEDVAVYYCQ

7432
chain variable

QCLSSPCTFGQGTKLEIK

region variant 2

(BK199)

SEQ
6C10
EIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQKPGQAP

ID NO:
humanized light
KLLIYSTSNLASGIPDRFSGSGSGTDYTLTISRLEPEDFAVYYCQQ

7435
chain variable

CLSSPCTFGQGTKLEIK

region variant 3

(BK200)

SEQ
6C10_
EIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQKPGQAP

ID NO:
hum2_scFv VL
KLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAVYYCQQ

7440
(BKM0162)

CLSSPCTFGQGTKLEIK

SEQ
6C10_hum3_sc
DIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQKPGKAP

ID NO:
Fv VL
KLLIYSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQ

7441
(BKM0163)

SLSSPSTFGQGTKLEIK

SEQ
6C10_hum4_sc
EIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQKPGQAP

ID NO:
Fv VL
KLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAVYYCQQ

7442
(BKM0164)

SLSSPSTFGQGTKLEIK

In some embodiments, the calreticulin-targeting antigen binding domain comprises an scFv comprising an amino acid sequence as listed in Table 25, or an amino acid sequence having at least 750%, 80%, 85%, 90%, 950%, 960%, 97%, 980%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises an scFv comprising a VH amino acid sequence as listed in Table 25, or an amino acid sequence having at least 75%, 80%, 850%, 90%, 95%, 960%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises an scFv comprising a VL amino acid sequence as listed in Table 25, or an amino acid sequence having at least 750%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises an scFv comprising a spacer amino acid sequence as listed in Table 25, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

TABLE 25

Exemplary scFv sequences for calreticulin-targeting antigen binding (underlining

indicates CDR sequences)

SEQ ID

NO:
Description
Sequence

7499
6C10_hum1_
EVQLVESGGGLVQPGRSLRLSCTASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSIVYLQMNSLK

(BKM0161)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSDIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQ

KPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFAT

YYCQQCLSSPCTFGQGTKLEIK

7500
6C10_
EVQLVESGGGLVQPGRSLRLSCTASGFTFSEYWMNWLRQAPGKG

hum2_scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSIVYLQMNSLK

(BKM0162)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSEIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQ

KPGQAPKLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAV

YYCQQCLSSPCTFGQGTKLEIK

7501
6C10_hum3_
EVQLVESGGGLVQPGRSLRLSCTASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSIVYLQMNSLK

(BKM0163)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSDIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQ

KPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFAT

YYCQQSLSSPSTFGQGTKLEIK

7502
6C10_hum4_
EVQLVESGGGLVQPGRSLRLSCTASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSIVYLQMNSLK

(BKM0164)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSEIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQ

KPGQAPKLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAV

YYCQQSLSSPSTFGQGTKLEIK

7503
6C10_hum5_
EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMNSLK

(BKM0165)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSDIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQ

KPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFAT

YYCQQCLSSPCTFGQGTKLEIK

7504
6C10_hum6_
EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMNSLK

(BKM0166)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSEIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQ

KPGQAPKLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAV

YYCQQCLSSPCTFGQGTKLEIK

7505
6C10_hum7_
EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMNSLK

(BKM0167)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSDIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQ

KPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFAT

YYCQQSLSSPSTFGQGTKLEIK

7506
6C10_hum8_
EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYWMNWLRQAPGKG

scFv
LEWVGVIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMNSLK

(BKM0168)
TEDTAVYYCARGRDVQDYWGQGTMVTVSSGGGGSGGGGSGGG

GSGGGGSEIVLTQSPATLSLSPGERATLSCSTSSSVTTNYLHWYQQ

KPGQAPKLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLQPEDFAV

YYCQQSLSSPSTFGQGTKLEIK

In some embodiments, the calreticulin-targeting antigen binding domain comprises an Fc region. In some embodiments, the Fc region is chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is chosen from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is chosen from the heavy chain constant region of IgG1 or IgG2 (e.g., human IgG1, or IgG2). In some embodiments, the heavy chain constant region is human IgG2a. In some embodiment, the heavy chain constant region comprises a murine IgG2a sequence, e.g., SEQ ID NO: 7448 below, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto:

(SEQ ID NO: 7448)

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV

HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPR

GPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS

EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGK

EFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC

MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNW

VERNSYSCSVVHEGLHNHHTTKSFSRTPGK

In some embodiments, the Fc region comprises a Fc region variant, e.g., as described herein. In some embodiments, the Fc region comprises one or more mutations. In some embodiments, the Fc region comprises an LALAPG mutation. In some embodiments, the Fc region comprises the amino acid sequence of a murine IgG2a-LALAPG variant, e.g., the sequence of SEQ ID NOs: 7449 or 7450 below, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto:

(SEQ ID NO: 7449)

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV

HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPR

GPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS

EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGK

EFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC

MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNW

VERNSYSCSVVHEGLHNHHTTKSFSRTPGK

(SEQ ID NO: 7450)

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGV

HTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPR

GPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS

EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGK

EFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPCEEEMTKKQVTLWC

MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNW

VERNSYSCSVVHEGLHNHHTTKSFSRTPGK

In some embodiments, the Fc region comprises the amino acid sequence of a human IgG2a N2976A variant, e.g., the sequence of SEQ ID NO: 7453 below, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto:

(SEQ ID NO: 7453)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP

KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC

LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, the calreticulin-targeting antigen binding domain comprises a light chain constant region, e.g., a CL kappa region, e.g., a human CL kappa region. In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 7451 below, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto:

(SEQ ID NO: 7451)

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ

NGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI

VKSFNRNEC

In some embodiments, the light chain constant region comprises the amino acid sequence of SEQ ID NO: 7454 below, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto:

(SEQ ID NO: 7454)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

TKSFNRGEC

Additional Calreticulin-Targeting Antigen Binding Domains

In some embodiments, the calreticulin-targeting antigen binding domain comprises any CDR amino acid sequence or variable region amino acid sequence disclosed in Tables 16-19. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6253 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 243 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6253, a VHCDR2 amino acid sequence of SEQ ID NO: 243, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6255. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6259, a VLCDR2 amino acid sequence of SEQ ID NO: 6260, and a VLCDR3 amino acid sequence of SEQ ID NO: 6261. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 244 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 245 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 244 and/or a VL comprising the amino acid sequence of SEQ ID NO: 245. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372, 234, 235, 236, or 237, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 238, 239, 240, 241, or 242, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 238 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 238. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 239 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 239. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 240 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 240. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 241 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 241. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6372 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 234 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 235 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 242. In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 242 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 237 and a VL comprising the amino acid sequence of SEQ ID NO: 242.

In some embodiments, the calreticulin-targeting antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 236 and a VL comprising the amino acid sequence of SEQ ID NO: 238.

TABLE 16

Exemplary variable regions of additional calreticulin-targeting antigen binding domains

SEQ ID

NO
Description
Sequence

SEQ ID
BJ092
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQG

NO: 6372
(VH)
LEWIGYISAYNGASSYNQKFKGRATFTVDTSTSTAYMELRSLRSDD

MAVYYCASSMDYWGQGTLVTVSS

SEQ ID
BJ093
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQG

NO: 234
(VH)
LEWIGYISAYNGASSYNQKFKGRATFTVDTSTSTAYMELRSLRSDD

TAVYYCASSMDYWGQGTLVTVSS

SEQ ID
BJ094
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGKG

NO: 235
(VH)
LEWIGYISAYNGASSYNQKFKGRATFTVDTSTSTAYMELSSLRSED

TAVYYCASSMDYWGQGTLVTVSS

SEQ ID
BJ095
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQG

NO: 236
(VH)
LEWIGYISAYNGASSYNQKFKGRATFTVDTSISTAYMELSRLRSDD

TAVYYCASSMDYWGQGTLVTVSS

SEQ ID
BJ096
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGQG

NO: 237
(VH)
LEWIGYISAYNGASSYNQKFKGRATFTVDTSTSTAYMELSSLRSED

TAVYYCASSMDYWGQGTLVTVSS

SEQ ID
VH
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYYIHWVRQAPGX₁G

NO: 244
consensus
LEWIGYISAYNGASSYNQKFKGRATFTVDTSX₂STAYMELX₃X₄LRS

DDX₅AVYYCASSMDYWGQGTLVTVSS, wherein:

X₁ is Q or K,

X₂ is I or T,

X₃ is S or R,

X₄ is R or S, or

X₅ is Tor M

SEQ ID
BJ097
DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPG

NO: 238
(VL)
QSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHC

WQGTHFPYTFGQGTKLEIK

SEQ ID
BJ098
DVVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPG

NO: 239
(VL)
QSPKLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHC

WQGTHFPYTFGQGTKLEIK

SEQ ID
BJ099
DVVMTQTPLSLSVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPG

NO: 240
(VL)
QPPKLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHC

WQGTHEPYTFGQGTKLEIK

SEQ ID
BJ100
DVVMTQTPLSSPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPG

NO: 241
(VL)
QPPKLLIYLVSKLDSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYH

CWQGTHEPYTFGQGTKLEIK

SEQ ID
BJ101
DVVMTQSPLSLPVTPGEPASISCKSSQSLLDSDGKTYLNWLLQKPG

NO: 242
(VL)
QSPKLLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHC

WQGTHFPYTFGQGTKLEIK

SEQ ID
VL
DVVMTQX₁PLSX₂X₃VTX₄GX₅PASISCKSSQSLLDSDGKTYLNWLX₆

NO: 245
consensus
QX₇PGQX₈PKX₉LIYLVSKLDSGVPDRFSGSGX1₀GTDFTLKISRVEAE

DVGVYHCWQGTHEPYTFGQGTKLEIK, wherein:

X₁ is S or T,

X₂ is L or S,

X₃ is P or S,

X₄ is L or P,

X₅ is Q or E,

X₆ is Q or L,

X₇ is R or K,

X₈ is S or P,

X₉ is R or L, or

X₁₀ is S or A

TABLE 17

Exemplary heavy chain CDRs of calreticulin-targeting antigen binding domains

VH (SEQ ID NO)
VHCDR1 (SEQ ID NO)
VHCDR2 (SEQ ID NO)
VHCDR3 (SEQ ID NO)

BJ092 (SEQ ID
YSFTGYYIH (SEQ ID
YISAYNGASSYNQKF
SSMDY (SEQ ID NO:

NO: 6372)
NO: 6253)
KG (SEQ ID NO: 243)
6255)

BJ093 (SEQ ID
YSFTGYYIH (SEQ ID
YISAYNGASSYNQKF
SSMDY (SEQ ID NO:

NO: 234)
NO: 6253)
KG (SEQ ID NO: 243)
6255)

BJ094 (SEQ ID
YSFTGYYIH (SEQ ID
YISAYNGASSYNQKF
SSMDY (SEQ ID NO:

NO: 235)
NO: 6253)
KG (SEQ ID NO: 243)
6255)

BJ095 (SEQ ID
YSFTGYYIH (SEQ ID
YISAYNGASSYNQKF
SSMDY (SEQ ID NO:

NO: 236)
NO: 6253)
KG (SEQ ID NO: 243)
6255)

BJ096 (SEQ ID
YSFTGYYIH (SEQ ID
YISAYNGASSYNQKF
SSMDY (SEQ ID NO:

NO: 237)
NO: 6253)
KG (SEQ ID NO: 243)
6255)

TABLE 18

Exemplary light chain CDRs of calreticulin-targeting antigen binding domains

VL (SEQ ID NO)
VLCDR1 (SEQ ID NO)
VLCDR2 (SEQ ID NO)
VLCDR3 (SEQ ID NO)

BJ097 (SEQ ID
KSSQSLLDSDGKTYLN
LVSKLDS (SEQ ID
WQGTHFPYT (SEQ

NO: 238)
(SEQ ID NO: 6259)
NO: 6260)
ID NO: 6261)

BJ098 (SEQ ID
KSSQSLLDSDGKTYLN
LVSKLDS (SEQ ID
WQGTHFPYT (SEQ

NO: 239)
(SEQ ID NO: 6259)
NO: 6260)
ID NO: 6261)

BJ099 (SEQ ID
KSSQSLLDSDGKTYLN
LVSKLDS (SEQ ID
WQGTHFPYT (SEQ

NO: 240)
(SEQ ID NO: 6259)
NO: 6260)
ID NO: 6261)

BJ100 (SEQ ID
KSSQSLLDSDGKTYLN
LVSKLDS (SEQ ID
WQGTHFPYT (SEQ

NO: 241)
(SEQ ID NO: 6259)
NO: 6260)
ID NO: 6261)

BJ101 (SEQ ID
KSSQSLLDSDGKTYLN
LVSKLDS (SEQ ID
WQGTHFPYT (SEQ

NO: 242)
(SEQ ID NO: 6259)
NO: 6260)
ID NO: 6261)

TABLE 19

Exemplary calreticulin-targeting antigen binding domains

Antibody

code
VH code
VH germline
VL code
VL germline

BJM0040
BJ092 (SEQ
IGHV1-
BJ097 (SEQ
IGKV2-

ID NO: 6372)
18*03
ID NO: 238)
30*01

BJM0041
BJ093 (SEQ
IGHV1-
BJ097 (SEQ
IGKV2-

ID NO: 234)
18*01
ID NO: 238)
30*01

BJM0042
BJ094 (SEQ
IGHV1-
BJ097 (SEQ
IGKV2-

ID NO: 235)
2*02
ID NO: 238)
30*01

BJM0043
BJ095 (SEQ
IGHV1-
BJ097 (SEQ
IGKV2-

ID NO: 236)
2*02
ID NO: 238)
30*01

BJM0044
BJ096 (SEQ
IGHV1-
BJ097 (SEQ
IGKV2-

ID NO: 237)
2*02
ID NO: 238)
30*01

BJM0045
BJ092 (SEQ
IGHV1-
BJ098 (SEQ
IGKV2-

ID NO: 6372)
18*03
ID NO: 239)
29*02

BJM0046
BJ093 (SEQ
IGHV1-
BJ098 (SEQ
IGKV2-

ID NO: 234)
18*01
ID NO: 239)
29*02

BJM0047
BJ094 (SEQ
IGHV1-
BJ098 (SEQ
IGKV2-

ID NO: 235)
2*02
ID NO: 239)
29*02

BJM0048
BJ095 (SEQ
IGHV1-
BJ098 (SEQ
IGKV2-

ID NO: 236)
2*02
ID NO: 239)
29*02

BJM0049
BJ096 (SEQ
IGHV1-
BJ098 (SEQ
IGKV2-

ID NO: 237)
2*02
ID NO: 239)
29*02

BJM0050
BJ092 (SEQ
IGHV1-
BJ099 (SEQ
IGKV2D-

ID NO: 6372)
18*03
ID NO: 240)
29*01

BJM0051
BJ093 (SEQ
IGHV1-
BJ099 (SEQ
IGKV2D-

ID NO: 234)
18*01
ID NO: 240)
29*01

BJM0052
BJ094 (SEQ
IGHV1-
BJ099 (SEQ
IGKV2D-

ID NO: 235)
2*02
ID NO: 240)
29*01

BJM0053
BJ095 (SEQ
IGHV1-
BJ099 (SEQ
IGKV2D-

ID NO: 236)
2*02
ID NO: 240)
29*01

BJM0054
BJ096 (SEQ
IGHV1-
BJ099 (SEQ
IGKV2D-

ID NO: 237)
2*02
ID NO: 240)
29*01

BJM0055
BJ092 (SEQ
IGHV1-
BJ100 (SEQ
IGKV2-

ID NO: 6372)
18*03
ID NO: 241)
24*01

BJM0056
BJ093 (SEQ
IGHV1-
BJ100 (SEQ
IGKV2-

ID NO: 234)
18*01
ID NO: 241)
24*01

BJM0057
BJ094 (SEQ
IGHV1-
BJ100 (SEQ
IGKV2-

ID NO: 235)
2*02
ID NO: 241)
24*01

BJM0058
BJ095 (SEQ
IGHV1-
BJ100 (SEQ
IGKV2-

ID NO: 236)
2*02
ID NO: 241)
24*01

BJM0059
BJ096 (SEQ
IGHV1-
BJ100 (SEQ
IGKV2-

ID NO: 237)
2*02
ID NO: 241)
24*01

BJM0060
BJ092 (SEQ
IGHV1-
BJ101 (SEQ
IGKV2-

ID NO: 6372)
18*03
ID NO: 242)
28*01

BJM0061
BJ093 (SEQ
IGHV1-
BJ101 (SEQ
IGKV2-

ID NO: 234)
18*01
ID NO: 242)
28*01

BJM0062
BJ094 (SEQ
IGHV1-
BJ101 (SEQ
IGKV2-

ID NO: 235)
2*02
ID NO: 242)
28*01

BJM0063
BJ095 (SEQ
IGHV1-
BJ101 (SEQ
IGKV2-

ID NO: 236)
2*02
ID NO: 242)
28*01

BJM0064
BJ096 (SEQ
IGHV1-
BJ101 (SEQ
IGKV2-

ID NO: 237)
2*02
ID NO: 242)
28*01

Immune Cell Engagers

The immune cell engagers of the multispecific or multifunctional molecules disclosed herein can mediate binding to, and/or activation of, an immune cell, e.g., an immune effector cell. In some embodiments, the immune cell is chosen from a T cell, an NK cell, a B cell, a dendritic cell, or a macrophage cell engager, or a combination thereof. In some embodiments, the immune cell engager is chosen from one, two, three, or all of a T cell engager, NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager, or a combination thereof. The immune cell engager can be an agonist of the immune system. In some embodiments, the immune cell engager can be an antibody molecule, a ligand molecule (e.g., a ligand that further comprises an immunoglobulin constant region, e.g., an Fc region), a small molecule, or a nucleotide molecule.

T Cell Engagers

The present disclosure provides, inter alia, multispecific (e.g., bi-, tri-, quad-specific) or multifunctional molecules, that are engineered to contain one or more T cell engagers that mediate binding to and/or activation of a T cell. Accordingly, in some embodiments, the T cell engager is selected from an antigen binding domain or ligand that binds to (e.g., and in some embodiments activates) one or more of the variable chain of the beta subunit of a TCR (e.g., TCRβV), CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In other embodiments, the T cell engager is selected from an antigen binding domain or ligand that binds to and does not activate one or more of TCRβV, CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell engager binds to TCRβV.

In some embodiments, the T cell engager binds to CD3 (e.g., comprises an antigen binding domain that binds to CD3). In some embodiments, the multispecific or multifunctional molecule comprises a T cell engager that binds to CD3 (e.g., comprises an antigen binding domain that binds to CD3) (e.g., comprises an antigen binding domain that binds to CD3) and a calreticulin-targeting antigen binding domain, e.g., as described herein.

In some embodiments, a multispecific or multifunctional molecule (e.g., as described herein) comprises an antigen binding domain that binds to CD3. In some embodiments, the multispecific or multifunctional molecule comprises an antigen binding domain that binds to CD3 and a calreticulin-targeting antigen binding domain, e.g., as described herein.

In some embodiments, the antigen binding domain that binds to CD3 comprises one or more CDRs (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3) disclosed in Table 26 and/or Table 42, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to CD3 comprises one or more framework regions (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and/or VLFWR4) disclosed in Table 26 and/or Table 42, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to CD3 comprises a VH and/or a VL disclosed in Table 27, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto.

TABLE 26

Exemplary heavy chain CDRs and FWRs of CD3-targeting antigen binding domains

Ab ID
VHFWR1
VHCDR1
VHFWR2
VHCDR2
VHFWR3
VHCDR3
VHFWR4

BJM1210
QVQLQQ

FTTYW

WVKQR

NFNPNN

KATLTVD

DDYGR

WGQG

(murine)
PGTELVK

MH

PGHGLE

GDTNY

KSSSTAY

YYFDY

TTLTV

PGASVKL
(SEQ
WIG

NEKFKT

MQLSSLT
(SEQ ID
SS (SEQ

SCKASGY
ID NO:
(SEQ ID
(SEQ ID
SEDSAVY
NO:
ID NO:

T (SEQ ID
7456)
NO:
NO:
YCAR
7460)
7461)

NO: 7455)

7457)
7458)
(SEQ ID

NO: 7459)

BKM0020
QVQLVQ

FTTYW

WVRQA

NFNPNN

RVTMTV

DDYGR

WGQG

(humanized)
SGAEVK

MH

PGQGLE

GDTNY

DKSTSTA

YYFDY

TLVTV

KPGASVK
(SEQ
WMG

NEKFKT

YMELRSL
(SEQ ID
SS (SEQ

VSCKASG
ID NO:
(SEQ ID
(SEQ ID
RSDDMA
NO:
ID NO:

YT (SEQ
7456)
NO:
NO:
VYYCAR
7460)
228)

ID NO:

6027)
7458)
(SEQ ID

7467)

NO: 7468)

BKM0025
QVQLVQ

FTTYW

WVRQA

NFNPNN

RVTMTV

DDYGR

WGQG

(humanized)
SGAEVK

MH

PGQGLE

GDINY

DKSTSTA

YYFDY

TLVTV

KPGASVK
(SEQ
WMG

NEKFKT

YMELRSL
(SEQ ID
SS (SEQ

VSCKASG
ID NO:
(SEQ ID
(SEQ ID
RSDDMA
NO:
ID NO:

YT (SEQ
7456)
NO:
NO:
VYYCAR
7460)
228)

ID NO:

6027)
7458)
(SEQ ID

7467)

NO: 7468)

BKM0028
QVQLVQ

FTTYW

WVRQA

NFNPNN

RVTMTV

DDYGR

WGQG

(humanized)
SGAEVK

MH

PGKGLE

GDTNY

DKSTSTA

YYFDY

TLVTV

KPGASVK
(SEQ
WMG

NEKFKT

YMELSSL
(SEQ ID
SS (SEQ

VSCKASG
ID NO:
(SEQ ID
(SEQ ID
RSEDTAV
NO:
ID NO:

YT (SEQ
7456)
NO:
NO:
YYCAR
7460)
228)

ID NO:

7473)
7458)
(SEQ ID

7467)

NO: 7474)

BKM0038
QVQLVQ

FTTYW

WVRQA
NFNPNN
RVTMTV

DDYGR

WGQG

(humanized)
SGAEVK

MH

PGKGLE

GDTNY

DKSTSTA

YYFDY

TLVTV

KPGASVK
(SEQ
WMG

NEKFKT

YMELSSL
(SEQ ID
SS (SEQ

VSCKASG
ID NO:
(SEQ ID
(SEQ ID
RSEDTAV
NO:
ID NO:

YT (SEQ
7456)
NO:
NO:
YYCAR
7460)
228)

ID NO:

7473)
7458)
(SEQ ID

7467)

NO: 7474)

TABLE 42

Exemplary light chain CDRs and FWRs of CD3-targeting antigen binding domains

Ab ID
VLFWR1
VLCDR1
VLFWR2
VLCDR2
VLFWR3
VLCDR3
VLFWR4

BJM1210
DIVMSQS

KSSQSL

WYQQ

WAFTR

GVPDRFT

KQSFIL

FGGGT

(murine)
PSSLAVS

LNSRTR

KPGQA

ES (SEQ
GSGSGTD

RT (SEQ
KLEIK

AGEKVT

KNYLA

PKLLIY
ID NO:
FTLTISSV
ID NO:
(SEQ ID

MSC (SEQ
(SEQ ID
(SEQ ID
7464)
QAEDLAI
7466)
NO:

ID NO:
NO:
NO:

YYC (SEQ

6284)

7462)
7463)
7433)

ID NO:

7465)

BKM0020
DIOMTQS

KSSQSL

WYQQ

WAFTR

GVPSRFSG

KQSFIL

FGGGT

(humanized)
PSTLSAS

LNSRTR

KPGKA

ES (SEQ
SGSGTEFT

RT (SEQ
KVEIK

VGDRVTI

KNYLA

PKLLIY
ID NO:
LTISSLQP
ID NO:
(SEQ ID

TC (SEQ
(SEQ ID
(SEQ ID
7464)
DDFATYY
7466)
NO:

ID NO:
NO:
NO:

C (SEQ ID

233)

6093)
7463)
280)

NO: 7469)

BKM0025
EIVMTQS

KSSQSL

WYQQ

WAFTR

GIPDRFSG

KQSFIL

FGGGT

(humanized)
PATLSLS

LNSRTR

KPGLA

ES (SEQ
SGSGTDFT

RT (SEQ
KVEIK

PGERATL

KNYLA

PRLLIY
ID NO:
LTISRLEP
ID NO:
(SEQ ID

SC (SEQ
(SEQ ID
(SEQ ID
7464)
EDFAVYY
7466)
NO:

ID NO:
NO:
NO:

C (SEQ ID

233)

7470)
7463)
7471)

NO: 7472)

BKM0028
EIVMTQS

KSSQSL

WYQQ

WAFTR

GIPDRESG

KQSFIL

FGGGT

(humanized)
PATLSLS

LNSRTR

KPGLA

ES (SEQ
SGSGTDFT

RT (SEQ
KVEIK

PGERATL

KNYLA

PRLLIY
ID NO:
LTISRLEP
ID NO:
(SEQ ID

SC (SEQ
(SEQ ID
(SEQ ID
7464)
EDFAVYY
7466)
NO:

ID NO:
NO:
NO:

C (SEQ ID

233)

7470)
7463)
7471)

NO: 7472)

BKM0038
DIQMTQS

KSSQSL

WYQQ

WAFTR

GVPSRFSG

KQSFIL

FGGGT

(humanized)
PSSLSAS

LNSRTR

KPGKA

ES (SEQ
SGSGTDFT

RT (SEQ
KVEIK

VGDRVTI

KNYLA

PKLLIY
ID NO:
LTISSLQP
ID NO:
(SEQ ID

TC (SEQ
(SEQ ID
(SEQ ID
7464)
EDFATYY
7466)
NO:

ID NO:
NO:
NO:

C (SEQ ID

233)

7475)
7463)
280)

NO: 7476)

TABLE 27

Exemplary variable regions of CD3-targeting antigen binding domains

SEQ

ID NO
Ab ID
Sequence

7477
BJM1210
QVQLQQPGTELVKPGASVKLSCKASGYTFTTYWMHWVKQRPGH

(murine) VH
GLEWIGNFNPNNGDTNYNEKFKTKATLTVDKSSSTAYMQLSSLTS

EDSAVYYCARDDYGRYYFDYWGQGTTLTVSS

7478
BKM0020
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQ

(humanized)
GLEWMGNFNPNNGDTNYNEKFKTRVTMTVDKSTSTAYMELRSL

VH
RSDDMAVYYCARDDYGRYYFDYWGQGTLVTVSS

7478
BKM0025
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQ

(humanized)
GLEWMGNFNPNNGDTNYNEKFKTRVTMTVDKSTSTAYMELRSL

VH
RSDDMAVYYCARDDYGRYYFDYWGQGTLVTVSS

7479
BKM0028
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGK

(humanized)
GLEWMGNFNPNNGDTNYNEKFKTRVTMTVDKSTSTAYMELSSLR

VH
SEDTAVYYCARDDYGRYYFDYWGQGTLVTVSS

7479
BKM0038
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGK

(humanized)
GLEWMGNFNPNNGDTNYNEKFKTRVTMTVDKSTSTAYMELSSLR

VH
SEDTAVYYCARDDYGRYYFDYWGQGTLVTVSS

7480
BJM1210
DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQK

(murine) VL
PGQAPKLLIYWAFTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIY

YCKQSFILRTFGGGTKLEIK

7481
BKM0020
DIQMTQSPSTLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQK

(humanized)
PGKAPKLLIYWAFTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATY

VL
YCKQSFILRTFGGGTKVEIK

7482
BKM0025
EIVMTQSPATLSLSPGERATLSCKSSQSLLNSRTRKNYLAWYQQKP

(humanized)
GLAPRLLIYWAFTRESGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY

VL
CKQSFILRTFGGGTKVEIK

7482
BKM0028
EIVMTQSPATLSLSPGERATLSCKSSQSLLNSRTRKNYLAWYQQKP

(humanized)
GLAPRLLIYWAFTRESGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY

VL
CKQSFILRTFGGGTKVEIK

7483
BKM0038
DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKP

(humanized)
GKAPKLLIYWAFTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYY

VL
CKQSFILRTFGGGTKVEIK

In some embodiments, the multifunctional molecule (e.g., an scFv, e.g., a bispecific scFv) comprises the amino acid sequence (or a CDR, VH, or VL sequence comprised therein) below, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity thereto:

(SEQ ID NO: 7452)

EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVA

YITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCAR

FDWDKNYWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSL

PASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPS

RFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKGG

GGSTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVD

VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM

SGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVCVLPPPEEEMTKKQV

TLSCAVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMVSKLRV

EKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

In some embodiments, the multifunctional molecule (e.g., a bispecific antibody molecule) comprises the amino acid sequence (or a CDR (underlined), VH, or constant region sequence comprised therein), below, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity thereto:

(SEQ ID NO: 7484)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSEYWMNWLRQAPGKGLEWVG

VIKYKYSNYATEFAESVKGRFTISRDDSKSSVYLQMNSLKTEDTAVYYC

ARGRDVQDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

PREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPGK

In some embodiments, the multifunctional molecule (e.g., a bispecific antibody molecule) comprises the amino acid sequence (or a CDR (underlined), VL, or constant region sequence comprised therein), below, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity thereto:

(SEQ ID NO: 7485)

DIQLTQSPSFLSASVGDRVTITCSTSSSVTTNYLHWYQQKPGKAPKLLI

YSTSNLASGVPSRFSGSGSGTEYTLTISSLQPEDFATYYCQQCLSSPCT

FGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE

VTHQGLSSPVTKSFNRGEC

In some embodiments, the multifunctional molecule (e.g., an scFv, e.g., a bispecific scFv) comprises the amino acid sequence (or a CDR (underlined), VH, or VL sequence comprised therein), below, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity thereto:

(SEQ ID NO: 7486)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWVRQAPGQGLEWMG

NFNPNNGDTNYNEKFKTRVTMTVDKSTSTAYMELRSLRSDDMAVYYCAR

DDYGRYYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSP

STLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWA

FTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCKQSFILRTFGGG

TKVEIKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQD

WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKN

QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL

TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human T Cell Receptor (TCR) Complex

T cell receptors (TCR) can be found on the surface of T cells. TCRs recognize antigens, e.g., peptides, presented on, e.g., bound to, major histocompatibility complex (MHC) molecules on the surface of cells, e.g., antigen-presenting cells. TCRs are heterodimeric molecules and can comprise an alpha chain, a beta chain, a gamma chain or a delta chain. TCRs comprising an alpha chain and a beta chain are also referred to as TCRαβ. The TCR beta chain consists of the following regions (also known as segments): variable (V), diversity (D), joining (J) and constant (C) (see Mayer G. and Nyland J. (2010) Chapter 10: Major Histocompatibility Complex and T-cell Receptors-Role in Immune Responses. In: Microbiology and Immunology on-line, University of South Carolina School of Medicine). The TCR alpha chain consists of V, J and C regions. The rearrangement of the T-cell receptor (TCR) through somatic recombination of V (variable), D (diversity), J (joining), and C (constant) regions is a defining event in the development and maturation of a T cell. TCR gene rearrangement takes place in the thymus.

TCRs can comprise a receptor complex, known as the TCR complex, which comprises a TCR heterodimer comprising of an alpha chain and a beta chain, and dimeric signaling molecules, e.g., CD3 co-receptors, e.g., CD3δ/ε, and/or CD3γ/ε.

TCR Beta V (TCRBV)

Diversity in the immune system enables protection against a huge array of pathogens. Since the germline genome is limited in size, diversity is achieved not only by the process of V(D)J recombination but also by junctional (junctions between V-D and D-J segments) deletion of nucleotides and addition of pseudo-random, non-templated nucleotides. The TCR beta gene undergoes gene arrangement to generate diversity.

The TCR V beta repertoire varies between individuals and populations because of, e.g., 7 frequently occurring inactivating polymorphisms in functional gene segments and a large insertion/deletion-related polymorphism encompassing 2 V beta gene segments.

This disclosure provides, inter alia, antibody molecules and fragments thereof, that bind, e.g., specifically bind, to a human TCR beta V chain (TCRβV), e.g., a TCRβV gene family (also referred to as a group), e.g., a TCRβV subfamily (also referred to as a subgroup), e.g., as described herein. TCR beta V families and subfamilies are known in the art, e.g., as described in Yassai et al., (2009) Immunogenetics 61(7)pp:493-502; Wei S. and Concannon P. (1994) Human Immunology 41(3) pp: 201-206. The antibodies described herein can be recombinant antibodies, e.g., recombinant non-murine antibodies, e.g., recombinant human or humanized antibodies.

In an aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to human TCRβV, e.g., a TCRβV family, e.g., gene family or a variant thereof. In some embodiments a TCRBV gene family comprises one or more subfamilies, e.g., as described herein, e.g., in FIG. 3, Table 28 or Table 29. In some embodiments, the TCRβV gene family comprises: a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCRβ V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, a TCRβ V29 subfamily, a TCRβ V1 subfamily, a TCRβ V17 subfamily, a TCRβ V21 subfamily, a TCRβ V23 subfamily, or a TCRβ V26 subfamily.

In some embodiments, TCRβ V6 subfamily is also known as TCRβ V13.1. In some embodiments, the TCRβ V6 subfamily comprises: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-8*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-2*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-3*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-1*01, or a variant thereof.

In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6, e.g., TCRβ V6-5*01, is recognized, e.g., bound, by SEQ ID NO: 1 and/or SEQ ID NO: 2. In some embodiments, TCRβ V6, e.g., TCRβ V6-5*01, is recognized, e.g., bound, by SEQ ID NO: 9 and/or SEQ ID NO: 10. In some embodiments, TCRβ V6 is recognized, e.g., bound, by SEQ ID NO: 9 and/or SEQ ID NO: 11.

In some embodiments, TCRβ V10 subfamily is also known as TCRβ V12. In some embodiments, the TCRβ V10 subfamily comprises: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01, or a variant thereof.

In some embodiments, TCRβ V12 subfamily is also known as TCRβ V8.1. In some embodiments, the TCRβ V12 subfamily comprises: TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01, or a variant thereof. In some embodiments, TCRβ V12 is recognized, e.g., bound, by SEQ ID NO: 15 and/or SEQ ID NO: 16. In some embodiments, TCRβ V12 is recognized, e.g., bound, by any one of SEQ ID NOs 23-25, and/or any one of SEQ ID NO: 26-30:

In some embodiments, the TCRβ V5 subfamily is chosen from: TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01, or a variant thereof.

In some embodiments, the TCRβ V7 subfamily comprises TCRβ V7-7*01, TCRβ V7-6*01, TCRβ V7-8*02, TCRβ V7-4*01, TCRβ V7-2*02, TCRβ V7-2*03, TCRβ V7-2*01, TCRβ V7-3*01, TCRβ V7-9*03, or TCRβ V7-9*01, or a variant thereof.

In some embodiments, the TCRβ V11 subfamily comprises: TCRβ V11-1*01, TCRβ V11-2*01 or TCRβ V11-3*01, or a variant thereof.

In some embodiments, the TCRβ V14 subfamily comprises TCRβ V14*01, or a variant thereof.

In some embodiments, the TCRβ V16 subfamily comprises TCRβ V16*01, or a variant thereof.

In some embodiments, the TCRβ V18 subfamily comprises TCRβ V18*01, or a variant thereof.

In some embodiments, the TCRβ V9 subfamily comprises TCRβ V9*01 or TCRβ V9*02, or a variant thereof.

In some embodiments, the TCRβ V13 subfamily comprises TCRβ V13*01, or a variant thereof.

In some embodiments, the TCRβ V4 subfamily comprises TCRβ V4-2*01, TCRβ V4-3*01, or TCRβ V4-1*01, or a variant thereof.

In some embodiments, the TCRβ V3 subfamily comprises TCRβ V3-1*01, or a variant thereof.

In some embodiments, the TCRβ V2 subfamily comprises TCRβ V2*01, or a variant thereof.

In some embodiments, the TCRβ V15 subfamily comprises TCRβ V15*01, or a variant thereof.

In some embodiments, the TCRβ V30 subfamily comprises TCRβ V30*01, or TCRβ V30*02, or a variant thereof.

In some embodiments, the TCRβ V19 subfamily comprises TCRβ V19*01, or TCRβ V19*02, or a variant thereof.

In some embodiments, the TCRβ V27 subfamily comprises TCRβ V27*01, or a variant thereof.

In some embodiments, the TCRβ V28 subfamily comprises TCRβ V28*01, or a variant thereof.

In some embodiments, the TCRβ V24 subfamily comprises TCRβ V24-1*01, or a variant thereof.

In some embodiments, the TCRβ V20 subfamily comprises TCRβ V20-1*01, or TCRβ V20-1*02, or a variant thereof.

In some embodiments, the TCRβ V25 subfamily comprises TCRβ V25-1*01, or a variant thereof.

In some embodiments, the TCRβ V29 subfamily comprises TCRβ V29-1*01, or a variant thereof.

TABLE 28

List of TCRβV subfamilies and subfamily members

Reference

in FIG. 3
Subfamily
Subfamily members

A
TCRβ V6
TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-

Also referred to as:
8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01,

TCR Vβ 13.1
TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01.

B
TCRβ V10
TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 or

Also referred to as:
TCRβ V10-2*01

TCRB V12

C
TCRβ V12
TCRβ V12-4*01, TCRβ V12-3*01, or TCRβ V12-5*01

Also referred to as:

TCRβ V8.1

D
TCRβ V5
TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-

8*01, TCRβ V5-1*01

E
TCRβ V7
TCRβ V7-7*01, TCRβ V7-6*01, TCRβ V7 -8*02, TCRβ V7-

4*01, TCRβ V7-2*02, TCRβ V7-2*03, TCRβ V7-2*01,

TCRβ V7-3*01, TCRβ V7-9*03, or TCRβ V7-9*01

F
TCRβ V11
TCRβ V11-1*01, TCRβ V11-2*01 or TCRβ V11-3*01

G
TCRβ V14
TCRβ V14*01

H
TCRβ V16
TCRβ V16*01

I
TCRβ V18
TCRβ V18*01

J
TCRβ V9
TCRβ V9*01 or TCRβ V9*02

K
TCRβ V13
TCRβ V13*01

L
TCRβ V4
TCRβ V4-2*01, TCRβ V4-3*01, or TCRβ V4-1*01

M
TCRβ V3
TCRβ V3-1*01

N
TCRβ V2
TCRβ V2*01

O
TCRβ V15
TCRβ V15*01

P
TCRβ V30
TCRβ V30*01, or TCRβ V30*02

Q
TCRβ V19
TCRβ V19*01, or TCRβ V19*02

R
TCRβ V27
TCRβ V27*01.

S
TCRβ V28
TCRβ V28*01.

T
TCRβ V24
TCRβ V24-1*01

U
TCRβ V20
TCRβ V20-1*01, or TCRβ V20-1*02

V
TCRβ V25
TCRβ V25-1*01

W
TCRβ V29
TCRβ V29-1*01

TABLE 29

Additional TCRβV subfamilies

Subfamily

TCRβ V1

TCRβ V17

TCRβ V21

TCRβ V23

TCRβ V26

Anti-TCRβV Antibodies

Disclosed herein, is the discovery of a novel class of antibodies, i.e. anti-TCRβV antibody molecules disclosed herein, which despite having low sequence similarity (e.g., low sequence identity among the different antibody molecules that recognize different TCRβV subfamilies), recognize a structurally conserved region, e.g., domain, on the TCRβV protein and have a similar function (e.g., a similar cytokine profile). Thus, the anti-TCRβV antibody molecules disclosed herein share a structure-function relationship.

In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., bind to, an interface of a TCRβV:TCRalpha complex.

In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., bind to, a constant region of a TCRβV protein. An exemplary antibody that binds to a constant region of a TCRβV region is JOVI.1 as described in Viney et al., (Hybridoma. 1992 December; 11(6):701-13).

In some embodiments, the anti-TCRβV antibody molecules disclosed herein do not recognize, e.g., bind to, one or more (e.g., all) of a complementarity determining region (e.g., CDR1, CDR2 and/or CDR3) of a TCRβV protein.

In some embodiments, the anti-TCRβV antibody molecules disclosed herein binds (e.g., specifically binds) to a TCRβV region. In some embodiments, binding of anti-TCRβV antibody molecules disclosed herein results in a cytokine profile that differs from a cytokine profile of a T cell engager that binds to a receptor or molecule other than a TCRβV region (“a non-TCRβV-binding T cell engager”). In some embodiments, the non-TCRβV-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., CD3 epsilon (CD3e) molecule); or a TCR alpha (TCRα) molecule. In some embodiments, the non-TCRβV-binding T cell engager is an OKT3 antibody or an SP34-2 antibody.

In an aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to human TCRβV, e.g., a TCRβV gene family, e.g., one or more of a TCRβV subfamily, e.g., as described herein, e.g., in FIG. 3, Table 28, or Table 29. In some embodiments, the anti-TCRβV antibody molecule binds to one or more TCRβV subfamilies chosen from: a TCRβ V6 subfamily, a TCRβ V10 subfamily, a TCRβ V12 subfamily, a TCRβ V5 subfamily, a TCRβ V7 subfamily, a TCRβ V11 subfamily, a TCRβ V14 subfamily, a TCRβ V16 subfamily, a TCRβ V18 subfamily, a TCRβ V9 subfamily, a TCR P V13 subfamily, a TCRβ V4 subfamily, a TCRβ V3 subfamily, a TCRβ V2 subfamily, a TCRβ V15 subfamily, a TCRβ V30 subfamily, a TCRβ V19 subfamily, a TCRβ V27 subfamily, a TCRβ V28 subfamily, a TCRβ V24 subfamily, a TCRβ V20 subfamily, TCRβ V25 subfamily, a TCRβ V29 subfamily, a TCRβ V1 subfamily, a TCRβ V17 subfamily, a TCRβ V21 subfamily, a TCRβ V23 subfamily, or a TCRβ V26 subfamily, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβ V6 subfamily comprising: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01, or a variant thereof. In some embodiments the TCRβ V6 subfamily comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-8*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-2*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-3*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-1*01, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβ V10 subfamily comprising: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβ V12 subfamily comprising: TCRβ V12-4*01, TCRβ V12-3*01 or TCRβ V12-5*01, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβ V5 subfamily comprising: TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01, or a variant thereof.

In some embodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V12, or binds to TCRβ V12 with an affinity and/or binding specificity that is less than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificity of the 16G8 murine antibody or a humanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V12 with an affinity and/or binding specificity that is greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificity of the 16G8 murine antibody or a humanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV region other than TCRβ V12 (e.g., TCRβV region as described herein, e.g., TCRβ V6 subfamily (e.g., TCRβ V6-5*01) with an affinity and/or binding specificity that is greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificity of the 16G8 murine antibody or a humanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule does not bind to TCRβ V5-5*01 or TCRβ V5-1*01, or binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and/or binding specificity that is less than (e.g., less than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificity of the TM23 murine antibody or a humanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to TCRβ V5-5*01 or TCRβ V5-1*01 with an affinity and/or binding specificity that is greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificity of the TM23 murine antibody or a humanized version thereof as described in U.S. Pat. No. 5,861,155.

In some embodiments, the anti-TCRβV antibody molecule binds to a TCRβV region other than TCRβ V5-5*01 or TCRβ V5-1*01 (e.g., TCRβV region as described herein, e.g., TCR P V6 subfamily (e.g., TCRβ V6-5*01) with an affinity and/or binding specificity that is greater than (e.g., greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 2-, 5-, or 10-fold) the affinity and/or binding specificity of the TM23 murine antibody or a humanized version thereof as described in U.S. Pat. No. 5,861,155.

Anti-TCRβ V6 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to human TCRβ V6, e.g., a TCRβ V6 subfamily comprising: TCRβ V6-4*01, TCRβ V6-4*02, TCRβ V6-9*01, TCRβ V6-8*01, TCRβ V6-5*01, TCRβ V6-6*02, TCRβ V6-6*01, TCRβ V6-2*01, TCRβ V6-3*01 or TCRβ V6-1*01. In some embodiments the TCRβ V6 subfamily comprises TCRβ V6-5*01 or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-4*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-9*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-8*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-5*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*02, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-6*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-2*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-3*01, or a variant thereof. In some embodiments, TCRβ V6 comprises TCRβ V6-1*01, or a variant thereof.

In some embodiments, TCRβ V6-5*01 is encoded by the nucleic acid sequence of SEQ ID NO: 43, or a sequence having 85%, 90%, 95%, 99% or more identity thereof.

SEQ ID NO: 43

ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAG

GTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAA

GACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAA

TACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTC

ATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGG

CTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTG

TCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACT

C

In some embodiments, TCRβ V6-5*01 comprises the amino acid sequence of SEQ ID NO: 1044, or an amino acid sequence having 85%, 90%, 95%, 99% or more identity thereof.

SEQ ID NO: 1044

MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHE

YMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLL

SAAPSQTSVYFCASSY

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a non-murine antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is a human antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is a humanized antibody molecule.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is isolated or recombinant.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three or four variable regions from an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody molecule described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule comprises a heavy chain variable region (VH) having a consensus sequence of SEQ ID NO: 231 or 3290.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule comprises a light chain variable region (VL) having a consensus sequence of SEQ ID NO: 230 or 3289.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain constant region for an IgG4, e.g., a human IgG4. In still another embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes a heavy chain constant region for an IgG1, e.g., a human IgG1. In one embodiment, the heavy chain constant region comprises an amino sequence set forth in Table 32, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises an amino sequence set forth in Table 32, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region (VH) of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in TABLE 30, or encoded by a nucleotide sequence shown in TABLE 30. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in TABLE 30, or encoded by a nucleotide sequence shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, or three complementarity determining regions (CDRs) from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence shown in TABLE 30, or encoded by a nucleotide sequence shown in TABLE 30. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in TABLE 30, or encoded by a nucleotide sequence shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, three, four, five or six CDRs (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in TABLE 30, or encoded by a nucleotide sequence shown in TABLE 30. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in TABLE 30, or encoded by a nucleotide sequence shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, molecule includes all six CDRs from an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30, or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in TABLE 30) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in TABLE 30) from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat definition as set out in TABLE 30) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Kabat et al. shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat definition as set out in TABLE 30) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al. shown in TABLE 30.

In one embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of an antibody described herein, e.g., an antibody chosen from chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, e.g., the same canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of an antibody described herein. See, e.g., Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798 for descriptions of hypervariable loop canonical structures. These structures can be determined by inspection of the tables described in these references.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in TABLE 30) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or as described in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in TABLE 30) from a light chain variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes at least one, two, three, four, five, or six CDRs according to Chothia et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Chothia definition as set out in TABLE 30) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by the nucleotide sequence in TABLE 30; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Chothia et al. shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes all six CDRs according to Chothia et al. (e.g., all six CDRs according to the Chothia definition as set out in TABLE 30) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or an antibody described in TABLE 30, or encoded by a nucleotide sequence in TABLE 30; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Chothia et al. shown in TABLE 30. In one embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can contain any combination of CDRs or hypervariable loops according to the Kabat and Chothia definitions.

In some embodiments, a combined CDR as set out in TABLE 30 is a CDR that comprises a Kabat CDR and a Chothia CDR.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, molecule includes a combination of CDRs or hypervariable loops identified as combined CDRs in TABLE 30. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can contain any combination of CDRs or hypervariable loops according the “combined” CDRs are described in TABLE 30.

In an embodiment, e.g., an embodiment comprising a variable region, a CDR (e.g., a combined CDR, Chothia CDR or Kabat CDR), or other sequence referred to herein, e.g., in TABLE 30, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic antibody molecule, or an antibody molecule that comprises an antigen binding fragment of an antibody, e.g., a half antibody or antigen binding fragment of a half antibody. In certain embodiments, the antibody molecule comprises a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCR P V6 (e.g., anti-TCR P V6-5*01) antibody molecule includes:

- (i) one, two or all of a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 2, SEQ ID NO: 10 or SEQ ID NO: 11, and/or
- (ii) one, two or all of a heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 1 or SEQ ID NO: 9.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 2, and a HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 1.

In some embodiments the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 10, and a HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

In some embodiments, the anti-TCR PV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO: 11, and a HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO: 9.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCR P V6 (e.g., anti-TCR P V6-5*01) antibody molecule comprises:

- (i) a LC CDR1 amino acid sequence of SEQ ID NO: 6, a LC CDR2 amino acid sequence of SEQ ID NO: 7, or a LC CDR3 amino acid sequence of SEQ ID NO: 8; and/or
- (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 3, a HC CDR2 amino acid sequence of SEQ ID NO: 4, or a HC CDR3 amino acid sequence of SEQ ID NO: 5.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

- (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 6, a LC CDR2 amino acid sequence of SEQ ID NO: 7, or a LC CDR3 amino acid sequence of SEQ ID NO: 8; and/or
- (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 3, a HC CDR2 amino acid sequence of SEQ ID NO: 4, or a HC CDR3 amino acid sequence of SEQ ID NO: 5.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCR P V6 (e.g., anti-TCR P V6-5*01) antibody molecule comprises:

- (i) a LC CDR1 amino acid sequence of SEQ ID NO: 51, a LC CDR2 amino acid sequence of SEQ ID NO: 52, or a LC CDR3 amino acid sequence of SEQ ID NO: 53; and/or
- (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 45, a HC CDR2 amino acid sequence of SEQ ID NO: 46, or a HC CDR3 amino acid sequence of SEQ ID NO: 47.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβV6-5*01) antibody molecule comprises:

- (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 51, a LC CDR2 amino acid sequence of SEQ ID NO: 52, or a LC CDR3 amino acid sequence of SEQ ID NO: 53; and/or
- (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 45, a HC CDR2 amino acid sequence of SEQ ID NO: 46, or a HC CDR3 amino acid sequence of SEQ ID NO: 47.

In an embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCR P V6 (e.g., anti-TCR P V6-5*01) antibody molecule comprises:

- (i) a LC CDR1 amino acid sequence of SEQ ID NO: 54, a LC CDR2 amino acid sequence of SEQ ID NO: 55, or a LC CDR3 amino acid sequence of SEQ ID NO: 56; and/or
- (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 48, a HC CDR2 amino acid sequence of SEQ ID NO: 49, or a HC CDR3 amino acid sequence of SEQ ID NO: 50.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises:

- (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 54, a LC CDR2 amino acid sequence of SEQ ID NO: 55, or a LC CDR3 amino acid sequence of SEQ ID NO: 56; and/or
- (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 48, a HC CDR2 amino acid sequence of SEQ ID NO: 49, or a HC CDR3 amino acid sequence of SEQ ID NO: 50.

In one embodiment, the light or the heavy chain variable framework (e.g., the region encompassing at least FR1, FR2, FR3, and optionally FR4) of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule can be chosen from: (a) a light or heavy chain variable framework including at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a light or heavy chain variable framework including from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a non-human framework that has been modified, e.g., to remove antigenic or cytotoxic determinants, e.g., deimmunized, or partially humanized. In one embodiment, the light or heavy chain variable framework region (particularly FR1, FR2 and/or FR3) includes a light or heavy chain variable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the frameworks of a VL or VH segment of a human germline gene.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, e.g., amino acid substitutions or deletions, from an amino acid sequence of any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, e.g., the amino acid sequence of the FR region in the entire variable region, e.g., shown in FIG. 1A, or in SEQ ID NO: 9.

Alternatively, or in combination with the heavy chain substitutions described herein, the anti-TCRβV antibody molecule, e.g., anti-TCR P V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more amino acid changes, e.g., amino acid substitutions or deletions, from an amino acid sequence of any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, e.g., the amino acid sequence of the FR region in the entire variable region, e.g., shown in FIG. 1B, or in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes one, two, three, or four heavy chain framework regions shown in FIG. 1A, or a sequence substantially identical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes one, two, three, or four light chain framework regions shown in FIG. 1B, or a sequence substantially identical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the light chain framework region 1 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the light chain framework region 2 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the light chain framework region 3 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the light chain framework region 4 of A-H.1 or A-H.2, e.g., as shown in FIG. 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 1 (FR1), comprising a change, e.g., a substitution (e.g., a conservative substitution) at position 10 according to Kabat numbering. In some embodiments, the FR1 comprises a Phenylalanine at position 10, e.g., a Serine to Phenyalanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 2 (FR2), comprising a change, e.g., a substitution (e.g., a conservative substitution) at a position disclosed herein according to Kabat numbering. In some embodiments, FR2 comprises a Histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a Tyrosine to Histidine substitution. In some embodiments, FR2 comprises an Alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., an Arginine to Alanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising a framework region, e.g., framework region 3 (FR3), comprising a change, e.g., a substitution (e.g., a conservative substitution) at a position disclosed herein according to Kabat numbering. In some embodiments, FR3 comprises a Phenyalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a Tyrosine to Phenyalanine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) a framework region 1 (FR1) comprising a Phenylalanine at position 10, e.g., a substitution at position 10 according to Kabat numbering, e.g., a Serine to Phenyalanine substitution; (b) a framework region 2 (FR2) comprising a Histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a Tyrosine to Histidine substitution, and a Alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., a Arginine to Alanine substitution; and (c) a framework region 3 (FR3) comprising a Phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a Tyrosine to Phenyalanine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 10. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) a framework region 2 (FR2) comprising a Histidine at position 36, e.g., a substitution at position 36 according to Kabat numbering, e.g., a Tyrosine to Histidine substitution, and a Alanine at position 46, e.g., a substitution at position 46 according to Kabat numbering, e.g., a Arginine to Alanine substitution; and (b) a framework region 3 (FR3) comprising a Phenylalanine at position 87, e.g., a substitution at position 87 according to Kabat numbering, e.g., a Tyrosine to Phenyalanine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 11. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a light chain variable domain comprising: (a) a framework region 1 (FR1) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) positions disclosed herein according to Kabat numbering, (b) a framework region 2 (FR2) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) position disclosed herein according to Kabat numbering and (c) a framework region 3 (FR3) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) position disclosed herein according to Kabat numbering. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework region 1 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework region 2 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework region 3 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework region 4 of A-H.1 or A-H.2, e.g., as shown in FIG. 1A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain variable domain comprising a framework region, e.g., framework region 3 (FR3), comprising a change, e.g., a substitution (e.g., a conservative substitution) at a position disclosed herein according to Kabat numbering. In some embodiments, FR3 comprises a Threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a Glutamic Acid to Threonine substitution. In some embodiments, FR3 comprises a Glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., an Arginine to Glycine substitution. In some embodiments, the substitution is relative to a human germline heavy chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises a heavy chain variable domain comprising a framework region 3 (FR3) comprising a Threonine at position 73, e.g., a substitution at position 73 according to Kabat numbering, e.g., a Glutamic Acid to Threonine substitution, and a Glycine at position 94, e.g., a substitution at position 94 according to Kabat numbering, e.g., a Arginine to Glycine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework regions 1-4 of A-H.1 or A-H.2, e.g., SEQ ID NO: 9, or as shown in FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the light chain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 10, or as shown in FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the light chain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 11, or as shown in FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 9; and the light chain framework regions 1-4 of A-H.1, e.g., SEQ ID NO: 10, or as shown in FIGS. 1A and 1B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises the heavy chain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 9; and the light chain framework regions 1-4 of A-H.2, e.g., SEQ ID NO: 11, or as shown in FIGS. 1A and 1B.

In some embodiments, the heavy or light chain variable domain, or both, of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, includes an amino acid sequence, which is substantially identical to an amino acid disclosed herein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical to a variable region of an antibody described herein, e.g., an antibody chosen from any one of A-H.1 to A-H.85, e.g., A-H.1, A-H.2 or A-H.68, or as described in TABLE 30, or encoded by the nucleotide sequence in TABLE 30; or which differs at least 1 or 5 residues, but less than 40, 30, 20, or 10 residues, from a variable region of an antibody described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence as set forth in TABLE 30, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the sequences shown in TABLE 30. In another embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule includes a VH and/or VL domain encoded by a nucleic acid having a nucleotide sequence as set forth in TABLE 30, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in TABLE 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 9; and/or
- a VL domain comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 9; and/or
- a VL domain comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is a full antibody or fragment thereof (e.g., a Fab, F(ab′)₂, Fv, or a single chain Fv fragment (scFv)). In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is a monoclonal antibody or an antibody with single specificity. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can also be a humanized, chimeric, camelid, shark, or an in vitro-generated antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is a humanized antibody molecule. The heavy and light chains of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, can be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab′)₂, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody).

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, is in the form of a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule, has a heavy chain constant region (Fc) chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is chosen from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is chosen from the heavy chain constant region of IgG1 or IgG2 (e.g., human IgG1, or IgG2). In some embodiments, the heavy chain constant region is human IgG1. In some embodiments, the Fc region comprises a Fc region variant, e.g., as described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβV6-5*01) antibody molecule, has a light chain constant region chosen from, e.g., the light chain constant regions of kappa or lambda, preferably kappa (e.g., human kappa). In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the constant region is mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding (e.g., the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314 (N to F) of SEQ ID NOs: 212 or 214; or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NOs: 215, 216, 217 or 218), e.g., relative to human IgG1.

Antibody A-H.1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 72. Antibody A-H.2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3278 and a light chain comprising the amino acid sequence of SEQ ID NO: 3279. Antibody A-H.68 comprises the amino acid sequence of SEQ ID NO: 1337, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

Additional exemplary humanized anti-TCRB V6 antibodies are provided in TABLE 30. In some embodiments, the anti-TCRβ V6 is antibody A, e.g., humanized antibody A (antibody A-H), as provided in TABLE 30. In some embodiments, the anti-TCRβV antibody comprises one or more (e.g., all three) of a LC CDR1, LC CDR2, and LC CDR3 provided in TABLE 30; and/or one or more (e.g., all three) of a HC CDR1, HC CDR2, and HC CDR3 provided in TABLE 30, or a sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, antibody A comprises a variable heavy chain (VH) and/or a variable light chain (VL) provided in TABLE 30, or a sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH and/or a VL of an antibody described in Table 1, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCR P V6 (e.g., anti-TCR P V6-5*01) antibody molecule comprises a VH of an antibody described in Table 1, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VL of an antibody described in Table 1, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH and a VL of an antibody described in Table 1, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VL of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.1, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.1, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and a VL of A-H.1, A-H.2, A-H.3, A-H.4, A-H.5, A-H.6, A-H.7, A-H.8, A-H.9, A-H.10, A-H.11, A-H.12, A-H.13, A-H.14, A-H.15, A-H.16, A-H.17, A-H.18, A-H.19, A-H.20, A-H.21, A-H.22, A-H.23, A-H.24, A-H.25, A-H.26, A-H.27, A-H.28, A-H.29, A-H.30, A-H.31, A-H.32, A-H.33, A-H.34, A-H.35, A-H.36, A-H.37, A-H.38, A-H.39, A-H.40, A-H.1, A-H.42, A-H.43, A-H.44, A-H.45, A-H.46, A-H.47, A-H.48, A-H.49, A-H.50, A-H.51, A-H.52, A-H.53, A-H.54, A-H.55, A-H.56, A-H.57, A-H.58, A-H.59, A-H.60, A-H.61, A-H.62, A-H.63, A-H.64, A-H.65, A-H.66, A-H.67, A-H.68, A-H.69, A-H.70, A-H.71, A-H.72, A-H.73, A-H.74, A-H.75, A-H.76, A-H.77, A-H.78, A-H.79, A-H.80, A-H.81, A-H.82, A-H.83, A-H.84, or A-H.85, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

TABLE 30

Amino acid and nucleotide sequences for murine, chimeric and humanized antibody

molecules which bind to TCRVB 6, e.g., TCRVB 6-5. The antibody molecules include murine mAb

Antibody A, and humanized mAb Antibody A-H Clones A-H.1 to A-H.85. The amino acid the heavy

and light chain CDRs, and the amino acid and nucleotide sequences of the heavy and light

chain variable regions, and the heavy and light chains are shown.

Antibody A (murine), also referred to as H131, binds to TCRVB 6-5

SEQ ID NO: 3
HC CDR1 (Combined)
GYSFTTYYIH

SEQ ID NO: 4
HC CDR2 (Combined)
WFFPGSGNIKYNEKFKG

SEQ ID NO: 5
HC CDR3 (Combined)
SYYSYDVLDY

SEQ ID NO: 45
HC CDR1 (Kabat)
TYYIH

SEQ ID NO: 46
HC CDR2 (Kabat)
WFFPGSGNIKYNEKFKG

SEQ ID NO: 47
HC CDR3 (Kabat)
SYYSYDVLDY

SEQ ID NO: 48
HC CDR1 (Chothia)
GYSFTTY

SEQ ID NO: 49
HC CDR2 (Chothia)
FPGSGN

SEQ ID NO: 50
HC CDR3 (Chothia)
SYYSYDVLDY

SEQ ID NO: 1
VH
QVQLQQSGPELVKPGTSVKISCKASGYSFTTYYI

HWVKQRPGQGLEWIGWFFPGSGNIKYNEKFKG

KATLTADTSSSTAYMQLSSLTSEESAVYFCAGSY

YSYDVLDYWGHGTTLTVSS

SEQ ID NO: 6
LC CDR1 (Combined)
KASQNVGINVV

SEQ ID NO: 7
LC CDR2 (Combined)
SSSHRYS

SEQ ID NO: 8
LC CDR3 (Combined)
QQFKSYPLT

SEQ ID NO: 51
LC CDR1 (Kabat)
KASQNVGINVV

SEQ ID NO: 52
LC CDR2 (Kabat)
SSSHRYS

SEQ ID NO: 53
LC CDR3 (Kabat)
QQFKSYPLT

SEQ ID NO: 54
LC CDR1 (Chothia)
KASQNVGINVV

SEQ ID NO: 55
LC CDR2 (chothia)
SSSHRYS

SEQ ID NO: 56
LC CDR3 (chothia)
QQFKSYPLT

SEQ ID NO: 2
VL
DILMTQSQKFMSTSLGDRVSVSCKASQNVGINV

VWHQQKPGQSPKALIYSSSHRYSGVPDRFTGSG

SGTDFTLTINNVQSEDLAEYFCQQFKSYPLTFGA

GTKLELK

Antibody A humanized (A-H antibody)

A-H.1 antibody

SEQ ID NO: 3
HC CDR1 (Combined)
GYSFTTYYIH

SEQ ID NO: 4
HC CDR2 (Combined)
WFFPGSGNIKYNEKFKG

SEQ ID NO: 5
HC CDR3 (Combined)
SYYSYDVLDY

SEQ ID NO: 9
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO: 12
DNA VH
CAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGT

GAAGAAACCTGGCTCCTCCGTGAAGGTGTCCT

GCAAGGCTTCCGGCTACTCCTTCACCACCTAC

TACATCCACTGGGTCCGACAGGCCCCTGGACA

AGGATTGGAATGGATGGGCTGGTTCTTCCCCG

GCTCCGGCAACATCAAGTACAACGAGAAGTTC

AAGGGCCGCGTGACCATCACCGCCGACACCTC

TACCTCTACCGCCTACATGGAACTGTCCAGCC

TGAGATCTGAGGACACCGCCGTGTACTACTGC

GCCGGCTCCTACTACTCTTACGACGTGCTGGA

TTACTGGGGCCAGGGCACCACAGTGACAGTGT

CCTCT

SEQ ID NO: 69
VH-IgM constant delta
METDTLLLWVLLLWVPGSTGQVQLVQSGAEVK

CDC
KPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGL

EWMGWFFPGSGNIKYNEKFKGRVTITADTSTST

AYMELSSLRSEDTAVYYCAGSYYSYDVLDYWG

QGTTVTVSSGSASAPTLFPLVSCENSPSDTSSVA

VGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSV

LRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQ

HPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFG

NPRKSKLICQATGFSPRQIQVSWLREGKQVGSG

VTTDQVQAEAKESGPTTYKVTSTLTIKESDWLG

QSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRV

FAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWT

RQNGEAVKTHTNISESHPNATFSAVGEASICEDD

WNSGERFTCTVTHTDLASSLKQTISRPKGVALH

RPDVYLLPPAREQLNLRESATITCLVTGFSPADV

FVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYF

AHSILTVSEEEWNTGETYTCVVAHEALPNRVTE

RTVDKSTGKPTLYNVSLVMSDTAGTCY

SEQ ID NO: 70
VH-IgGA1
METDTLLLWVLLLWVPGSTGQVQLVQSGAEVK

KPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGL

EWMGWFFPGSGNIKYNEKFKGRVTITADTSTST

AYMELSSLRSEDTAVYYCAGSYYSYDVLDYWG

QGTTVTVSSASPTSPKVFPLSLCSTQPDGNVVIA

CLVQGFFPQEPLSVTWSESGQGVTARNFPPSQD

ASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYT

NPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRL

SLHRPALEDLLLGSEANLTCTLTGLRDASGVTFT

WTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAE

PWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRP

EVHLLPPPSEELALNELVTLTCLARGFSPKDVLV

RWLQGSQELPREKYLTWASRQEPSQGTTTFAVT

SILRVAAEDWKKGDTFSCMVGHEALPLAFTQKT

IDRLAGKPTHVNVSVVMAEVDGTCY

SEQ ID NO: 71
VH-IgGA2
METDTLLLWVLLLWVPGSTGQVQLVQSGAEVK

KPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGL

EWMGWFFPGSGNIKYNEKFKGRVTITADTSTST

AYMELSSLRSEDTAVYYCAGSYYSYDVLDYWG

QGTTVTVSSASPTSPKVFPLSLDSTPQDGNVVVA

CLVQGFFPQEPLSVTWSESGQNVTARNFPPSQD

ASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYT

NSSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLL

LGSEANLTCTLTGLRDASGATFTWTPSSGKSAV

QGPPERDLCGCYSVSSVLPGCAQPWNHGETFTC

TAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEE

LALNELVTLTCLARGFSPKDVLVRWLQGSQELP

REKYLTWASRQEPSQGTTTYAVTSILRVAAEDW

KKGETFSCMVGHEALPLAFTQKTIDRMAGKPTH

INVSVVMAEADGTCY

SEQ ID NO:
Heavy chain
METDTLLLWVLLLWVPGSTGQVQLVQSGAEVK

3278

KPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGL

EWMGWFFPGSGNIKYNEKFKGRVTITADTSTST

AYMELSSLRSEDTAVYYCAGSYYSYDVLDYWG

QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGK

SEQ ID NO: 6
LC CDR1 (Combined)
KASQNVGINVV

SEQ ID NO: 7
LC CDR2 (Combined)
SSSHRYS

SEQ ID NO: 8
LC CDR3 (Combined)
QQFKSYPLT

SEQ ID NO: 10
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGINVV

WHQQKPGKAPKALIYSSSHRYSGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 13
DNA VL
GACATCCAGATGACCCAGTCTCCATCCTTCCT

GTCCGCCTCTGTGGGCGACAGAGTGACCATCA

CATGCAAGGCCTCTCAGAACGTGGGCATCAAC

GTCGTGTGGCACCAGCAGAAGCCTGGCAAGG

CTCCTAAGGCTCTGATCTACTCCTCCAGCCACC

GGTACTCTGGCGTGCCCTCTAGATTTTCCGGCT

CTGGCTCTGGCACCGAGTTTACCCTGACAATC

TCCAGCCTGCAGCCTGAGGACTTCGCCACCTA

CTTTTGCCAGCAGTTCAAGAGCTACCCTCTGA

CCTTTGGCCAGGGCACCAAGCTGGAAATCAAG

SEQ ID NO: 72
VL and kappa constant
METDTLLLWVLLLWVPGSTGDIQMTQSPSFLSA

region/light chain
SVGDRVTITCKASQNVGINVVWHQQKPGKAPK

ALIYSSSHRYSGVPSRFSGSGSGTEFTLTISSLQPE

DFATYFCQQFKSYPLTFGQGTKLEIKRTVAAPSV

FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

A-H.2 antibody

SEQ ID NO: 3
HC CDR1 (Combined)
GYSFTTYYIH

SEQ ID NO: 4
HC CDR2 (Combined)
WFFPGSGNIKYNEKFKG

SEQ ID NO: 5
HC CDR3 (Combined)
SYYSYDVLDY

SEQ ID NO: 9
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO: 12
DNA VH
CAGGTGCAGCTGGTTCAGTCTGGCGCCGAAGT

GAAGAAACCTGGCTCCTCCGTGAAGGTGTCCT

GCAAGGCTTCCGGCTACTCCTTCACCACCTAC

TACATCCACTGGGTCCGACAGGCCCCTGGACA

AGGATTGGAATGGATGGGCTGGTTCTTCCCCG

GCTCCGGCAACATCAAGTACAACGAGAAGTTC

AAGGGCCGCGTGACCATCACCGCCGACACCTC

TACCTCTACCGCCTACATGGAACTGTCCAGCC

TGAGATCTGAGGACACCGCCGTGTACTACTGC

GCCGGCTCCTACTACTCTTACGACGTGCTGGA

TTACTGGGGCCAGGGCACCACAGTGACAGTGT

CCTCT

SEQ ID NO:
Heavy chain
METDTLLLWVLLLWVPGSTGQVQLVQSGAEVK

3278

KPGSSVKVSCKASGYSFTTYYIHWVRQAPGQGL

EWMGWFFPGSGNIKYNEKFKGRVTITADTSTST

AYMELSSLRSEDTAVYYCAGSYYSYDVLDYWG

QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ

SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT

KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV

LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGK

SEQ ID NO: 6
LC CDR1 (Combined)
KASQNVGINVV

SEQ ID NO: 7
LC CDR2 (Combined)
SSSHRYS

SEQ ID NO: 8
LC CDR3 (Combined)
QQFKSYPLT

SEQ ID NO: 11
VL
DIQMTQSPSSLSASVGDRVTITCKASQNVGINVV

WHQQKPGKVPKALIYSSSHRYSGVPSRFSGSGS

GTDFTLTISSLQPEDVATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 14
DNA VL
GACATCCAGATGACCCAGTCTCCATCCTCTCT

GTCCGCCTCTGTGGGCGACAGAGTGACCATCA

CATGCAAGGCCTCTCAGAACGTGGGCATCAAC

GTCGTGTGGCACCAGCAGAAACCTGGCAAGGT

GCCCAAGGCTCTGATCTACTCCTCCAGCCACA

GATACTCCGGCGTGCCCTCTAGATTCTCCGGC

TCTGGCTCTGGCACCGACTTTACCCTGACAAT

CTCCAGCCTGCAGCCTGAGGACGTGGCCACCT

ACTTTTGCCAGCAGTTCAAGAGCTACCCTCTG

ACCTTTGGCCAGGGCACCAAGCTGGAAATCAA

G

SEQ ID NO:
Light chain
METDTLLLWVLLLWVPGSTGDIQMTQSPSSLSA

3279

SVGDRVTITCKASQNVGINVVWHQQKPGKVPK

ALIYSSSHRYSGVPSRFSGSGSGTDFTLTISSLQPE

DVATYFCQQFKSYPLTFGQGTKLEIKRTVAAPSV

FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK

VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

A-H.3 antibody

SEQ ID NO: 80
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVEDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 81
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA

WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 82
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.4

SEQ ID NO: 83
VH+VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVEDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 84
VI
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA

WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 85
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.5

SEQ ID NO: 86
VH+VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRDF

YIHWVRQAPGQGLEWMGRVYPGSGSYRYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 87
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 88
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRDF

YIHWVRQAPGQGLEWMGRVYPGSGSYRYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.6

SEQ ID NO: 89
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

YIHWVRQAPGQGLEWMGRISAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDNRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 90
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 91
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

YIHWVRQAPGQGLEWMGRISAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.7

SEQ ID NO: 92
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVENKVAWHQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 93
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVENKVA

WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 94
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.8

SEQ ID NO: 95
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRIFAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 96
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 97
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRIFAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.9

SEQ ID NO: 98
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGNRVAWYQQKPGKAPKALIYSSSHRYS

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 99
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGNRV

AWYQQKPGKAPKALIYSSSHRYSGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 100
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.10

SEQ ID NO: 101
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRIFAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIKs

SEQ ID NO: 102
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 103
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRIFAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.11

SEQ ID NO: 104
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRVSPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 105
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 106
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRVSPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.12

SEQ ID NO: 107
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRVSAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGNRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 108
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGNRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 109
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRVSAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.13 (also referred to as A-H.69)

SEQ ID NO: 110
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDNRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 111
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 112
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.14

SEQ ID NO: 113
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 114
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 115
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.15

SEQ ID NO: 116
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTY

IHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDNKVAWHQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 117
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNKV

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 118
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTY

IHWVRQAPGQGLEWMGRVSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.16

SEQ ID NO: 119
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTY

IHWVRQAPGQGLEWMGRVYPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 120
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 121
VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTY

IHWVRQAPGQGLEWMGRVYPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.17

SEQ ID NO: 122
VH+VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTY

IHWVRQAPGQGLEWMGRIFPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 123
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 124
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTY

IHWVRQAPGQGLEWMGRIFPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.18

SEQ ID NO: 125
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVEDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 126
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA

WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 127
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.19

SEQ ID NO: 128
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 129
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 130
VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFRLTY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.20

SEQ ID NO: 131
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGGTFDKT

YIHWVRQAPGQGLEWMGRISAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 132
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 133
VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFDKT

YIHWVRQAPGQGLEWMGRISAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.21

SEQ ID NO: 134
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRISAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 135
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 136
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRISAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.22

SEQ ID NO: 137
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDNKVAWHQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 138
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNKV

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 139
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.23

SEQ ID NO: 140
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVADRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 141
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 142
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.24

SEQ ID NO: 143
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLW

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDNKVAWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 144
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNKV

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 145
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLW

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.25

SEQ ID NO: 146
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLW

YIHWVRQAPGQGLEWMGRVFAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVEDKVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 147
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDKVA

WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 148
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLW

YIHWVRQAPGQGLEWMGRVFAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.26

SEQ ID NO: 149
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 150
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 151
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRIFPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.27

SEQ ID NO: 153
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGNRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 154
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGNRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 155
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.28

SEQ ID NO: 156
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 157
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 158
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.29

SEQ ID NO: 159
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLW

YIHWVRQAPGQGLEWMGRISPGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGDRVAWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 160
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 161
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFHLW

YIHWVRQAPGQGLEWMGRISPGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.31

SEQ ID NO: 162
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

YIHWVRQAPGQGLEWMGRISAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 163
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 164
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

YIHWVRQAPGQGLEWMGRISAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.31

SEQ ID NO: 165
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFHLW

YIHWVRQAPGQGLEWMGRVFAGSGSYRYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 166
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 167
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFHLW

YIHWVRQAPGQGLEWMGRVFAGSGSYRYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.32

SEQ ID NO: 168
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVADRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 169
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 170
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.33

SEQ ID NO: 171
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVEDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 172
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA

WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 173
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.34

SEQ ID NO: 174
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTY

IHWVRQAPGQGLEWMGRISPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGNRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 175
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGNRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 176
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFRLTY

IHWVRQAPGQGLEWMGRISPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.35

SEQ ID NO: 177
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKT

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVEDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 178
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA

WYQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 179
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKT

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.36

SEQ ID NO: 180
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

YIHWVRQAPGQGLEWMGRVSPGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVEDRVAWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 181
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVA

WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

SEQ ID NO: 182
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

YIHWVRQAPGQGLEWMGRVSPGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.37

SEQ ID NO: 183
VH+VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKT

YIHWVRQAPGQGLEWMGRIYPGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVADRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 184
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 185
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKT

YIHWVRQAPGQGLEWMGRIYPGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.38

SEQ ID NO: 186
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKT

YIHWVRQAPGQGLEWMGRISAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 187
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 188
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKT

YIHWVRQAPGQGLEWMGRISAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.39

SEQ ID NO: 189
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 190
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 191
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.40

SEQ ID NO: 192
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGDRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 193
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 194
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKIY

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.41

SEQ ID NO: 195
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGGTFKLTY

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDDRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 196
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDDRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 197
VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFKLTY

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.42

SEQ ID NO: 198
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDNRVAWHQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

SEQ ID NO: 199
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 200
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

IHWVRQAPGQGLEWMGRISPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.43

SEQ ID NO: 201
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDNRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 202
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO: 203
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.44

SEQ ID NO: 204
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKFY

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGDRVVWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

SEQ ID NO: 205
VH
QVQLVQSGAEVKKPGSSVKVSCKASGTDFDKFY

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.45

SEQ ID NO: 206
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSAGSGNTKYNEKF

KGRVTITADISTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGINVVWHQQKPGKAPKALIYSSSHRYSG

VPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFK

SYPLTFGQGTKLEIK

SEQ ID NO: 207
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

A-H.46

SEQ ID NO: 208
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSAGSGNTKYNEKF

KGRVTITADISTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGINVVWHQQKPGKAPKALIYSSSHRYSG

VPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFK

SYPLTFGQGTKLEIK

SEQ ID NO: 209
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.47

SEQ ID NO: 210
VH + VL
QVQLVQSGABVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFFPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGINVVWHQQKPGKAPKALIYSSSHRYSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSY

PLTFGQGTKLEIK

SEQ ID NO: 211
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFFPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.48

SEQ ID NO: 212
VH + VL
QVQLVQSGABVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGINVVWHQQKPGKAPKALIYSSSHRYSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSY

PLTFGQGTKLEIK

SEQ ID NO: 213
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSS

A-H.49

SEQ ID NO: 214
VH + VL
QVQLVQSGABVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGINVVWHQQKPGKAPKALIYSSSHRYSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSY

PLTFGQGTKLEIK

SEQ ID NO: 215
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFSPGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.50

SEQ ID NO: 216
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGRIFPGSGNIKYNEKFKG

RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY

YSYDVLDYWGQGTTVTVSSGGGGSGGGGSGGG

GSGGGGSDIQMTQSPSFLSASVGDRVTITCKASQ

NVGINVVWHQQKPGKAPKALTYSSSHRYSGVPS

RFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSYP

LTFGQGTKLEIK

SEQ ID NO: 217
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGRIFPGSGNIKYNEKFKG

RVTITADTSTSTAYMELSSLRSEDTAVYYCAGSY

YSYDVLDYWGQGTTVTVSS

A-H.51

SEQ ID NO: 218
VH + VL
QVQLVQSGABVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

IYSAGVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGINVVWHQQKPGKAPKALIYSSSHRYSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSY

PLTFGQGTKLEIK

SEQ ID NO: 219
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

IYSAGVLDYWGQGTTVTVSS

A-H.52

SEQ ID NO: 220
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTLGY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGINVVWHQQKPGKAPKALIYSSSHRYSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSY

PLTFGQGTKLEIK

SEQ ID NO: 221
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTLGY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.53

SEQ ID NO: 222
VH + VL
QVQLVQSGABVKKPGSSVKVSCKASGYSFRLTY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGINVVWHQQKPGKAPKALIYSSSHRYSGVP

SRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKSY

PLTFGQGTKLEIK

SEQ ID NO: 223
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFRLTY

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

A-H.54

SEQ ID NO: 224
VH + VL
QVQLVQSGAEVKKPGSSVKVSCKASGYSFHNW

YIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGINVVWHQQKPGKAPKALIYSSSHRYSG

VPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFK

SYPLTFGQGTKLEIK

SEQ ID NO: 225
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFHNW

YIHWVRQAPGQGLEWMGWFFPGSGNIKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSS

A-H.55 antibody

SEQ ID NO: 3
HC CDR1 (Combined)
GYSFTTYYIH

SEQ ID NO: 4
HC CDR2 (Combined)
WFFPGSGNIKYNEKFKG

SEQ ID NO: 5
HC CDR3 (Combined)
SYYSYDVLDY

SEQ ID NO: 45
HC CDR1 (Kabat)
TYYIH

SEQ ID NO: 46
HC CDR2 (Kabat)
WFFPGSGNIKYNEKFKG

SEQ ID NO: 47
HC CDR3 (Kabat)
SYYSYDVLDY

SEQ ID NO: 48
HC CDR1 (Chothia)
GYSFTTY

SEQ ID NO: 49
HC CDR2 (Chothia)
FPGSGN

SEQ ID NO: 50
HC CDR3 (Chothia)
SYYSYDVLDY

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGYSFTTYY

1100

IHWVRQAPGQGLEWMGWFFPGSGNIKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO: 6
LC CDR1 (Combined)
KASQNVGINVV

SEQ ID NO: 7
LC CDR2 (Combined)
SSSHRYS

SEQ ID NO: 8
LC CDR3 (Combined)
QQFKSYPLT

SEQ ID NO: 51
LC CDR1 (Kabat)
KASQNVGINVV

SEQ ID NO: 52
LC CDR2 (Kabat)
SSSHRYS

SEQ ID NO: 53
LC CDR3 (Kabat)
QQFKSYPLT

SEQ ID NO: 54
LC CDR1 (Chothia)
KASQNVGINVV

SEQ ID NO: 55
LC CDR2 (Chothia)
SSSHRYS

SEQ ID NO: 56
LC CDR3 (Chothia)
QQFKSYPLT

SEQ ID NO:
VL
QSVLTQPPSVSEAPRQRVTISCKASQNVGINVVW

1101

HQQLPGKAPKALIYSSSHRYSGVSDRFSGSGSGT

SFSLAISGLQSEDEADYFCQQFKSYPLTFGTGTK

VTVL

A-H.56

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFDKF

1309

YIHWVRQAPGQGLEWMGRVSAGSGNVKYNEK

FKGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGNRVAWYQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.57

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1326

IHWVRQAPGQGLEWMGRVSAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVGDRVVWHQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

A-H.58

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1327

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGNRVVWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.59

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1328

IHWVRQAPGQGLEWMGRIYAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVADRVVWHQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

A-H.60

SEQ ID NO:
V H+ VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1329

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGDRVAWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.61

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1330

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVDNRVAWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.62

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1331

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVADRVVWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.63

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1332

IHWVRQAPGQGLEWMGRVYAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVEDRVVWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.64

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1333

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVADRVVWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.65

SEQ ID NO:
V H+ VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1334

YIHWVRQAPGQGLEWMGRISAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGDRVVWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.66

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1335

YIHWVRQAPGQGLEWMGRIYAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSSGGGGSGGGGS

GGGGSGGGGSDIQMTQSPSFLSASVGDRVTITCK

ASQNVGDRVVWHQQKPGKAPKALIYSSSHRYK

GVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQF

KSYPLTFGQGTKLEIK

A-H.67

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

1336

IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVDNRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

A-H.68

SEQ ID NO:
VH + VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1337

IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDIQMTQSPSFLSASVGDRVTITCKAS

QNVADRVAWYQQKPGKAPKALIYSSSHRYKGV

PSRFSGSGSGTEFTLTISSLQPEDFATYFCQQFKS

YPLTFGQGTKLEIK

A-H.69 (also referred to as A-H.13)

SEQ ID NO: 110
VH+ VL (ScFv)
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLT

YIHWVRQAPGQGLEWMGRIFPGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

GSYYSYDVLDYWGQGTTVTVSSGGGGGGGG

SGGGGSGGGGSDIQMTQSPSFLSASVGDRVTI

TCKASQNVDNRVAWYQQKPGKAPKALIYSSSH

RYKGVPSRFSGSGSGTEFTLTISSLQPEDFATY

FCQQFKSYPLTFGQGTKLEIK

A-H humanized-matured VH

SEQ ID NO:
VH-humanized
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

1310
matured 1
IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAGS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VH-humanized
QVQLVQSGAEVKKPGSSVKVSCKASGTDFKLTY

1311
matured 2
IHWVRQAPGQGLEWMGRIFPGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VH-humanized
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1312
matured 3
IHWVRQAPGQGLEWMGRISAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSS

A-H humanized-matured VL

SEQ ID NO:
VL-humanized matured
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

1313
1
AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

SEQ ID NO:
VL-humanized matured
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

1314
2
AWYQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.70

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1346
(CDRs underlined)

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGNRV

1347
(CDRs underlined)

VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.71

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1348
(CDRs underlined)

IHWVRQAPGQGLEWMGRIYAGSGNVKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

1349
(CDRs underlined)

VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.72

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1350
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

1351
(CDRs underlined)

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.73

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1350
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

1353
(CDRs underlined)

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.74

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1346
(CDRs underlined)

IHWVRQAPGQGLEWMGRVSAGSGNVKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

1349
(CDRs underlined)

VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.75

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1356
(CDRs underlined)

IHWVRQAPGQGLEWMGRVYAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVEDRVV

1357
(CDRs underlined)
WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

A-H.76

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1350
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVADRV

1349
(CDRs underlined)
VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.77

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1360
(CDRs underlined)

YIHWVRQAPGQGLEWMGRISAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

1361
(CDRs underlined)

VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.78

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1362
(CDRs underlined)

YIHWVRQAPGQGLEWMGRIYAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

1361
(CDRs underlined)

VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.79

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1350
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCRASQNVDNRLG

1365
(CDRs underlined)
WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

A-H.80

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1350
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

1367
(CDRs underlined)

AWHQQKPGKAPKALIYAASSLQKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.81

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1350
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNTKYNEKF

KGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

1369
(CDRs underlined)

AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCLQHNSYPLTFGQG

TKLEIK

A-H.82

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1370
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNVNYAQK

FQGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCRASQNVDNRLG

1365
(CDRs underlined)
WHQQKPGKAPKALIYSSSHRYKGVPSRFSGSGS

GTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQGT

KLEIK

A-H.83

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1370
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNVNYAQK

FQGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

1367
(CDRs underlined)

AWHQQKPGKAPKALIYAASSLQKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

A-H.84

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFKLT

1370
(CDRs underlined)

YIHWVRQAPGQGLEWMGRVSAGSGNVNYAQK

FQGRVTITADTSTSTAYMELSSLRSEDTAVYYCA

VSYYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVDNRV

1369
(CDRs underlined)
AWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCLQHNSYPLTFGQG

TKLEIK

A-H.85

SEQ ID NO:
VH
QVQLVQSGAEVKKPGSSVKVSCKASGHDFRLTY

1344
(CDRs underlined)

IHWVRQAPGQGLEWMGRVSAGSGNTKYNEKFK

GRVTITADTSTSTAYMELSSLRSEDTAVYYCAVS

YYSYDVLDYWGQGTTVTVSS

SEQ ID NO:
VL
DIQMTQSPSFLSASVGDRVTITCKASQNVGDRV

1361
(CDRs underlined)

VWHQQKPGKAPKALIYSSSHRYKGVPSRFSGSG

SGTEFTLTISSLQPEDFATYFCQQFKSYPLTFGQG

TKLEIK

In some embodiments, the anti-TCRβ3V antibody molecule, e.g., anti-TCRβ3 V6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH and/or a VL of an antibody described in TABLE 30, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβV6 (e.g., anti-TCRβ V6-5*01) antibody molecule comprises a VH and a VL of an antibody described in TABLE 30, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, an anti-TCRVb antibody disclosed herein has an antigen binding domain having a VL having a consensus sequence of SEQ ID NO: 230, wherein position 30 is G, E, A or D; position 31 is N or D; position 32 is R or K; position 36 is Y or H; and/or position 56 is K or S.

In some embodiments, an anti-TCRVb antibody disclosed herein has an antigen binding domain having a VH having a consensus sequence of SEQ ID NO: 231, wherein: position 27 is H or T or G or Y; position 28 is D or T or S; position 30 is H or R or D or K or T; position 31 is L or D or K or T or N; position 32 is W or F or T or I or Y or G; position 49 is R or W; position 50 is V or I or F; position 51 is F or S or Y; position 52 is A or P; position 56 is N or S; position 57 is T or V or Y or I; position 58 is K or R; position 97 is G or V; position 99 is Y or I; position 102 is Y or A; and/or position 103 is D or G.

Anti-TCRβ V12 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to human TCRβ V12, e.g., a TCRβ V12 subfamily comprising: TCRβ V12-4*01, TCRβ V12-3*01 or TCRβ V12-5*01. In some embodiments the TCRβ V12 subfamily comprises TCRβ V12-4*01. In some embodiments the TCRβ V12 subfamily comprises TCRβ V12-3*01.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, is a non-murine antibody molecule, e.g., a human or humanized antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule is a human antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule is a humanized antibody molecule.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, is isolated or recombinant.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, comprises at least one antigen-binding region, e.g., a variable region or an antigen-binding fragment thereof, from an antibody described herein, e.g., an antibody described in Table 31, or encoded by a nucleotide sequence in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, comprises at least one, two, three or four variable regions from an antibody described herein, e.g., an antibody as described in Table 31, or encoded by a nucleotide sequence in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, comprises at least one or two heavy chain variable regions from an antibody described herein, e.g., an antibody as described in Table 31, or encoded by a nucleotide sequence in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, comprises at least one or two light chain variable regions from an antibody described herein, e.g., an antibody as described in Table 31, or encoded by a nucleotide sequence in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, comprises a heavy chain constant region for an IgG4, e.g., a human IgG4. In still another embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes a heavy chain constant region for an IgG1, e.g., a human IgG1. In one embodiment, the heavy chain constant region comprises an amino sequence set forth in Table 32, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes a kappa light chain constant region, e.g., a human kappa light chain constant region. In one embodiment, the light chain constant region comprises an amino sequence set forth in Table 32, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes at least one, two, or three complementarity determining regions (CDRs) from a heavy chain variable region of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes at least one, two, or three CDRs (or collectively all of the CDRs) from a heavy chain variable region comprising an amino acid sequence shown in Table 31, or encoded by a nucleotide sequence shown in Table 31. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 31, or encoded by a nucleotide sequence shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes at least one, two, or three complementarity determining regions (CDRs) from a light chain variable region of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes at least one, two, or three CDRs (or collectively all of the CDRs) from a light chain variable region comprising an amino acid sequence shown in Table 31, or encoded by a nucleotide sequence shown in Table 31. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 31, or encoded by a nucleotide sequence shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, includes at least one, two, three, four, five or six CDRs (or collectively all of the CDRs) from a heavy and light chain variable region comprising an amino acid sequence shown in Table 31, or encoded by a nucleotide sequence shown in Table 31. In one embodiment, one or more of the CDRs (or collectively all of the CDRs) have one, two, three, four, five, six or more changes, e.g., amino acid substitutions or deletions, relative to the amino acid sequence shown in Table 31, or encoded by a nucleotide sequence shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, molecule includes all six CDRs from an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31, or closely related CDRs, e.g., CDRs which are identical or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions). In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Table 31) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen as described in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three CDRs according to Kabat et al. (e.g., at least one, two, or three CDRs according to the Kabat definition as set out in Table 31) from a light chain variable region of an antibody described herein, e.g., an antibody as described in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Kabat et al. shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, three, four, five, or six CDRs according to Kabat et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Kabat definition as set out in Table 31) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Kabat et al. shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes all six CDRs according to Kabat et al. (e.g., all six CDRs according to the Kabat definition as set out in Table 31) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or encoded by the nucleotide sequence in Table 31; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Kabat et al. shown in Table 31. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three hypervariable loops that have the same canonical structures as the corresponding hypervariable loop of an antibody described herein, e.g., an antibody described in Table 31, e.g., the same canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light chain variable domains of an antibody described herein. See, e.g., Chothia et al., (1992) J. Mol. Biol. 227:799-817; Tomlinson et al., (1992) J. Mol. Biol. 227:776-798 for descriptions of hypervariable loop canonical structures. These structures can be determined by inspection of the tables described in these references.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in Table 31) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen as described in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three CDRs according to Chothia et al. (e.g., at least one, two, or three CDRs according to the Chothia definition as set out in Table 31) from a light chain variable region of an antibody described herein, e.g., an antibody as described in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to Chothia et al. shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, three, four, five, or six CDRs according to Chothia et al. (e.g., at least one, two, three, four, five, or six CDRs according to the Chothia definition as set out in Table 31) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to Chothia et al. shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes all six CDRs according to Chothia et al. (e.g., all six CDRs according to the Chothia definition as set out in Table 31) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or encoded by the nucleotide sequence in Table 31; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to Chothia et al. shown in Table 31. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule may include any CDR described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three CDRs according to a combined CDR (e.g., at least one, two, or three CDRs according to the combined CDR definition as set out in Table 31) from a heavy chain variable region of an antibody described herein, e.g., an antibody chosen as described in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to combined CDR shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, or three CDRs according to a combined CDR (e.g., at least one, two, or three CDRs according to the combined CDR definition as set out in Table 31) from a light chain variable region of an antibody described herein, e.g., an antibody as described in Table 31, or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, or three CDRs according to a combined CDR shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes at least one, two, three, four, five, or six CDRs according to a combined CDR. (e.g., at least one, two, three, four, five, or six CDRs according to the combined CDR definition as set out in Table 31) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to one, two, three, four, five, or six CDRs according to a combined CDR shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes all six CDRs according to a combined CDR (e.g., all six CDRs according to the combined CDR definition as set out in Table 31) from the heavy and light chain variable regions of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or encoded by the nucleotide sequence in Table 31; or a sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical) to any of the aforesaid sequences; or which have at least one amino acid alteration, but not more than two, three or four alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) relative to all six CDRs according to a combined CDR shown in Table 31. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule may include any CDR described herein.

In some embodiments, a combined CDR as set out in TABLE 31 is a CDR that comprises a Kabat CDR and a Chothia CDR.

In some embodiments, the anti-TCRβV antibody molecule, e e.g., anti-TCRβ V12 antibody molecule, molecule includes a combination of CDRs or hypervariable loops identified as combined CDRs in TABLE 31. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, can contain any combination of CDRs or hypervariable loops according the “combined” CDRs are described in TABLE 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes a combination of CDRs or hypervariable loops defined according to the Kabat et al. and Chothia et al., or as described in TABLE 31

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule can contain any combination of CDRs or hypervariable loops according to the Kabat and Chothia definitions.

In an embodiment, e.g., an embodiment comprising a variable region, a CDR (e.g., a combined CDR, Chothia CDR or Kabat CDR), or other sequence referred to herein, e.g., in Table 31, the antibody molecule is a monospecific antibody molecule, a bispecific antibody molecule, a bivalent antibody molecule, a biparatopic antibody molecule, or an antibody molecule that comprises an antigen binding fragment of an antibody, e.g., a half antibody or antigen binding fragment of a half antibody. In some embodiments, the antibody molecule comprises a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes:

- (i) one, two or all of a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3) of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, and/or
- (ii) one, two or all of a heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- (i) a LC CDR1 amino acid sequence of SEQ ID NO: 20, a LC CDR2 amino acid sequence of SEQ ID NO: 21, or a LC CDR3 amino acid sequence of SEQ ID NO: 22; and/or
- (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 17, a HC CDR2 amino acid sequence of SEQ ID NO: 18, or a HC CDR3 amino acid sequence of SEQ ID NO: 19.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 20, a LC CDR2 amino acid sequence of SEQ ID NO: 21, and a LC CDR3 amino acid sequence of SEQ ID NO: 2; and/or
- (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 17, a HC CDR2 amino acid sequence of SEQ ID NO: 18, and a HC CDR3 amino acid sequence of SEQ ID NO: 19.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- (i) a LC CDR1 amino acid sequence of SEQ ID NO: 63, a LC CDR2 amino acid sequence of SEQ ID NO: 64, or a LC CDR3 amino acid sequence of SEQ ID NO: 65; and/or
- (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 57, a HC CDR2 amino acid sequence of SEQ ID NO: 58, or a HC CDR3 amino acid sequence of SEQ ID NO: 59.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 63, a LC CDR2 amino acid sequence of SEQ ID NO: 64, or a LC CDR3 amino acid sequence of SEQ ID NO: 65; and/or
- (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 57, a HC CDR2 amino acid sequence of SEQ ID NO: 58, or a HC CDR3 amino acid sequence of SEQ ID NO: 59.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- (i) a LC CDR1 amino acid sequence of SEQ ID NO: 66, a LC CDR2 amino acid sequence of SEQ ID NO: 67, or a LC CDR3 amino acid sequence of SEQ ID NO: 68; and/or
- (ii) a HC CDR1 amino acid sequence of SEQ ID NO: 60, a HC CDR2 amino acid sequence of SEQ ID NO: 61, or a HC CDR3 amino acid sequence of SEQ ID NO: 62.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- (i) a light chain variable region (VL) comprising a LC CDR1 amino acid sequence of SEQ ID NO: 63, a LC CDR2 amino acid sequence of SEQ ID NO: 64, or a LC CDR3 amino acid sequence of SEQ ID NO: 65; and/or
- (ii) a heavy chain variable region (VH) comprising a HC CDR1 amino acid sequence of SEQ ID NO: 57, a HC CDR2 amino acid sequence of SEQ ID NO: 58, or a HC CDR3 amino acid sequence of SEQ ID NO: 59.

In one embodiment, the light or the heavy chain variable framework (e.g., the region encompassing at least FR1, FR2, FR3, and optionally FR4) of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule can be chosen from: (a) a light or heavy chain variable framework including at least 80%, 85%, 87% 90%, 92%, 93%, 95%, 97%, 98%, or 100% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (b) a light or heavy chain variable framework including from 20% to 80%, 40% to 60%, 60% to 90%, or 70% to 95% of the amino acid residues from a human light or heavy chain variable framework, e.g., a light or heavy chain variable framework residue from a human mature antibody, a human germline sequence, or a human consensus sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a non-human framework that has been modified, e.g., to remove antigenic or cytotoxic determinants, e.g., deimmunized, or partially humanized. In one embodiment, the light or heavy chain variable framework region (particularly FR1, FR2 and/or FR3) includes a light or heavy chain variable framework sequence at least 70, 75, 80, 85, 87, 88, 90, 92, 94, 95, 96, 97, 98, 99% identical or identical to the frameworks of a VL or VH segment of a human germline gene.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, comprises a heavy chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more changes, e.g., amino acid substitutions or deletions, from an amino acid sequence described in Table 31.e.g., the amino acid sequence of the FR region in the entire variable region, e.g., shown in FIGS. 2A and 2B, or in SEQ ID NOs: 23-25.

Alternatively, or in combination with the heavy chain substitutions described herein the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain variable domain having at least one, two, three, four, five, six, seven, ten, fifteen, twenty or more amino acid changes, e.g., amino acid substitutions or deletions, from an amino acid sequence of an antibody described herein e.g., the amino acid sequence of the FR region in the entire variable region, e.g., shown in FIGS. 2A and 2B, or in SEQ ID NOs: 26-30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes one, two, three, or four heavy chain framework regions shown in FIG. 2A, or a sequence substantially identical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes one, two, three, or four light chain framework regions shown in FIG. 2B, or a sequence substantially identical thereto.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the light chain framework region 1 e.g., as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the light chain framework region 2 e.g., as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the light chain framework region 3, e.g., as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the light chain framework region 4, e.g., as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 1 (FR1), comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more, e.g., all, position disclosed herein according to Kabat numbering. In some embodiments, FR1 comprises an Aspartic Acid at position 1, e.g., a substitution at position 1 according to Kabat numbering, e.g., an Alanine to Aspartic Acid substitution. In some embodiments, FR1 comprises an Asparagine at position 2, e.g., a substitution at position 2 according to Kabat numbering, e.g., an Isoleucine to Asparagine substitution, Serine to Asparagine substitution or Tyrosine to Asparagine substitution. In some embodiments, FR1 comprises a Leucine at position 4, e.g., a substitution at position 4 according to Kabat numbering, e.g., a Methionine to Leucine substitution.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 1 (FR1), comprising a substitution at position 1 according to Kabat numbering, e.g., an Alanine to Aspartic Acid substitution, a substitution at position 2 according to Kabat numbering, e.g., an Isoleucine to Asparagine substitution, Serine to Asparagine substitution or Tyrosine to Asparagine substitution, and a substitution at position 4 according to Kabat numbering, e.g., a Methionine to Leucine substitution. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 1 (FR1), comprising a substitution at position 1 according to Kabat numbering, e.g., an Alanine to Aspartic Acid substitution, and a substitution at position 2 according to Kabat numbering, e.g., an Isoleucine to Asparagine substitution, Serine to Asparagine substitution or Tyrosine to Asparagine substitution. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 1 (FR1), comprising a substitution at position 1 according to Kabat numbering, e.g., an Alanine to Aspartic Acid substitution, and a substitution at position 4 according to Kabat numbering, e.g., a Methionine to Leucine substitution. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 1 (FR1), comprising a substitution at position 2 according to Kabat numbering, e.g., an Isoleucine to Asparagine substitution, Serine to Asparagine substitution or Tyrosine to Asparagine substitution, and a substitution at position 4 according to Kabat numbering, e.g., a Methionine to Leucine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 3 (FR3), comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more, e.g., all, position disclosed herein according to Kabat numbering. In some embodiments, FR3 comprises a Glycine at position 66, e.g., a substitution at position 66 according to Kabat numbering, e.g., a Lysine to Glycine substitution, or a Serine to Glycine substitution. In some embodiments, FR3 comprises an Asparagine at position 69, e.g., a substitution at position 69 according to Kabat numbering, e.g., a Tyrosine to Asparagine substitution. In some embodiments, FR3 comprises a Tyrosine at position 71, e.g., a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, or an Alanine to Tyrosine substitution.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 3 (FR3), comprising a substitution at position 66 according to Kabat numbering, e.g., a Lysine to Glycine substitution, or a Serine to Glycine substitution, and a substitution at position 69 according to Kabat numbering, e.g., a Tyrosine to Asparagine substitution. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 3 (FR3), comprising a substitution at position 66 according to Kabat numbering, e.g., Lysine to Glycine substitution, or a Serine to Glycine substitution, and a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, or an Alanine to Tyrosine substitution. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 3 (FR3), comprising a substitution at position 69 according to Kabat numbering, e.g., a Tyrosine to Asparagine substitution and a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, or an Alanine to Tyrosine substitution. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising a framework region, e.g., framework region 3 (FR3), comprising a substitution at position 66 according to Kabat numbering, e.g., a Lysine to Glycine substitution, or a Serine to Glycine substitution, a substitution at position 69 according to Kabat numbering, e.g., a Tyrosine to Asparagine substitution and a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, or an Alanine to Tyrosine substitution. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising: a framework region 1 (FR1) comprising a substitution at position 2 according to Kabat numbering, e.g., a Isoleucine to Asparagine substitution; and a framework region 3 (FR3), comprising a substitution at position 69 according to Kabat numbering, e.g., a Threonine to Asparagine substitution and a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 26. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising: (a) a framework region 1 (FR1) comprising a substitution at position 1 according to Kabat numbering, e.g., a Alanine to Aspartic Acid substitution, and a substitution at position 2 according to Kabat numbering, e.g., a Isoleucine to Asparagine substitution; and (b) a framework region 3 (FR3), comprising a substitution at position 69 according to Kabat numbering, e.g., a Threonine to Asparagine substitution and a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 27 In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising: (a) a framework region 1 (FR1) comprising a substitution at position 2 according to Kabat numbering, e.g., a Serine to Asparagine substitution; and a substitution at position 4 according to Kabat numbering, e.g., a Methionine to Leucine substitution; and (b) a framework region 3 (FR3), comprising a substitution at position 69 according to Kabat numbering, e.g., a Threonine to Asparagine substitution and a substitution at position 71 according to Kabat numbering, e.g., a Phenylalanine to Tyrosine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 28 In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising: (a) a framework region 1 (FR1) comprising a substitution at position 2 according to Kabat numbering, e.g., a Serine to Asparagine substitution; and (b) a framework region 3 (FR3) comprising a substitution at position 66 according to Kabat numbering, e.g., a Lysine to Glycine substitution; a substitution at position 69 according to Kabat numbering, e.g., a Threonine to Asparagine substitution; and a substitution at position 71 according to Kabat numbering, e.g., a Alanine to Tyrosine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 29. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain comprising: (a) a framework region 1 (FR1) comprising a substitution at position 2 according to Kabat numbering, e.g., a Tyrosine to Asparagine substitution; and (b) a framework region 3 (FR3) comprising a substitution at position 66 according to Kabat numbering, e.g., a Serine to Glycine substitution; a substitution at position 69 according to Kabat numbering, e.g., a Threonine to Asparagine substitution; and a substitution at position 71 according to Kabat numbering, e.g., a Alanine to Tyrosine substitution, e.g., as shown in the amino acid sequence of SEQ ID NO: 29. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises a light chain variable domain comprising: (a) a framework region 1 (FR1) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) positions disclosed herein according to Kabat numbering, and (b) a framework region 3 (FR3) comprising a change, e.g., a substitution (e.g., a conservative substitution) at one or more (e.g., all) position disclosed herein according to Kabat numbering. In some embodiments, the substitution is relative to a human germline light chain framework region sequence.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the heavy chain framework region 1, e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the heavy chain framework region 2, e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the heavy chain framework region 3, e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the heavy chain framework region 4, e.g., as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the heavy chain framework regions 1-4, e.g., SEQ ID NOS: 20-23, or as shown in FIG. 2A.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the light chain framework regions 1-4, e.g., SEQ ID NOs: 26-30, or as shown in FIG. 2B.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises the heavy chain framework regions 1-4, e.g., SEQ ID NOs: 23-25; and the light chain framework regions 1-4, e.g., SEQ ID NOs: 26-30, or as shown in FIGS. 2A and 2B.

In some embodiments, the heavy or light chain variable domain, or both, of, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes an amino acid sequence, which is substantially identical to an amino acid disclosed herein, e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identical to a variable region of an antibody described herein, e.g., an antibody as described in Table 31, or encoded by the nucleotide sequence in Table 31; or which differs at least 1 or 5 residues, but less than 40, 30, 20, or 10 residues, from a variable region of an antibody described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises at least one, two, three, or four antigen-binding regions, e.g., variable regions, having an amino acid sequence as set forth in Table 31, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the sequences shown in Table 31. In another embodiment, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule includes a VH and/or VL domain encoded by a nucleic acid having a nucleotide sequence as set forth in Table 31, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in Table 31.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising an amino acid sequence chosen from the amino acid sequence of SEQ ID NO: 23, SEQ ID NO:24 or SEQ ID NO:25, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 23, SEQ ID NO:24 or SEQ ID NO:25, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 23, SEQ ID NO:24 or SEQ ID NO:25; and/or
- a VL domain comprising an amino acid sequence chosen from the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 23, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 23; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 26, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 23, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 23; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 27, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 23, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 23; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 28, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 23, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 23; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 29, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 23, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 23, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 23; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 30, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 24, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 24; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 26, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 24, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 24; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 27, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 24, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 24; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 28, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 24, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 24; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 29, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 24, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 24, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 24; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 30, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 25, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 25; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 26, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 26, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 26.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 25, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 25; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 27, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 27, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 25, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 25; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 28, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 28.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 25, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 25; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 29, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 29.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule comprises:

- a VH domain comprising the amino acid sequence of SEQ ID NO: 25, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 25, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 25; and
- a VL domain comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence at least about 85%, 90%, 95%, 99% or more identical to the amino acid sequence SEQ ID NO: 30, or an amino acid sequence which differs by no more than 1, 2, 5, 10, or 15 amino acid residues from the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule is a full antibody or fragment thereof (e.g., a Fab, F(ab′)₂, Fv, or a single chain Fv fragment (scFv)). In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V6 (e.g., anti-TCRβ V6-5*01) antibody molecule is a monoclonal antibody or an antibody with single specificity. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule, can also be a humanized, chimeric, camelid, shark, or an in vitro-generated antibody molecule. In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule is a humanized antibody molecule. The heavy and light chains of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule can be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment (e.g., a Fab, F(ab′)₂, Fv, a single chain Fv fragment, a single domain antibody, a diabody (dAb), a bivalent antibody, or bispecific antibody or fragment thereof, a single domain variant thereof, or a camelid antibody).

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule is in the form of a multispecific molecule, e.g., a bispecific molecule, e.g., as described herein.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule has a heavy chain constant region (Fc) chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the Fc region is chosen from the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc region is chosen from the heavy chain constant region of IgG1 or IgG2 (e.g., human IgG1, or IgG2). In some embodiments, the heavy chain constant region is human IgG1.

In some embodiments, the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule has a light chain constant region chosen from, e.g., the light chain constant regions of kappa or lambda, preferably kappa (e.g., human kappa). In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the anti-TCRβV antibody molecule, e.g., anti-TCRβ V12 antibody molecule (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the constant region is mutated at positions 296 (M to Y), 298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) to alter Fc receptor binding (e.g., the mutated positions correspond to positions 132 (M to Y), 134 (S to T), 136 (T to E), 313 (H to K) and 314 (N to F) of SEQ ID NOs: 212 or 214; or positions 135 (M to Y), 137 (S to T), 139 (T to E), 316 (H to K) and 317 (N to F) of SEQ ID NOs: 215, 216, 217 or 218).

Antibody B-H.1 comprises a first chain comprising the amino acid sequence of SEQ ID NO: 3280 and a second chain comprising the amino acid sequence of SEQ ID NO: 3281.

Additional exemplary anti-TCRβ V12 antibodies of the disclosure are provided in Table 31. In some embodiments, the anti-TCRβ V12 is antibody B, e.g., humanized antibody B (antibody B-H), as provided in Table 31. In some embodiments, the anti-TCRβV antibody comprises one or more (e.g., all three) of a LC CDR1, LC CDR2, and LC CDR3 provided in Table 31; and/or one or more (e.g., all three) of a HC CDR1, HC CDR2, and HC CDR3 provided in Table 31, or a sequence with at least 95% identity thereto. In some embodiments, antibody B comprises a variable heavy chain (VH) and/or a variable light chain (VL) provided in Table 31, or a sequence with at least 95% identity thereto.

TABLE 31

Amino acid and nucleotide sequences for murine and humanized antibody molecules which

bind to TCRVB 12, e.g., TCRVB 12-3 or TCRVB 12-4. The antibody molecules include murine mAb

Antibody B and humanized mAb Antibody B-H.1 to B-H.6. The amino acid the heavy and light chain

CDRs, and the amino acid and nucleotide sequences of the heavy and light chain variable

regions, and the heavy and light chains are shown.

Antibody B (murine), also referred to as 16G8

SEQ ID NO: 17
HC CDR1 (Combined)
GFTFSNFGMH

SEQ ID NO: 18
HC CDR2 (Combined)
YISSGSSTIYYADTLKG

SEQ ID NO: 19
HC CDR3 (Combined)
RGEGAMDY

SEQ ID NO: 57
HC CDR1 (Kabat)
NFGMH

SEQ ID NO: 58
HC CDR2 (Kabat)
YISSGSSTIYYADTLKG

SEQ ID NO: 59
HC CDR3 (Kabat)
RGEGAMDY

SEQ ID NO: 60
HC CDR1 (Chothia)
GFTFSNF

SEQ ID NO: 61
HC CDR2 (Chothia)
SSGSST

SEQ ID NO: 62
HC CDR3 (Chothia)
RGEGAMDY

SEQ ID NO: 15
VH
DVQLVESGGGLVQPGGSRKLSCAASGFTFSNFG

MHWVRQAPDKGLEWVAYISSGSSTIYYADTLK

GRFTISRDNPKNTLFLQMTSLRSEDTAMYYCAR

RGEGAMDYWGQGTSVTVSS

SEQ ID NO: 20
LC CDR1 (Combined)
RASSSVNYIY

SEQ ID NO: 21
LC CDR2 (Combined)
YTSNLAP

SEQ ID NO: 22
LC CDR3(Combined)
QQFTSSPFT

SEQ ID NO: 63
LC CDR1 (Kabat)
RASSSVNYIY

SEQ ID NO: 64
LC CDR2 (Kabat)
YTSNLAP

SEQ ID NO: 65
LC CDR3 (Kabat)
QQFTSSPFT

SEQ ID NO: 66
LC CDR1 (Chothia)
RASSSVNYIY

SEQ ID NO: 67
LC CDR2 (Chothia)
YTSNLAP

SEQ ID NO: 68
LC CDR3 (Chothia)
QQFTSSPFT

SEQ ID NO: 16
VL
ENVLTQSPAIMSASLGEKVTMSCRASSSVNYIY

WYQQKSDASPKLWIYYTSNLAPGVPTRFSGSGS

GNSYSLTISSMEGEDAATYYCQQFTSSPFTFGSG

TKLEIK

Antibody B humanized (B-H)

Antibody B-H.1A HC-1

SEQ ID NO: 17
HC CDR1 (Combined)
GFTFSNFGMH

SEQ ID NO: 18
HC CDR2 (Combined)
YISSGSSTIYYADTLKG

SEQ ID NO: 19
HC CDR3 (Combined)
RGEGAMDY

SEQ ID NO: 23
VH
EVOLVESGGGLVQPGGSLRLSCAASGFTFSNFG

MHWVRQAPGKGLEWVSYISSGSSTIYYADTLKG

RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARR

GEGAMDYWGQGTTVTVSS

SEQ ID NO: 31
DNA VH
GAGGTGCAGCTGGTTGAATCTGGCGGAGGATT

GGTTCAGCCTGGCGGCTCTCTGAGACTGTCTT

GTGCCGCTTCTGGCTTCACCTTCTCCAACTTCG

GCATGCACTGGGTCCGACAGGCCCCTGGAAAA

GGACTGGAATGGGTGTCCTACATCTCCTCCGG

CTCCTCCACCATCTACTACGCTGACACCCTGA

AGGGCAGATTCACCATCTCTCGGGACAACGCC

AAGAACTCCCTGTACCTGCAGATGAACAGCCT

GAGAGCCGAGGACACCGCCGTGTACTACTGTG

CTAGAAGAGGCGAGGGCGCCATGGATTATTG

GGGCCAGGGAACCACAGTGACCGTGTCTAGC

Antibody B-H.1B HC-2

SEQ ID NO: 17
HC CDR1 (Combined)
GFTFSNFGMH

SEQ ID NO: 18
HC CDR2 (Combined)
YISSGSSTIYYADTLKG

SEQ ID NO: 19
HC CDR3 (Combined)
RGEGAMDY

SEQ ID NO: 24
VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFG

MHWVRQAPGKGLEWVSYISSGSSTIYYADTLKG

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR

GEGAMDYWGQGTTVTVSS

SEQ ID NO: 32
DNA VH
GAGGTGCAGCTGGTTGAATCTGGCGGAGGATT

GGTTCAGCCTGGCGGCTCTCTGAGACTGTCTT

GTGCCGCTTCTGGCTTCACCTTCTCCAACTTCG

GCATGCACTGGGTCCGACAGGCCCCTGGAAAA

GGACTGGAATGGGTGTCCTACATCTCCTCCGG

CTCCTCCACCATCTACTACGCTGACACCCTGA

AGGGCAGATTCACCATCAGCCGGGACAACTCC

AAGAACACCCTGTACCTGCAGATGAACTCCCT

GAGAGCCGAGGACACCGCCGTGTACTACTGTG

CTAGAAGAGGCGAGGGCGCCATGGATTATTG

GGGCCAGGGAACCACAGTGACCGTGTCTAGC

Antibody B-H.1C HC-3

SEQ ID NO: 17
HC CDR1 (Combined)
GFTFSNFGMH

SEQ ID NO: 18
HC CDR2 (Combined)
YISSGSSTIYYADTLKG

SEQ ID NO: 19
HC CDR3 (Combined)
RGEGAMDY

SEQ ID NO: 25
VH
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFG

MHWVRQAPGKGLEWVAYISSGSSTIYYADTLK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

RGEGAMDYWGQGTTVTVSS

SEQ ID NO: 33
DNA VH
CAGGTGCAGCTGGTGGAATCTGGTGGCGGAGT

TGTGCAGCCTGGCAGATCCCTGAGACTGTCTT

GTGCCGCCTCTGGCTTCACCTTCTCCAACTTCG

GCATGCACTGGGTCCGACAGGCCCCTGGAAAA

GGATTGGAGTGGGTCGCCTACATCTCCTCCGG

CTCCTCCACCATCTACTACGCTGACACCCTGA

AGGGCAGATTCACCATCAGCCGGGACAACTCC

AAGAACACCCTGTACCTGCAGATGAACTCCCT

GAGAGCCGAGGACACCGCCGTGTACTACTGTG

CTAGAAGAGGCGAGGGCGCCATGGATTATTG

GGGCCAGGGAACCACAGTGACCGTGTCTAGC

Antibody B-H.1D LC-1

SEQ ID NO: 20
LC CDR1 (Combined)
RASSSVNYIY

SEQ ID NO: 21
LC CDR2 (Combined)
YTSNLAP

SEQ ID NO: 22
LC CDR3(Combined)
QQFTSSPFT

SEQ ID NO: 26
VL
DNQLTQSPSFLSASVGDRVTITCRASSSVNYIYW

YQQKPGKAPKLLIYYTSNLAPGVPSRFSGSGSGN

EYTLTISSLQPEDFATYYCQQFTSSPFTFGQGTKL

EIK

SEQ ID NO: 34
DNA VL
GATAACCAGCTGACCCAGTCTCCTAGCTTCCT

GTCTGCCTCTGTGGGCGACAGAGTGACAATTA

CCTGCCGGGCCTCCTCCTCCGTGAACTACATCT

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCT

AAGCTGCTGATCTACTACACCTCCAATCTGGC

CCCTGGCGTGCCCTCTAGATTTTCCGGATCTGG

CTCCGGCAACGAGTATACCCTGACAATCTCCA

GCCTGCAGCCTGAGGACTTCGCCACCTACTAC

TGCCAGCAGTTCACCTCCTCTCCATTCACCTTT

GGCCAGGGCACCAAGCTGGAAATCAAA

Antibody B-H.1E LC-2

SEQ ID NO: 20
LC CDR1 (Combined)
RASSSVNYIY

SEQ ID NO: 21
LC CDR2 (Combined)
YTSNLAP

SEQ ID NO: 22
LC CDR3(Combined)
QQFTSSPFT

SEQ ID NO: 27
VL
DNQLTQSPSSLSASVGDRVTITCRASSSVNYIYW

YQQKPGKAPKLLIYYTSNLAPGVPSRFSGSGSGN

DYTLTISSLQPEDFATYYCQQFTSSPFTFGQGTKL

EIK

SEQ ID NO: 35
DNA VL
ATAACCAGCTGACCCAGTCTCCTTCCAGCCTG

TCTGCTTCTGTGGGCGACAGAGTGACAATTAC

CTGCCGGGCCTCCTCCTCCGTGAACTACATCT

ACTGGTATCAGCAGAAGCCCGGCAAGGCCCCT

AAGCTGCTGATCTACTACACCTCCAATCTGGC

CCCTGGCGTGCCCTCTAGATTTTCCGGATCTGG

CTCCGGCAACGACTATACCCTGACAATCTCCA

GCCTGCAGCCTGAGGACTTCGCCACCTACTAC

TGCCAGCAGTTCACCTCCTCTCCATTCACCTTT

GGCCAGGGCACCAAGCTGGAAATCAAA

Antibody B-H.1F LC-3

SEQ ID NO: 20
LC CDR1 (Combined)
RASSSVNYIY

SEQ ID NO: 21
LC CDR2 (Combined)
YTSNLAP

SEQ ID NO: 22
LC CDR3(Combined)
QQFTSSPFT

SEQ ID NO: 28
VL
ENVLTQSPATLSVSPGERATLSCRASSSVNYIYW

YQQKPGQAPRLLIYYTSNLAPGIPARFSGSGSGN

EYTLTISSLQSEDFAVYYCQQFTSSPFTFGQGTKL

EIK

SEQ ID NO: 36
DNA VL
GAGAATGTGCTGACCCAGTCTCCTGCCACACT

GTCTGTTAGCCCTGGCGAGAGAGCTACCCTGA

GCTGCAGAGCCTCTTCCTCCGTGAACTACATC

TACTGGTATCAGCAGAAGCCCGGCCAGGCTCC

TAGACTGCTGATCTACTACACCTCCAATCTGG

CCCCTGGCATCCCTGCCAGATTTTCCGGATCTG

GCTCCGGCAACGAGTATACCCTGACCATCTCC

AGCCTGCAGTCCGAGGACTTTGCTGTGTACTA

TTGCCAGCAGTTCACAAGCAGCCCTTTCACCT

TTGGCCAGGGCACCAAGCTGGAAATCAAA

Antibody B-H.1G LC-4

SEQ ID NO: 20
LC CDR1 (Combined)
RASSSVNYIY

SEQ ID NO: 21
LC CDR2 (Combined)
YTSNLAP

SEQ ID NO: 22
LC CDR3(Combined)
QQFTSSPFT

SEQ ID NO: 29
VL
QNVLTQPPSASGTPGQRVTISCRASSSVNYIYWY

QQLPGTAPKLLIYYTSNLAPGVPDRFSGSGSGNS

YSLAISGLRSEDEADYYCQQFTSSPFTFGTGTKV

TVL

SEQ ID NO: 37
DNA VL
CAGAATGTGCTGACCCAACCTCCTTCCGCCTC

TGGCACACCTGGACAGAGAGTGACAATCTCCT

GCCGGGCCTCCTCCTCCGTGAACTACATCTAC

TGGTATCAGCAGCTGCCCGGCACCGCTCCTAA

ACTGCTGATCTACTACACCTCCAATCTGGCCC

CTGGCGTGCCCGATAGATTTTCCGGATCTGGC

TCCGGCAACTCCTACAGCCTGGCTATCTCTGG

CCTGAGATCTGAGGACGAGGCCGACTACTACT

GCCAGCAGTTCACCTCCTCTCCATTCACCTTTG

GCACCGGCACCAAAGTGACAGTTCTT

Antibody B-H.1H LC-5

SEQ ID NO: 20
LC CDR1 (Combined)
RASSSVNYIY

SEQ ID NO: 21
LC CDR2 (Combined)
YTSNLAP

SEQ ID NO: 22
LC CDR3 (Combined)
QQFTSSPFT

SEQ ID NO: 30
VL
SNELTQPPSVSVSPGQTARITCRASSSVNYIYWY

QQKSGQAPVLVIYYTSNLAPGIPERFSGSGSGNM

YTLTISGAQVEDEADYYCQQFTSSPFTFGTGTKV

TVL

SEQ ID NO: 38
DNA VL
TCTAATGAGCTGACCCAGCCTCCTTCCGTGTCC

GTGTCTCCTGGACAGACCGCCAGAATTACCTG

CCGGGCCTCCTCCTCCGTGAACTACATCTACT

GGTATCAGCAGAAGTCCGGCCAGGCTCCTGTG

CTCGTGATCTACTACACCTCCAATCTGGCCCCT

GGCATCCCTGAGAGATTCTCCGGATCTGGCTC

CGGCAACATGTACACCCTGACCATCTCTGGCG

CCCAGGTGGAAGATGAGGCCGACTACTACTGC

CAGCAGTTCACCTCCTCTCCATTCACCTTTGGC

ACCGGCACCAAAGTGACAGTTCTT

Antibody B-H.1

SEQ ID NO:
Chain1: Fc only
METDTLLLWVLLLWVPGSTGDKTHTCPPCPAPE

3280

LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

HEDPEVKFNWYVDGVEVHNAKTKPREEQYNST

YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP

IEKTISKAKGQPREPQVYTLPPCREEMTKNQVSL

WCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNRFTQKSLSLSPGK

SEQ ID NO:
Chain2: humanized B-H
METDTLLLWVLLLWVPGSTGEVQLVESGGGLV

3281
scFv
QPGGSLRLSCAASGFTFSNFGMHWVRQAPGKGL

EWVSYISSGSSTIYYADTLKGRFTISRDNSKNTLY

LQMNSLRAEDTAVYYCARRGEGAMDYWGQGT

TVTVSSGGGGSGGGGSGGGGSGGGGSDNQLTQ

SPSFLSASVGDRVTITCRASSSVNYIYWYQQKPG

KAPKLLIYYTSNLAPGVPSRFSGSGSGNEYTLTIS

SLQPEDFATYYCQQFTSSPFTFGQGTKLEIKGGG

GSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVCTL

PPSREEMTKNQVSLSCAVKGFYPSDIAVEWESN

GQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGKGG

GGSGGGGSGLNDIFEAQKIEWHE

SEQ ID NO:
scFv
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFG

7492

MHWVRQAPGKGLEWVSYISSGSSTIYYADTLKG

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR

GEGAMDYWGQGTTVTVSSGGGGSGGGGSGGG

GSGGGGSDNQLTQSPSFLSASVGDRVTITCRASS

SVNYIYWYQQKPGKAPKLLIYYTSNLAPGVPSR

FSGSGSGNEYTLTISSLQPEDFATYYCQQFTSSPF

TFGQGTKLEIK

Antibody B-H.2

SEQ ID NO:
scFv
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFG

1338

MHWVRQAPGKGLEWVSYISSGSSTIYYADTLKG

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR

GEGAMDYWGQGTTVTVSSGGGGSGGGGSGGG

GSGGGGSDNQLTQSPSSLSASVGDRVTITCRASS

SVNYIYWYQQKPGKAPKLLIYYTSNLAPGVPSR

FSGSGSGNDYTLTISSLQPEDFATYYCQQFTSSPF

TFGQGTKLEIK

Antibody B-H.3

SEQ ID NO:
scFv
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFG

1339

MHWVRQAPGKGLEWVSYISSGSSTIYYADTLKG

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARR

GEGAMDYWGQGTTVTVSSGGGGSGGGGSGGG

GSGGGGSSNELTQPPSVSVSPGQTARITCRASSS

VNYIYWYQQKSGQAPVLVIYYTSNLAPGIPERFS

GSGSGNMYTLTISGAQVEDEADYYCQQFTSSPF

TFGTGTKVTVL

Antibody B-H.4

SEQ ID NO:
scFv
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFG

1340

MHWVRQAPGKGLEWVAYISSGSSTIYYADTLK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

RGEGAMDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDNQLTQSPSFLSASVGDRVTITCRAS

SSVNYIYWYQQKPGKAPKLLIYYTSNLAPGVPS

RFSGSGSGNEYTLTISSLQPEDFATYYCQQFTSSP

FTFGQGTKLEIK

Antibody B-H.5

SEQ ID NO:
scFv
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFG

1341

MHWVRQAPGKGLEWVAYISSGSSTIYYADTLK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

RGEGAMDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSDNQLTQSPSSLSASVGDRVTITCRAS

SSVNYIYWYQQKPGKAPKLLIYYTSNLAPGVPS

RFSGSGSGNDYTLTISSLQPEDFATYYCQQFTSSP

FTFGQGTKLEIK

Antibody B-H.6

SEQ ID NO:
scFv
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNFG

1342

MHWVRQAPGKGLEWVAYISSGSSTIYYADTLK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

RGEGAMDYWGQGTTVTVSSGGGGSGGGGSGG

GGSGGGGSSNELTQPPSVSVSPGQTARITCRASS

SVNYIYWYQQKSGQAPVLVIYYTSNLAPGIPERF

SGSGSGNMYTLTISGAQVEDEADYYCQQFTSSPF

TFGTGTKVTVL

TABLE 32

Constant region amino acid sequences of human IgG heavy chains and human kappa

light chain

Human kappa
LC
RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ

constant region

WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE

SEQ ID NO: 39

KHKVYACEVT HQGLSSPVTK SFNRGEC

IgG4 (S228P)
HC
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA

mutant constant

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS

region (EU

NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT

Numbering)

PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST

SEQ ID NO: 40

YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP

REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE

NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL

HNHYTQKSLSLSLG

IgG1 wild type
HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

SEQ ID NO: 41

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN

TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

IgG1 (N297A)
HC
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA

mutant constant

LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN

region (EU

TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

Numbering)

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA

SEQ ID NO: 42

STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ

PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE

ALHNHYTQKSLSLSPGK

IgM constant
HC
GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKN

delta CDC

NSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKV

(P311A, P313S)

QHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQ

SEQ ID NO: 73

ATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVT

STLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIR

VFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKT

HTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSL

KQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPAD

VFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEE

WNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSD

TAGTCY

IgGA1
HC
ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQ

SEQ ID NO: 74

GVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHY

TNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDL

LLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGC

YSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFR

PEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELP

REKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVG

HEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

IgGA2
HC
ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESG

SEQ ID NO: 75

QNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKH

YTNSSQDVTVPCRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCT

LTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCA

QPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEE

LALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQ

EPSQGTTTYAVTSILRVAAEDWKKGETFSCMVGHEALPLAFTQK

TIDRMAGKPTHINVSVVMAEADGTCY

Human Ig_J
HC
MKNHLLFWGVLAVFIKAVHVKAQEDERIVLVDNKCKCARITSRII

chain

RSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCK

SEQ ID NO: 76

KCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVP

LVYGGETKMVETALTPDACYPD

Anti-TCRβ V5 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to human TCRβ V5. In some embodiments, the TCRβ V5 subfamily comprises TCRβ V5-5*01, TCRβ V5-6*01, TCRβ V5-4*01, TCRβ V5-8*01, TCRβ V5-1*01, or a variant thereof.

Exemplary anti-TCRβ V5 antibodies of the disclosure are provided in Table 33. In some embodiments, the anti-TCRβ3 VS is antibody C, e.g., humanized antibody C (antibody C-H), as provided in Table 33. In some embodiments, the anti-TCRβV antibody comprises one or more (e.g., all three) of a LC CDR, LC CDR2, and LC CDR3 provided in Table 33; and/or one or more (e.g., all three) of a HC CDR1, HC CDR2, and HC CDR3 provided in Table 33, or a sequence with at least 95% identity thereto. In some embodiments, antibody C comprises a variable heavy chain (VH) and/or a variable light chain (VL) provided in Table 33, or a sequence with at least 95% identity thereto.

TABLE 33

Amino acid sequences for anti TCRβ V5 antibodies

Amino acid and nucleotide sequences for murine and humanized antibody molecules which bind to

TCRVB 5 (e.g., TCRVB 5-5 or TCRVB 5-6). The amino acid the heavy and light chain CDRs, and the

amino acid and nucleotide sequences of the heavy and light chain variable regions, and the

heavy and light chains are shown.

Murine antibody C, also referred to as 4H11

SEQ ID NO: 1315
HC CDR1 (Kabat)
AYGVN

SEQ ID NO: 1316
HC CDR2 (Kabat)
MIWGDGNTDYNSALKS

SEQ ID NO: 1317
HC CDR3 (Kabat)
DRVTATLYAMDY

SEQ ID NO: 1318
HC CDR1 (Chothia)
GFSLTAY

SEQ ID NO: 1319
HC CDR2 (Chothia)
WGDGN

SEQ ID NO: 1317
HC CDR3 (Chothia)
DRVTATLYAMDY

SEQ ID NO: 1320
HC CDR1 (Combined)
GFSLTAYGVN

SEQ ID NO: 1316
HC CDR2 (Combined)
MIWGDGNTDYNSALKS

SEQ ID NO: 1317
HC CDR3(Combined)
DRVTATLYAMDY

SEQ ID NO: 1321
LC CDR1 (Kabat)
SASQGISNYLN

SEQ ID NO: 1322
LC CDR2 (Kabat)
YTSSLHS

SEQ ID NO: 1323
LC CDR3 (Kabat)
QQYSKLPRT

SEQ ID NO: 1321
LC CDR1 (Chothia)
SASQGISNYLN

SEQ ID NO: 1322
LC CDR2 (Chothia)
YTSSLHS

SEQ ID NO: 1323
LC CDR3 (Chothia)
QQYSKLPRT

SEQ ID NO: 1321
LC CDR1 (Combined)
SASQGISNYLN

SEQ ID NO: 1322
LC CDR2 (Combined)
YTSSLHS

SEQ ID NO: 1323
LC CDR3(Combined)
QQYSKLPRT

SEQ ID NO: 232
VH
DIQMTQTTSSLSASLGDRVTISCSASQGISNY

LNWYQQKPDGTVKLLIYYTSSLHSGVPSRFS

GSGSGTDYSLTISNLEPEDIATYYCQQYSKLP

RTFGGGTKVEIK

SEQ ID NO: 7488
VL
QVQLKESGPGLVAPSQSLSITCTVSGFSLTAY

GVNWVRQPPGKGLEWLGMIWGDGNTDYN

SALKSRLSISKDNSKSQVFLKMNSLQTDDTA

RYYCARDRVTATLYAMDYWGQGTSVTVSS

Humanized antibody C

C-H-1 antibody

SEQ ID NO: 1315
HC CDR1 (Kabat)
AYGVN

SEQ ID NO: 1316
HC CDR2 (Kabat)
MIWGDGNTDYNSALKS

SEQ ID NO: 1317
HC CDR3 (Kabat)
DRVTATLYAMDY

SEQ ID NO: 1318
HC CDR1 (Chothia)
GFSLTAY

SEQ ID NO: 1319
HC CDR2 (Chothia)
WGDGN

SEQ ID NO: 1317
HC CDR3 (Chothia)
DRVTATLYAMDY

SEQ ID NO: 1320
HC CDR1 (Combined)
GFSLTAYGVN

SEQ ID NO: 1316
HC CDR2 (Combined)
MIWGDGNTDYNSALKS

SEQ ID NO: 1317
HC CDR3(Combined)
DRVTATLYAMDY

SEQ ID NO: 1321
LC CDR1 (Kabat)
SASQGISNYLN

SEQ ID NO: 1322
LC CDR2 (Kabat)
YTSSLHS

SEQ ID NO: 1323
LC CDR3 (Kabat)
QQYSKLPRT

SEQ ID NO: 1321
LC CDR1 (Chothia)
SASQGISNYLN

SEQ ID NO: 1322
LC CDR2 (Chothia)
YTSSLHS

SEQ ID NO: 1323
LC CDR3 (Chothia)
QQYSKLPRT

SEQ ID NO: 1321
LC CDR1 (Combined)
SASQGISNYLN

SEQ ID NO: 1322
LC CDR2 (Combined)
YTSSLHS

SEQ ID NO: 1323
LC CDR3(Combined)
QQYSKLPRT

SEQ ID NO: 1324
VL
DIQMTQSPSSLSASVGDRVTITCSASQGISNYL

NWYQQTPGKAPKLLIYYTSSLHSGVPSRFSGS

GSGTDYTFTISSLQPEDIATYYCQQYSKLPRT

FGQGTKLQIT

SEQ ID NO: 1325
VH
QVQLQESGPGLVRPSQTLSLTCTVSGFSLTA

YGVNWVRQPPGRGLEWLGMIWGDGNTDY

NSALKSRVTMLKDTSKNQFSLRLSSVTAAD

TAVYYCARDRVTATLYAMDYW

GQGSLVTVSS

Humanized antibody C Variable light chain (VL)

SEQ ID NO: 3000
VL
C-H-VL.1
DIQMTQSPSFLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKLLIYYTSSLHSGVPSRFS

GSGSGTEYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3001
VL
C-H-VL.2
DIQMTQSPSFLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3002
VL
C-H-VL.3
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKVVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDVATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3003
VL
C-H-VL.4
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDVATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3004
VL
C-H-VL.5
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTFTISSLQPEDIATYYCQQYSKLP

RTFGGGTKVEIK

SEQ ID NO: 3005
VL
C-H-VL.6
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKTVKLLIYYTSSLHSGIPSRFS

GSGSGTDYTLTIRSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3006
VL
C-H-VL.7
AIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3007
VL
C-H-VL.8
DIQMTQSPSSVSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3008
VL
C-H-VL.9
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKRLIYYTSSLHSGVPSRF

SGSGSGTEYTLTISNLQPEDFATYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3009
VL
C-H-VL.10
AIRMTQSPFSLSASVGDRVTITCSASQGISNY

LNWYQQKPAKAVKLFIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3010
VL
C-H-VL.11
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKRLIYYTSSLHSGVPSRF

SGSGSGTEYTLTISSLQPEDFATYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3011
VL
C-H-VL.12
DIQMTQSPSTLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKLLIYYTSSLHSGVPSRFS

GSGSGTEYTLTISSLQPDDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO:3012
VL
C-H-VL.13
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKSLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3013
VL
C-H-VL.14
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPGKAVKSLIYYTSSLHSGVPSKFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3014
VL
C-H-VL.15
DIQMTQSPSSLSASVGDRVTITCSASQGISNY

LNWYQQKPEKAVKSLIYYTSSLHSGVPSRFS

GSGSGTDYTLTISSLQPEDFATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3015
VL
C-H-VL.16
DIQMTQSPSAMSASVGDRVTITCSASQGISN

YLNWYQQKPGKVVKRLIYYTSSLHSGVPSR

FSGSGSGTEYTLTISSLQPEDFATYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3016
VL
C-H-VL.17
DIVMTQSPDSLAVSLGERATINCSASQGISNY

LNWYQQKPGQPVKLLIYYTSSLHSGVPDRFS

GSGSGTDYTLTISSLQAEDVAVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3017
VL
C-H-VL.18
EIVMTQSPGTLSLSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPDRFS

GSGSGTDYTLTISRLEPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3018
VL
C-H-VL.19
EIVMTQSPPTLSLSPGERVTLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGSGTDYTLTISSLQPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3019
VL
C-H-VL.20
EIVMTQSPPTLSLSPGERVTLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSSIPARFS

GSGSGTDYTLTISSLQPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3020
VL
C-H-VL.21
EIVMTQSPATLSLSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGSGTDYTLTISSLEPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3021
VL
C-H-VL.22
EIVMTQSPATLSLSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGSGTDYTLTISRLEPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3022
VL
C-H-VL.23
EIVMTQSPATLSLSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPDRFS

GSGSGTDYTLTISRLEPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3023
VL
C-H-VL.24
EIVMTQSPATLSLSPGERATLSCSASQGISNY

LNWYQQKPGLAVKLLIYYTSSLHSGIPDRFS

GSGSGTDYTLTISRLEPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3024
VL
C-H-VL.25
DIQMIQSPSFLSASVGDRVSIICSASQGISNYL

NWYLQKPGKSVKLFIYYTSSLHSGVSSRFSG

RGSGTDYTLTIISLKPEDFAAYYCQQYSKLP

RTFGGGTKVEIK

SEQ ID NO: 3025
VL
C-H-VL.26
EIVMTQSPATLSLSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGSGTDYTLTISSLQPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3026
VL
C-H-VL.27
EIVMTQSPATLSLSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGPGTDYTLTISSLEPEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3027
VL
C-H-VL.28
DIVMTQTPLSLSVTPGQPASISCSASQGISNY

LNWYLQKPGQSVKLLIYYTSSLHSGVPDRFS

GSGSGTDYTLKISRVEAEDVGVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3028
VL
C-H-VL.29
DIVMTQTPLSLSVTPGQPASISCSASQGISNY

LNWYLQKPGQPVKLLIYYTSSLHSGVPDRFS

GSGSGTDYTLKISRVEAEDVGVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3029
VL
C-H-VL.30
DIVMTQSPAFLSVTPGEKVTITCSASQGISNY

LNWYQQKPDQAVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTFTISSLEAEDAATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3030
VL
C-H-VL.31
DIVMTQSPLSLPVTPGEPASISCSASQGISNYL

NWYLQKPGQSVKLLIYYTSSLHSGVPDRFSG

SGSGTDYTLKISRVEAEDVGVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3031
VL
C-H-VL.32
DIVMTQTPLSLPVTPGEPASISCSASQGISNY

LNWYLQKPGQSVKLLIYYTSSLHSGVPDRFS

GSGSGTDYTLKISRVEAEDVGVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3032
VL
C-H-VL.33
EIVMTQSPATLSVSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGSGTEYTLTISILQSEDFAVYYCQQYSKLP

RTFGGGTKVEIK

SEQ ID NO: 3033
VL
C-H-VL.34
EIVMTQSPATLSVSPGERATLSCSASQGISNY

LNWYQQKPGQAVKLLIYYTSSLHSGIPARFS

GSGSGTEYTLTISSLQSEDFAVYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3034
VL
C-H-VL.35
DIVMTQSPLSLPVTLGQPASISCSASQGISNY

LNWYQQRPGQSVKRLIYYTSSLHSGVPDRFS

GSGSGTDYTLKISRVEAEDVGVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3035
VL
C-H-VL.36
EITMTQSPAFMSATPGDKVNISCSASQGISNY

LNWYQQKPGEAVKFIIYYTSSLHSGIPPRFSG

SGYGTDYTLTINNIESEDAAYYYCQQYSKLP

RTFGGGTKVEIK

SEQ ID NO: 3036
VL
C-H-VL.37
DIVMTQTPLSSPVTLGQPASISCSASQGISNY

LNWYQQRPGQPVKLLIYYTSSLHSGVPDRFS

GSGAGTDYTLKISRVEAEDVGVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3037
VL
C-H-VL.38
EIVMTQSPDFQSVTPKEKVTITCSASQGISNY

LNWYQQKPDQSVKLLIYYTSSLHSGVPSRFS

GSGSGTDYTLTINSLEAEDAATYYCQQYSKL

PRTFGGGTKVEIK

SEQ ID NO: 3038
VL
C-H-VL.39
EIVMTQTPLSLSITPGEQASISCSASQGISNYL

NWYLQKARPVVKLLIYYTSSLHSGVPDRFS

GSGSGTDYTLKISRVEAEDFGVYYCQQYSK

LPRTFGGGTKVEIK

SEQ ID NO: 3039
VL
C-H-VL.40
EIVMTQTPLSLSITPGEQASMSCSASQGISNY

LNWYLQKARPVVKLLIYYTSSLHSGVPDRFS

GSGSGTDYTLKISRVEAEDFGVYYCQQYSK

LPRTFGGGTKVEIK

Humanized antibody C Variable HEAVY chain (VH)

SEQ ID NO: 3040
VH
C-H-VH.1
QVTLKESGPVLVKPTETLTLTCTVSGFSLTA

YGVNWVRQPPGKALEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVVLTMTNMDPVD

TATYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3041
VH
C-H-VH.2
QVTLKESGPALVKPTETLTLTCTVSGFSLTA

YGVNWVRQPPGKALEWLGMIWGDGNTDY

NSALKSRLIISKDNSKSQVVLTMTNMDPVDT

ATYYCARDRVTATLYAMDYWGQGTLVTVS

S

SEQ ID NO: 3042
VH
C-H-VH.3
QVTLKESGPALVKPTQTLTLTCTVSGFSLTA

YGVNWVRQPPGKALEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVVLTMTNMDPVD

TATYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3043
VH
C-H-VH.4
QVQLQESGPGLVKPSGTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3044
VH
C-H-VH.5
QVTLKESGPTLVKPTQTLTLTCTVSGFSLTA

YGVNWVRQPPGKALEWLGMIWGDGNTDY

NSALKSRLTITKDNSKSQVVLTMTNMDPVD

TATYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3045
VH
C-H-VH.6
QVTLKESGPALVKPTQTLTLTCTVSGFSLTA

YGVNWVRQPPGKALEWLGMIWGDGNTDY

NSALKSRLTITKDNSKSQVVLTMTNMDPVD

TATYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3046
VH
C-H-VH.7
QVQLQESGPGLVKPSQTLSLTCTVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3047
VH
C-H-VH.8
QVQLQESGPGLVKPSETLSLTCTVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3048
VH
C-H-VH.9
QVQLQESGPGLVKPSQTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3049
VH
C-H-VH.10
QVQLQESGPGLVKPSDTLSLTCTVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3050
VH
C-H-VH.11
QVQLQESGPGLVKPSQTLSLTCTVSGFSLTA

YGVNWVRQHPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3051
VH
C-H-VH.12
QVQLQESGPGLVKPSQTLSLTCTVSGFSLTA

YGVNWVRQPAGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3052
VH
C-H-VH.13
QVQLQESGPGLVKPSQTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAVDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3053
VH
C-H-VH.14
QVQLQESGPGLVKPSETLSLTCTVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSHVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3054
VH
C-H-VH.15
QVQLQESGPGLVKPSETLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3055
VH
C-H-VH.16
QVQLQESGPGLVKPSQTLSLTCAVYGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3056
VH
C-H-VH.17
RVQLQESGPGLVKPSETLSLTCTVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVPLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3057
VH
C-H-VH.18
QVQLQESGPGLVKPSQTLSLTCTVSGFSLTA

YGVNWVRQHPGKGLEWLGMIWGDGNTDY

NSALKSLLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3058
VH
C-H-VH.19
QVQLQESGPGLVKPSDTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTALDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3059
VH
C-H-VH.20
QVQLQESGPGLVKPSDTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAVDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3060
VH
C-H-VH.21
QVQLQESGSGLVKPSQTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3061
VH
C-H-VH.22
EVOLVESGGGLVQPGRSLRLSCTVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSIVYLQMNSLKTEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3062
VH
C-H-VH.23
EVOLVESGGGLVQPGPSLRLSCTVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSIVYLQMNSLKTEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3063
VH
C-H-VH.24
QVQLQESGSGLVKPSQTLSLTCAVSGFSLTA

YGVNWVRQSPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3064
VH
C-H-VH.25
QVQLQESGPGLVKPSETLSLTCTVSGFSLTA

YGVNWVRQPAGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3065
VH
C-H-VH.26
EVOLVESGGGLVKPGRSLRLSCTVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSIVYLQMNSLKTEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3066
VH
C-H-VH.27
QVQLQESGPGLVKPSETLSLTCAVYGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVYLKLSSVTAADT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3067
VH
C-H-VH.28
QVQLQESGPGLVKPSDTLSLTCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSQVSLKLSSVTAVDT

GVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3068
VH
C-H-VH.29
EVQLVESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSSVYLQMNSLKTEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3069
VH
C-H-VH.30
EVOLVESGGGLVKPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSTVYLQMNSLKTEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3070
VH
C-H-VH.31
QVQLQQSGPGLVKPSQTLSLTCAVSGFSLTA

YGVNWVRQSPSRGLEWLGMIWGDGNTDY

NSALKSRLTINKDNSKSQVSLQLNSVTPEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3071
VH
C-H-VH.32
QVQLVESGGGLVQPGGSLRLSCSVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSTVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3072
VH
C-H-VH.33
QVQLQQWGAGLLKPSETLSLTCAVYGFSLT

AYGVNWVRQPPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNSKSQVSLKLSSVTAAD

TAVYYCARDRVTATLYAMDYWGQGTLVT

VSS

SEQ ID NO: 3073
VH
C-H-VH.34
QVQLVESGGGVVQPGRSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNSTSTVFLQMNSLRAED

TAVYYCARDRVTATLYAMDYWGQGTLVT

VSS

SEQ ID NO: 3074
VH
C-H-VH.35
EVOLVESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSTVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3075
VH
C-H-VH.36
EVOLVESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNAKSSVYLQMNSLRDEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3076
VH
C-H-VH.37
EVQLLESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSTVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3077
VH
C-H-VH.38
QVQLVESGGGLVKPGGSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNAKSSVYLQMNSLRAE

DTAVYYCARDRVTATLYAMDYWGQGTLV

TVSS

SEQ ID NO: 3078
VH
C-H-VH.39
EVOLVESGGGLVQPGGSLKLSCAVSGFSLTA

YGVNWVRQASGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSTVYLQMNSLKTEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3079
VH
C-H-VH.40
QVQLLESGGGLVKPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNAKSSVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3080
VH
C-H-VH.41
QVQLVESGGGVVQPGRSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNSKSTVYLQMNSLRAED

TAVYYCARDRVTATLYAMDYWGQGTLVT

VSS

SEQ ID NO: 3081
VH
C-H-VH.42
QVQLVESGGGVVQPGRSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNSKSRVYLQMNSLRAE

DTAVYYCARDRVTATLYAMDYWGQGTLV

TVSS

SEQ ID NO: 3082
VH
C-H-VH.43
QVQLVESGGGVVQPGRSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLAISKDNSKSTVYLQMNSLRAE

DTAVYYCARDRVTATLYAMDYWGQGTLV

TVSS

SEQ ID NO: 3083
VH
C-H-VH.44
QVQLVESGGGVVQPGGSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNSKSTVYLQMNSLRAED

TAVYYCARDRVTATLYAMDYWGQGTLVT

VSS

SEQ ID NO: 3084
VH
C-H-VH.45
EVOLVESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNAKSTVYLQMNSLRAED

TAVYYCARDRVTATLYAMDYWGQGTLVT

VSS

SEQ ID NO: 3085
VH
C-H-VH.46
EVOLVESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNAKSSVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3086
VH
C-H-VH.47
EVOLVESGGVVVQPGGSLRLSCAVSGFSLT

AYGVNWVRQAPGKGLEWLGMIWGDGNTD

YNSALKSRLTISKDNSKSSVYLQMNSLRTED

TALYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3087
VH
C-H-VH.48
EVOLVESGGGLVQPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKHNSKSTVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3088
VH
C-H-VH.49
EVOLVESGGGLVKPGGSLRLSCAVSGFSLTA

YGVNWVRQAPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNAKSSVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

SEQ ID NO: 3089
VH
C-H-VH.50
EVOLVESGGGLIQPGGSLRLSCAVSGFSLTA

YGVNWVRQPPGKGLEWLGMIWGDGNTDY

NSALKSRLTISKDNSKSTVYLQMNSLRAEDT

AVYYCARDRVTATLYAMDYWGQGTLVTV

SS

Exemplary anti-TCRβ V5 antibodies of the disclosure are provided in Table 11. In some embodiments, the anti-TCRβ V5 is antibody E, e.g., humanized antibody E (antibody E-H), as provided in Table 11. In some embodiments, the anti-TCRβV antibody comprises one or more (e.g., all three) of a LC CDR1, LC CDR2, and LC CDR3 provided in Table 11; and/or one or more (e.g., all three) of a HC CDR1, HC CDR2, and HC CDR3 provided in Table 11, or a sequence with at least 95 identity thereto. In some embodiments, antibody E comprises a variable heavy chain (VH) and/or a variable light chain (VL) provided in Table 11, or a sequence with at least 95% identity thereto.

In some embodiments, antibody E comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 3284 and/or a light chain comprising the amino acid sequence of SEQ ID NO: 3285, or a sequence with at least 95% identity thereto.

TABLE 11

Amino acid sequences for anti TCRβ V5 antibodies

Amino acid and nucleotide sequences for murine and humanized antibody molecules which bind to

TCRVB 5 (e.g., TCRVB 5-5 or TCRVB 5-6). The amino acid the heavy and light chain CDRs, and

the amino acid and nucleotide sequences of the heavy and light chain variable regions, and

the heavy and light chains are shown.

Murine antibody E, also referred to as MH3-2

SEQ ID NO: 1298
HC CDR1 (Kabat)
SSWMN

SEQ ID NO: 1299
HC CDR2 (Kabat)
RIYPGDGDTKYNGKFKG

SEQ ID NO: 1300
HC CDR3 (Kabat)
RGTGGWYFDV

SEQ ID NO: 1302
HC CDR1 (Chothia)
GYAFSSS

SEQ ID NO: 1303
HC CDR2 (Chothia)
YPGDGD

SEQ ID NO: 1301
HC CDR3 (Chothia)
RGTGGWYFDV

SEQ ID NO: 1304
HC CDR1 (Combined)
GYAFSSSWMN

SEQ ID NO: 1299
HC CDR2 (Combined))
RIYPGDGDTKYNGKFKG

SEQ ID NO: 1301
HC CDR3(Combined)
RGTGGWYFDV

SEQ ID NO: 1305
LC CDR1 (Kabat)
RASESVDSSGNSFMH

SEQ ID NO: 1306
LC CDR2 (Kabat)
RASNLES

SEQ ID NO: 1307
LC CDR3 (Kabat)
QQSFDDPFT

SEQ ID NO: 1308
LC CDR1 (Chothia)
SESVDSSGNSF

SEQ ID NO: 1306
LC CDR2 (Chothia)
RASNLES

SEQ ID NO: 1307
LC CDR3 (Chothia)
QQSFDDPFT

SEQ ID NO: 1305
LC CDR1 (Combined)
RASESVDSSGNSFMH

SEQ ID NO: 1306
LC CDR2 (Combined)
RASNLES

SEQ ID NO: 1307
LC CDR3(Combined)
QQSFDDPFT

SEQ ID NO: 3091
VH
QVQLQQSGPELVKPGASVKISCKASGYAFSSS

WMNWVKQRPGQGLEWIGRIYPGDGDTKYN

GKFKGKATLTADKSSSTAYMHLSSLTSVDSA

VYFCARRGTGGWYFDVWGAGTTVTVSS

SEQ ID NO: 3284
Heavy chain
METDTLLLWVLLLWVPGSTGQVQLQQSGPEL

VKPGASVKISCKASGYAFSSSWMNWVKQRP

GQGLEWIGRIYPGDGDTKYNGKFKGKATLTA

DKSSSTAYMHLSSLTSVDSAVYFCARRGTGG

WYFDVWGAGTTVTVSSAKTTAPSVYPLAPV

CGDTTGSSVTLGCLVKGYFPEPVTLTWNSGS

LSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPS

QSITCNVAHPASSTKVDKKIEPRGPTIKPCPPC

KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTC

VVVDVSEDDPDVQISWFVNNVEVHTAQTQT

HREDYNSTLRVVSALPIQHQDWMSGKEFKCK

VNNKDLPAPIERTISKPKGSVRAPQVYVLPPPE

EEMTKKQVTLTCMVTDFMPEDIYVEWTNNG

KTELNYKNTEPVLDSDGSYFMYSKLRVEKKN

WVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

SEQ ID NO: 3092
VL
DIVLTQSPASLAVSLGQRATISCRASESVDSSG

NSFMHWYQQKPGQPPQLLIYRASNLESGIPAR

FSGSGSRTDFTLTINPVEADDVATFYCQQSFD

DPFTFGSGTKLEIK

SEQ ID NO: 3285
Light chain
METDTLLLWVLLLWVPGSTGDIVLTQSPASL

AVSLGQRATISCRASESVDSSGNSFMHWYQQ

KPGQPPQLLIYRASNLESGIPARFSGSGSRTDF

TLTINPVEADDVATFYCQQSFDDPFTFGSGTK

LEIKRADAAPTVSIFPPSSEQLTSGGASVVCFL

NNFYPKDINVKWKIDGSERQNGVLNSWTDQ

DSKDSTYSMSSTLTLTKDEYERHNSYTCEAT

HKTSTSPIVKSFNRNEC

Humanized antibody E (E-H antibody)

Variable light chain (VL)

SEQ ID NO: 3093
VL
E-H.1
DIVLTQSPDSLAVSLGERATINCRASESVDSSG

NSFMHWYQQKPGQPPQLLIYRASNLESGVPD

RFSGSGSRTDFTLTISSLQAEDVAVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3094
VL
E-H.2
EIVLTQSPATLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGSRTDFTLTISSLEPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3095
VL
E-H.3
EIVLTQSPATLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGSRTDFTLTISRLEPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3096
VL
E-H.4
EIVLTQSPATLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGSRTDFTLTISSLQPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3097
VL
E-H.5
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDVATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3098
VL
E-H.6
EIVLTQSPATLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGPRTDFTLTISSLEPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3099
VL
E-H.7
EIVLTQSPATLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPD

RFSGSGSRTDFTLTISRLEPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3100
VL
E-H.8
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKVPQLLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDVATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3101
VL
E-H.9
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKTPQLLIYRASNLESGIPSR

FSGSGSRTDFTLTIRSLQPEDFATYYCQQSFDD

PFTFGQGTKLEIK

SEQ ID NO: 3102
VL
E-H.10
EIVLTQSPGTLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPD

RFSGSGSRTDFTLTISRLEPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3103
VL
E-H.11
EIVLTQSPATLSLSPGERATLSCRASESVDSSG

NSFMHWYQQKPGLAPQLLIYRASNLESGIPDR

FSGSGSRTDFTLTISRLEPEDFAVYYCQQSFDD

PFTFGQGTKLEIK

SEQ ID NO: 3104
VL
E-H.12
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQLLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3105
VL
E-H.13
DIQLTQSPSSVSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQLLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3106
VL
E-H.14
AIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQLLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3107
VL
E-H.15
DIQLTQSPSFLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQLLIYRASNLESGVPS

RFSGSGSRTEFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3108
VL
E-H.16
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQLLIYRASNLESGVPS

RFSGSGSRTDFTFTISSLQPEDIATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3109
VL
E-H.17
EIVLTQSPATLSVSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGSRTEFTLTISILQSEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3110
VL
E-H.18
EIVLTQSPATLSVSPGERATLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGSRTEFTLTISSLQSEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3111
VL
E-H.19
AIRLTQSPFSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPAKAPQLFIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3112
VL
E-H.20
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQSLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3113
VL
E-H.21
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQRLIYRASNLESGVPS

RFSGSGSRTEFTLTISNLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3114
VL
E-H.22
DIQLTQSPSTLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQLLIYRASNLESGVPS

RFSGSGSRTEFTLTISSLQPDDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3115
VL
E-H.23
EIVLTQSPDFQSVTPKEKVTITCRASESVDSSG

NSFMHWYQQKPDQSPQLLIYRASNLESGVPS

RFSGSGSRTDFTLTINSLEAEDAATYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3116
VL
E-H.24
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQSLIYRASNLESGVPS

KFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3117
VL
E-H.25
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPGKAPQRLIYRASNLESGVPS

RFSGSGSRTEFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3118
VL
E-H.26
DIVLTQTPLSLSVTPGQPASISCRASESVDSSG

NSFMHWYLQKPGQPPQLLIYRASNLESGVPD

RFSGSGSRTDFTLKISRVEAEDVGVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3119
VL
E-H.27
DIQLTQSPSSLSASVGDRVTITCRASESVDSSG

NSFMHWYQQKPEKAPQSLIYRASNLESGVPS

RFSGSGSRTDFTLTISSLQPEDFATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3120
VL
E-H.28
EIVLTQSPPTLSLSPGERVTLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESGIPA

RFSGSGSRTDFTLTISSLQPEDFAVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3121
VL
E-H.29
DIQLTQSPSAMSASVGDRVTITCRASESVDSS

GNSFMHWYQQKPGKVPQRLIYRASNLESGVP

SRFSGSGSRTEFTLTISSLQPEDFATYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO:3122
VL
E-H.30
DIVLTQSPLSLPVTPGEPASISCRASESVDSSGN

SFMHWYLQKPGQSPQLLIYRASNLESGVPDR

FSGSGSRTDFTLKISRVEAEDVGVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3123
VL
E-H.31
DIVLTQTPLSLPVTPGEPASISCRASESVDSSG

NSFMHWYLQKPGQSPQLLIYRASNLESGVPD

RFSGSGSRTDFTLKISRVEAEDVGVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3124
VL
E-H.32
DIVLTQTPLSLSVTPGQPASISCRASESVDSSG

NSFMHWYLQKPGQSPQLLIYRASNLESGVPD

RFSGSGSRTDFTLKISRVEAEDVGVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3125
VL
E-H.33
EIVLTQSPPTLSLSPGERVTLSCRASESVDSSG

NSFMHWYQQKPGQAPQLLIYRASNLESSIPAR

FSGSGSRTDFTLTISSLQPEDFAVYYCQQSFDD

PFTFGQGTKLEIK

SEQ ID NO: 3126
VL
E-H.34
DIVLTQSPLSLPVTLGQPASISCRASESVDSSG

NSFMHWYQQRPGQSPQRLIYRASNLESGVPD

RFSGSGSRTDFTLKISRVEAEDVGVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3127
VL
E-H.35
DIVLTQTPLSSPVTLGQPASISCRASESVDSSG

NSFMHWYQQRPGQPPQLLIYRASNLESGVPD

RFSGSGARTDFTLKISRVEAEDVGVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3128
VL
E-H.36
DIVLTQSPAFLSVTPGEKVTITCRASESVDSSG

NSFMHWYQQKPDQAPQLLIYRASNLESGVPS

RFSGSGSRTDFTFTISSLEAEDAATYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3129
VL
E-H.37
DIQLIQSPSFLSASVGDRVSIICRASESVDSSGN

SFMHWYLQKPGKSPQLFIYRASNLESGVSSRF

SGRGSRTDFTLTIISLKPEDFAAYYCQQSFDDP

FTFGQGTKLEIK

SEQ ID NO: 3130
VL
E-H.38
EIVLTQTPLSLSITPGEQASISCRASESVDSSGN

SFMHWYLQKARPVPQLLIYRASNLESGVPDR

FSGSGSRTDFTLKISRVEAEDFGVYYCQQSFD

DPFTFGQGTKLEIK

SEQ ID NO: 3131
VL
E-H.39
EIVLTQTPLSLSITPGEQASMSCRASESVDSSG

NSFMHWYLQKARPVPQLLIYRASNLESGVPD

RFSGSGSRTDFTLKISRVEAEDFGVYYCQQSF

DDPFTFGQGTKLEIK

SEQ ID NO: 3132
VL
E-H.40
EITLTQSPAFMSATPGDKVNISCRASESVDSSG

NSFMHWYQQKPGEAPQFIIYRASNLESGIPPR

FSGSGYRTDFTLTINNIESEDAAYYYCQQSFD

DPFTFGQGTKLEIK

Variable HEAVY chain (VH)

SEQ ID NO: 3133
VH
E-H.1
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELSSLRSEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3134
VH
E-H.2
QVQLVQSGAEVKKPGSSVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELSSLRSEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3135
VH
E-H.3
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELSSLRSEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3136
VH
E-H.4
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQELEWIGRIYPGDGDTKYN

GKFKGRATLTADKSISTAYMELSSLRSEDTAT

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3137
VH
E-H.5
EVQLVQSGAEVKKPGATVKISCKASGYAFSSS

WMNWVQQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELSSLRSEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3138
VH
E-H.6
QVQLVQSGAEVKKTGSSVKVSCKASGYAFSS

SWMNWVRQAPGQALEWIGRIYPGDGDTKYN

GKFKGRATLTADKSMSTAYMELSSLRSEDTA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3139
VH
E-H.7
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQRLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSASTAYMELSSLRSEDMA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3140
VH
E-H.8
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELRSLRSDDMA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3141
VH
E-H.9
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQRLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSASTAYMELSSLRSEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3142
VH
E-H.10
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELRSLRSDDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3143
VH
E-H.11
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSISTAYMELSRLRSDDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3144
VH
E-H.12
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSISTAYMELSRLRSDDTV

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3145
VH
E-H.13
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGWATLTADKSISTAYMELSRLRSDDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3146
VH
E-H.14
QVQLVQSGAEVKKPGASVKVSCKASGYAFSS

SWMNWVRQATGQGLEWIGRIYPGDGDTKYN

GKFKGRATLTANKSISTAYMELSSLRSEDTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3147
VH
E-H.15
QVQLVQSGSELKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRAVLSADKSVSTAYLQISSLKAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3148
VH
E-H.16
QVQLVQSGPEVKKPGTSVKVSCKASGYAFSS

SWMNWVRQARGQRLEWIGRIYPGDGDTKYN

GKFKGRATLTADKSTSTAYMELSSLRSEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3149
VH
E-H.17
EVQLVQSGAEVKKPGESLKISCKASGYAFSSS

WMNWVRQMPGKGLEWIGRIYPGDGDTKYN

GKFKGQATLSADKSISTAYLQWSSLKASDTA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3150
VH
E-H.18
QVQLVQSGSELKKPGASVKVSCKASGYAFSS

SWMNWVRQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRAVLSADKSVSMAYLQISSLKAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3151
VH
E-H.19
QVQLVQSGHEVKQPGASVKVSCKASGYAFSS

SWMNWVPQAPGQGLEWIGRIYPGDGDTKYN

GKFKGRAVLSADKSASTAYLQISSLKAEDMA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3152
VH
E-H.20
EVQLVQSGAEVKKPGESLKISCKASGYAFSSS

WMNWVRQMPGKGLEWIGRIYPGDGDTKYN

GKFKGQATLSADKPISTAYLQWSSLKASDTA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3153
VH
E-H.21
EVQLVQSGAEVKKPGESLRISCKASGYAFSSS

WMNWVRQMPGKGLEWIGRIYPGDGDTKYN

GKFKGQATLSADKSISTAYLQWSSLKASDTA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3154
VH
E-H.22
EVQLVQSGAEVKKPGESLRISCKASGYAFSSS

WMNWVRQMPGKGLEWIGRIYPGDGDTKYN

GKFKGHATLSADKSISTAYLQWSSLKASDTA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3155
VH
E-H.23
QVQLVQSGAEVKKTGSSVKVSCKASGYAFSS

SWMNWVRQAPRQALEWIGRIYPGDGDTKYN

GKFKGRATLTADKSMSTAYMELSSLRSEDTA

MYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3156
VH
E-H.24
EVQLVESGGGLVQPGRSLRLSCTASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSIAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3157
VH
E-H.25
EVQLVESGGGLVQPGPSLRLSCTASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSIAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3158
VH
E-H.26
QVQLQESGPGLVKPSQTLSLTCTASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAADTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3159
VH
E-H.27
QVQLQESGPGLVKPSGTLSLTCAASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAADTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3160
VH
E-H.28
EVQLVESGGGLVKPGRSLRLSCTASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSIAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3161
VH
E-H.29
EVQLVESGGGLVQPGGSLKLSCAASGYAFSSS

WMNWVRQASGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3162
VH
E-H.30
QVQLQESGPGLVKPSQTLSLTCAASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAADTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3163
VH
E-H.31
EVQLVESGGGLVKPGGSLRLSCAASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3164
VH
E-H.32
EVQLVESGGALVKPGGSLRLSCAASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3165
VH
E-H.33
QVQLQESGPGLVKPSQTLSLTCAAYGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAADTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3166
VH
E-H.34
QVQLQESGSGLVKPSQTLSLTCAASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAADTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3167
VH
E-H.35
EVQLVESGGGLVQPGGSLRLSCAASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSSAYLQMNSLKTEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3168
VH
E-H.36
QVQLQESGPGLVKPSDTLSLTCTASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAADTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3169
VH
E-H.37
QVQLQESGPGLVKPSQTLSLTCTASGYAFSSS

WMNWVRQHPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSQASLKLSSVTAADTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3170
VH
E-H.38
QVQLQESGPGLVKPSQTLSLTCTASGYAFSSS

WMNWVRQHPGKGLEWIGRIYPGDGDTKYN

GKFKGLATLSADKSKSQASLKLSSVTAADTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3171
VH
E-H.39
QVQLVESGGGVVQPGRSLRLSCAASGYAFSS

SWMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMSSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3172
VH
E-H.40
QVQLVESGGGLVKPGGSLRLSCAASGYAFSS

SWMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKAKSSAYLQMNSLRAEDT

AVYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3173
VH
E-H.41
QVQLVESGGGLVQPGGSLRLSCSASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3174
VH
E-H.42
QVQLLESGGGLVKPGGSLRLSCAASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKAKSSAYLQMNSLRAEDT

AVYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3175
VH
E-H.43
EVQLVESGGGLVQPGGSLRLSCSASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMSSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3176
VH
E-H.44
QVQLQESGPGLVKPSDTLSLTCAASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAVDTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3177
VH
E-H.45
QVQLQESGPGLVKPSQTLSLTCAASGYAFSSS

WMNWVRQPPGKGLEWIGRIYPGDGDTKYNG

KFKGRATLSADKSKSQASLKLSSVTAVDTAV

YYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3178
VH
E-H.46
EVQLVESGGGLVQPGGSLRLSCSASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYVQMSSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3179
VH
E-H.47
QVQLVDSGGGVVQPGRSLRLSCAASGYAFSS

SWMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3180
VH
E-H.48
QVQLVESGGGVVQPGRSLRLSCAASGYAFSS

SWMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLRAEGTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3181
VH
E-H.49
QVQLVESGGGVVQPGRSLRLSCAASGYAFSS

SWMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

SEQ ID NO: 3182
VH
E-H.50
EVQLVESGGGLVQPGGSLRLSCAASGYAFSSS

WMNWVRQAPGKGLEWIGRIYPGDGDTKYN

GKFKGRATLSADKSKSTAYLQMNSLRAEDTA

VYYCARRGTGGWYFDVWGQGTTVTVSS

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VH and/or a VL of an antibody described in Table 33, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VH and a VL of an antibody described in Table 33, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VH and/or a VL of an antibody described in Table 11, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V5 antibody molecule comprises a VH and a VL of an antibody described in Table 11, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

Anti-TCRβ V10 Antibodies

Accordingly, in one aspect, the disclosure provides an anti-TCRβV antibody molecule that binds to a human TCRβ V10 subfamily member. In some embodiments, TCRβ V10 subfamily is also known as TCRβ V12. In some embodiments, the TCRβ V10 subfamily comprises: TCRβ V10-1*01, TCRβ V10-1*02, TCRβ V10-3*01 or TCRβ V10-2*01, or a variant thereof.

Exemplary anti-TCRβ V10 antibodies of the disclosure are provided in Table 12. In some embodiments, the anti-TCRβ V10 is antibody D, e.g., humanized antibody D (antibody D-H), as provided in Table 12. In some embodiments, antibody D comprises one or more (e.g., three) light chain CDRs and/or one or more (e.g., three) heavy chain CDRs provided in Table 12, or a sequence with at least 95% identity thereto. In some embodiments, antibody D comprises a variable heavy chain (VH) and/or a variable light chain (VL) provided in Table 12, or a sequence with at least 95% identity thereto.

TABLE 12

Amino acid sequences for anti TCRβ V10 antibodies

Amino acid and nucleotide sequences for murine and humanized antibody molecules which bind to

TCRBV 10 (e.g., TCRBV 10-1, TCRBV 10-2 or TCRBV 10-3). The amino acid the heavy and light

chain CDRs, and the amino acid and nucleotide sequences of the heavy and light chain variable regions,

and the heavy and light chains are shown.

Murine antibody D, also referred to as S511 antibody

SEQ ID NO: 1288
HC CDR1 (Kabat)
SYGMS

SEQ ID NO: 1289
HC CDR2 (Kabat)
LISSGGSYTYYTDSVKG

SEQ ID NO: 1290
HC CDR3 (Kabat)
HGGNFFDY

SEQ ID NO: 1291
HC CDR1 (Chothia)
GFTFRSY

SEQ ID NO: 1292
HC CDR2 (Chothia)
SSGGSY

SEQ ID NO: 1290
HC CDR3 (Chothia)
HGGNFFDY

SEQ ID NO: 1293
HC CDR1 (Combined)
GFTFRSYGMS

SEQ ID NO: 1289
HC CDR2 (Combined))
LISSGGSYTYYTDSVKG

SEQ ID NO: 1290
HC CDR3(Combined)
HGGNFFDY

SEQ ID NO: 1294
LC CDR1 (Kabat)
SVSSSVSYMH

SEQ ID NO: 1295
LC CDR2 (Kabat)
DTSKLAS

SEQ ID NO: 1296
LC CDR3 (Kabat)
QQWSSNPQYT

SEQ ID NO: 1297
LC CDR1 (Chothia)
SSSVSY

SEQ ID NO: 1295
LC CDR2 (Chothia)
DTSKLAS

SEQ ID NO: 1296
LC CDR3 (Chothia)
QQWSSNPQYT

SEQ ID NO: 1294
LC CDR1 (Combined)
SVSSSVSYMH

SEQ ID NO: 1295
LC CDR2 (Combined)
DTSKLAS

SEQ ID NO: 1296
LC CDR3 (Combined)
QQWSSNPQYT

SEQ ID NO: 3183
VH
EVQLVESGGDLVKPGGSLKLSCAVSGFTFRSY

GMSWVRQTPDKRLEWVALISSGGSYTYYTDS

VKGRFTISRDNAKNTLYLQMSSLKSEDTAIYY

CSRHGGNFFDYWGQGTTLTVSS

SEQ ID NO: 3184
VL
QIVLTQSPSIMSASPGEKVTMTCSVSSSVSYM

HWYQQKSGTSPKRWIYDTSKLASGVPARFSG

SGSGTSYSLTISSMEAEDAATYYCQQWSSNPQ

YTFGGGTKLEIK

Humanized antibody D (D-H antibody)

Variable light chain (VL)

SEQ ID NO: 3185
VL
D-VL-
DIVLTQSPAFLSVTPGEKVTITCSVSSSVSYMHWYQQK

H.1
PDQAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSL

EAEDAATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3186
VL
D-VL-
AIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.2
PGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3187
VL
D-VL-
DIQLTQSPSFLSASVGDRVTITCSVSSSVSYMHWYQQK

H.3
PGKAPKLLIYDTSKLASGVPSRFSGSGSGTEYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3188
VL
D-VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.4
PGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3189
VL
D-VL-
DIQLTQSPSSVSASVGDRVTITCSVSSSVSYMHWYQQ

H.5
KPGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISS

LQPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3190
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.6
PGKVPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDVATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3191
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.7
PGQAPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDVATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3192
VL
D- VL-
EIVLTQSPDFQSVTPKEKVTITCSVSSSVSYMHWYQQK

H.8
PDQSPKLLIYDTSKLASGVPSRFSGSGSGTDYTLTINSL

EAEDAATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3193
VL
D- VL-
AIRLTQSPFSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.9
PAKAPKLFIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3194
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.10
PGKAPKLLIYDTSKLASGVPSRFSGSGSGTDYTFTISSL

QPEDIATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3195
VL
D- VL-
EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQK

H.11
PGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISSL

EPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3196
VL
D- VL-
DIQLTQSPSTLSASVGDRVTITCSVSSSVSYMHWYQQ

H.12
KPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEYTLTISS

LQPDDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3197
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.13
PGKTPKLLIYDTSKLASGIPSRFSGSGSGTDYTLTIRSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3198
VL
D- VL-
EIVLTQSPPTLSLSPGERVTLSCSVSSSVSYMHWYQQK

H.14
PGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISSL

QPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3199
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.15
PGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3200
VL
D- VL-
EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQK

H.16
PGQAPKLLIYDTSKLASGIPARFSGSGPGTDYTLTISSL

EPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3201
VL
D- VL-
EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQK

H.17
PGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISRL

EPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3202
VL
D- VL-
EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQK

H.18
PGQAPKLLIYDTSKLASGIPARFSGSGSGTDYTLTISSL

QPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3203
VL
D- VL-
EIVLTQSPATLSVSPGERATLSCSVSSSVSYMHWYQQ

H.19
KPGQAPKLLIYDTSKLASGIPARFSGSGSGTEYTLTISS

LQSEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3204
VL
D- VL-
EIVLTQSPATLSVSPGERATLSCSVSSSVSYMHWYQQ

H.20
KPGQAPKLLIYDTSKLASGIPARFSGSGSGTEYTLTISIL

QSEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3205
VL
D- VL-
EIVLTQSPPTLSLSPGERVTLSCSVSSSVSYMHWYQQK

H.21
PGQAPKLLIYDTSKLASSIPARFSGSGSGTDYTLTISSL

QPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3206
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.22
PGKAPKSLIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3207
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.23
PGKAPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISNL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3208
VL
D- VL-
DIQLTQSPSAMSASVGDRVTITCSVSSSVSYMHWYQQ

H.24
KPGKVPKRLIYDTSKLASGVPSRFSGSGSGTEYTLTISS

LQPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3209
VL
D- VL-
EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQK

H.25
PGQAPKLLIYDTSKLASGIPDRFSGSGSGTDYTLTISRL

EPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3210
VL
D- VL-
EIVLTQSPATLSLSPGERATLSCSVSSSVSYMHWYQQK

H.26
PGLAPKLLIYDTSKLASGIPDRFSGSGSGTDYTLTISRL

EPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3211
VL
D- VL-
EIVLTQSPGTLSLSPGERATLSCSVSSSVSYMHWYQQK

H.27
PGQAPKLLIYDTSKLASGIPDRFSGSGSGTDYTLTISRL

EPEDFAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3212
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.28
PGKAPKSLIYDTSKLASGVPSKFSGSGSGTDYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3213
VL
D- VL-
DIQLTQSPSSLSASVGDRVTITCSVSSSVSYMHWYQQK

H.29
PEKAPKSLIYDTSKLASGVPSRFSGSGSGTDYTLTISSL

QPEDFATYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3214
VL
D- VL-
DIVLTQSPDSLAVSLGERATINCSVSSSVSYMHWYQQ

H.30
KPGQPPKLLIYDTSKLASGVPDRFSGSGSGTDYTLTISS

LQAEDVAVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3215
VL
D- VL-
EIVLTQTPLSLSITPGEQASMSCSVSSSVSYMHWYLQK

H.31
ARPVPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISR

VEAEDFGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3216
VL
D- VL-
EIVLTQTPLSLSITPGEQASISCSVSSSVSYMHWYLQKA

H.32
RPVPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISRV

EAEDFGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3217
VL
D- VL-
DIVLTQSPLSLPVTPGEPASISCSVSSSVSYMHWYLQK

H.33
PGQSPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISR

VEAEDVGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3218
VL
D- VL-
DIVLTQSPLSLPVTLGQPASISCSVSSSVSYMHWYQQR

H.34
PGQSPKRLIYDTSKLASGVPDRFSGSGSGTDYTLKISR

VEAEDVGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3219
VL
D- VL-
DIVLTQTPLSLPVTPGEPASISCSVSSSVSYMHWYLQK

H.35
PGQSPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISR

VEAEDVGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3220
VL
D- VL-
DIVLTQTPLSLSVTPGQPASISCSVSSSVSYMHWYLQK

H.36
PGQSPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISR

VEAEDVGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3221
VL
D- VL-
DIVLTQTPLSLSVTPGQPASISCSVSSSVSYMHWYLQK

H.37
PGQPPKLLIYDTSKLASGVPDRFSGSGSGTDYTLKISR

VEAEDVGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3222
VL
D- VL-
DIQLIQSPSFLSASVGDRVSIICSVSSSVSYMHWYLQKP

H.38
GKSPKLFIYDTSKLASGVSSRFSGRGSGTDYTLTIISLK

PEDFAAYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3223
VL
D- VL-
DIVLTQTPLSSPVTLGQPASISCSVSSSVSYMHWYQQR

H.39
PGQPPKLLIYDTSKLASGVPDRFSGSGAGTDYTLKISR

VEAEDVGVYYCQQWSSNPQYTFGQGTKLEIK

SEQ ID NO: 3224
VL
D- VL-
EITLTQSPAFMSATPGDKVNISCSVSSSVSYMHWYQQ

H.40
KPGEAPKFIIYDTSKLASGIPPRFSGSGYGTDYTLTINNI

ESEDAAYYYCQQWSSNPQYTFGQGTKLEIK

Variable HEAVY chain (VH)

SEQ ID NO: 3225
VH
D-VH-
EVQLVESGGGLVKPGGSLRLSCAVSGFTFRSYGMSW

H.1
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLKTEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3226
VH
D- VH-
EVQLVESGGALVKPGGSLRLSCAVSGFTFRSYGMSW

H.2
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLKTEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3227
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.3
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3228
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.4
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3229
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.5
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNSLYLQMNSLKTEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3230
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.6
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDMAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3231
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.7
VRQAPGKGLEWVALISSGGSYTYYTDSVKGQFTISRD

NAKNTLYLQMNSLRAEDMAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3232
VH
D- VH-
EVQLVESGGGLVKPGRSLRLSCTVSGFTFRSYGMSWV

H.8
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNILYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQG

TTVTVSS

SEQ ID NO: 3233
VH
D- VH-
EVQLVESGGGLVKPGGSLRLSCAVSGFTFRSYGMSW

H.9
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3234
VH
D- VH-
EVQLVESGGGLVQPGGSLKLSCAVSGFTFRSYGMSW

H.10
VRQASGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLKTEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3235
VH
D- VH-
QVQLVESGGGVVQPGGSLRLSCAVSGFTFRSYGMSW

H.11
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3236
VH
D- VH-
QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.12
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMSSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3237
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCPVSGFTFRSYGMSWV

H.13
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

ANNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3238
VH
D- VH-
EVQLVESGGGLVQPGRSLRLSCTVSGFTFRSYGMSWV

H.14
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNILYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQG

TTVTVSS

SEQ ID NO: 3239
VH
D- VH-
EVQLVESGGGLVQPGPSLRLSCTVSGFTFRSYGMSWV

H.15
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNILYLQMNSLKTEDTAVYYCSRHGGNFFDYWGQG

TTVTVSS

SEQ ID NO: 3240
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.16
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3241
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.17
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRDEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3242
VH
D- VH-
QVQLVESGGGLVKPGGSLRLSCAVSGFTFRSYGMSW

H.18
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3243
VH
D- VH-
QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.19
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3244
VH
D- VH-
EVQLLESGGGLVQPGGSLRLSCAVSGFTFRSYGMSWV

H.20
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3245
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.21
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRH

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3246
VH
D- VH-
EVQLVESGGGLIQPGGSLRLSCAVSGFTFRSYGMSWV

H.22
RQPPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDNS

KNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQG

TTVTVSS

SEQ ID NO: 3247
VH
D- VH-
EVQLVESGGGLIQPGGSLRLSCAVSGFTFRSYGMSWV

H.23
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3248
VH
D- VH-
EVQLVESGGGLVQPGRSLRLSCAVSGFTFRSYGMSW

H.24
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDTALYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3249
VH
D- VH-
QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.25
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNRLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3250
VH
D- VH-
QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.26
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLRAEGTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3251
VH
D- VH-
QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.27
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFAISRD

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3252
VH
D- VH-
QVQLVDSGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.28
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3253
VH
D- VH-
EVQLVESGGGVVRPGGSLRLSCAVSGFTFRSYGMSW

H.29
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDTALYHCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3254
VH
D- VH-
EVQLVESGGVVVQPGGSLRLSCAVSGFTFRSYGMSW

H.30
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNSLYLQMNSLRAEDTALYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3255
VH
D- VH-
EVQLVESGGGVVQPGGSLRLSCAVSGFTFRSYGMSW

H.31
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNSLYLQMNSLRTEDTALYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3256
VH
D- VH-
EVQLVESGGVVVQPGGSLRLSCAVSGFTFRSYGMSW

H.32
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNSLYLQMNSLRTEDTALYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3257
VH
D- VH-
EVQLVETGGGLIQPGGSLRLSCAVSGFTFRSYGMSWV

H.33
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3258
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.34
VRQATGKGLEWVALISSGGSYTYYTDSVKGRFTISRE

NAKNSLYLQMNSLRAGDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3259
VH
D- VH-
EVQLVESRGVLVQPGGSLRLSCAVSGFTFRSYGMSW

H.35
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLHLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3260
VH
D- VH-
EVQLVESGGGLVQPGRSLRLSCAVSGFTFRSYGMSW

H.36
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDMALYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3261
VH
D- VH-
QVQLVESGGGLVQPGGSLRLSCSVSGFTFRSYGMSW

H.37
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3262
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCSVSGFTFRSYGMSWV

H.38
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNTLYLQMSSLRAEDTAVYYCSRHGGNFFDYWGQG

TTVTVSS

SEQ ID NO: 3263
VH
D- VH-
QVQLVESGGGVVQPGRSLRLSCAVSGFTFRSYGMSW

H.39
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSTNTLFLQMNSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3264
VH
D- VH-
QVQLLESGGGLVKPGGSLRLSCAVSGFTFRSYGMSW

H.40
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NAKNSLYLQMNSLRAEDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3265
VH
D- VH-
EVQLVESGEGLVQPGGSLRLSCAVSGFTFRSYGMSWV

H.41
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNTLYLQMGSLRAEDMAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3266
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.42
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMGSLRAEDMAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3267
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCSVSGFTFRSYGMSWV

H.43
RQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRDN

SKNTLYVQMSSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3268
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.44
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFIISRD

NSRNSLYLQKNRRRAEDMAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3269
VH
D- VH-
EVQLVESGGGLVQPGGSLRLSCAVSGFTFRSYGMSW

H.45
VHQAPGKGLEWVALISSGGSYTYYTDSVKGRFIISRD

NSRNTLYLQTNSLRAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3270
VH
D- VH-
EVHLVESGGGLVQPGGALRLSCAVSGFTFRSYGMSW

H.46
VRQATGKGLEWVALISSGGSYTYYTDSVKGRFTISRE

NAKNSLYLQMNSLRAGDTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3271
VH
D- VH-
EVQLVESGGGLVQPRGSLRLSCAVSGFTFRSYGMSW

H.47
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNNLRAEGTAVYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3272
VH
D- VH-
EVQLVESGGGLVQPRGSLRLSCAVSGFTFRSYGMSW

H.48
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTISRD

NSKNTLYLQMNNLRAEGTAAYYCSRHGGNFFDYWG

QGTTVTVSS

SEQ ID NO: 3273
VH
D- VH-
QVQLVQSGAEVKKPGASVKVSCKVSGFTFRSYGMSW

H.49
VRQAPGKGLEWVALISSGGSYTYYTDSVKGRFTITRD

NSTNTLYMELSSLRSEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

SEQ ID NO: 3274
VH
D- VH-
QVQLVQSGSELKKPGASVKVSCKVSGFTFRSYGMSW

H.50
VRQAPGQGLEWVALISSGGSYTYYTDSVKGRFVISRD

NSVNTLYLQISSLKAEDTAVYYCSRHGGNFFDYWGQ

GTTVTVSS

In some embodiments, the anti-TCRβ V10 antibody molecule comprises a VH or a VL of an antibody described in Table 12, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In some embodiments, the anti-TCRβ V10 antibody molecule comprises a VH and a VL of an antibody described in Table 12, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

Additional Anti-TCRVβ Antibodies

Additional exemplary anti-TCRβV antibodies of the disclosure are provided in Table 13. In some embodiments, the anti-TCRβV antibody is a humanized antibody, e.g., as provided in Table 13. In some embodiments, the anti-TCRβV antibody comprises one or more (e.g., all three) of a LC CDR1, LC CDR2, and LC CDR3 provided in Table 13; and/or one or more (e.g., all three) of a HC CDR1, HC CDR2, and HC CDR3 provided in Table 13, or a sequence with at least 95% identity thereto. In some embodiments, the anti-TCRβV antibody comprises a variable heavy chain (VH) and/or a variable light chain (VL) provided in Table 13, or a sequence with at least 95% identity thereto.

TABLE 13

Amino acid sequences for additional anti-TCRβ V antibodies

Amino acid and nucleotide sequences for murine and humanized antibody molecules which bind to

various TCRVB families are disclosed. The amino acid the heavy and light chain CDRs, and the amino

acid and nucleotide sequences of the heavy and light chain variable regions, and the heavy and light

chains are shown. Antibodies disclosed in the table include, MPB2D5, CAS1.1.3, IMMU222, REA1062,

JOVI-3, IMMU546 and MR5-2. MPB2D5 binds human TCRβV 20-1 (TCRβV2 per old nomenclature).

CAS1.1.3 binds human TCRβV 27 (TCRβV14 per old nomenclature). IMMU 222 binds human TCRβV

6-5, TCRβV 6-6, or TCRβV 6-9 (TCRβV13.1 per old nomenclature). REA1062 binds human TCRβV 5-

1). JOVI-3 binds human TCRβV 28 (TCRβV3.1 per old nomenclature). IMMU546 binds human TCRβV

2. MR5-2 binds human TCRVβ 13-2.

Binds to human TCRVB 20-1

MPB2D5 (murine), also referred to here as BJ1188, BJ1190 and REA654; or Antibody G

Binds to human TCRVβ 20-1

SEQ ID NO: 1102
HC CDR1 (Kabat)
SAYMH

SEQ ID NO: 1103
HC CDR2 (Kabat)
RIDPATGKTKYAPKFQA

SEQ ID NO: 1104
HC CDR3 (Kabat)
SLNWDYGLDY

SEQ ID NO: 1105
HC CDR1 (Chothia)
GFNIKSA

SEQ ID NO: 1106
HC CDR2 (Chothia)
DPATGK

SEQ ID NO: 1104
HC CDR3 (Chothia)
SLNWDYGLDY

SEQ ID NO: 1107
HC CDR1 (Combined)
GFNIKSAYMH

SEQ ID NO: 1103
HC CDR2 (Combined)
RIDPATGKTKYAPKFQA

SEQ ID NO: 1104
HC CDR3 (Combined)
SLNWDYGLDY

SEQ ID NO: 7489
LC CDR1 (Kabat)
RASKSVSILGTHLIH

SEQ ID NO: 1108
LC CDR2 (Kabat)
AASNLES

SEQ ID NO: 1109
LC CDR3 (Kabat)
QQSIEDPWT

SEQ ID NO: 1110
LC CDR1 (Chothia)
SKSVSILGTHL

SEQ ID NO: 1108
LC CDR2 (Chothia)
AASNLES

SEQ ID NO: 1109
LC CDR3 (Chothia)
QQSIEDPWT

SEQ ID NO: 7489
LC CDR1 (Combined)
RASKSVSILGTHLIH

SEQ ID NO: 1108
LC CDR2 (Combined)
AASNLES

SEQ ID NO: 1109
LC CDR3(Combined)
QQSIEDPWT

SEQ ID NO: 1111
VL
DIVLTQSPASLAVSLGQRATISCRASKSVSILGTH

LIHWYQQKPGQPPKLLIYAASNLESGVPARFSGS

GSETVFTLNIHPVEEEDAATYFCQQSIEDPWTFG

GGTKLGIK

SEQ ID NO: 1112
VH
EVQLQQSVADLVRPGASLKLSCTASGFNIKSAY

MHWVIQRPDQGPECLGRIDPATGKTKYAPKFQA

KATITADTSSNTAYLQLSSLTSEDTAIYYCTRSLN

WDYGLDYWGQGTSVTVSS

VH for MPB2D5 (humanized) also referred to as Antibody G-H (humanized)

Binds to human TCRVβ 20-1

SEQ ID NO: 1113
VH - 1
QVQLVQSGAEVKKPGASVKVSCKASGFNIKSAY

MHWVRQAPGQGLEWMGRIDPATGKTKYAPKF

QARVTMTADTSTNTAYMELSSLRSEDTAVYYC

ARSLNWDYGLDYWGQGTLVTVSS

SEQ ID NO: 1114
VH - 2
QVQLVQSGAEVKKPGASVKVSCKASGFNIKSAY

MHWVRQAPGQEPGCMGRIDPATGKTKYAPKFQ

ARVTMTADTSINTAYTELSSLRSEDTATYYCARS

LNWDYGLDYWGQGTLVTVSS

SEQ ID NO: 1115
VH - 3
QVQLVQSGAEVKKPGSSVKVSCKASGFNIKSAY

MHWVRQAPGQGLEWMGRIDPATGKTKYAPKF

QARVTITADTSTNTAYMELSSLRSEDTAVYYCA

RSLNWDYGLDYWGQGTLVTVSS

SEQ ID NO: 1116
VH - 4
QVQLVQSGAEVKKPGASVKVSCKASGFNIKSAY

MHWVRQAPGQRLEWMGRIDPATGKTKYAPKF

QARVTITADTSANTAYMELSSLRSEDTAVYYCA

RSLNWDYGLDYWGQGTLVTVSS

VL for MPB2D5 (humanized) also referred to as Antibody G-H (humanized)

Binds to human TCRVβ 20-1

SEQ ID NO: 1117
VL - 1
EIVLTQSPATLSLSPGERATLSCRASKSVSILGTH

LIHWYQQKPGQAPRLLIYAASNLESGIPARFSGS

GSETDFTLTISSLEPEDFAVYFCQQSIEDPFGGGT

KVEIK

SEQ ID NO: 1118
VL - 2
EIVLTQSPATLSLSPGERATLSCRASKSVSILGTH

LIHWYQQKPGLAPRLLIYAASNLESGIPDRESGS

GSETDFTLTISRLEPEDFAVYFCQQSIEDPFGGGT

KVEIK

SEQ ID NO: 1119
VL - 3
EIVLTQSPGTLSLSPGERATLSCRASKSVSILGTH

LIHWYQQKPGQAPRLLIYAASNLESGIPDRESGS

GSETDFTLTISRLEPEDFAVYFCQQSIEDPFGGGT

KVEIK

CAS1.1.3 (murine) also referred to herein as BJ1460; or Antibody H

Binds to human TCRVβ 27

SEQ ID NO: 1120
HC CDR1 (Kabat)
DTYMY

SEQ ID NO: 1121
HC CDR2 (Kabat)
RIDPANGNTKYDPKFQD

SEQ ID NO: 1122
HC CDR3 (Kabat)
GSYYYAMDY

SEQ ID NO: 1123
HC CDR1 (Chothia)
GFKTEDT

SEQ ID NO: 1124
HC CDR2 (Chothia)
DPANGN

SEQ ID NO: 1122
HC CDR3 (Chothia)
GSYYYAMDY

SEQ ID NO: 1125
HC CDR1 (Combined)
GFKTEDTYMY

SEQ ID NO: 1121
HC CDR2 (Combined)
RIDPANGNTKYDPKFQD

SEQ ID NO: 1122
HC CDR3(Combined)
GSYYYAMDY

SEQ ID NO: 1126
LC CDR1 (Kabat)
RASESVDSYGNSFMH

SEQ ID NO: 1127
LC CDR2 (Kabat)
RASNLES

SEQ ID NO: 1128
LC CDR3 (Kabat)
QQSNEDPYT

SEQ ID NO: 1129
LC CDR1 (Chothia)
SESVDSYGNSF

SEQ ID NO: 1127
LC CDR2 (Chothia)
RASNLES

SEQ ID NO: 1128
LC CDR3 (Chothia)
QQSNEDPYT

SEQ ID NO: 1126
LC CDR1 (Combined)
RASESVDSYGNSFMH

SEQ ID NO: 1127
LC CDR2 (Combined)
RASNLES

SEQ ID NO: 1128
LC CDR3(Combined)
QQSNEDPYT

SEQ ID NO: 7490
VL
DIVLTQSPASLAVSLGQRATISCRASESVDSYGN

SFMHWYQQKPGQPPKLLIYRASNLESGIPARFSG

SGSRTDFTLTINPVEADDVATYYCQQSNEDPYTF

GGGTKLEIK

SEQ ID NO: 1130
VH
EVQLQQSGAELVKPGASVKLSCTASGFKTEDTY

MYWVKQRPEQGLEWIGRIDPANGNTKYDPKFQ

DKATITADSSSNTAYLQLSSLPSEDTAVYYCARG

SYYYAMDYWGQGTSVTVSS

VH for CAS1.1.3 (humanized) also referred to as Antibody H-H (humanized)

Binds to human TCRVβ 27

SEQ ID NO: 1131
VH - 1
QVQLVQSGAEVKKPGSSVKVSCKASGFKTEDTY

MYWVRQAPGQGLEWIGRIDPANGNTKYDPKFQ

DRATITADSSTNTAYMELSSLRSEDTAVYYCAR

GSYYYAMDYWGQGTLVTVSS

SEQ ID NO: 1132
VH - 2
QVQLVQSGAEVKKPGASVKVSCKASGFKTEDT

YMYWVRQAPGQRLEWIGRIDPANGNTKYDPKF

QDRATITADSSANTAYMELSSLRSEDTAVYYCA

RGSYYYAMDYWGQGTLVTVSS

SEQ ID NO: 1133
VH - 3
EVQLVESGGGLVQPGGSLKLSCAASGFKTEDTY

MYWVRQASGKGLEWIGRIDPANGNTKYDPKFQ

DRATISADSSKNTAYLQMNSLKTEDTAVYYCAR

GSYYYAMDYWGQGTLVTVSS

SEQ ID NO: 1134
VH - 4
EVQLVQSGAEVKKPGESLRISCKASGFKTEDTY

MYWVRQMPGKGLEWIGRIDPANGNTKYDPKFQ

DQATISADSSINTAYLQWSSLKASDTAMYYCAR

GSYYYAMDYWGQGTLVTVSS

SEQ ID NO: 1135
VH - 5
QVQLVQSGSELKKPGASVKVSCKASGFKTEDTY

MYWVRQAPGQGLEWIGRIDPANGNTKYDPKFQ

DRAVISADSSVNTAYLQISSLKAEDTAVYYCAR

GSYYYAMDYWGQGTLVTVSS

VL for CAS1.1.3 (humanized) also referred to as Antibody H-H (humanized)

Binds to human TCRVβ 27

SEQ ID NO: 1136
VL - 1
DIVLTQSPDSLAVSLGERATINCRASESVDSYGN

SFMHWYQQKPGQPPKLLIYRASNLESGVPDRFS

GSGSRTDFTLTISSLQAEDVAVYYCQQSNEDPYT

FGQGTKLEIK

SEQ ID NO: 1137
VL - 2
EIVLTQSPATLSLSPGERATLSCRASESVDSYGNS

FMHWYQQKPGQAPKLLIYRASNLESGIPARFSGS

GSRTDFTLTISRLEPEDFAVYYCQQSNEDPYTFG

QGTKLEIK

SEQ ID NO: 1138
VL - 3
DIQLTQSPSSLSASVGDRVTITCRASESVDSYGNS

FMHWYQQKPGQAPKLLIYRASNLESGVPSRFSG

SGSRTDFTLTISSLQPEDVATYYCQQSNEDPYTF

GQGTKLEIK

SEQ ID NO: 1139
VL - 4
AIQLTQSPSSLSASVGDRVTITCRASESVDSYGNS

FMHWYQQKPGKAPKLLIYRASNLESGVPSRFSG

SGSRTDFTLTISSLQPEDFATYYCQQSNEDPYTFG

QGTKLEIK

SEQ ID NO: 1140
VL - 5
EIVLTQSPDFQSVTPKEKVTITCRASESVDSYGNS

FMHWYQQKPDQSPKLLIYRASNLESGVPSRFSG

SGSRTDFTLTINSLEAEDAATYYCQQSNEDPYTF

GQGTKLEIK

IMMU222 (murine) also referred to as BJ1461; or Antibody I

Binds to human TCRVβ 6-5,6-6,6-9

SEQ ID NO: 1141
HC CDR1 (Kabat)
SYAMS

SEQ ID NO: 1142
HC CDR2 (Kabat)
HISNGGDYIYYADTVKG

SEQ ID NO: 1143
HC CDR3 (Kabat)
PSYYSDPWFFDV

SEQ ID NO: 1144
HC CDR1 (Chothia)
GFTFRSY

SEQ ID NO: 1145
HC CDR2 (Chothia)
SNGGDY

SEQ ID NO: 1143
HC CDR3 (Chothia)
PSYYSDPWFFDV

SEQ ID NO: 1146
HC CDR1 (Combined)
GFTFRSYAMS

SEQ ID NO: 1142
HC CDR2 (Combined)
HISNGGDYIYYADTVKG

SEQ ID NO: 1143
HC CDR3(Combined)
PSYYSDPWFFDV

SEQ ID NO: 1147
LC CDR1 (Kabat)
SAGSSVSFMH

SEQ ID NO: 1148
LC CDR2 (Kabat)
DTSKLAS

SEQ ID NO: 1149
LC CDR3 (Kabat)
LOGSGFPLT

SEQ ID NO: 1150
LC CDR1 (Chothia)
GSSVSF

SEQ ID NO: 1148
LC CDR2 (Chothia)
DTSKLAS

SEQ ID NO: 1149
LC CDR3 (Chothia)
LQGSGFPLT

SEQ ID NO: 1147
LC CDR1 (Combined)
SAGSSVSFMH

SEQ ID NO: 1148
LC CDR2 (Combined)
DTSKLAS

SEQ ID NO: 1149
LC CDR3(Combined)
LQGSGFPLT

SEQ ID NO: 1151
VL
ENVLTQSPAIMSASPGEKVTMTCSAGSSVSFMH

WYQQKSSTSPKLWIYDTSKLASGVPGRFSGSGS

GNSFSLTISSMEAEDVAIYYCLQGSGFPLTFGSGT

KLEIK

SEQ ID NO: 1152
VH
DVKLVESGEGLVKPGGSLKLSCAASGFTFRSYA

MSWVRQTPEKRLEWVAHISNGGDYIYYADTVK

GRFTISRDNARNTLYLQMSSLKSEDTAMYYCTR

PSYYSDPWFFDVWGTGTTVTVSS

VH for IMMU222 (humanized) also referred to as Antibody I-H

Binds to human TCRVβ 6-5,6-6,6-9

SEQ ID NO: 1153
VH - 1
EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYA

MSWVRQAPGKGLEWVAHISNGGDYIYYADTVK

GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTR

PSYYSDPWFFDVWGQGTTVTVSS

SEQ ID NO: 1154
VH - 2
QVQLVESGGGVVQPGRSLRLSCAASGFTFRSYA

MSWVRQAPGKGLEWVAHISNGGDYIYYADTVK

GRFTISRDNSKNTLYLQMSSLRAEDTAVYYCTR

PSYYSDPWFFDVWGQGTTVTVSS

SEQ ID NO: 1155
VH - 3
EVQLVESGGGLVQPGGSLRLSCAASGFTFRSYA

MSWVRQAPGKGLEWVAHISNGGDYIYYADTVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTR

PSYYSDPWFFDVWGQGTTVTVSS

SEQ ID NO: 1156
VH - 4
QVQLVQSGSELKKPGASVKVSCKASGFTFRSYA

MSWVRQAPGQGLEWVAHISNGGDYIYYADTVK

GRFVISRDNSVNTLYLQISSLKAEDTAVYYCTRP

SYYSDPWFFDVWGQGTTVTVSS

SEQ ID NO: 1157
VH - 5
QVQLVQSGAEVKKPGASVKVSCKASGFTFRSYA

MSWVRQAPGQRLEWVAHISNGGDYIYYADTVK

GRFTITRDNSANTLYMELSSLRSEDTAVYYCTRP

SYYSDPWFFDVWGQGTTVTVSS

VL for IMMU222 (humanized)) also referred to as Antibody I-H

Binds to human TCRVβ 6-5,6-6,6-9

SEQ ID NO: 1158
VL - 1
ENVLTQSPATLSLSPGERATLSCSAGSSVSFMHW

YQQKPGQAPKLLIYDTSKLASGIPARFSGSGSGN

DFTLTISSLEPEDFAVYYCLQGSGFPLTFGQGTKL

EIK

SEQ ID NO: 1159
VL - 2
ENVLTQSPDFQSVTPKEKVTITCSAGSSVSFMHW

YQQKPDQSPKLLIYDTSKLASGVPSRFSGSGSGN

DFTLTINSLEAEDAATYYCLQGSGFPLTFGQGTK

LEIK

SEQ ID NO: 1160
VL - 3
DNQLTQSPSSLSASVGDRVTITCSAGSSVSFMHW

YQQKPGKVPKLLIYDTSKLASGVPSRFSGSGSGN

DFTLTISSLQPEDVATYYCLQGSGFPLTFGQGTK

LEIK

SEQ ID NO: 1161
VL - 4
ANQLTQSPSSLSASVGDRVTITCSAGSSVSFMHW

YQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGN

DFTLTISSLQPEDFATYYCLQGSGFPLTFGQGTKL

EIK

SEQ ID NO: 1162
VL - 5
DNVLTQSPDSLAVSLGERATINCSAGSSVSFMH

WYQQKPGQPPKLLIYDTSKLASGVPDRFSGSGS

GNDFTLTISSLQAEDVAVYYCLQGSGFPLTFGQG

TKLEIK

REA1062 (murine), also referred to as BJ1189 or as Antibody J

Binds to human TCRVβ 5-1

SEQ ID NO: 1163
HC CDR1 (Kabat)
DYNIH

SEQ ID NO: 1164
HC CDR2 (Kabat)
YINPYNGRTGYNQKFKA

SEQ ID NO: 1165
HC CDR3 (Kabat)
WDGSSYFDY

SEQ ID NO: 1166
HC CDR1 (Chothia)
GYTFTDYNIH

SEQ ID NO: 1167
HC CDR2 (Chothia)
NPYNGR

SEQ ID NO: 1165
HC CDR3 (Chothia)
WDGSSYFDY

SEQ ID NO: 1166
HC CDR1 (Combined)
GYTFTDYNIH

SEQ ID NO: 1164
HC CDR2 (Combined)
YINPYNGRTGYNQKFKA

SEQ ID NO: 1165
HC CDR3(Combined)
WDGSSYFDY

SEQ ID NO: 1168
LC CDR1 (Kabat)
SASSSVSYMH

SEQ ID NO: 1169
LC CDR2 (Kabat)
EISKLAS

SEQ ID NO: 1170
LC CDR3 (Kabat)
QQWNYPLLT

SEQ ID NO: 1171
LC CDR1 (Chothia)
SSSVSY

SEQ ID NO: 1169
LC CDR2 (Chothia)
EISKLAS

SEQ ID NO: 1170
LC CDR3 (Chothia)
QQWNYPLLT

SEQ ID NO: 1168
LC CDR1 (Combined)
SASSSVSYMH

SEQ ID NO: 1169
LC CDR2 (Combined)
EISKLAS

SEQ ID NO: 1170
LC CDR3(Combined)
QQWNYPLLT

SEQ ID NO: 7491
VL
EIVLTQSPAITAASLGQKVTITCSASSSVSYMHW

YQQKSGTSPKPWIYEISKLASGVPARFSGSGSGT

SYSLTISSMEAEDAAIYYCQQWNYPLLTFGAGT

KLELK

SEQ ID NO: 1172
VH
EVQLQQSGPVLVKPGASVRMSCKASGYTFTDY

NIHWVKQSHGRSLEWVGYINPYNGRTGYNQKF

KAKATLTVDKSSSTAYMDLRSLTSEDSAVYYCA

RWDGSSYFDYWGQGTTLTVSS

VH for REA1062 (humanized) also referred to as Antibody J-H

Binds to human TCRVβ 5-1

SEQ ID NO: 1173
VH - 1
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDY

NIHWVRQAPGQGLEWVGYINPYNGRTGYNQKF

KARATLTVDKSTSTAYMELSSLRSEDTAVYYCA

RWDGSSYFDYWGQGTTVTVSS

SEQ ID NO: 1174
VH - 2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDY

NIHWVRQAPGQGLEWVGYINPYNGRTGYNQKF

KARATLTVDKSTSTAYMELRSLRSDDMAVYYC

ARWDGSSYFDYWGQGTTVTVSS

SEQ ID NO: 1175
VH - 3
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDY

NIHWVRQATGQGLEWVGYINPYNGRTGYNQKF

KARATLTVNKSISTAYMELSSLRSEDTAVYYCA

RWDGSSYFDYWGQGTTVTVSS

SEQ ID NO: 1176
VH - 4
EVQLVESGGGLVQPGRSLRLSCTASGYTFTDYNI

HWVRQAPGKGLEWVGYINPYNGRTGYNQKFK

ARATLSVDKSKSIAYLQMNSLKTEDTAVYYCAR

WDGSSYFDYWGQGTTVTVSS

SEQ ID NO: 1177
VH - 5
QVQLVQSGSELKKPGASVKVSCKASGYTFTDYN

IHWVRQAPGQGLEWVGYINPYNGRTGYNQKFK

ARAVLSVDKSVSTAYLQISSLKAEDTAVYYCAR

WDGSSYFDYWGQGTTVTVSS

VL for REA1062 (humanized) also referred to as Antibody J-H

Binds to human TCRVβ 5-1

SEQ ID NO: 1178
VL - 1
EIVLTQSPATLSLSPGERATLSCSASSSVSYMHW

YQQKPGQAPKLLIYEISKLASGIPARFSGSGSGTD

YTLTISSLEPEDFAVYYCQQWNYPLLTFGQGTKL

EIK

SEQ ID NO: 1179
VL - 2
EIVLTQSPATLSLSPGERATLSCSASSSVSYMHW

YQQKPGQAPKLLIYEISKLASGIPARFSGSGSGTD

YTLTISRLEPEDFAVYYCQQWNYPLLTFGQGTK

LEIK

SEQ ID NO: 1180
VL - 3
EIVLTQSPDFQSVTPKEKVTITCSASSSVSYMHW

YQQKPDQSPKLLIYEISKLASGVPSRFSGSGSGTD

YTLTINSLEAEDAATYYCQQWNYPLLTFGQGTK

LEIK

SEQ ID NO: 1181
VL - 4
DIQLTQSPSFLSASVGDRVTITCSASSSVSYMHW

YQQKPGKAPKLLIYEISKLASGVPSRFSGSGSGTE

YTLTISSLQPEDFATYYCQQWNYPLLTFGQGTKL

EIK

SEQ ID NO: 1182
VL - 5
AIQLTQSPSSLSASVGDRVTITCSASSSVSYMHW

YQQKPGKAPKLLIYEISKLASGVPSRFSGSGSGT

DYTLTISSLQPEDFATYYCQQWNYPLLTFGQGT

KLEIK

SEQ ID NO: 1183
VL - 6
AIRLTQSPFSLSASVGDRVTITCSASSSVSYMHW

YQQKPAKAPKLFIYEISKLASGVPSRFSGSGSGT

DYTLTISSLQPEDFATYYCQQWNYPLLTFGQGT

KLEIK

SEQ ID NO: 1184
VL - 7
DIVLTQSPDSLAVSLGERATINCSASSSVSYMHW

YQQKPGQPPKLLIYEISKLASGVPDRFSGSGSGT

DYTLTISSLQAEDVAVYYCQQWNYPLLTFGQGT

KLEIK

JOVI-3 (murine), also referred to as BJ1187 or Antibody K

Binds to human TCRVβ 28

SEQ ID NO: 1185
HC CDR1 (Kabat)
GSWMN

SEQ ID NO: 1186
HC CDR2 (Kabat)
RIYPGDGDTDYSGKFKG

SEQ ID NO: 1187
HC CDR3 (Kabat)
SGYFNYVPVFDY

SEQ ID NO: 1188
HC CDR1 (Chothia)
GYTFSGS

SEQ ID NO: 1189
HC CDR2 (Chothia)
YPGDGD

SEQ ID NO: 1187
HC CDR3 (Chothia)
SGYFNYVPVFDY

SEQ ID NO: 1190
HC CDR1 (Combined)
GYTFSGSWMN

SEQ ID NO: 1186
HC CDR2 (Combined)
RIYPGDGDTDYSGKFKG

SEQ ID NO: 1187
HC CDR3(Combined)
SGYFNYVPVFDY

SEQ ID NO: 1191
LC CDR1 (Kabat)
SANSTVGYIH

SEQ ID NO: 1192
LC CDR2 (Kabat)
TTSNLAS

SEQ ID NO: 1193
LC CDR3 (Kabat)
HQWSFYPT

SEQ ID NO: 1194
LC CDR1 (Chothia)
NSTVGY

SEQ ID NO: 1192
LC CDR2 (Chothia)
TTSNLAS

SEQ ID NO: 1193
LC CDR3 (Chothia)
HQWSFYPT

SEQ ID NO: 1191
LC CDR1 (Combined)
SANSTVGYIH

SEQ ID NO: 1192
LC CDR2 (Combined)
TTSNLAS

SEQ ID NO: 1193
LC CDR3(Combined)
HQWSFYPT

SEQ ID NO: 1195
VL
QIVLTQSPAIMSASLGEEIALTCSANSTVGYIHW

YQQKSGTSPKLLIYTTSNLASGVPSRFSGSGSGTF

YSLTISSVEAEDAADYFCHQWSFYPTFGGGTKLE

IK

SEQ ID NO: 1196
VH
QIQLQQSGPEVVKPGASVQISCKASGYTFSGSW

MNWVKQRPGKGLEWIGRIYPGDGDTDYSGKFK

GRATLTADKSSSTAYMRLSSLTSEDSAVYFCARS

GYFNYVPVFDYWGQGTTLSVSS

VH for JOVI-3 (humanized) also referred to as Antibody K-H

Binds to human TCRVβ 28

SEQ ID NO: 1197
VH - 1
QIQLVQSGAEVKKPGASVKVSCKASGYTFSGSW

MNWVRQAPGQGLEWIGRIYPGDGDTDYSGKFK

GRATLTADKSTSTAYMELSSLRSEDTAVYYCAR

SGYFNYVPVFDYWGQGTTVTVSS

SEQ ID NO: 1198
VH - 2
QIQLVQSGAEVKKPGSSVKVSCKASGYTFSGSW

MNWVRQAPGQGLEWIGRIYPGDGDTDYSGKFK

GRATLTADKSTSTAYMELSSLRSEDTAVYYCAR

SGYFNYVPVFDYWGQGTTVTVSS

SEQ ID NO: 1199
VH - 3
EIQLVQSGAEVKKPGESLKISCKASGYTFSGSWM

NWVRQMPGKGLEWIGRIYPGDGDTDYSGKFKG

QATLSADKSISTAYLQWSSLKASDTAMYYCARS

GYFNYVPVFDYWGQGTTVTVSS

SEQ ID NO: 1200
VH - 4
QIQLVQSGSELKKPGASVKVSCKASGYTFSGSW

MNWVRQAPGQGLEWIGRIYPGDGDTDYSGKFK

GRAVLSADKSVSTAYLQISSLKAEDTAVYYCAR

SGYFNYVPVFDYWGQGTTVTVSS

SEQ ID NO: 1201
VH - 5
QIQLVQSGSELKKPGASVKVSCKASGYTFSGSW

MNWVRQAPGQGLEWIGRIYPGDGDTDYSGKFK

GRAVLSADKSVSMAYLQISSLKAEDTAVYYCAR

SGYFNYVPVFDYWGQGTTVTVSS

SEQ ID NO: 1202
VH - 6
EIQLVESGGGLVQPGRSLRLSCTASGYTFSGSW

MNWVRQAPGKGLEWIGRIYPGDGDTDYSGKFK

GRATLSADKSKSIAYLQMNSLKTEDTAVYYCAR

SGYFNYVPVFDYWGQGTTVTVSS

VL for JOVI-3 (humanized) also referred to as Antibody K-H

Binds to human TCRVβ 28

SEQ ID NO: 1203
VL - 1
EIVLTQSPATLSLSPGERATLSCSANSTVGYIHW

YQQKPGQAPKLLIYTTSNLASGIPARFSGSGSGT

DYTLTISSLEPEDFAVYFCHQWSFYPTFGQGTKL

EIK

SEQ ID NO: 1204
VL - 2
DIQLTQSPSFLSASVGDRVTITCSANSTVGYIHW

YQQKPGKAPKLLIYTTSNLASGVPSRFSGSGSGT

EYTLTISSLQPEDFATYFCHQWSFYPTFGQGTKL

EIK

SEQ ID NO: 1205
VL - 3
EIVLTQSPATLSLSPGERATLSCSANSTVGYIHW

YQQKPGQAPKLLIYTTSNLASGIPARFSGSGPGT

DYTLTISSLEPEDFAVYFCHQWSFYPTFGQGTKL

EIK

SEQ ID NO: 1206
VL - 4
DIVLTQSPDSLAVSLGERATINCSANSTVGYIHW

YQQKPGQPPKLLIYTTSNLASGVPDRFSGSGSGT

DYTLTISSLQAEDVAVYFCHQWSFYPTFGQGTK

LEIK

SEQ ID NO: 1207
VL - 5
EIVLTQSPDFQSVTPKEKVTITCSANSTVGYIHW

YQQKPDQSPKLLIYTTSNLASGVPSRFSGSGSGT

DYTLTINSLEAEDAATYFCHQWSFYPTFGQGTK

LEIK

ZOE (murine), also referred to as BJ1538 or as Antibody L

Binds to human TCRVβ 4-1,4-2,4-3

SEQ ID NO: 1208
HC CDR1 (Kabat)
DYYMY

SEQ ID NO: 1209
HC CDR2 (Kabat)
TISGGGSYTYSPDSVKG

SEQ ID NO: 1210
HC CDR3 (Kabat)
ERDIYYGNFNAMVY

SEQ ID NO: 1211
HC CDR1 (Chothia)
GFTFSDY

SEQ ID NO: 1212
HC CDR2 (Chothia)
SGGGSY

SEQ ID NO: 1210
HC CDR3 (Chothia)
ERDIYYGNFNAMVY

SEQ ID NO: 1213
HC CDR1 (Combined)
GFTFSDYYMY

SEQ ID NO: 1209
HC CDR2 (Combined)
TISGGGSYTYSPDSVKG

SEQ ID NO: 1210
HC CDR3(Combined)
ERDIYYGNFNAMVY

SEQ ID NO: 1214
LC CDR1 (Kabat)
RASKSVSTSGYSYMH

SEQ ID NO: 1215
LC CDR2 (Kabat)
LASNLES

SEQ ID NO: 1216
LC CDR3 (Kabat)
QHSRDLPWT

SEQ ID NO: 1217
LC CDR1 (Chothia)
SKSVSTSGYSY

SEQ ID NO: 1215
LC CDR2 (Chothia)
LASNLES

SEQ ID NO: 1216
LC CDR3 (Chothia)
QHSRDLPWT

SEQ ID NO: 1214
LC CDR1 (Combined)
RASKSVSTSGYSYMH

SEQ ID NO: 1215
LC CDR2 (Combined)
LASNLES

SEQ ID NO: 1216
LC CDR3(Combined)
QHSRDLPWT

SEQ ID NO: 1218
VL
DIVLTQSPVSLTVSLGQRATISCRASKSVSTSGYS

YMHWYQQKPGQPPKLLIYLASNLESGVPARFSG

SGSGTDFTLNIHPVEEEDAATYYCQHSRDLPWTF

GGGTKLEIK

SEQ ID NO: 1219
VH
EVQLVESGGGLVKPGGSLKLSCAASGFTFSDYY

MYWVRQTPEKRLEWVATISGGGSYTYSPDSVK

GRFTISRDNAKNNLYLQMSSLRSEDTAMYFCAR

ERDIYYGNFNAMVYWGRGTSVTVSS

VH for ZOE (humanized) also referred to as Antibody L-H

Binds to human TCRVβ 4-1,4-2,4-3

SEQ ID NO: 1220
VH - 1
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYY

MYWVRQAPGKGLEWVATISGGGSYTYSPDSVK

GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR

ERDIYYGNFNAMVYWGRGTLVTVSS

SEQ ID NO: 1221
VH - 2
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYY

MYWVRQAPGKGLEWVATISGGGSYTYSPDSVK

GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR

ERDIYYGNFNAMVYWGRGTLVTVSS

SEQ ID NO: 1222
VH - 3
QVQLVESGGGVVQPGRSLRLSCAASGFTFSDY

YMYWVRQAPGKGLEWVATISGGGSYTYSPDS

VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY

CARERDIYYGNFNAMVYWGRGTLVTVSS

SEQ ID NO: 1223
VH - 4
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYY

MYWIRQAPGKGLEWVATISGGGSYTYSPDSVK

GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR

ERDIYYGNFNAMVYWGRGTLVTVSS

VL for ZOE (humanized) also referred to as Antibody L-H

Binds to human TCRVβ 4-1,4-2,4-3

SEQ ID NO: 1224
VL - 1
EIVLTQSPGTLSLSPGERATLSCRASKSVSTSGYS

YMHWYQQKPGQAPRLLIYLASNLESGIPDRFSG

SGSGTDFTLTISRLEPEDFAVYYCQHSRDLPWTF

GGGTKVEIK

SEQ ID NO: 1225
VL - 2
EIVLTQSPATLSLSPGERATLSCRASKSVSTSGYS

YMHWYQQKPGQAPRLLIYLASNLESGIPARFSG

SGSGTDFTLTISSLEPEDFAVYYCQHSRDLPWTF

GGGTKVEIK

SEQ ID NO: 1226
VL - 3
DIQLTQSPSTLSASVGDRVTITCRASKSVSTSGYS

YMHWYQQKPGKAPKLLIYLASNLESGVPSRFSG

SGSGTEFTLTISSLQPDDFATYYCQHSRDLPWTF

GGGTKVEIK

SEQ ID NO: 1227
VL - 4
AIQLTQSPSSLSASVGDRVTITCRASKSVSTSGYS

YMHWYQQKPGKAPKLLIYLASNLESGVPSRFSG

SGSGTDFTLTISSLQPEDFATYYCQHSRDLPWTF

GGGTKVEIK

Anti-TCRvb19 (murine), also referred to as BJ1465; or Antibody M

Binds to human TCRVβ 19

SEQ ID NO: 1229
HC CDR1 (Kabat)
GYFWN

SEQ ID NO: 1230
HC CDR2 (Kabat)
YISYDGSNNYNPSLKN

SEQ ID NO: 1231
HC CDR3 (Kabat)
PSPGTGYAVDY

SEQ ID NO: 1232
HC CDR1 (Chothia)
GYSITSGY

SEQ ID NO: 1233
HC CDR2 (Chothia)
SYDGSN

SEQ ID NO: 1231
HC CDR3 (Chothia)
PSPGTGYAVDY

SEQ ID NO: 1234
HC CDR1 (Combined)
GYSITSGYFWN

SEQ ID NO: 1230
HC CDR2 (Combined)
YISYDGSNNYNPSLKN

SEQ ID NO: 1231
HC CDR3(Combined)
PSPGTGYAVDY

SEQ ID NO: 1235
LC CDR1 (Kabat)
RSSQSLVHSNGNTYLH

SEQ ID NO: 1236
LC CDR2 (Kabat)
KVSNRFS

SEQ ID NO: 1237
LC CDR3 (Kabat)
SQSTHVPFT

SEQ ID NO: 1238
LC CDR1 (Chothia)
SQSLVHSNGNTY

SEQ ID NO: 1236
LC CDR2 (Chothia)
KVSNRFS

SEQ ID NO: 1237
LC CDR3 (Chothia)
SQSTHVPFT

SEQ ID NO: 1235
LC CDR1 (Combined)
RSSQSLVHSNGNTYLH

SEQ ID NO: 1236
LC CDR2 (Combined)
KVSNRFS

SEQ ID NO: 1237
LC CDR3(Combined)
SQSTHVPFT

SEQ ID NO: 1239
VL
NVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNG

NTYLHWYLQKPGQSPKFLIYKVSNRFSGVPDRF

SGGGSGTEFTLKISRVEAEDLGVYFCSQSTHVPF

TFGSGTKLEIK

SEQ ID NO: 1240
|VH
NVQLQESGPGLVKPSQSLSLTCSVAGYSITSGYF

WNWIRQFPGNKLEWMGYISYDGSNNYNPSLKN

RISITRDTSKNQFFLKLNSVTTEDTATYYCASPSP

GTGYAVDYWGQGTSVTVSS

VH for Anti-TCRvb19 (humanized) also referred to as Antibody M-H

Binds to human TCRVβ 19

SEQ ID NO: 1241
VH - 1
QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYF

WNWIRQPPGKGLEWIGYISYDGSNNYNPSLKNR

VTISRDTSKNQFSLKLSSVTAADTAVYYCASPSP

GTGYAVDYWGQGTLVTVSS

SEQ ID NO: 1242
VH - 2
QVQLQESGPGLVKPSETLSLTCTVSGYSITSGYF

WNWIRQPPGKGLEWIGYISYDGSNNYNPSLKNR

VTISRDTSKNQFSLKLSSVTAADTAVYYCASPSP

GTGYAVDYWGQGTLVTVSS

SEQ ID NO: 1243
VH - 3
QVQLVESGGGLVQPGGSLRLSCSVSGYSITSGYF

WNWVRQAPGKGLEWVGYISYDGSNNYNPSLK

NRFTISRDTSKNTFYLQMNSLRAEDTAVYYCAS

PSPGTGYAVDYWGQGTLVTVSS

VL for Anti-TCRvb19 (humanized) also referred to as Antibody M-H

Binds to human TCRVβ 19

SEQ ID NO: 1244
VL - 1
VVMTQSPGTLSLSPGERATLSCRSSQSLVHSNGN

TYLHWYQQKPGQAPRFLIYKVSNRFSGIPDRFSG

SGSGTDFTLTISRLEPEDFAVYFCSQSTHVPFTFG

QGTKLEIK

SEQ ID NO: 1245
VL - 2
EVVMTQSPATLSLSPGERATLSCRSSQSLVHSNG

NTYLHWYQQKPGQAPRFLIYKVSNRFSGIPARFS

GSGSGTDFTLTISSLEPEDFAVYFCSQSTHVPFTF

GQGTKLEIK

SEQ ID NO: 1246
VL - 3
EVVMTQSPATLSVSPGERATLSCRSSQSLVHSNG

NTYLHWYQQKPGQAPRFLIYKVSNRFSGIPARFS

GSGSGTEFTLTISSLQSEDFAVYFCSQSTHVPFTF

GQGTKLEIK

SEQ ID NO: 1247
VL - 4
DVQMTQSPSSLSASVGDRVTITCRSSQSLVHSNG

NTYLHWYQQKPGKAPKFLIYKVSNRFSGVPSRF

SGSGSGTDFTFTISSLQPEDIATYFCSQSTHVPFTF

GQGTKLEIK

BL37.2 (murine), also referred to as BJ1539 or Antibody N

Binds to human TCRVβ 9

SEQ ID NO: 1248
HC CDR1 (Kabat)
DYIVH

SEQ ID NO: 1249
HC CDR2 (Kabat)
WINTYTGTPTYADDFEG

SEQ ID NO: 1250
HC CDR3 (Kabat)
SWRRGIRGIGFDY

SEQ ID NO: 1251
HC CDR1 (Chothia)
GYTFTDY

SEQ ID NO: 1252
HC CDR2 (Chothia)
NTYTGT

SEQ ID NO: 1250
HC CDR3 (Chothia)
SWRRGIRGIGFDY

SEQ ID NO: 1253
HC CDR1 (Combined)
GYTFTDYIVH

SEQ ID NO: 1249
HC CDR2 (Combined)
WINTYTGTPTYADDFEG

SEQ ID NO: 1250
HC CDR3(Combined)
SWRRGIRGIGFDY

SEQ ID NO: 1254
LC CDR1 (Kabat)
KASKSINKYLA

SEQ ID NO: 1255
LC CDR2 (Kabat)
DGSTLQS

SEQ ID NO: 1256
LC CDR3 (Kabat)
QQHNEYPPT

SEQ ID NO: 1257
LC CDR1 (Chothia)
SKSINKY

SEQ ID NO: 1255
LC CDR2 (Chothia)
DGSTLQS

SEQ ID NO: 1256
LC CDR3 (Chothia)
QQHNEYPPT

SEQ ID NO: 1254
LC CDR1 (Combined)
KASKSINKYLA

SEQ ID NO: 1255
LC CDR2 (Combined)
DGSTLQS

SEQ ID NO: 1256
LC CDR3(Combined)
QQHNEYPPT

SEQ ID NO: 1258
VL
DVQMTQSPYNLAASPGESVSINCKASKSINKYLA

WYQQKPGKPNKLLIYDGSTLQSGIPSRFSGSGSG

TDFTLTIRGLEPEDFGLYYCQQHNEYPPTFGAGT

KLELK

SEQ ID NO: 1259
VH
QLQLVQSGPELREPGESVKISCKASGYTFTDYIV

HWVKQAPGKGLKWMGWINTYTGTPTYADDFE

GRFVFSLEASASTANLQISNLKNEDTATYFCARS

WRRGIRGIGFDYWGQGVMVTVSS

VH for BL37.2 (humanized) also referred to as Antibody N-H

Binds to human TCRVβ 9

SEQ ID NO: 1260
VH - 1
QLQLVQSGAEVKKPGASVKVSCKASGYTFTDYI

VHWVRQAPGQGLEWMGWINTYTGTPTYADDF

EGWVTMTLDASISTAYMELSRLRSDDTAVYYC

ARSWRRGIRGIGFDYWGQGTMVTVSS

SEQ ID NO: 1261
VH - 2
QLQLVQSGAEVKKPGASVKVSCKASGYTFTDYI

VHWVRQAPGQGLEWMGWINTYTGTPTYADDF

EGRVTMTLDASTSTAYMELSSLRSEDTAVYYCA

RSWRRGIRGIGFDYWGQGTMVTVSS

SEQ ID NO: 1262
VH - 3
QLQLVQSGAEVKKPGASVKVSCKASGYTFTDYI

VHWVRQAPGQRLEWMGWINTYTGTPTYADDF

EGRVTITLDASASTAYMELSSLRSEDMAVYYCA

RSWRRGIRGIGFDYWGQGTMVTVSS

SEQ ID NO: 1263
VH - 4
QLQLVQSGAEVKKPGASVKVSCKASGYTFTDYI

VHWVRQATGQGLEWMGWINTYTGTPTYADDF

EGRVTMTLNASISTAYMELSSLRSEDTAVYYCA

RSWRRGIRGIGFDYWGQGTMVTVSS

VL for BL37.2 (humanized) also referred to as Antibody N-H

Binds to human TCRVβ 9

SEQ ID NO: 1264
VL - 1
EVVMTQSPGTLSLSPGERATLSCKASKSINKYLA

WYQQKPGQAPRLLIYDGSTLQSGIPDRFSGSGSG

TDFTLTISRLEPEDFAVYYCQQHNEYPPTFGQGT

KLEIK

SEQ ID NO: 1265
VL - 2
EVVMTQSPATLSLSPGERATLSCKASKSINKYLA

WYQQKPGQAPRLLIYDGSTLQSGIPARFSGSGSG

TDFTLTISSLEPEDFAVYYCQQHNEYPPTFGQGT

KLEIK

SEQ ID NO: 1266
VL - 3
DVQMTQSPSSLSASVGDRVTITCKASKSINKYLA

WYQQKPGKAPKLLIYDGSTLQSGVPSRFSGSGS

GTDFTLTISSLQPEDFATYYCQQHNEYPPTFGQG

TKLEIK

SEQ ID NO: 1267
VL - 4
AVRMTQSPSSFSASTGDRVTITCKASKSINKYLA

WYQQKPGKAPKLLIYDGSTLQSGVPSRFSGSGS

GTDFTLTISCLOSEDFATYYCQQHNEYPPTFGQG

TKLEIK

IG125 (murine) binds to TRVβ 11-2; also referred to as Antibody O

SEQ ID NO: 1268
HC CDR1 (Kabat)
NYGVH

SEQ ID NO: 1269
HC CDR2 (Kabat)
VIWSDGSTDYDTAFIS

SEQ ID NO: 1270
HC CDR3 (Kabat)
RAVVADFDY

SEQ ID NO: 1271
HC CDR1 (Chothia)
GFSLTN

SEQ ID NO: 1272
HC CDR2 (Chothia)
VIWSDGSTD

SEQ ID NO: 1270
HC CDR3 (Chothia)
RAVVADFDY

SEQ ID NO: 1273
HC CDR1 (combined)
GFSLTNYGVH

SEQ ID NO: 1269
HC CDR2 (combined)
VIWSDGSTDYDTAFIS

SEQ ID NO: 1270
HC CDR3 (combined)
RAVVADFDY

SEQ ID NO: 1274
VH
QVQLKQSGPGLLQPSQSLSITCTVSGFSLTNYGV

HWVRQSPGKGLEWLGVIWSDGSTDYDTAFISRL

SISKDNSKSQVFFKLNSLQADDTAIYYCARRAV

VADFDYWGQGTTLTVSS

SEQ ID NO:1275
LC CDR1 (Kabat)
KASKEVTIFGSISALH

SEQ ID NO: 1276
LC CDR2 (Kabat)
NGAKLES

SEQ ID NO: 1277
LC CDR3 (Kabat)
LQNKEVPFT

SEQ ID NO:1275
LC CDR1 (Chothia)
KASKEVTIFGSISALH

SEQ ID NO:1276
LC CDR2 (Chothia)
NGAKLES

SEQ ID NO: 1277
LC CDR3 (Chothia)
LQNKEVPFT

SEQ ID NO: 1275
LC CDR1 (combined)
KASKEVTIFGSISALH

SEQ ID NO: 1276
LC CDR2 (combined)
NGAKLES

SEQ ID NO: 1277
LC CDR3 (combined)
LQNKEVPFT

SEQ ID NO: 1278
VL
DIVLTQSPASLAVSLGQKATISCKASKEVTIFGSI

SALHWYQQKPGQPPKLIYNGAKLESGVSARFS

DSGSQNRSPFGNQLSFTLTIAPVEADDAATYYC

LQNKEVPFTFGSGTKLEIK

VL for IG125 (humanized) also referred to as Antibody O-H

binds to TRβV 11-2

SEQ ID NO: 1279
VL-1
DIVLTQSPDSLAVSLGERATINCKASKEVTIFGSI

SALHWYQQKPGQPPKLLYNGAKLESGVSARFG

VPDRFSRSGSGLDFTLTISSLQAEDVAVYYCLQ

NKEVPFTFGQGTKLEIK

SEQ ID NO: 1280
VL-2
EIVLTQSPDFQSVTPKEKVTITCKASKEVTIFGSI

SALHWYQQKPDQSPKLLYNGAKLESGVSARFG

VPSRFSRSGSGLDFTLTINSLEAEDAATYYCLQN

KEVPFTFGQGTKLEIK

SEQ ID NO: 1281
VL-3
AIQLTQSPSSLSASVGDRVTITCKASKEVTIFGSI

SALHWYQQKPGKAPKLLYNGAKLESGVSARF

GVPSRFSRSGSGLDFTLTISSLQPEDFATYYCLQ

NKEVPFTFGQGTKLEIK

SEQ ID NO: 1282
VL-4
DIVLTQTPLSLSVTPGQPASISCKASKEVTIFGSIS

ALHWYLQKPGQPPKLLYNGAKLESGVSARFGV

PDRFSRSGSGLDFTLKISRVEAEDVGVYYCLQN

KEVPFTFGQGTKLEIK

VH for IG125 (humanized) also referred to as Antibody O-H

binds to TRVβ 11-2

SEQ ID NO: 1283
VH-1
QVTLKESGPVLVKPTETLTLTCTVSGFSLTNYG

VHWVRQPPGKALEWLGVIWSDGSTDYDTAFIS

RLTISKDNSKSQVVLTMTNMDPVDTATYYCAR

RAVVADFDYWGQGTTVTVSS

SEQ ID NO: 1284
VH-2
QVQLQESGPGLVKPSGTLSLTCAVSGFSLTNYG

VHWVRQPPGKGLEWLGVIWSDGSTDYDTAFIS

RLTISKDNSKSQVSLKLSSVTAADTAVYYCARR

AVVADFDYWGQGTTVTVSS

SEQ ID NO: 1285
VH-3
QVQLQQSGPGLVKPSQTLSLTCAVSGFSLTNYG

VHWVRQSPSRGLEWLGVIWSDGSTDYDTAFIS

RLTINKDNSKSQVSLQLNSVTPEDTAVYYCARR

AVVADFDYWGQGTTVTVSS

SEQ ID NO: 1286
VH-4
EVQLVESGGGLVQPGPSLRLSCTVSGFSLTNYG

VHWVRQAPGKGLEWLGVIWSDGSTDYDTAFIS

RLTISKDNSKSIVYLQMNSLKTEDTAVYYCARR

AVVADFDYWGQGTTVTVSS

SEQ ID NO: 1287
VH-5
EVQLVQSGAEVKKPGESLRISCKVSGFSLTNYG

VHWVRQMPGKGLEWLGVIWSDGSTDYDTAFI

SQLTISKDNSISTVYLQWSSLKASDTAMYYCAR

RAVVADFDYWGQGTTVTVSS

MR5-2 (murine), Binds to human TCRVβ 13-2

SEQ ID NO: 1376
SCFV (VH + VL)
QVQLQQSGTELMKPGASVKISCKASGYTFSNY

WIEWIKQRPGHGLEWVGEILPGAGPTNYNEKF

KGKATFTADSSSNTAYMQLSSLTSEDSAVYYC

ARTDYDYDWFAYWGQGTLVTVSAGGGGSGG

GGSGGGGSGGGGSDIVMSQSPSSLAVSVGEKV

TMSCKSSQSLLYSGNQKNYLAWYQQKPGQSPK

LLIYWASTRESGVPDRFTGSGSGTDFTLTINSVK

AEDLTVYYCQQYYGYPRTFGGGTKVEIK

Anti-TCRVβ Antibody Effector Function and Fc Variants

In some embodiments, an anti-TCRVβ antibody disclosed herein comprises an Fc region, e.g., as described herein. In some embodiments, the Fc region is a wildtype Fc region, e.g., a wildtype human Fc region. In some embodiments, the Fc region comprises a variant, e.g., an Fc region comprising an addition, substitution, or deletion of at least one amino acid residue in the Fc region which results in, e.g., reduced or ablated affinity for at least one Fc receptor.

The Fc region of an antibody interacts with a number of receptors or ligands including Fc Receptors (e.g., FcγRI, FcγRIIA, FcγRIIIA), the complement protein CIq, and other molecules such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events including: antibody dependent cell-mediated cytotoxicity (ADCC), Antibody-dependent cellular phagocytosis (ADCP) and complement dependent cytotoxicity (CDC).

In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has reduced, e.g., ablated, affinity for an Fc receptor, e.g., an Fc receptor described herein. In some embodiments, the reduced affinity is compared to an otherwise similar antibody with a wildtype Fc region.

In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has one or more of the following properties: (1) reduced effector function (e.g., reduced ADCC, ADCP and/or CDC); (2) reduced binding to one or more Fc receptors; and/or (3) reduced binding to C1q complement. In some embodiments, the reduction in any one, or all of properties (1)-(3) is compared to an otherwise similar antibody with a wildtype Fc region.

In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has reduced affinity to a human Fc receptor, e.g., FcγR I, FcγR II and/or FcγR III. In some embodiments, the anti-TCRVβ antibody comprising a variant Fc region comprises a human IgG1 region or a human IgG4 region.

In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region activates and/or expands T cells, e.g., as described herein. In some embodiments, an anti-TCRVβ antibody comprising a variant Fc region has a cytokine profile described herein, e.g., a cytokine profile that differs from a cytokine profile of a T cell engager that binds to a receptor or molecule other than a TCRβV region (“a non-TCRβV-binding T cell engager”). In some embodiments, the non-TCRβV-binding T cell engager comprises an antibody that binds to a CD3 molecule (e.g., CD3 epsilon (CD3e) molecule); or a TCR alpha (TCRα) molecule.

Exemplary Fc region variants are provided in Table 14 and also disclosed in Saunders 0, (2019) Frontiers in Immunology; vol 10, article1296, the entire contents of which is hereby incorporated by reference.

In some embodiments, an anti-TCRVβ antibody disclosed herein comprises any one or all, or any combination of Fc region variants, e.g., mutations, disclosed in Table 14. In some embodiments, an anti-TCRVβ antibody disclosed herein comprise an Asn297Ala (N297A) mutation. In some embodiments, an anti-TCRVβ antibody disclosed herein comprise a Leu234Ala/Leu235Ala (LALA) mutation.

TABLE 14

Exemplary Fc modifications

Modification or mutation
Altered effector function

Leu235Glu
ADCC;

Leu234Ala/Leu235Ala (LALA)
ADCC; ADCP; CDC

Ser228Pro/Leu235Glu

Leu234Ala/Leu235Ala/Pro329Gly
ADCP

Pro331Ser/Leu234Glu/Leu235Phe
CDC

Asp265Ala
ADCC, ADCP

Gly237Ala
ADCP

Glu318Ala
ADCP

Glu233Pro

Gly236Arg/Leu328Arg
ADCC

His268Gln/Val309Leu/Ala330Ser/Pro331Ser
ADCC; ADCP; CDC

Val234Ala/Gly237Ala/Pro238Ser/
ADCC; ADCP; CDC

His268Ala/Val309Leu/Ala330Ser/Pro331Ser

Leu234Ala/L235Ala/Gly237Ala/P238Ser/
ADCC; CDC

His268Ala/Ala330Ser/Pro331Ser

Ala330Leu
CDC

Asp270Ala
CDC

Lys322Ala
CDC

Pro329Ala
CDC

Pro331Ala
CDC

Val264Ala
CDC

High mannose glycosylation
CDC

Phe241Ala
CDC

Asn297Ala or Gly or Gln
ADCC; ADCP; CDC

S228P/Phe234Ala/Leu235Ala
ADCC; CDC

Natural Killer Cell Engagers

Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. NKG2D is a receptor that provides both stimulatory and costimulatory innate immune responses on activated killer (NK) cells, leading to cytotoxic activity. DNAM1 is a receptor involved in intercellular adhesion, lymphocyte signaling, cytotoxicity and lymphokine secretion mediated by cytotoxic T-lymphocyte (CTL) and NK cell. DAP10 (also known as HCST) is a transmembrane adapter protein which associates with KLRK1 to form an activation receptor KLRK1-HCST in lymphoid and myeloid cells; this receptor plays a major role in triggering cytotoxicity against target cells expressing cell surface ligands such as MHC class I chain-related MICA and MICB, and U(optionally L1)6-binding proteins (ULBPs); it KLRK1-HCST receptor plays a role in immune surveillance against tumors and is required for cytolysis of tumors cells; indeed, melanoma cells that do not express KLRK1 ligands escape from immune surveillance mediated by NK cells. CD16 is a receptor for the Fc region of IgG, which binds complexed or aggregated IgG and also monomeric IgG and thereby mediates antibody-dependent cellular cytotoxicity (ADCC) and other antibody-dependent responses, such as phagocytosis.

The present disclosure provides, inter alia, multispecific (e.g., bi-, tri-, quad-specific) or multifunctional molecules, that are engineered to contain one or more NK cell engagers that mediate binding to and/or activation of an NK cell. Accordingly, in some embodiments, the NK cell engager is selected from an antigen binding domain or ligand that binds to (e.g., activates): NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160.

In some embodiments, the NK cell engager is an antigen binding domain that binds to NKp30 (e.g., NKp30 present, e.g., expressed or displayed, on the surface of an NK cell) and comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Tables 7, 8, 35, 36, 9, 10, or 34. In some embodiments, the NK cell engager is an antigen binding domain that binds to NKp30 (e.g., NKp30 present, e.g., expressed or displayed, on the surface of an NK cell) and comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in U.S. Pat. Nos. 6,979,546, 9,447,185, PCT Application No. WO2015121383A1, PCT Application No.

WO2016110468A1, PCT Application No. WO2004056392A1, or U.S. Application Publication No. US20070231322A1, the sequences of which are hereby incorporated by reference. In some embodiments, binding of the NK cell engager, e.g., antigen binding domain that binds to NKp30, to the NK cell activates the NK cell. An antigen binding domain that binds to NKp30 (e.g., NKp30 present, e.g., expressed or displayed, on the surface of an NK cell) may be said to target NKp30, the NK cell, or both.

In some embodiments, the antigen binding domain that binds to NKp30 comprises one or more CDRs (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3) disclosed in Table 7, Table 34, or Table 8, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to NKp30 comprises one or more framework regions (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and/or VLFWR4) disclosed in Table 7, Table 34, or Table 8, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, the antigen binding domain that binds to NKP30 comprises one or more CDRs (e.g., VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3) disclosed in Table 35 and/or Table 36, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the antigen binding domain that binds to NKP30 comprises one or more framework regions (e.g., VHFWR1, VHFWR2, VHFWR3, VHFWR4, VLFWR1, VLFWR2, VLFWR3, and/or VLFWR4) disclosed in Table 35 and/or Table 36, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, the antigen binding domain that binds to NKp30 comprises a VH and/or a VL disclosed in Table 9, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto. In some embodiments, any of the VH domains disclosed in Table 9 may be paired with any of the VL domains disclosed in Table 9 to form the antigen binding domain that binds to NKp30. In some embodiments, the antigen binding domain that binds to NKp30 comprises an amino acid sequence disclosed in Table 10, or a sequence having at least 85%, 90%, 95%, or 99% identity thereto.

In some embodiments, the antigen binding domain that binds to NKp30 comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1), a VHCDR2, and a VHCDR3, and a VL comprising a light chain complementarity determining region 1 (VLCDR1), a VLCDR2, and a VLCDR3.

In some embodiments, the VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 6001, and 7315, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 6001, and 6002, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 6008, and 6009, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 7385, and 7315, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, and VHCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 7318, and 6009, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

In some embodiments, the VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7326, 7327, and 7329, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 6063, 6064, and 7293, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 6070, 6071, and 6072, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 6070, 6064, and 7321, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 6001, 7315, 7326, 7327, and 7329, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 6001, 6002, 6063, 6064, and 7293, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 6008, 6009, 6070, 6071, and 6072, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 7385, 7315, 6070, 6064, and 7321, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3 comprise the amino acid sequences of SEQ ID NOs: 7313, 7318, 6009, 6070, 6064, and 7321, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7298 or 7300-7304 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto) and/or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7299 or 7305-7309 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 7302 and 7305, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 7302 and 7309, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6121 or 6123-6128 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto) and/or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7294 or 6137-6141 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6122 or 6129-6134 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto) and/or the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6136 or 6142-6147 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 7295 and 7296, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 7297 and 7296, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 6122 and 6136, respectively (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

In some embodiments, the antigen binding domain that binds to NKp30 comprises the amino acid sequence of SEQ ID NO: 7310 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the antigen binding domain that binds to NKp30 comprises the amino acid sequence of SEQ ID NO: 7311 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto). In some embodiments, the antigen binding domain that binds to NKp30 comprises the amino acid sequence of SEQ ID NO: 6187, 6188, 6189 or 6190 (or a sequence having at least 85%, 90%, 95%, or 99% identity thereto).

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6064 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6064 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the NKp30 antigen binding domain comprises a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6071 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6072 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6070, a VLCDR2 amino acid sequence of SEQ ID NO: 6071, and a VLCDR3 amino acid sequence of SEQ ID NO: 6072.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6008 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6009 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6071 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6072 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the NKp30 antigen binding domain comprises a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6007, a VHCDR2 amino acid sequence of SEQ ID NO: 6008, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6009, and a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6070, a VLCDR2 amino acid sequence of SEQ ID NO: 6071, and a VLCDR3 amino acid sequence of SEQ ID NO: 6072.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, a VHFWR2 amino acid sequence of SEQ ID NO: 6004, a VHFWR3 amino acid sequence of SEQ ID NO: 6005, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6006, and a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6066 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6067 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 7292 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6003 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6004 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6005 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6006, and a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6066 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6067 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 7292 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073, a VLFWR2 amino acid sequence of SEQ ID NO: 6074, a VLFWR3 amino acid sequence of SEQ ID NO: 6075, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6076.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6010, a VHFWR2 amino acid sequence of SEQ ID NO: 6011, a VHFWR3 amino acid sequence of SEQ ID NO: 6012, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6013, and a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073, a VLFWR2 amino acid sequence of SEQ ID NO: 6074, a VLFWR3 amino acid sequence of SEQ ID NO: 6075, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6076.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6073 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6074 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6075 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6076.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6010 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6011 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6012 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6013, and a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6073 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6074 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6075 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6076.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6014, a VHFWR2 amino acid sequence of SEQ ID NO: 6015, a VHFWR3 amino acid sequence of SEQ ID NO: 6016, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6017.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6014 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6015 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6016 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6017.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6077, a VLFWR2 amino acid sequence of SEQ ID NO: 6078, a VLFWR3 amino acid sequence of SEQ ID NO: 6079, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6080.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6077 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6078 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6079 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6080.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6018, a VHFWR2 amino acid sequence of SEQ ID NO: 6019, a VHFWR3 amino acid sequence of SEQ ID NO: 6020, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6021.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6018 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6019 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6020 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6021.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6081, a VLFWR2 amino acid sequence of SEQ ID NO: 6082, a VLFWR3 amino acid sequence of SEQ ID NO: 6083, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6084.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6081 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6082 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6083 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6084.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6022, a VHFWR2 amino acid sequence of SEQ ID NO: 6023, a VHFWR3 amino acid sequence of SEQ ID NO: 6024, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6025.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6022 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6023 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6024 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6025.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6085, a VLFWR2 amino acid sequence of SEQ ID NO: 6086, a VLFWR3 amino acid sequence of SEQ ID NO: 6087, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6088.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6085 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6086 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6087 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6088.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6026, a VHFWR2 amino acid sequence of SEQ ID NO: 6027, a VHFWR3 amino acid sequence of SEQ ID NO: 6028, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6029.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6026 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6027 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6028 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6029.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6089, a VLFWR2 amino acid sequence of SEQ ID NO: 6090, a VLFWR3 amino acid sequence of SEQ ID NO: 6091, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6092.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6089 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6090 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6091 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6092.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6030, a VHFWR2 amino acid sequence of SEQ ID NO: 6032, a VHFWR3 amino acid sequence of SEQ ID NO: 6033, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6034.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6030 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6032 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6033 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6034.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6093, a VLFWR2 amino acid sequence of SEQ ID NO: 6094, a VLFWR3 amino acid sequence of SEQ ID NO: 6095, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6096.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6093 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6094 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6095 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6096.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6035, a VHFWR2 amino acid sequence of SEQ ID NO: 6036, a VHFWR3 amino acid sequence of SEQ ID NO: 6037, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6038.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6035 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6036 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6037 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6038.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6039, a VHFWR2 amino acid sequence of SEQ ID NO: 6040, a VHFWR3 amino acid sequence of SEQ ID NO: 6041, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6042.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6039 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6040 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6041 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6042.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6097, a VLFWR2 amino acid sequence of SEQ ID NO: 6098, a VLFWR3 amino acid sequence of SEQ ID NO: 6099, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6100.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6097 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6098 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6099 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6100.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6043, a VHFWR2 amino acid sequence of SEQ ID NO: 6044, a VHFWR3 amino acid sequence of SEQ ID NO: 6045, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6046.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6043 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6044 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6045 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6046.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6101, a VLFWR2 amino acid sequence of SEQ ID NO: 6102, a VLFWR3 amino acid sequence of SEQ ID NO: 6103, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6104.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6101 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6102 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6103 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6104.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6047, a VHFWR2 amino acid sequence of SEQ ID NO: 6048, a VHFWR3 amino acid sequence of SEQ ID NO: 6049, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6050.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6047 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6048 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6049 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6050.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6105, a VLFWR2 amino acid sequence of SEQ ID NO: 6106, a VLFWR3 amino acid sequence of SEQ ID NO: 6107, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6108.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6105 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6106 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6107 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6108.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6051, a VHFWR2 amino acid sequence of SEQ ID NO: 6052, a VHFWR3 amino acid sequence of SEQ ID NO: 6053, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6054.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6051 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6052 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6053 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6054.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6109, a VLFWR2 amino acid sequence of SEQ ID NO: 6110, a VLFWR3 amino acid sequence of SEQ ID NO: 6111, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6112.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6109 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6110 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6111 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6112.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6055, a VHFWR2 amino acid sequence of SEQ ID NO: 6056, a VHFWR3 amino acid sequence of SEQ ID NO: 6057, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6058.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6055 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6056 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6057 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6058.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6113, a VLFWR2 amino acid sequence of SEQ ID NO: 6114, a VLFWR3 amino acid sequence of SEQ ID NO: 6115, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6116.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6113 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6114 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6115 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6116.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6059, a VHFWR2 amino acid sequence of SEQ ID NO: 6060, a VHFWR3 amino acid sequence of SEQ ID NO: 6061, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6062.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6059 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6060 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6061 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6062.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6117, a VLFWR2 amino acid sequence of SEQ ID NO: 6118, a VLFWR3 amino acid sequence of SEQ ID NO: 6119, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6120.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6117 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6118 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6119 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6120.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6148 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6148). In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6149 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6149). In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6150). In some embodiments, antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6148. In some embodiments, antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6149. In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising the amino acid sequence of SEQ ID NO: 6150.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6148, and a VL comprising the amino acid sequence of SEQ ID NO: 6150. In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6149, and a VL comprising the amino acid sequence of SEQ ID NO: 6150.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6151 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6151). In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6152 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6152). In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising the amino acid sequence of SEQ ID NO: 6153 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6153). In some embodiments, antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6151. In some embodiments, antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6152. In some embodiments, the antigen binding domain that targets NKp30 comprises a VL comprising the amino acid sequence of SEQ ID NO: 6153.

In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6151, and a VL comprising the amino acid sequence of SEQ ID NO: 6153. In some embodiments, the antigen binding domain that targets NKp30 comprises a VH comprising the amino acid sequence of SEQ ID NO: 6152, and a VL comprising the amino acid sequence of SEQ ID NO: 6153.

In some embodiments, the antigen binding domain that targets NKp30 comprises an scFv. In some embodiments, the scFv comprises an amino acid sequence selected from SEQ ID NOs: 6187-6190, or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto.

TABLE 7

Exemplary heavy chain CDRs and FWRs of NKp30-targeting antigen binding domains

Ab ID
VHFWR1
VHCDR1
VHFWR2
VHCDR2
VHFWR3
VHCDR3
VHFWR4

9G1-
QIQLQES
TGGYH
WIRQFP
YIYSSGS
RISITRDTS
GNWHY
WGQGT

HC
GPGLVKP
WN
GKKLE
TSYNPSL
KNQFFLQ
FDF (SEQ
MVTVSS

SQSLSLT
(SEQ ID
WMG
KS (SEQ
LNSVTTE
ID NO:
(SEQ ID

CSVTGFSI
NO:
(SEQ ID
ID NO:
DTATYYC
6002)
NO:

N (SEQ ID
6000)
NO:
6001)
AR (SEQ

6006)

NO: 6003)

6004)

ID NO:

6005)

15H6-
QIQLQES
TGGYH
WIRQFP
YIYSSGT
RISITRDTS
GNWHY
WGQGT

HC
GPGLVKP
WN
GKKLE
TRYNPS
KNQFFLQ
FDY
LVAVSS

SQSLSLT
(SEQ ID
WMG
LKS
LNSVTPED
(SEQ ID
(SEQ ID

CSVTGFSI
NO:
(SEQ ID
(SEQ ID
TATYYCT
NO: 6009)
NO:

N (SEQ ID
6007)
NO:
NO: 6008)
R (SEQ ID

6013)

NO: 6010)

6011)

NO: 6012)

9G1-
QIQLQES
TGGYH
WIRQPA
YIYSSGS
RVTMSRD
GNWHY
WGQGT

HC_1
GPGLVKP
WN
GKGLE
TSYNPSL
TSKNQFSL
FDF (SEQ
MVTVSS

SETLSLT
(SEQ ID
WIG
KS (SEQ
KLSSVTA
ID NO:
(SEQ ID

CTVSGFSI
NO:
(SEQ ID
ID NO:
ADTAVYY
6002)
NO:

N (SEQ ID
6000)
NO:
6001)
CAR (SEQ

6017)

NO: 6014)

6015)

ID NO:

6016)

9G1-
QIQLQES
TGGYH
WIRQHP
YIYSSGS
LVTISRDT
GNWHY
WGQGT

HC_2
GPGLVKP
WN
GKGLE
TSYNPSL
SKNQFSL
FDF (SEQ
MVTVSS

SQTLSLT
(SEQ ID
WIG
KS (SEQ
KLSSVTA
ID NO:
(SEQ ID

CTVSGFSI
NO:
(SEQ ID
ID NO:
ADTAVYY
6002)
NO:

N (SEQ ID
6000)
NO:
6001)
CAR (SEQ

6021)

NO: 6018)

6019)

ID NO:

9G1-
EIQLLES
TGGYH
WVRQA
YIYSSGS
RFTISRDT
GNWHY
WGQGT

HC_3
GGGLVQ
WN
PGKGLE
TSYNPSL
SKNTFYL
FDF (SEQ
MVTVSS

PGGSLRL
(SEQ ID
WVG
KS (SEQ
QMNSLRA
ID NO:
(SEQ ID

SCAVSGF
NO:
(SEQ ID
ID NO:
EDTAVYY
6002)
NO:

SIN (SEQ
6000)
NO:
6001)
CAR (SEQ

6025)

ID NO:

6023)

ID NO:

6022)

6024)

9G1-
QIQLVQS
TGGYH
WVRQA
YIYSSGS
RVTITRDT
GNWHY
WGQGT

HC_4
GAEVKK
WN
PGQGLE
TSYNPSL
STNTFYM
FDF (SEQ
MVTVSS

PGSSVKV
(SEQ ID
WMG
KS (SEQ
ELSSLRSE
ID NO:
(SEQ ID

SCKVSGF
NO:
(SEQ ID
ID NO:
DTAVYYC
6002)
NO:

SIN (SEQ
6000)
NO
6001)
AR (SEQ

6029)

ID NO:

6027)

ID NO:

6026)

6028)

9G1-
EIQLVES
TGGYH
WVRQA
YIYSSGS
RFTISRDT
GNWHY
WGQGT

HC_5
GGGLVQ
WN
PGKGLE
TSYNPSL
AKNSFYL
FDF (SEQ
MVTVSS

PGGSLRL
(SEQ ID
WVG
KS (SEQ
QMNSLRA
ID NO:
(SEQ ID

SCAVSGF
NO:
(SEQ ID
ID NO:
EDTAVYY
6002)
NO

SIN (SEQ
6000)
NO:
6001)
CAR (SEQ

6034)

ID NO:

6032)

ID NO:

6030)

6033)

9G1-
QIQLVQS
TGGYH
WVRQA
YIYSSGS
RVTMTRD
GNWHY
WGQGT

HC_6
GAEVKK
WN
PGQGLE
TSYNPSL
TSTNTFY
FDF (SEQ
MVTVSS

PGASVKV
(SEQ ID
WMG
KS (SEQ
MELSSLRS
ID NO:
(SEQ ID

SCKVSGF
NO:
(SEQ ID
ID NO:
EDTAVYY
6002)
NO:

SIN (SEQ
6000)
NO:
6001)
CAR (SEQ

6038)

ID NO:

6036)

ID NO:

6035)

6037)

15H6-
QIQLQES
TGGYH
WIRQHP
YIYSSGT
LVTISRDT
GNWHY
WGQGT

HC_1
GPGLVKP
WN
GKGLE
TRYNPS
SKNQFSL
FDY
LVTVSS

SQTLSLT
(SEQ ID
WIG
LKS
KLSSVTA
(SEQ ID
(SEQ ID

CTVSGFSI
NO:
(SEQ ID
(SEQ ID
ADTAVYY
NO: 6009)
NO:

N (SEQ ID
6007)
NO:
NO: 6008)
CAR (SEQ

6042)

NO: 6039)

6040)

ID NO:

6041)

15H6-
QIQLQES
TGGYH
WIRQPA
YIYSSGT
RVTMSRD
GNWHY
WGQGT

HC_2
GPGLVKP
WN
GKGLE
TRYNPS
TSKNQFSL
FDY
LVTVSS

SETLSLT
(SEQ ID
WIG
LKS
KLSSVTA
(SEQ ID
(SEQ ID

CTVSGFSI
NO:
(SEQ ID
(SEQ ID
ADTAVYY
NO: 6009)
NO

N (SEQ ID
6007)
NO:
NO: 6008)
CAR (SEQ

6046)

NO: 6043)

6044)

ID NO:

6045)

15H6-
EIQLLES
TGGYH
WVRQA
YIYSSGT
RFTISRDT
GNWHY
WGQGT

HC_3
GGGLVQ
WN
PGKGLE
TRYNPS
SKNTFYL
FDY
LVTVSS

PGGSLRL
(SEQ ID
WVG
LKS
QMNSLRA
(SEQ ID
(SEQ ID

SCAVSGF
NO:
(SEQ ID
(SEQ ID
EDTAVYY
NO: 6009)
NO:

SIN (SEQ
6007)
NO
NO: 6008)
CAR (SEQ

6050)

ID NO:

6048)

ID NO:

6047)

6049)

15H6-
QIQLVES
TGGYH
WIRQAP
YIYSSGT
RFTISRDT
GNWHY
WGQGT

HC_4
GGGLVK
WN
GKGLE
TRYNPS
AKNSFYL
FDY
LVTVSS

PGGSLRL
(SEQ ID
WVG
LKS
QMNSLRA
(SEQ ID
(SEQ ID

SCAVSGF
NO:
(SEQ ID
(SEQ ID
EDTAVYY
NO: 6009)
NO:

SIN (SEQ
6007)
NO:
NO: 6008)
CAR (SEQ

6054)

ID NO:

6052)

ID NO:

6051)

6053)

15H6-
QIQLVQS
TGGYH
WVRQA
YIYSSGT
RVTMTRD
GNWHY
WGQGT

HC_5
GAEVKK
WN
PGQGLE
TRYNPS
TSTNTFY
FDY
LVTVSS

PGASVKV
(SEQ ID
WMG
LKS
MELSSLRS
(SEQ ID
(SEQ ID

SCKVSGF
NO:
(SEQ ID
(SEQ ID
EDTAVYY
NO: 6009)
NO:

SIN (SEQ
6007)
NO:
NO: 6008)
CAR (SEQ

6058)

ID NO:

6056)

ID NO:

6055)

6057)

15H6-
EIQLVQS
TGGYH
WVQQA
YIYSSGT
RVTITRDT
GNWHY
WGQGT

HC_6
GAEVKK
WN
PGKGLE
TRYNPS
STNTFYM
FDY
LVTVSS

PGATVKI
(SEQ ID
WMG
LKS
ELSSLRSE
(SEQ ID
(SEQ ID

SCKVSGF
NO:
(SEQ ID
(SEQ ID
DTAVYYC
NO: 6009)
NO:

SIN (SEQ
6007)
NO:
NO: 6008)
AR (SEQ

6062)

ID NO:

6060)

ID NO:

6059)

6061)

TABLE 34

Exemplary heavy chain CDRs and FWRs of NKp30-targeting antigen binding domains

(according to the Kabat numbering scheme)

Ab ID
VHFWR1
VHCDR1
VHFWR2
VHCDR2
VHFWR3
VHCDR3
VHFWR4

9G1-HC
QIQLQE
GYHWN
WIRQFP
YIYSSGS
RISITRD
GNWHY
WGQGT

SGPGLV
(SEQ ID
GKKLE
TSYNPS
TSKNQF
FDF
MVTVSS

KPSQSL
NO:
WMG
LKS
FLQLNS
(SEQ ID
(SEQ ID

SLTCSV
7313)
(SEQ ID
(SEQ ID
VTTEDT
NO:
NO:

TGFSINT

NO:
NO:
ATYYCA
6002)
6006)

G (SEQ

6004)
6001)
R (SEQ

ID NO:

ID NO:

7317)

6005)

15H6-HC
QIQLQE
GYHWN
WIRQFP
YIYSSG
RISITRD
GNWHY
WGQGT

SGPGLV
(SEQ ID
GKKLE
TTRYNP
TSKNQF
FDY
LVAVSS

KPSQSL
NO:
WMG
SLKS
FLQLNS
(SEQ ID
(SEQ ID

SLTCSV
7313)
(SEQ ID
(SEQ ID
VTPEDT
NO:
NO:

TGFSINT

NO:
NO:
ATYYCT
6009)
6013)

G (SEQ

6011)
6008)
R (SEQ

ID NO:

ID NO:

7317)

6012)

9G1-HC_1
QIQLQE
GYHWN
WIRQPA
YIYSSGS
RVTMSR
GNWHY
WGQGT

SGPGLV
(SEQ ID
GKGLE
TSYNPS
DTSKNQ
FDF
MVTVSS

KPSETL
NO:
WIG
LKS
FSLKLSS
(SEQ ID
(SEQ ID

SLTCTV
7313)
(SEQ ID
(SEQ ID
VTAADT
NO:
NO:

SGFSINT

NO:
NO:
AVYYC
6002)
6017)

G (SEQ

6015)
6001)
AR (SEQ

ID NO:

ID NO:

7371)

6016)

9G1-HC_2
QIQLQE
GYHWN
WIRQHP
YIYSSGS
LVTISR
GNWHY
WGQGT

SGPGLV
(SEQ ID
GKGLE
TSYNPS
DTSKNQ
FDF
MVTVSS

KPSQTL
NO:
WIG
LKS
FSLKLSS
(SEQ ID
(SEQ ID

SLTCTV
7313)
(SEQ ID
(SEQ ID
VTAADT
NO:
NO:

SGFSINT

NO:
NO:
AVYYC
6002)
6021)

G (SEQ

6019)
6001)
AR (SEQ

ID NO:

ID NO:

7372)

6020)

9G1-HC_3
EIQLLES
GYHWN
WVRQA
YIYSSGS
RFTISRD
GNWHY
WGQGT

GGGLV
(SEQ ID
PGKGLE
TSYNPS
TSKNTF
FDF
MVTVSS

QPGGSL
NO:
WVG
LKS
YLQMN
(SEQ ID
(SEQ ID

RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6002)
6025)

G (SEQ

6023)
6001)
AR (SEQ

ID NO:

ID NO:

7373)

6024)

9G1-HC_4
QIQLVQ
GYHWN
WVRQA
YIYSSGS
RVTITR
GNWHY
WGQGT

SGAEVK
(SEQ ID
PGQGLE
TSYNPS
DTSTNT
FDF
MVTVSS

KPGSSV
NO:
WMG
LKS
FYMELS
(SEQ ID
(SEQ ID

KVSCKV
7313)
(SEQ ID
(SEQ ID
SLRSED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6002)
6029)

G (SEQ

6027)
6001)
AR (SEQ

ID NO:

ID NO:

7374)

6028)

9G1-HC_5
EIQLVES
GYHWN
WVRQA
YIYSSGS
RFTISRD
GNWHY
WGQGT

GGGLV
(SEQ ID
PGKGLE
TSYNPS
TAKNSF
FDF
MVTVSS

QPGGSL
NO:
WVG
LKS
YLQMN
(SEQ ID
(SEQ ID

RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6002)
6034)

G (SEQ

6032)
6001)
AR (SEQ

ID NO:

ID NO:

7375)

6033)

9G1-HC_6
QIQLVQ
GYHWN
WVRQA
YIYSSGS
RVTMTR
GNWHY
WGQGT

SGAEVK
(SEQ ID
PGQGLE
TSYNPS
DTSTNT
FDF
MVTVSS

KPGASV
NO:
WMG
LKS
FYMELS
(SEQ ID
(SEQ ID

KVSCKV
7313)
(SEQ ID
(SEQ ID
SLRSED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6002)
6038)

G (SEQ

6036)
6001)
AR (SEQ

ID NO:

ID NO:

7376)

6037)

15H6-
QIQLQE
GYHWN
WIRQHP
YIYSSG
LVTISR
GNWHY
WGQGT

HC_1
SGPGLV
(SEQ ID
GKGLE
TTRYNP
DTSKNQ
FDY
LVTVSS

KPSQTL
NO:
WIG
SLKS
FSLKLSS
(SEQ ID
(SEQ ID

SLTCTV
7313)
(SEQ ID
(SEQ ID
VTAADT
NO:
NO:

SGFSINT

NO:
NO:
AVYYC
6009)
6042)

G (SEQ

6040)
6008)
AR (SEQ

ID NO:

ID NO:

7372)

6041)

15H6-
QIQLQE
GYHWN
WIRQPA
YIYSSG
RVTMSR
GNWHY
WGQGT

HC_2
SGPGLV
(SEQ ID
GKGLE
TTRYNP
DTSKNQ
FDY
LVTVSS

KPSETL
NO:
WIG
SLKS
FSLKLSS
(SEQ ID
(SEQ ID

SLTCTV
7313)
(SEQ ID
(SEQ ID
VTAADT
NO:
NO:

SGFSINT

NO:
NO:
AVYYC
6009)
6046)

G (SEQ

6044)
6008)
AR (SEQ

ID NO:

ID NO:

7371)

6045)

15H6-
EIQLLES
GYHWN
WVRQA
YIYSSG
RFTISRD
GNWHY
WGQGT

HC_3
GGGLV
(SEQ ID
PGKGLE
TTRYNP
TSKNTF
FDY
LVTVSS

QPGGSL
NO:
WVG
SLKS
YLQMN
(SEQ ID
(SEQ ID

RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSINT

NO:
NO
TAVYYC
6009)
6050)

G (SEQ

6048)
6008)
AR (SEQ

ID NO:

ID NO:

7373)

6049)

15H6-
QIQLVE
GYHWN
WIRQAP
YIYSSG
RFTISRD
GNWHY
WGQGT

HC_4
SGGGLV
(SEQ ID
GKGLE
TTRYNP
TAKNSF
FDY
LVTVSS

KPGGSL
NO:
WVG
SLKS
YLQMN
(SEQ ID
(SEQ ID

RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6009)
6054)

G (SEQ

6052)
6008)
AR (SEQ

ID NO:

ID NO:

7377)

6053)

15H6-
QIQLVQ
GYHWN
WVRQA
YIYSSG
RVTMTR
GNWHY
WGQGT

HC_5
SGAEVK
(SEQ ID
PGQGLE
TTRYNP
DTSTNT
FDY
LVTVSS

KPGASV
NO:
WMG
SLKS
FYMELS
(SEQ ID
(SEQ ID

KVSCKV
7313)
(SEQ ID
(SEQ ID
SLRSED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6009)
6058)

G (SEQ

6056)
6008)
AR (SEQ

ID NO:

ID NO:

7376)

6057)

15H6-
EIQLVQ
GYHWN
WVQQA
YIYSSG
RVTITR
GNWHY
WGQGT

HC_6
SGAEVK
(SEQ ID
PGKGLE
TTRYNP
DTSTNT
FDY
LVTVSS

KPGATV
NO:
WMG
SLKS
FYMELS
(SEQ ID
(SEQ ID

KISCKV
7313)
(SEQ ID
(SEQ ID
SLRSED
NO:
NO:

SGFSINT

NO:
NO:
TAVYYC
6009)
6062)

G (SEQ

6060)
6008)
AR (SEQ

ID NO:

ID NO:

7378)

6061)

9D9-HC
QIQLQE
GYHWN
WIRQFP
YIYSSG
RISITRD
GDWHY
WGQGT

SGPGLV
(SEQ ID
GKKVE
TTKYNP
TSKNQF
FDY
MVAVSS

KPSQSL
NO:
WMG
SLKS
FLQLNS
(SEQ ID
(SEQ ID

SLSCSV
7313)
(SEQ ID
(SEQ ID
VTTEDT
NO:
NO:

TGFSINT

NO:
NO:
ATYYCA
7315)
7316)

G (SEQ

7314)
7385)
R (SEQ

ID NO:

ID NO:

7312)

6005)

3A12-HC
QIQLQE
GYHWN
WIRQFP
YIYSSGS
RFSITRD
GNWHY
WGQGT

SGPGLV
(SEQ ID
GKKLE
TRYNPS
TSKNQF
FDY
LVAVSS

KPSQSL
NO:
WMG
LKS
FLQLNS
(SEQ ID
(SEQ ID

SLTCSV
7313)
(SEQ ID
(SEQ ID
VTTEDT
NO:
NO:

TGFSINT

NO:
NO:
ATYYCT
6009)
6013)

G (SEQ

6004)
7318)
R (SEQ

ID NO:

ID NO:

7317)

7319)

12D10-HC
QIQLQE
GYHWN
WIRQFP
YIYSSG
RISITRD
GNWHY
WGQGT

SGPGLV
(SEQ ID
GKKLE
TTRYNP
TSKNQF
FDY
LVAVSS

KPSQSL
NO:
WMG
SLKS
FLQLNS
(SEQ ID
(SEQ ID

SLTCSV
7313)
(SEQ ID
(SEQ ID
VTPEDT
NO:
NO:

TGFSINT

NO:
NO:
ATYYCT
6009)
6013)

G (SEQ

6004)
6008)
R (SEQ

ID NO:

ID NO:

7317)

6012)

15E1-HC
QIQLQE
GYHWN
WIRQFP
YIYSSGS
RFSITRD
GDWHY
WGPGT

SGPGLV
(SEQ ID
GKKLE
TSYNPS
TSKNQF
FDY
MVTVSS

KPSQSL
NO:
WMG
LKS
FLQLNS
(SEQ ID
(SEQ ID

SLSCSV
7313)
(SEQ ID
(SEQ ID
VTTEDT
NO:
NO:

TGFSITT

NO:
NO:
ATYYCA
7315)
7324)

T (SEQ

6004)
6001)
R (SEQ

ID NO:

ID NO:

7322)

7323)

15E1
QIQLQE
GYHWN
WIRQHP
YIYSSGS
LVTISR
GDWHY
WGQGT

Humanized
SGPGLV
(SEQ ID
GKGLE
TSYNPS
DTSKNQ
FDY
MVTVSS

variant_VH
KPSQTL
NO:
WIG
LKS
FSLKLSS
(SEQ ID
(SEQ ID

1
SLTCTV
7313)
(SEQ ID
(SEQ ID
VTAADT
NO:
NO:

SGFSITT

NO:
NO:
AVYYC
7315)
6006)

T (SEQ

6019)
6001)
AR (SEQ

ID NO:

ID NO:

7330)

6020)

15E1
QIQLVE
GYHWN
WIRQAP
YIYSSGS
RFTISRD
GDWHY
WGQGT

Humanized
SGGGLV
(SEQ ID
GKGLE
TSYNPS
TAKNSF
FDY
MVTVSS

variant_VH
KPGGSL
NO:
WVG
LKS
YLQMN
(SEQ ID
(SEQ ID

2
RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSITT

NO:
NO:
TAVYYC
7315)
6006)

T (SEQ

6052)
6001)
AR (SEQ

ID NO:

ID NO:

7331)

6033)

15E1
EIQLLES
GYHWN
WVRQA
YIYSSGS
RFTISRD
GDWHY
WGQGT

Humanized
GGGLV
(SEQ ID
PGKGLE
TSYNPS
TSKNTF
FDY
MVTVSS

variant_VH
QPGGSL
NO:
WVG
LKS
YLQMN
(SEQ ID
(SEQ ID

3
RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSITT

NO:
NO:
TAVYYC
7315)
6006)

T (SEQ

6023)
6001)
AR (SEQ

ID NO:

ID NO:

7332)

6024)

15E1
EIQLVES
GYHWN
WVRQA
YIYSSGS
RFTISRD
GDWHY
WGQGT

Humanized
GGGLV
(SEQ ID
PGKGLE
TSYNPS
TAKNSF
FDY
MVTVSS

variant_VH
QPGGSL
NO:
WVG
LKS
YLQMN
(SEQ ID
(SEQ ID

4
RLSCAV
7313)
(SEQ ID
(SEQ ID
SLRAED
NO:
NO:

SGFSITT

NO:
NO:
TAVYYC
7315)
6006)

T (SEQ

6023)
6001)
AR (SEQ

ID NO:

ID NO:

7333)

6033)

15E1
QIQLVQ
GYHWN
WVRQA
YIYSSGS
RVTMTR
GDWHY
WGQGT

Humanized
SGAEVK
(SEQ ID
PGQGLE
TSYNPS
DTSTNT
FDY
MVTVSS

variant_VH
KPGASV
NO:
WMG
LKS
FYMELS
(SEQ ID
(SEQ ID

5
KVSCKV
7313)
(SEQ ID
(SEQ ID
SLRSED
NO:
NO:

SGFSITT

NO:
NO:
TAVYYC
7315)
6006)

T (SEQ

6027)
6001)
AR (SEQ

ID NO:

ID NO:

7334)

6037)

TABLE 8

Exemplary light chain CDRs and FWRs of NKp30-targeting antigen binding domains

Ab ID
VLFWR1
VLCDR1
VLFWR2
VLCDR2
VLFWR3
VLCDR3
VLFWR4

9G1-LC
SYTLTQ
SGERLS
WYQQK
ENDKRP
GIPDQFS
QSWDST
FGSGTQ

PPLLSV
DKYVH
PGRAPV
S (SEQ
GSNSGN
NSAV
LTVL

ALGHK
(SEQ ID
MVIY
ID NO:
IATLTIS
(SEQ ID
(SEQ ID

ATITC
NO:
(SEQ ID
6064)
KAQAG
NO:
NO:

(SEQ ID
6063)
NO:

YEADY
7293)
6069)

NO:

6067)

YC (SEQ

6066)

ID NO:

7292)

15H6-LC
SYTLTQ
SGENLS
WYQQK
ENEKRP
GIPDQFS
HYWESI
FGSGTH

PPSLSV
DKYVH
PGRAPV
S (SEQ
GSNSGN
NSVV
LTVL

APGQKA
(SEQ ID
MVIY
ID NO:
IATLTIS
(SEQ ID
(SEQ ID

TIIC
NO:
(SEQ ID
6071)
KAQPGS
NO:
NO:

(SEQ ID
6070)
NO:

EADYYC
6072)
6076)

NO:

6074)

(SEQ ID

6073)

NO:

6075)

9G1-LC_1
QSVTTQ
SGERLS
WYQQL
ENDKRP
GVPDRF
QSWDST
FGGGTQ

PPSVSG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSAV
LTVL

APGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

TISC
NO:
(SEQ ID
6064)
ITGLQA
NO:
NO:

(SEQ ID
6063)
NO:

EDEADY
7293)
6080)

NO:

6078)

YC (SEQ

6077)

ID NO:

6079)

9G1-LC_2
QSVTTQ
SGERLS
WYQQL
ENDKRP
GVPDRF
QSWDST
FGGGTQ

PPSASG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSAV
LTVL

TPGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

TISC
NO:
(SEQ ID
6064)
ISGLQSE
NO:
NO:

(SEQ ID
6063)
NO

DEADY
7293)
6084)

NO:

6082)

YC (SEQ

6081)

ID NO:

6083)

9G1-LC_3
QSVTTQ
SGERLS
WYQQL
ENDKRP
GVPDRF
QSWDST
FGGGTQ

PPSASG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSAV
LTVL

TPGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

TISC
NO:
(SEQ ID
6064)
ISGLRSE
NO
NO:

(SEQ ID
6063)
NO:

DEADY
7293)
6088)

NO:

6086)

YC (SEQ

6085)

ID NO:

6087)

9G1-LC 4
SSETTQ
SGERLS
WYQQK
ENDKRP
GIPERFS
QSWDST
FGGGTQ

PHSVSV
DKYVH
PGQDPV
S (SEQ
GSNPGN
NSAV
LTVL

ATAQM
(SEQ ID
MVIY
ID NO:
TATLTIS
(SEQ ID
(SEQ ID

ARITC
NO:
(SEQ ID
6064)
RIEAGD
NO:
NO:

(SEQ ID
6063)
NO:

EADYYC
7293)
6092)

NO:

6090)

(SEQ ID

6089)

NO:

6091)

9G1-LC_5
DIQMTQ
SGERLS
WYQQK
ENDKRP
GVPSRF
QSWDST
FGQGTK

SPSTLSA
DKYVH
PGKAPK
S (SEQ
SGSNSG
NSAV
VEIK

SVGDRV
(SEQ ID
MLIY
ID NO:
NEATLT
(SEQ ID
(SEQ ID

TITC
NO:
(SEQ ID
6064)
ISSLQPD
NO:
NO:

(SEQ ID
6063)
NO:

DFATYY
7293)
6096)

NO:

6094)

C (SEQ

6093)

ID NO:

6095)

15H6-
QYVLTQ
SGENLS
WYQQL
ENEKRP
GVPDRF
HYWESI
FGEGTE

LC_1
PPSASG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSVV
LTVL

TPGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

TISC
NO:
(SEQ ID
6071)
ISGLQSE
NO:
NO:

(SEQ ID
6070)
NO:

DEADY
6072)
6100)

NO:

6098)

YC (SEQ

6097)

ID NO:

6099)

15H6-
QYVLTQ
SGENLS
WYQQL
ENEKRP
GVPDRF
HYWESI
FGEGTE

LC_2
PPSASG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSVV
LTVL

TPGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

TISC
NO:
(SEQ ID
6071)
ISGLRSE
NO:
NO:

(SEQ ID
6070)
NO:

DEADY
6072)
6104)

NO:

6102)

YC (SEQ

6101)

ID NO:

6103)

15H6-
SYELTQ
SGENLS
WYQQK
ENEKRP
GIPERFS
HYWESI
FGEGTE

LC_3
PPSVSV
DKYVH
PGQSPV
S (SEQ
GSNSGN
NSVV
LTVL

SPGQTA
(SEQ ID
MVIY
ID NO:
TATLTIS
(SEQ ID
(SEQ ID

SITC
NO:
(SEQ ID
6071)
GTQAM
NO:
NO:

(SEQ ID
6070)
NO:

DEADY
6072)
6108)

NO:

6106)

YC (SEQ

6105)

ID NO:

6107)

15H6-
DYVLTQ
SGENLS
WYLQK
ENEKRP
GVPDRF
HYWESI
FGQGTK

LC_4
SPLSLPV
DKYVH
PGQSPQ
S (SEQ
SGSNSG
NSVV
VEIK

TPGEPA
(SEQ ID
MLIY
ID NO:
NDATLK
(SEQ ID
(SEQ ID

SISC
NO:
(SEQ ID
6071)
ISRVEA
NO:
NO:

(SEQ ID
6070)
NO:

EDVGV
6072)
6112)

NO:

6110)

YYC

6109)

(SEQ ID

NO:

6111)

15H6-
AYQLTQ
SGENLS
WYQQK
ENEKRP
GVPSRF
HYWESI
FGQGTK

LC_5
SPSSLSA
DKYVH
PGKAPK
S (SEQ
SGSNSG
NSVV
VEIK

SVGDRV
(SEQ ID
MLIY
ID NO:
NDATLT
(SEQ ID
(SEQ ID

TITC
NO:
(SEQ ID
6071)
ISSLQPE
NO:
NO:

(SEQ ID
6070)
NO:

DFATYY
6072)
6116)

NO:

6114)

C (SEQ

6113)

ID NO:

6115)

15H6-
EYVLTQ
SGENLS
WYQQK
ENEKRP
GIPARFS
HYWESI
FGQGTK

LC_6
SPATLS
DKYVH
PGQAPR
S (SEQ
GSNSGN
NSVV
VEIK

VSPGER
(SEQ ID
MLIY
ID NO:
EATLTIS
(SEQ ID
(SEQ ID

ATLSC
NO:
(SEQ ID
6071)
SLQSED
NO:
NO:

(SEQ ID
6070)
NO:

FAVYYC
6072)
6120)

NO:

6118)

(SEQ ID

6117)

NO:

6119)

9D9-LC
SYTLTQ
SGENLS
WYQQK
ENDKRP
GIPDQFS
HCWDS
FGSGTH

PPLVSV
DKYVH
PGRAPV
S (SEQ
GSNSGN
TNSAV
LTVL

ALGQK
(SEQ ID
MVIY
ID NO:
IATLTIS
(SEQ ID
(SEQ ID

ATIIC
NO:
(SEQ ID
6064)
KAQAG
NO:
NO:

(SEQ ID
6070)
NO:

YEADY
7321)
6076)

NO:

6067)

YC (SEQ

7320)

ID NO:

7292)

3A12-LC
SYTLTQ
SGENLS
WYQQK
ENDKRP
GIPDQFS
HCWDS
FGSGTH

PPLVSV
DKYVH
PGRAPV
S (SEQ
GSNSGN
TNSAV
LTVL

ALGQK
(SEQ ID
MVIY
ID NO:
IATLTIS
(SEQ ID
(SEQ ID

ATIIC
NO:
(SEQ ID
6064)
KAQAG
NO:
NO:

(SEQ ID
6070)
NO:

YEADY
7321)
6076)

NO:

6067)

YC (SEQ

7320)

ID NO:

7292)

12D10-LC
SYTLTQ
SGENLS
WYQQK
ENEKRP
GIPDQFS
HYWESI
FGSGTH

PPSLSV
DKYVH
PGRAPV
S (SEQ
GSNSGN
NSVV
LTVL

APGQKA
(SEQ ID
MVIY
ID NO:
IATLTIS
(SEQ ID
(SEQ ID

TIIC
NO:
(SEQ ID
6071)
KAQPGS
NO:
NO:

(SEQ ID
6070)
NO:

EADYYC
6072)
6076)

NO:

6074)

(SEQ ID

6073)

NO:

6075)

15E1-LC
SFTLTQ
SGEKLS
WYQQK
ENDRRP
GIPDQFS
QFWDST
FGGGTQ

PPLVSV
DKYVH
PGRAPV
S (SEQ
GSNSGN
NSAV
LTVL

AVGQV
(SEQ ID
MVIY
ID NO:
IASLTIS
(SEQ ID
(SEQ ID

ATITC
NO:
(SEQ ID
7327)
KAQAG
NO:
NO:

(SEQ ID
7326)
NO:

DEADYF
7329)
6080)

NO:

6067)

C (SEQ

7325)

ID NO:

7328)

15E1
SSETTQ
SGEKLS
WYQQK
ENDRRP
GIPERFS
QFWDST
FGGGTQ

Humanized
PPSVSV
DKYVH
PGQSPV
S (SEQ
GSNSGN
NSAV
LTVL

variant_VL
SPGQTA
(SEQ ID
MVIY
ID NO:
TATLTIS
(SEQ ID
(SEQ ID

1
SITC
NO:
(SEQ ID
7327)
GTQAM
NO:
NO:

(SEQ ID
7326)
NO:

DEADYF
7329)
6080)

NO:

6106)

C (SEQ

7335)

ID NO:

7336)

15E1
SSETTQ
SGEKLS
WYQQK
ENDRRP
GIPERFS
QFWDST
FGGGTQ

Humanized
PHSVSV
DKYVH
PGQDPV
S (SEQ
GSNPGN
NSAV
LTVL

variant_VL
ATAQM
(SEQ ID
MVIY
ID NO:
TATLTIS
(SEQ ID
(SEQ ID

2
ARITC
NO:
(SEQ ID
7327)
RIEAGD
NO:
NO:

(SEQ ID
7326)
NO:

EADYFC
7329)
6080)

NO:

6090)

(SEQ ID

6089)

NO:

7337)

15E1
QSVTTQ
SGEKLS
WYQQL
ENDRRP
GVPDRF
QFWDST
FGGGTQ

Humanized
PPSASG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSAV
LTVL

variant_VL
TPGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

3
TISC
NO:
(SEQ ID
7327)
ISGLRSE
NO:
NO:

(SEQ ID
7326)
NO:

DEADYF
7329)
6080)

NO:

6078)

C (SEQ

6081)

ID NO:

7338)

15E1
QSVTTQ
SGEKLS
WYQQL
ENDRRP
GVPDRF
QFWDST
FGGGTQ

Humanized
PPSVSG
DKYVH
PGTAPK
S (SEQ
SGSNSG
NSAV
LTVL

variant_VL
APGQRV
(SEQ ID
MLIY
ID NO:
NSASLA
(SEQ ID
(SEQ ID

4
TISC
NO:
(SEQ ID
7327)
ITGLQA
NO:
NO:

(SEQ ID
7326)
NO:

EDEADY
7329)
6080)

NO:

6078)

FC (SEQ

6077)

ID NO:

7339)

15E1
DSVTTQ
SGEKLS
WYQQR
ENDRRP
GVPDRF
QFWDST
FGGGTK

Humanized
SPLSLPV
DKYVH
PGQSPR
S (SEQ
SGSNSG
NSAV
VEIK

variant_VL
TLGQPA
(SEQ ID
MLIY
ID NO:
NDATLK
(SEQ ID
(SEQ ID

5
SISC
NO:
(SEQ ID
7327)
ISRVEA
NO:
NO: 233)

(SEQ ID
7326)
NO:

EDVGV
7329)

NO:

7341)

YFC

7340)

(SEQ ID

NO:

7342)

TABLE 35

Exemplary heavy chain CDRs and FWRs of NKp30-targeting antigen binding domains

Ab

ID
VHFWR1
VHCDR1
VHFWR2
VHCDR2
VHFWR3
VHCDR3
VHFWR4

BKM
EIQLLES

ITTTGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYF

WGQGT

0138
GGGLVQ

WN (SEQ
GKGLEW

TSYNPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
6001)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTTGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYF

WGQGT

0139
GGGLVQ

WN (SEQ
GKGLEW

TSYNPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
6001)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTIGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYE

WGQGT

0140
GGGLVQ

WN (SEQ
GKGLEW

ISYNPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
6001)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTIGYH

WVRQAP

YTYSSGS

RFTISRD

GDWHYF

WGQGT

0141
GGGLVQ

WN (SEQ
GKGLEW

TSYNPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
6001)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTTGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYF

WGQGT

0142
GGGLVQ

WN (SEQ
GKGLEW

TSYAPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
7437)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTIGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYF

WGQGT

0143
GGGLVQ

WN (SEQ
GKGLEW

TSYAPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
7437)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTTGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYF

WGQGT

0144
GGGLVQ

WN (SEQ
GKGLEW

TSYAPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
7437)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

BKM
EIQLLES

ITTTGYH

WVRQAP

YIYSSGS

RFTISRD

GDWHYF

WGQGT

0145
GGGLVQ

WN (SEQ
GKGLEW

TSYAPSL

TSKNTFY

DY (SEQ
MVTVSS

PGGSLRL
ID NO:
VG (SEQ

KS (SEQ
LQMNSL
ID NO:
(SEQ ID

SCAVSGF
7498)
ID NO:
ID NO:
RAEDTA
7315)
NO: 6006)

S (SEQ ID

6023)
7437)
VYYCAR

NO: 7497)

(SEQ ID

NO: 6024)

TABLE 36

Exemplary light chain CDRs and FWRs of NKp30-targeting antigen binding domains

Ab

ID
VLFWR1
VLCDR1
VLFWR2
VLCDR2
VLFWR3
VLCDR3
VLFWR4

BKM
DSVTTQS

SGEKLSD

WYQQRP
ENDRRPS
GVPDRES

QFWDST

FGGGTK

0138
PLSLPVT

KYVH

GQSPRM
(SEQ ID
GSNSGN

ASAV

VEIK

LGQPASI
(SEQ ID
LIY (SEQ
NO: 7327)
DATLKIS
(SEQ ID
(SEQ ID

SC (SEQ
NO: 7326)
ID NO:

RVEAED
NO: 7416)
NO: 233)

ID NO:

7341)

VGVYFC

7493)

(SEQ ID

NO: 7342)

BKM
DSVTTQS

SGEKLSD

WYQQRP

ENDRRPS

GVPDRFS

QFWAST

FGGGTK

0139
PLSLPVT

KYVH

GQSPRM
(SEQ ID
GSNSGN

NSAV

VEIK

LGQPASI
(SEQ ID
LIY (SEQ
NO: 7496)
DATLKIS
(SEQ ID
(SEQ ID

SC (SEQ
NO: 7494)
ID NO:

RVEAED
NO: 44)
NO: 233)

ID NO:

7495)

VGVYFC

7493)

(SEQ ID

NO: 7342)

BKM
SSETTQP

SGEKLSD

WYQQKP

ENDRRPS

GIPERFS

QFWAST

FGGGTQ

0140
PSVSVSP

KYVH

GQSPVM
(SEQ ID
GSNSGN

NSAV

LTVL

GQTASIT
(SEQ ID
VIY (SEQ
NO: 7327)
TATLTIS
(SEQ ID
(SEQ ID

C (SEQ ID
NO: 7326)
ID NO:

GTQAMD
NO: 44)
NO: 6080)

NO: 7335)

6106)

BADYFC

(SEQ ID

NO: 7336)

BKM
SSETTQP

SGEKLSD

WYQQKP

ENDRRPS

GIPERFS

QFWDST

FGGGTQ

0141
PSVSVSP

KYVH

GQSPVM
(SEQ ID
GSNSGN

ASAV

LTVL

GQTASIT
(SEQ ID
VIY (SEQ
NO: 7327)
TATLTIS
(SEQ ID
(SEQ ID

C (SEQ ID
NO: 7326)
ID NO:

GTQAMD
NO: 7416)
NO: 6080)

NO: 7335)

6106)

EADYFC

(SEQ ID

NO: 7336)

BKM
DSVTTQS

SGEKLSD

WYQQRP

ENDRRPS

GVPDRFS

QFWDST

FGGGTK

0142
PLSLPVT

KYVH

GQSPRM
(SEQ ID
GSNSGN

NSAV

VEIK

LGQPASI
(SEQ ID
LIY (SEQ
NO: 7327)
DATLKIS
(SEQ ID
(SEQ ID

SC (SEQ
NO: 7326)
ID NO:

RVEAED
NO: 7329)
NO: 233)

ID NO:

7341)

VGVYFC

7493)

(SEQ ID

NO: 7342)

BKM
SSETTQP

SGEKLSD

WYQQKP

ENDRRPS

GIPERFS

QFWDST

FGGGTQ

0143
PSVSVSP

KYVH

GQSPVM
(SEQ ID
GSNSGN

NSAV

LTVL

GQTASIT
(SEQ ID
VIY (SEQ
NO: 7327)
TATLTIS
(SEQ ID
(SEQ ID

C (SEQ ID
NO: 7326)
ID NO:

GTQAMD
NO: 7329)
NO: 6080)

NO: 7335)

6106)

EADYFC

(SEQ ID

NO: 7336)

BKM
DSVTTQS

SGEKLSD

WYQQRP

ENDRRPS

GVPDRFS

QFWAST

FGGGTK

0144
PLSLPVT

KYVH

GQSPRM
(SEQ ID
GSNSGN

ASAV

VEIK

LGQPASI
(SEQ ID
LIY (SEQ
NO: 7327)
DATLKIS
(SEQ ID
(SEQ ID

SC (SEQ
NO: 7326)
ID NO:

RVEAED
NO: 7447)
NO: 233)

ID NO:

7341)

VGVYFC

7493)

(SEQ ID

NO: 7342)

BKM
SSETTQP

SGEKLSD

WYQQKP

ENDRRPS

GIPERFS

QFWAST

FGGGTQ

0145
PSVSVSP

KYVH

GQSPVM
(SEQ ID
GSNSGN

ASAV

LTVL

GQTASIT
(SEQ ID
VIY (SEQ
NO: 7327)
TATLTIS
(SEQ ID
(SEQ ID

C (SEQ ID
NO: 7326)
ID NO:

GTQAMD
NO: 7447)
NO: 6080)

NO: 7335)

6106)

EADYFC

(SEQ ID

NO: 7336)

TABLE 9

Exemplary variable regions of NKp30-targeting antigen binding domains

SEQ ID

NO
Ab ID
Description
Sequence

SEQ ID
9G1-HC
9G1 heavy chain
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYH

NO: 6121

variable region
WNWIRQFPGKKLEWMGYIYSSGSTSYNPSLKSRI

SITRDTSKNQFFLQLNSVTTEDTATYYCARGNWH

YFDFWGQGTMVTVSS

SEQ ID
15H6-HC
15H6 heavy
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYH

NO: 6122

chain variable
WNWIRQFPGKKLEWMGYIYSSGTTRYNPSLKSRI

region
SITRDTSKNQFFLQLNSVTPEDTATYYCTRGNWH

YFDYWGQGTLVAVSS

SEQ ID
9G1-HC_1
9G1 heavy chain
QIQLQESGPGLVKPSETLSLTCTVSGFSINTGGYH

NO: 6123

variable region
WNWIRQPAGKGLEWIGYIYSSGSTSYNPSLKSRV

humanized
TMSRDTSKNQFSLKLSSVTAADTAVYYCARGNW

variant 1
HYFDFWGQGTMVTVSS

SEQ ID
9G1-HC_2
9G1 heavy chain
QIQLQESGPGLVKPSQTLSLTCTVSGFSINTGGYH

NO: 6124

variable region
WNWIRQHPGKGLEWIGYIYSSGSTSYNPSLKSLV

humanized
TISRDTSKNQFSLKLSSVTAADTAVYYCARGNW

variant 2
HYFDFWGQGTMVTVSS

SEQ ID
9G1-HC_3
9G1 heavy chain
EIQLLESGGGLVQPGGSLRLSCAVSGFSINTGGYH

NO: 6125

variable region
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

humanized
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGN

variant 3
WHYFDFWGQGTMVTVSS

SEQ ID
9G1-HC_4
9G1 heavy chain
QIQLVQSGAEVKKPGSSVKVSCKVSGFSINTGGY

NO: 6126

variable region
HWNWVRQAPGQGLEWMGYIYSSGSTSYNPSLKS

humanized
RVTITRDTSTNTFYMELSSLRSEDTAVYYCARGN

variant 4
WHYFDFWGQGTMVTVSS

SEQ ID
9G1-HC_5
9G1 heavy chain
EIQLVESGGGLVQPGGSLRLSCAVSGFSINTGGYH

NO: 6127

variable region
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

humanized
FTISRDTAKNSFYLQMNSLRAEDTAVYYCARGN

variant 5
WHYFDFWGQGTMVTVSS

SEQ ID
9G1-HC_6
9G1 heavy chain
QIQLVQSGAEVKKPGASVKVSCKVSGFSINTGGY

NO: 6128

variable region
HWNWVRQAPGQGLEWMGYIYSSGSTSYNPSLKS

humanized
RVTMTRDTSTNTFYMELSSLRSEDTAVYYCARG

variant 6
NWHYFDFWGQGTMVTVSS

SEQ ID
15H6-
15H6 heavy
QIQLQESGPGLVKPSQTLSLTCTVSGFSINTGGYH

NO: 6129
HC_1
chain variable
WNWIRQHPGKGLEWIGYIYSSGTTRYNPSLKSLV

region
TISRDTSKNQFSLKLSSVTAADTAVYYCARGNW

humanized
HYFDYWGQGTLVTVSS

variant 1

SEQ ID
15H6-
15H6 heavy
QIQLQESGPGLVKPSETLSLTCTVSGFSINTGGYH

NO: 6130
HC_2
chain variable
WNWIRQPAGKGLEWIGYIYSSGTTRYNPSLKSRV

region
TMSRDTSKNQFSLKLSSVTAADTAVYYCARGNW

humanized
HYFDYWGQGTLVTVSS

variant 2

SEQ ID
15H6-
15H6 heavy
EIQLLESGGGLVQPGGSLRLSCAVSGFSINTGGYH

NO: 6131
HC_3
chain variable
WNWVRQAPGKGLEWVGYIYSSGTTRYNPSLKSR

region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGN

humanized
WHYFDYWGQGTLVTVSS

variant 3

SEQ ID
15H6-
15H6 heavy
QIQLVESGGGLVKPGGSLRLSCAVSGFSINTGGY

NO: 6132
HC_4
chain variable
HWNWIRQAPGKGLEWVGYIYSSGTTRYNPSLKS

region
RFTISRDTAKNSFYLQMNSLRAEDTAVYYCARG

humanized
NWHYFDYWGQGTLVTVSS

variant 4

SEQ ID
15H6-
15H6 heavy
QIQLVQSGAEVKKPGASVKVSCKVSGFSINTGGY

NO: 6133
HC_5
chain variable
HWNWVRQAPGQGLEWMGYIYSSGTTRYNPSLK

region
SRVTMTRDTSTNTFYMELSSLRSEDTAVYYCARG

humanized
NWHYFDYWGQGTLVTVSS

variant 5

SEQ ID
15H6-
15H6 heavy
EIQLVQSGAEVKKPGATVKISCKVSGFSINTGGYH

NO: 6134
HC_6
chain variable
WNWVQQAPGKGLEWMGYIYSSGTTRYNPSLKS

region
RVTITRDTSTNTFYMELSSLRSEDTAVYYCARGN

humanized
WHYFDYWGQGTLVTVSS

variant 6

SEQ ID
9G1-LC
9G1 light chain
SYTLTQPPLLSVALGHKATITCSGERLSDKYVHW

NO: 7294

variable region
YQQKPGRAPVMVIYENDKRPSGIPDQFSGSNSGN

IATLTISKAQAGYEADYYCQSWDSTNSAVFGSGT

QLTVL

SEQ ID
15H6-LC
15H6 light chain
SYTLTQPPSLSVAPGQKATIICSGENLSDKYVHW

NO: 6136

variable region
YQQKPGRAPVMVIYENEKRPSGIPDQFSGSNSGNI

ATLTISKAQPGSEADYYCHYWESINSVVFGSGTH

LTVL

SEQ ID
9G1-LC_1
9G1 light chain
QSVTTQPPSVSGAPGQRVTISCSGERLSDKYVHW

NO: 6137

variable region
YQQLPGTAPKMLIYENDKRPSGVPDRFSGSNSGN

humanized
SASLAITGLQAEDEADYYCQSWDSTNSAVFGGG

variant 1
TQLTVL

SEQ ID
9G1-LC_2
9G1 light chain
QSVTTQPPSASGTPGQRVTISCSGERLSDKYVHW

NO: 6138

variable region
YQQLPGTAPKMLIYENDKRPSGVPDRFSGSNSGN

humanized
SASLAISGLQSEDEADYYCQSWDSTNSAVFGGGT

variant 2
QLTVL

SEQ ID
9G1-LC_3
9G1 light chain
QSVTTQPPSASGTPGQRVTISCSGERLSDKYVHW

NO: 6139

variable region
YQQLPGTAPKMLIYENDKRPSGVPDRFSGSNSGN

humanized
SASLAISGLRSEDEADYYCQSWDSTNSAVFGGGT

variant 3
QLTVL

SEQ ID
9G1-LC_4
9G1 light chain
SSETTQPHSVSVATAQMARITCSGERLSDKYVHW

NO: 6140

variable region
YQQKPGQDPVMVIYENDKRPSGIPERFSGSNPGN

humanized
TATLTISRIEAGDEADYYCQSWDSTNSAVFGGGT

variant 4
QLTVL

SEQ ID
9G1-LC_5
9G1 light chain
DIQMTQSPSTLSASVGDRVTITCSGERLSDKYVH

NO: 6141

variable region
WYQQKPGKAPKMLIYENDKRPSGVPSRFSGSNS

humanized
GNEATLTISSLQPDDFATYYCQSWDSTNSAVFGQ

variant 5
GTKVEIK

SEQ ID
15H6-
15H6 light chain
QYVLTQPPSASGTPGQRVTISCSGENLSDKYVHW

NO: 6142
LC_1
variable region
YQQLPGTAPKMLIYENEKRPSGVPDRFSGSNSGN

humanized
SASLAISGLQSEDEADYYCHYWESINSVVFGEGT

variant 1
ELTVL

SEQ ID
15H6-
15H6 light chain
QYVLTQPPSASGTPGQRVTISCSGENLSDKYVHW

NO: 6143
LC_2
variable region
YQQLPGTAPKMLIYENEKRPSGVPDRFSGSNSGN

humanized
SASLAISGLRSEDEADYYCHYWESINSVVFGEGT

variant 2
ELTVL

SEQ ID
15H6-
15H6 light chain
SYELTQPPSVSVSPGQTASITCSGENLSDKYVHW

NO: 6144
LC_3
variable region
YQQKPGQSPVMVIYENEKRPSGIPERFSGSNSGNT

humanized
ATLTISGTQAMDEADYYCHYWESINSVVFGEGTE

variant 3
LTVL

SEQ ID
15H6-
15H6 light chain
DYVLTQSPLSLPVTPGEPASISCSGENLSDKYVHW

NO: 6145
LC_4
variable region
YLQKPGQSPQMLIYENEKRPSGVPDRFSGSNSGN

humanized
DATLKISRVEAEDVGVYYCHYWESINSVVFGQG

variant 4
TKVEIK

SEQ ID
15H6-
15H6 light chain
AYQLTQSPSSLSASVGDRVTITCSGENLSDKYVH

NO: 6146
LC_5
variable region
WYQQKPGKAPKMLIYENEKRPSGVPSRFSGSNSG

humanized
NDATLTISSLQPEDFATYYCHYWESINSVVFGQG

variant 5
TKVEIK

SEQ ID
15H6-
15H6 light chain
EYVLTQSPATLSVSPGERATLSCSGENLSDKYVH

NO: 6147
LC_6
variable region
WYQQKPGQAPRMLIYENEKRPSGIPARFSGSNSG

humanized
NEATLTISSLQSEDFAVYYCHYWESINSVVFGQG

variant 6
TKVEIK

SEQ ID
9D9-HC
9D9 heavy chain
QIQLQESGPGLVKPSQSLSLSCSVTGFSINTGGYH

NO: 7295

variable region
WNWIRQFPGKKVEWMGYIYSSGTTKYNPSLKSRI

SITRDTSKNQFFLQLNSVTTEDTATYYCARGDWH

YFDYWGQGTMVAVSS

SEQ ID
9D9-LC
9D9 light chain
SYTLTQPPLVSVALGQKATIICSGENLSDKYVHW

NO: 7296

variable region
YQQKPGRAPVMVIYENDKRPSGIPDQFSGSNSGN

IATLTISKAQAGYEADYYCHCWDSTNSAVFGSGT

HLTVL

SEQ ID
3A12-HC
3A12 heavy
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYH

NO: 7297

chain variable
WNWIRQFPGKKLEWMGYIYSSGSTRYNPSLKSRF

region
SITRDTSKNQFFLQLNSVTTEDTATYYCTRGNWH

YFDYWGQGTLVAVSS

SEQ ID
3A12-LC
3A12 light chain
SYTLTQPPLVSVALGQKATIICSGENLSDKYVHW

NO: 7296

variable region
YQQKPGRAPVMVIYENDKRPSGIPDQFSGSNSGN

IATLTISKAQAGYEADYYCHCWDSTNSAVFGSGT

HLTVL

SEQ ID
12D10-HC
12D10 heavy
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYH

NO: 6122

chain variable
WNWIRQFPGKKLEWMGYIYSSGTTRYNPSLKSRI

region
SITRDTSKNQFFLQLNSVTPEDTATYYCTRGNWH

YFDYWGQGTLVAVSS

SEQ ID
12D10-LC
12D10 light
SYTLTQPPSLSVAPGQKATIICSGENLSDKYVHW

NO: 6136

chain variable
YQQKPGRAPVMVIYENEKRPSGIPDQFSGSNSGNI

region
ATLTISKAQPGSEADYYCHYWESINSVVFGSGTH

LTVL

SEQ ID
15E1-HC
15E1 heavy
QIQLQESGPGLVKPSQSLSLSCSVTGFSITTTGYH

NO: 7298

chain variable
WNWIRQFPGKKLEWMGYIYSSGSTSYNPSLKSRF

region
SITRDTSKNQFFLQLNSVTTEDTATYYCARGDWH

YFDYWGPGTMVTVSS

SEQ ID
15E1-LC
15E1 light chain
SFTLTQPPLVSVAVGQVATITCSGEKLSDKYVHW

NO: 7299

variable region
YQQKPGRAPVMVIYENDRRPSGIPDQFSGSNSGNI

ASLTISKAQAGDEADYFCQFWDSTNSAVFGGGT

QLTVL

SEQ ID
15E1_
15E1 heavy
QIQLQESGPGLVKPSQTLSLTCTVSGFSITTTGYH

NO: 7300
Humanized
chain variable
WNWIRQHPGKGLEWIGYIYSSGSTSYNPSLKSLV

variant_VH
region
TISRDTSKNQFSLKLSSVTAADTAVYYCARGDW

1
humanized
HYFDYWGQGTMVTVSS

variant 1

SEQ ID
15E1_
15E1 heavy
QIQLVESGGGLVKPGGSLRLSCAVSGFSITTTGYH

NO: 7301
Humanized
chain variable
WNWIRQAPGKGLEWVGYIYSSGSTSYNPSLKSRF

variant_VH
region
TISRDTAKNSFYLQMNSLRAEDTAVYYCARGDW

2
humanized
HYFDYWGQGTMVTVSS

variant 2

SEQ ID
15E1_
15E1 heavy
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7302
Humanized
chain variable
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

variant_VH
region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

3
humanized
WHYFDYWGQGTMVTVSS

(BJM0407
variant 3

VH and

BJM0411

VH)

SEQ ID
15E1_
15E1 heavy
EIQLVESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7303
Humanized
chain variable
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

variant_VH
region
FTISRDTAKNSFYLQMNSLRAEDTAVYYCARGD

4
humanized
WHYFDYWGQGTMVTVSS

variant 4

SEQ ID
15E1_
15E1 heavy
QIQLVQSGAEVKKPGASVKVSCKVSGFSITTTGY

NO: 7304
Humanized
chain variable
HWNWVRQAPGQGLEWMGYIYSSGSTSYNPSLKS

variant_VH
region
RVTMTRDTSTNTFYMELSSLRSEDTAVYYCARG

5
humanized
DWHYFDYWGQGTMVTVSS

variant 5

SEQ ID
15E1_
15E1 light chain
SSETTQPPSVSVSPGQTASITCSGEKLSDKYVHWY

NO: 7305
Humanized
variable region
QQKPGQSPVMVIYENDRRPSGIPERFSGSNSGNTA

variant_VL
humanized
TLTISGTQAMDEADYFCQFWDSTNSAVFGGGTQ

1
variant 1
LTVL

(BJM0407

VL)

SEQ ID
15E1_
15E1 light chain
SSETTQPHSVSVATAQMARITCSGEKLSDKYVHW

NO: 7306
Humanized
variable region
YQQKPGQDPVMVIYENDRRPSGIPERFSGSNPGN

variant_VL
humanized
TATLTISRIEAGDEADYFCQFWDSTNSAVFGGGT

2
variant 2
QLTVL

SEQ ID
15E1_
15E1 light chain
QSVTTQPPSASGTPGQRVTISCSGEKLSDKYVHW

NO: 7307
Humanized
variable region
YQQLPGTAPKMLIYENDRRPSGVPDRFSGSNSGN

variant_VL
humanized
SASLAISGLRSEDEADYFCQFWDSTNSAVFGGGT

3
variant 3
QLTVL

SEQ ID
15E1_
15E1 light chain
QSVTTQPPSVSGAPGQRVTISCSGEKLSDKYVHW

NO: 7308
Humanized
variable region
YQQLPGTAPKMLIYENDRRPSGVPDRFSGSNSGN

variant_VL
humanized
SASLAITGLQAEDEADYFCQFWDSTNSAVFGGGT

4
variant 4
QLTVL

SEQ ID
15E1_
15E1 light chain
DSVTTQSPLSLPVTLGQPASISCSGEKLSDKYVHW

NO: 7309
Humanized
variable region
YQQRPGQSPRMLIYENDRRPSGVPDRFSGSNSGN

variant_VL
humanized
DATLKISRVEAEDVGVYFCQFWDSTNSAVFGGG

5
variant 5
TKVEIK

(BJM0411

VL)

SEQ ID
BKM0138
BKM0138
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7302
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0139
BKM0139
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7302
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0140
BKM0140
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7302
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0141
BKM0141
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7302
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0142
BKM0142
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7390
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0143
BKM0143
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7390
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0144
BKM0144
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7390
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0145
BKM0145
EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYH

NO: 7390
VH
heavy chain
WNWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSR

variable region
FTISRDTSKNTFYLQMNSLRAEDTAVYYCARGD

WHYFDYWGQGTMVTVSS

SEQ ID
BKM0138
BKM0138 light
DSVTTQSPLSLPVTLGQPASISCSGEKLSDKYVHW

NO: 7395
VL
chain variable
YQQRPGQSPRMLIYENDRRPSGVPDRFSGSNSGN

region
DATLKISRVEAEDVGVYFCQFWDSTASAVFGGG

TKVEIK

SEQ ID
BKM0139
BKM0139 light
DSVTTQSPLSLPVTLGQPASISCSGEKLSDKYVHW

NO: 7397
VL
chain variable
YQQRPGQSPRMLIYENDRRPSGVPDRFSGSNSGN

region
DATLKISRVEAEDVGVYFCQFWASTNSAVFGGG

TKVEIK

SEQ ID
BKM0140
BKM0140 light
SSETTQPPSVSVSPGQTASITCSGEKLSDKYVHWY

NO: 7399
VL
chain variable
QQKPGQSPVMVIYENDRRPSGIPERFSGSNSGNTA

region
TLTISGTQAMDEADYFCQFWASTNSAVFGGGTQ

LTVL

SEQ ID
BKM0141
BKM0141 light
SSETTQPPSVSVSPGQTASITCSGEKLSDKYVHWY

NO: 7401
VL
chain variable
QQKPGQSPVMVIYENDRRPSGIPERFSGSNSGNTA

region
TLTISGTQAMDEADYFCQFWDSTASAVFGGGTQ

LTVL

SEQ ID
BKM0142
BKM0142 light
DSVTTQSPLSLPVTLGQPASISCSGEKLSDKYVHW

NO: 7309
VL
chain variable
YQQRPGQSPRMLIYENDRRPSGVPDRFSGSNSGN

region
DATLKISRVEAEDVGVYFCQFWDSTNSAVFGGG

TKVEIK

SEQ ID
BKM0143
BKM0143 light
SSETTQPPSVSVSPGQTASITCSGEKLSDKYVHWY

NO: 7305
VL
chain variable
QQKPGQSPVMVIYENDRRPSGIPERFSGSNSGNTA

region
TLTISGTQAMDEADYFCQFWDSTNSAVFGGGTQ

LTVL

SEQ ID
BKM0144
BKM0144 light
DSVTTQSPLSLPVTLGQPASISCSGEKLSDKYVHW

NO: 7404
VL
chain variable
YQQRPGQSPRMLIYENDRRPSGVPDRFSGSNSGN

region
DATLKISRVEAEDVGVYFCQFWASTASAVFGGG

TKVEIK

SEQ ID
BKM0145
BKM0145 light
SSETTQPPSVSVSPGQTASITCSGEKLSDKYVHWY

NO: 7405
VL
chain variable
QQKPGQSPVMVIYENDRRPSGIPERFSGSNSGNTA

region
TLTISGTQAMDEADYFCQFWASTASAVFGGGTQ

LTVL

TABLE 10

Exemplary NKp30-targeting antigen binding domains/antibody molecules

SEQ ID

NO
Ab ID
Description
Sequence

SEQ ID
Ch(anti-
9G1 heavy
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYHWN

NO:
NKp30
chain
WIRQFPGKKLEWMGYIYSSGSTSYNPSLKSRISITRD

6148
9G1)HC

TSKNQFFLQLNSVTTEDTATYYCARGNWHYFDFWG

N297A

QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC

LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEM

TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

SEQ ID
Ch(anti-
9G1 heavy
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYHWN

NO:
NKp30
chain
WIRQFPGKKLEWMGYIYSSGSTSYNPSLKSRISITRD

6149
9G1)HC

TSKNQFFLQLNSVTTEDTATYYCARGNWHYFDFWG

QGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC

LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEM

TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

SEQ ID
Ch(anti-
9G1 light
SYTLTQPPLLSVALGHKATITCSGERLSDKYVHWYQ

NO:
NKp30
chain
QKPGRAPVMVIYENDKRPSGIPDQFSGSNSGNIATLT

6150
9G1)LC

ISKAQAGYEADYYCQSWDSTNSAVFGSGTQLTVLG

QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAV

TVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLS

LTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID
Ch(anti-
15H6
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYHWN

NO:
NKp30
heavy
WIRQFPGKKLEWMGYIYSSGTTRYNPSLKSRISITRD

6151
15H6)HC
chain
TSKNQFFLQLNSVTPEDTATYYCTRGNWHYFDYWG

N297A

QGTLVAVSSASTKGPSVFPLAPSSKSTSGGTAALGC

LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEM

TKNQVSLSCAVKGFYPSDIA VEWESNGQPENNYKT

TPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

SEQ ID
Ch(anti-
15H6
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYHWN

NO:
NKp30
heavy
WIRQFPGKKLEWMGYIYSSGTTRYNPSLKSRISITRD

6152
15H6)HC
chain
TSKNQFFLQLNSVTPEDTATYYCTRGNWHYFDYWG

(hole)

QGTLVAVSSASTKGPSVFPLAPSSKSTSGGTAALGC

LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE

PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI

SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEM

TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKT

TPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPGK

SEQ ID
Ch(anti-
15H6 light
SYTLTQPPSLSVAPGQKATIICSGENLSDKYVHWYQ

NO:
NKp30
chain
QKPGRAPVMVIYENEKRPSGIPDQFSGSNSGNIATLT

6153
15H6)LC

ISKAQPGSEADYYCHYWESINSVVFGSGTHLTVLGQ

PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVT

VAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSL

TPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID
anti-NKp30
Hamster
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYHWN

NO:
9G1 scFv
anti-
WIRQFPGKKLEWMGYIYSSGSTSYNPSLKSRISITRD

6187
(VH-VL)
NKp30
TSKNQFFLQLNSVTTEDTATYYCARGNWHYFDFWG

scFv of
QGTMVTVSSGGGGSGGGGGGGGSGGGGSSYTLTQ

9G1 in VH
PPLLSVALGHKATITCSGERLSDKYVHWYQQKPGR

to VL
APVMVIYENDKRPSGIPDQFSGSNSGNIATLTISKAQ

orientation
AGYEADYYCQSWDSTNSAVFGSGTQLTVL

SEQ ID
anti-NKp30
Hamster
SYTLTQPPLLSVALGHKATITCSGERLSDKYVHWYQ

NO:
9G1 scFv
anti-
QKPGRAPVMVIYENDKRPSGIPDQFSGSNSGNIATLT

6188
(VL-VH)
NKp30
ISKAQAGYEADYYCQSWDSTNSAVFGSGTQLTVLG

scFv of
GGGSGGGGSGGGGSGGGGSQIQLQESGPGLVKPSQ

9G1 in VL
SLSLTCSVTGFSINTGGYHWNWIRQFPGKKLEWMG

to VH
YIYSSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTT

orientation
EDTATYYCARGNWHYFDFWGQGTMVTVSS

SEQ ID
anti-NKp30
Hamster
QIQLQESGPGLVKPSQSLSLTCSVTGFSINTGGYHWN

NO:
15H6 scFv
anti-
WIRQFPGKKLEWMGYIYSSGTTRYNPSLKSRISITRD

6189
(VH-VL)
NKp30
TSKNQFFLQLNSVTPEDTATYYCTRGNWHYFDYWG

scFv of
QGTLVAVSSGGGGSGGGGSGGGGSGGGGSSYTLTQ

15H6 in
PPSLSVAPGQKATIICSGENLSDKYVHWYQQKPGRA

VH to VL
PVMVIYENEKRPSGIPDQFSGSNSGNIATLTISKAQP

orientation
GSEADYYCHYWESINSVVFGSGTHLTVL

SEQ ID
anti-NKp30
Hamster
SYTLTQPPSLSVAPGQKATIICSGENLSDKYVHWYQ

NO:
15H6 scFv
anti-
QKPGRAPVMVIYENEKRPSGIPDQFSGSNSGNIATLT

6190
(VL-VH)
NKp30
ISKAQPGSEADYYCHYWESINSVVFGSGTHLTVLGG

scFv of
GGSGGGGSGGGGSGGGGSQIQLQESGPGLVKPSQSL

15H6 in
SLTCSVTGFSINTGGYHWNWIRQFPGKKLEWMGYI

VL to VH
YSSGTTRYNPSLKSRISITRDTSKNQFFLQLNSVTPED

orientation
TATYYCTRGNWHYFDYWGQGTLVAVSS

SEQ ID
BJM0859

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
lambda scFv

NWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTIS

7310

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSSS

ETTQPPSVSVSPGQTASITCSGEKLSDKYVHWYQQK

PGQSPVMVIYENDRRPSGIPERFSGSNSGNTATLTIS

GTQAMDEADYFCQFWDSTNSAVFGGGTQLTVL

SEQ ID
BJM0860

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
kappa scFv

NWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTIS

7311

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDS

VTTQSPLSLPVTLGQPASISCSGEKLSDKYVHWYQQ

RPGQSPRMLIYENDRRPSGVPDRFSGSNSGNDATLKI

SRVEAEDVGVYFCQFWDSTNSAVFGGGTKVEIK

SEQ ID
BKM0138

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTIS

7406

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDS

VTTQSPLSLPVTLGQPASISCSGEKLSDKYVHWYQQ

RPGQSPRMLIYENDRRPSGVPDRFSGSNSGNDATLKI

SRVEAEDVGVYFCQFWDSTASAVFGGGTKVEIK

SEQ ID
BKM0139

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTIS

7407

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDS

VTTQSPLSLPVTLGQPASISCSGEKLSDKYVHWYQQ

RPGQSPRMLIYENDRRPSGVPDRFSGSNSGNDATLKI

SRVEAEDVGVYFCQFWASTNSAVFGGGTKVEIK

SEQ ID
BKM0140

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTIS

7408

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSSS

ETTQPPSVSVSPGQTASITCSGEKLSDKYVHWYQQK

PGQSPVMVIYENDRRPSGIPERFSGSNSGNTATLTIS

GTQAMDEADYFCQFWASTNSAVFGGGTQLTVL

SEQ ID
BKM0141

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYNPSLKSRFTIS

7409

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSSS

ETTQPPSVSVSPGQTASITCSGEKLSDKYVHWYQQK

PGQSPVMVIYENDRRPSGIPERFSGSNSGNTATLTIS

GTQAMDEADYFCQFWDSTASAVFGGGTQLTVL

SEQ ID
BKM0142

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSRFTIS

7411

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDS

VTTQSPLSLPVTLGQPASISCSGEKLSDKYVHWYQQ

RPGQSPRMLIYENDRRPSGVPDRFSGSNSGNDATLKI

SRVEAEDVGVYFCQFWDSTNSAVFGGGTKVEIK

SEQ ID
BKM0143

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSRFTIS

7412

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSSS

ETTQPPSVSVSPGQTASITCSGEKLSDKYVHWYQQK

PGQSPVMVIYENDRRPSGIPERFSGSNSGNTATLTIS

GTQAMDEADYFCQFWDSTNSAVFGGGTQLTVL

SEQ ID
BKM0144

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSRFTIS

7413

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSDS

VTTQSPLSLPVTLGQPASISCSGEKLSDKYVHWYQQ

RPGQSPRMLIYENDRRPSGVPDRFSGSNSGNDATLKI

SRVEAEDVGVYFCQFWASTASAVFGGGTKVEIK

SEQ ID
BKM0145

EIQLLESGGGLVQPGGSLRLSCAVSGFSITTTGYHW

NO:
scFv

NWVRQAPGKGLEWVGYIYSSGSTSYAPSLKSRFTIS

7414

RDTSKNTFYLQMNSLRAEDTAVYYCARGDWHYFD

YWGQGTMVTVSSGGGGSGGGGSGGGGSGGGGSSS

ETTQPPSVSVSPGQTASITCSGEKLSDKYVHWYQQK

PGQSPVMVIYENDRRPSGIPERFSGSNSGNTATLTIS

GTQAMDEADYFCQFWASTASAVFGGGTQLTVL

In some embodiments, the NK cell engager is an antigen binding domain that binds to NKp46 (e.g., NKp46 present, e.g., expressed or displayed, on the surface of an NK cell) and comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Table 15. In some embodiments, binding of the NK cell engager, e.g., antigen binding domain that binds to NKp46, to the NK cell activates the NK cell. An antigen binding domain that binds to NKp46 (e.g., NKp46 present, e.g., expressed or displayed, on the surface of an NK cell) may be said to target NKp46, the NK cell, or both.

In some embodiments, the NK cell engager is an antigen binding domain that binds to NKG2D (e.g., NKG2D present, e.g., expressed or displayed, on the surface of an NK cell) and comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Table 15. In some embodiments, binding of the NK cell engager, e.g., antigen binding domain that binds to NKG2D, to the NK cell activates the NK cell. An antigen binding domain that binds to NKG2D (e.g., NKG2D present, e.g., expressed or displayed, on the surface of an NK cell) may be said to target NKG2D, the NK cell, or both. 006851 In some embodiments, the NK cell engager is an antigen binding domain that binds to CD16 (e.g., CD16 present, e.g., expressed or displayed, on the surface of an NK cell) and comprises any CDR amino acid sequence, framework region (FWR) amino acid sequence, or variable region amino acid sequence disclosed in Table 15. In some embodiments, binding of the NK cell engager, e.g., antigen binding domain that binds to CD16, to the NK cell activates the NK cell. An antigen binding domain that binds to CD16 (e.g., CD16 present, e.g., expressed or displayed, on the surface of an NK cell) may be said to target CD16, the NK cell, or both.

TABLE 15

Exemplary variable regions of NKp46, NKG2D, or CD16-targeting antigen binding

domains

SEQ

ID NO
Ab ID
Description
Sequence

SEQ
NKG2D_
scFv that binds
QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIR

ID NO:
1scFv
NKG2D
QPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQ

6175

FSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTV

SSGGGGSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPG

ERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASS

RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYG

SSPWTFGQGTKVEIK

SEQ
NKG2D_
VH that binds
QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIR

ID NO:
1VH
NKG2D
QPPGKGLEWIGHISYSGSANYNPSLKSRVTISVDTSKNQ

6176

FSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTV

SS

SEQ
NKG2D_
VL that binds
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ

ID NO:
1VL
NKG2D
KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRL

6177

EPEDFAVYYCQQYGSSPWTFGQGTKVEIK

SEQ
NKG2D_
scFv that binds
EVQLVQSGAEVKEPGESLKISCKNSGYSFTNYWVGWV

ID NO:
2scFv
NKG2D
RQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKS

6178

INTAYLQWSSLKASDTAMYYCGRLTMFRGIIIGYFDY

WGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQ

SPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAP

RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFA

VYYCQQRSNWPWTFGQGTKVEIK

SEQ
NKG2D_
VH that binds
EVQLVQSGAEVKEPGESLKISCKNSGYSFTNYWVGWV

ID NO:
2VH
NKG2D
RQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKS

6179

INTAYLQWSSLKASDTAMYYCGRLTMFRGIIIGYFDY

WGQGTLVTVSS

SEQ
NKG2D_
VL that binds
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ

ID NO:
2VL
NKG2D
KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL

6180

EPEDFAVYYCQQRSNWPWTFGQGTKVEIK

SEQ
NKp46
scFv that binds
QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWG

ID NO:
scFv
NKp46
KQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADK

6181

SSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWG

QGTSVTVSSGGGGGGGGSGGGGSGGGGSDIQMTQTT

SSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKL

LIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIAT

YFCQQGNTRPWTFGGGTKLEIK

SEQ
NKp46
VH that binds
QVQLQQSGPELVKPGASVKMSCKASGYTFTDYVINWG

ID NO:
VH
NKp46
KQRSGQGLEWIGEIYPGSGTNYYNEKFKAKATLTADK

6182

SSNIAYMQLSSLTSEDSAVYFCARRGRYGLYAMDYWG

QGTSVTVSS

SEQ
NKp46
VL that binds
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQ

ID NO:
VL
NKp46
KPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINN

6183

LEQEDIATYFCQQGNTRPWTFGGGTKLEIK

SEQ
CD16
scFv that binds
EVQLVESGG GVVRPGGSLR LSCAASGFTF

ID NO:
scFv
CD16
DDYGMSWVRQ APGKGLEWVS

6184

GINWNGGSTG YADSVKGRFT ISRDNAKNSL

YLQMNSLRAE DTAVYYCARG

RSLLFDYWGQ GTLVTVSRGG GGSGGGGSGG

GGSSELTQDP AVSVALGQTV

RITCQGDSLR SYYASWYQQK PGQAPVLVIY

GKNNRPSGIP DRFSGSSSGN

TASLTITGAQ AEDEADYYCN SRDSSGNHVV

FGGGTKLTVL

SEQ
CD16V
VH that binds
EVQLVESGG GVVRPGGSLR LSCAASGFTF

ID NO:
H
CD16
DDYGMSWVRQ APGKGLEWVS

6185

GINWNGGSTG YADSVKGRFT ISRDNAKNSL

YLQMNSLRAE DTAVYYCARG

RSLLFDYWGQ GTLVTVSR

SEQ
CD16V
VL that binds
SSELTQDP AVSVALGQTVRITCQGDSLR

ID NO:
L
CD16
SYYASWYQQK PGQAPVLVIY GKNNRPSGIP

6186

DRFSGSSSGNTASLTITGAQ AEDEADYYCN

SRDSSGNHVV FGGGTKLTVL

In one embodiment, the NK cell engager is a ligand of NKp30, e.g., is a B37-6, e.g., comprises the amino acid sequence of:

(SEQ ID NO: 6198)

DLKVEMMAGGTQITPLNDNVTIFCNIFYSQPLNITSMGITWFWKSLTFD

KEVKVFEFFGDHQEAFRPGAIVSPWRLKSGDASLRLPGIQLEEAGEYRC

EVVVTPLKAQGTVQLEVVASPASRLLLDQVGMKENEDKYMCESSGFYPE

AINITWEKQTQKFPHPIEISEDVITGPTIKNMDGTFNVTSCLKLNSSQE

DPGTVYQCVVRHASLHTPLRSNFTLTAARHSLSETEKTDNFS,

In other embodiments, the NK cell engager is a ligand of NKp44 or NKp46, which is a viral HA. Viral hemagglutinins (HA) are glyco proteins which are on the surface of viruses. HA proteins allow viruses to bind to the membrane of cells via sialic acid sugar moieties which contributes to the fusion of viral membranes with the cell membranes (see e.g., Eur J Immunol. 2001 Sep.; 31(9):2680-9 “Recognition of viral hemagglutinins by NKp44 but not by NKp30”; and Nature. 2001 Feb. 22; 409(6823):1055-60 “Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells” the contents of each of which are incorporated by reference herein).

In other embodiments, the NK cell engager is a ligand of NKG2D chosen from MICA, MICB, or ULBP1, e.g., wherein:

(i) MICA comprises the amino acid sequence:

(SEQ ID NO: 6199)

EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQW

AEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEI

HEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLK

EDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEG

NITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWV

ATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW,

- (ii) MICB comprises the amino acid sequence:

(SEQ ID NO: 6200)

AEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQ

WAEDVLGAKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCE

IHEDSSTRGSRHFYYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFW

KEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSE

GNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTW

VATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKVLVLQSQRTD,

- (iii) ULBP1 comprises the amino acid sequence:

(SEQ ID NO: 6201)

GWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAF

ASLGKKVNVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQA

RMSCEHEAHGHGRGSWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKW

EKNRDVTMFFQKISLGDCKMWLEEFLMYWEQMLDPTKPPSLAPG,

In other embodiments, the NK cell engager is a ligand of DNAM1 chosen from NECTIN2 or NECL5, e.g., wherein:

- (i) NECTIN2 comprises the amino acid sequence:

(SEQ ID NO: 6202)

QDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQ

NVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHG

LTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPT

TVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVP

SGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVSISGYDDNWYLGR

TDATLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNT

TFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGG,

- (ii) NECL5 comprises the amino acid sequence:

(SEQ ID NO: 6203)

WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARH

GESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDE

GNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCV

STGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDG

KNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLT

CDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNV

TNALGARQAELTVQVKEGPPSEHSGISRN,

In yet other embodiments, the NK cell engager is a ligand of DAP10, which is an adapter for NKG2D (see e.g., Proc Natl Acad Sci USA. 2005 May 24; 102(21): 7641-7646; and Blood, 15 Sep. 2011 Volume 118, Number 11, the full contents of each of which is incorporated by reference herein).

In other embodiments, the NK cell engager is a ligand of CD16, which is a CD16a/b ligand, e.g., a CD16a/b ligand further comprising an antibody Fc region (see e.g., Front Immunol. 2013; 4: 76 discusses how antibodies use the Fc to trigger NK cells through CD16, the full contents of which are incorporated herein).

In other embodiments, the NK cell engager is a ligand of CRTAM, which is NECL2, e.g., wherein NECL2 comprises the amino acid sequence:

(SEQ ID NO: 6204)

QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLK

DSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLV

PPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSE

VEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQ

YKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQ

HAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIP

PPTTTTTTTTTTTTTILTIITDSRAGEEGSIRAVDH,

In other embodiments, the NK cell engager is a ligand of CD27, which is CD70, e.g., wherein CD70 comprises the amino acid sequence:

(SEQ ID NO: 6205)

QRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLH

GPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPA

SRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETF

FGVQWVRP,

In other embodiments, the NK cell engager is a ligand of PSGL1, which is L-selectin (CD62L), e.g., wherein L-selectin comprises the amino acid sequence:

(SEQ ID NO: 6206)

WTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYY

WIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRN

KDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVG

YYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLT

GIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTS

ACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDY

N,

In other embodiments, the NK cell engager is a ligand of CD96, which is NECL5, e.g., wherein NECL5 comprises the amino acid sequence:

In other embodiments, the NK cell engager is a ligand of CD100 (SEMA4D), which is CD72, e.g., wherein CD72 comprises the amino acid sequence:

(SEQ ID NO: 6207)

RYLQVSQQLQQTNRVLEVINSSLRQQLRLKITQLGQSAEDLQGSRRELA

QSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLS

NMENRLKPFFTCGSADTCCPSGWIMHQKSCFYISLTSKNWQESQKQCET

LSSKLATFSEIYPQSHSYYFLNSLLPNGGSGNSYWTGLSSNKDWKLTDD

TQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYICEMTAFRFPD,

In other embodiments, the NK cell engager is a ligand of NKp80, which is CLEC2B (AICL), e.g., wherein CLEC2B (AICL) comprises the amino acid sequence:

(SEQ ID NO: 6208)

KLTRDSQSLCPYDWIGFQNKCYYFSKEEGDWNSSKYNCSTQHADLTIID

NIEEMNFLRRYKCSSDHWIGLKMAKNRTGQWVDGATFTKSFGMRGSEGC

AYLSDDGAATARCYTERKWICRKRIH,

In other embodiments, the NK cell engager is a ligand of CD244, which is CD48, e.g., wherein CD48 comprises the amino acid sequence:

(SEQ ID NO: 6209)

QGHLVHMTVVSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSK

YFESKFKGRVRLDPQSGALYISKVQKEDNSTYIMRVLKKTGNEQEWKIK

LQVLDPVPKPVIKIEKIEDMDDNCYLKLSCVIPGESVNYTWYGDKRPFP

KELQNSVLETTLMPHNYSRCYTCQVSNSVSSKNGTVCLSPPCTLARS,

In some embodiments, the NK cell engager is a viral hemagglutinin (HA), HA is a glycoprotein found on the surface of influenza viruses. It is responsible for binding the virus to cells with sialic acid on the membranes, such as cells in the upper respiratory tract or erythrocytes. HA has at least 18 different antigens. These subtypes are named H1 through H18. NCRs can recognize viral proteins. NKp46 has been shown to be able to interact with the HA of influenza and the HA-NA of Paramyxovirus, including Sendai virus and Newcastle disease virus. Besides NKp46, NKp44 can also functionally interact with HA of different influenza subtypes.

In some embodiments of any of the multifunctional molecules described herein, the immune cell engager is an NK cell engager, e.g., an NK cell engager that mediates binding to and activation of an NK cell, or an NK cell engager that mediates binding to but not activation of an NK cell. In certain embodiments, the NK cell engager is chosen from an antibody molecule, e.g., an antigen binding domain, or ligand that binds to (e.g., activates): NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160, e.g., the NK cell engager is an antibody molecule or ligand that binds to (e.g., activates) NKp30. In certain some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain.

In some embodiments, the NK cell engager is capable of engaging an NK cell.

In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKp30, NKp46, NKG2D, or CD16.

In some embodiments, the multifunctional molecule:

- (i) binds specifically to an epitope of NKp30, NKp46, NKG2D, or CD16, e.g., the same or similar epitope as the epitope recognized by an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein;
- (ii) shows the same or similar binding affinity or specificity, or both, as an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein;
- (iii) inhibits, e.g., competitively inhibits, the binding of an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein;
- (iv) binds the same or an overlapping epitope with an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule as described herein; or
- (v) competes for binding, and/or binds the same epitope, with an anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 molecule as described herein.

In some embodiments, the anti-NKp30, anti-NKp46, anti-NKG2D, or anti-CD16 antibody molecule comprises one or more CDRs, framework regions, variable domains, heavy or light chains, or an antigen binding domain chosen from Tables 7, 8, 35, 36, 9, 10, 15, or 34, or a sequence substantially identical thereto. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKp30. In some embodiments, lysis of the lymphoma cell or lymphocyte is mediated by NKp30. In some embodiments, the multifunctional molecule does not activate the NK cell when incubated with the NK cell in the absence of the tumor antigen on the lymphoma cell or TRBC1 or TRBC2 on the lymphocyte. In some embodiments, the multifunctional molecule activates the NK cell when the NK cell is a NKp30 expressing NK cell and either: (1) the tumor antigen on the lymphoma cell is also present or (2) TRBC1 or TRBC2 on the lymphocyte is also present. In some embodiments, the multifunctional molecule does not activate the NK cell when the NK cell is not a NKp30 expressing NK cell and either: (1) the tumor antigen on the lymphoma cell is also present or (2) TRBC1 or TRBC2 on the lymphocyte is also present.

In some embodiments, the NK cell engager comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6064 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the NK cell engager comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

In some embodiments, the NK cell engager comprises: (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6004 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6005 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6006 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), and/or (2) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6067 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 7292 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6069 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises:

- (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, a VHFWR2 amino acid sequence of SEQ ID NO: 6004, a VHFWR3 amino acid sequence of SEQ ID NO: 6005, or a VHFWR4 amino acid sequence of SEQ ID NO: 6006, and
- (3) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292, or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the NK cell engager comprises:

- (i) a VH comprising the amino acid sequence of SEQ ID NO: 6121 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6121), and/or
- (ii) a VL comprising the amino acid sequence of SEQ ID NO: 7294 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 7294).

In some embodiments, the NK cell engager comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6150).

In some embodiments, the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NOs: 6148 or 6149 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 6148 or 6149), and a light chain comprising the amino acid sequence of SEQ ID NO: 6150 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6150).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6014 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6015 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6016 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6017 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6014, a VHFWR2 amino acid sequence of SEQ ID NO: 6015, a VHFWR3 amino acid sequence of SEQ ID NO: 6016, or a VHFWR4 amino acid sequence of SEQ ID NO: 6017.

In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6123 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6123).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6018 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6019 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6020 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6021 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6018, a VHFWR2 amino acid sequence of SEQ ID NO: 6019, a VHFWR3 amino acid sequence of SEQ ID NO: 6020, or a VHFWR4 amino acid sequence of SEQ ID NO: 6021.

In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6124 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6124).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6022 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6023 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6024 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6025 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6022, a VHFWR2 amino acid sequence of SEQ ID NO: 6023, a VHFWR3 amino acid sequence of SEQ ID NO: 6024, or a VHFWR4 amino acid sequence of SEQ ID NO: 6025. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6125 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6125).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6026 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6027 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6028 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6029 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6026, a VHFWR2 amino acid sequence of SEQ ID NO: 6027, a VHFWR3 amino acid sequence of SEQ ID NO: 6028, or a VHFWR4 amino acid sequence of SEQ ID NO: 6029. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6126 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6126).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6030 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6032 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6033 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6034 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6030, a VHFWR2 amino acid sequence of SEQ ID NO: 6032, a VHFWR3 amino acid sequence of SEQ ID NO: 6033, or a VHFWR4 amino acid sequence of SEQ ID NO: 6034. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6127 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6127).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6035 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6036 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6037 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6038 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6035, a VHFWR2 amino acid sequence of SEQ ID NO: 6036, a VHFWR3 amino acid sequence of SEQ ID NO: 6037, or a VHFWR4 amino acid sequence of SEQ ID NO: 6038. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6128 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6128).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6077 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6078 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6079 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6080 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6077, a VLFWR2 amino acid sequence of SEQ ID NO: 6078, a VLFWR3 amino acid sequence of SEQ ID NO: 6079, or a VLFWR4 amino acid sequence of SEQ ID NO: 6080. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6137 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6137).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6081 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6082 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6083 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6084 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6081, a VLFWR2 amino acid sequence of SEQ ID NO: 6082, a VLFWR3 amino acid sequence of SEQ ID NO: 6083, or a VLFWR4 amino acid sequence of SEQ ID NO: 6084. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6138 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6138).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6085 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6086 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6087 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6088 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6085, a VLFWR2 amino acid sequence of SEQ ID NO: 6086, a VLFWR3 amino acid sequence of SEQ ID NO: 6087, or a VLFWR4 amino acid sequence of SEQ ID NO: 6088. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6139 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6139).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6089 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6090 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6091 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6092 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6089, a VLFWR2 amino acid sequence of SEQ ID NO: 6090, a VLFWR3 amino acid sequence of SEQ ID NO: 6091, or a VLFWR4 amino acid sequence of SEQ ID NO: 6092. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6140 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6140).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6093 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6094 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6095 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6096 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6093, a VLFWR2 amino acid sequence of SEQ ID NO: 6094, a VLFWR3 amino acid sequence of SEQ ID NO: 6095, or a VLFWR4 amino acid sequence of SEQ ID NO: 6096. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6141 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6141).

In some embodiments, the NK cell engager comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6008 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6009 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6071 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6072 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In certain embodiments, the NK cell engager comprises:
- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6007, a VHCDR2 amino acid sequence of SEQ ID NO: 6008, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6009, and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6070, a VLCDR2 amino acid sequence of SEQ ID NO: 6071, and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6072.

In some embodiments, the NK cell engager comprises: (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6010 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6011 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6012 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6013 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), and/or (2) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6074 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6075 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6076 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises: (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6010, a VHFWR2 amino acid sequence of SEQ ID NO: 6011, a VHFWR3 amino acid sequence of SEQ ID NO: 6012, or a VHFWR4 amino acid sequence of SEQ ID NO: 6013, and (3) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6073, a VLFWR2 amino acid sequence of SEQ ID NO: 6074, a VLFWR3 amino acid sequence of SEQ ID NO: 6075, or a VLFWR4 amino acid sequence of SEQ ID NO: 6076.

In some embodiments, the NK cell engager comprises:

- (i) a VH comprising the amino acid sequence of SEQ ID NO: 6122 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6122), and/or
- (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6136 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6136).

In some embodiments, the NK cell engager comprises a light chain comprising the amino acid sequence of SEQ ID NO: 6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6153).

In some embodiments, the NK cell engager comprises a heavy chain comprising the amino acid sequence of SEQ ID NOs: 6151 or 6152 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NOs: 6151 or 6152), and a light chain comprising the amino acid sequence of SEQ ID NO: 6153 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6153).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6039 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6040 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6041 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6042 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6039, a VHFWR2 amino acid sequence of SEQ ID NO: 6040, a VHFWR3 amino acid sequence of SEQ ID NO: 6041, or a VHFWR4 amino acid sequence of SEQ ID NO: 6042. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6129 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6129).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6043 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6044 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6045 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6046 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6043, a VHFWR2 amino acid sequence of SEQ ID NO: 6044, a VHFWR3 amino acid sequence of SEQ ID NO: 6045, or a VHFWR4 amino acid sequence of SEQ ID NO: 6046.

In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6130 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6130).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6047 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6048 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6049 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6050 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6047, a VHFWR2 amino acid sequence of SEQ ID NO: 6048, a VHFWR3 amino acid sequence of SEQ ID NO: 6049, or a VHFWR4 amino acid sequence of SEQ ID NO: 6050. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6131 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6131).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6051 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6052 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6053 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6054 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6051, a VHFWR2 amino acid sequence of SEQ ID NO: 6052, a VHFWR3 amino acid sequence of SEQ ID NO: 6053, or a VHFWR4 amino acid sequence of SEQ ID NO: 6054. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6132 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6132).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6055 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6056 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6057 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6058 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6055, a VHFWR2 amino acid sequence of SEQ ID NO: 6056, a VHFWR3 amino acid sequence of SEQ ID NO: 6057, or a VHFWR4 amino acid sequence of SEQ ID NO: 6058. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6133 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6133).

In some embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6059 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 amino acid sequence of SEQ ID NO: 6060 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 amino acid sequence of SEQ ID NO: 6061 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 amino acid sequence of SEQ ID NO: 6062 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6059, a VHFWR2 amino acid sequence of SEQ ID NO: 6060, a VHFWR3 amino acid sequence of SEQ ID NO: 6061, or a VHFWR4 amino acid sequence of SEQ ID NO: 6062. In certain embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6134 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6134).

In some embodiments, wherein the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6097 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6098 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6099 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6100 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6097, a VLFWR2 amino acid sequence of SEQ ID NO: 6098, a VLFWR3 amino acid sequence of SEQ ID NO: 6099, or a VLFWR4 amino acid sequence of SEQ ID NO: 6100. In certain embodiments, wherein the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6142 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6142).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6101 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6102 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6103 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6104 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6101, a VLFWR2 amino acid sequence of SEQ ID NO: 6102, a VLFWR3 amino acid sequence of SEQ ID NO: 6103, or a VLFWR4 amino acid sequence of SEQ ID NO: 6104. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6143 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6143).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6105 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6106 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6107 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6108 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6105, a VLFWR2 amino acid sequence of SEQ ID NO: 6106, a VLFWR3 amino acid sequence of SEQ ID NO: 6107, or a VLFWR4 amino acid sequence of SEQ ID NO: 6108. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6144 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6144).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6109 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6110 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6111 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6112 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6109, a VLFWR2 amino acid sequence of SEQ ID NO: 6110, a VLFWR3 amino acid sequence of SEQ ID NO: 6111, or a VLFWR4 amino acid sequence of SEQ ID NO: 6112. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6145 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6145).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6113 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6114 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6115 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6116 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6113, a VLFWR2 amino acid sequence of SEQ ID NO: 6114, a VLFWR3 amino acid sequence of SEQ ID NO: 6115, or a VLFWR4 amino acid sequence of SEQ ID NO: 6116. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6146 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6146).

In some embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6117 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 amino acid sequence of SEQ ID NO: 6118 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 amino acid sequence of SEQ ID NO: 6119 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 amino acid sequence of SEQ ID NO: 6120 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom). In certain embodiments, the NK cell engager comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6117, a VLFWR2 amino acid sequence of SEQ ID NO: 6118, a VLFWR3 amino acid sequence of SEQ ID NO: 6119, or a VLFWR4 amino acid sequence of SEQ ID NO: 6120. In certain embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6147 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6147).

In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKp46. In certain embodiments, lysis of the lymphoma cell is mediated by NKp46. In some embodiments, the multifunctional molecule does not activate the NK cell when incubated with the NK cell in the absence of the tumor antigen on the lymphoma cell. In some embodiments, the multifunctional molecule activates the NK cell when the NK cell is a NKp46 expressing NK cell and the tumor antigen on the lymphoma cell is also present. In some embodiments, the multifunctional molecule does not activate the NK cell when the NK cell is not a NKp46 expressing NK cell and the tumor antigen on the lymphoma cell is also present. In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6182 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6182). In some embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6183 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6183). In some embodiments, the NK cell engager comprises an scFv comprising the amino acid sequence of SEQ ID NO: 6181 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6181).

In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKG2D. In certain embodiments, lysis of the lymphoma cell is mediated by NKG2D. In some embodiments, the multifunctional molecule does not activate the NK cell when incubated with the NK cell in the absence of the tumor antigen on the lymphoma cell. In some embodiments, the multifunctional molecule activates the NK cell when the NK cell is a NKG2D expressing NK cell and the tumor antigen on the lymphoma cell is also present. In some embodiments, the multifunctional molecule does not activate the NK cell when the NK cell is not a NKG2D expressing NK cell and the tumor antigen on the lymphoma cell is also present. In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6176 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6176). In some embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6177 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6177). In some embodiments, the NK cell engager comprises an scFv comprising the amino acid sequence of SEQ ID NO: 6175 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6175). In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6179 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6179). In some embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6180 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6180). In some embodiments, the NK cell engager comprises an scFv comprising the amino acid sequence of SEQ ID NO: 6178 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6178).

In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to CD16. In some embodiments, lysis of the lymphoma cell is mediated by CD16. In some embodiments, the multifunctional molecule does not activate the NK cell when incubated with the NK cell in the absence of the tumor antigen on the lymphoma cell. In some embodiments, the multifunctional molecule activates the NK cell when the NK cell is a CD16 expressing NK cell and the tumor antigen on the lymphoma cell is also present. In some embodiments, the multifunctional molecule does not activate the NK cell when the NK cell is not a CD16 expressing NK cell and the tumor antigen on the lymphoma cell is also present. In some embodiments, the NK cell engager comprises a VH comprising the amino acid sequence of SEQ ID NO: 6185 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6185). In some embodiments, the NK cell engager comprises a VL comprising the amino acid sequence of SEQ ID NO: 6186 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6186). In some embodiments, the NK cell engager comprises an scFv comprising the amino acid sequence of SEQ ID NO: 6184 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6184).

In some embodiments, the NK cell engager is a ligand, optionally, the ligand further comprises an immunoglobulin constant region, e.g., an Fc region. In certain embodiments, the NK cell engager is a ligand of NKp44 or NKp46, e.g., a viral HA. In certain embodiments, the NK cell engager is a ligand of DAP10, e.g., a coreceptor for NKG2D. In certain embodiments, the NK cell engager is a ligand of CD16, e.g., a CD16a/b ligand, e.g., a CD16a/b ligand further comprising an antibody Fc region.

B Cell, Macrophage & Dendritic Cell Engagers

Broadly, B cells, also known as B lymphocytes, are a type of white blood cell of the lymphocyte subtype. They function in the humoral immunity component of the adaptive immune system by secreting antibodies. Additionally, B cells present antigen (they are also classified as professional antigen-presenting cells (APCs)) and secrete cytokines. Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, cancer cells via phagocytosis. Besides phagocytosis, they play important roles in nonspecific defense (innate immunity) and also help initiate specific defense mechanisms (adaptive immunity) by recruiting other immune cells such as lymphocytes. For example, they are important as antigen presenters to T cells. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Dendritic cells (DCs) are antigen-presenting cells that function in processing antigen material and present it on the cell surface to the T cells of the immune system.

The present disclosure provides, inter alia, multispecific (e.g., bi-, tri-, quad-specific) or multifunctional molecules, that include, e.g., are engineered to contain, one or more B cell, macrophage, and/or dendritic cell engager that mediate binding to and/or activation of a B cell, macrophage, and/or dendritic cell.

Accordingly, in some embodiments, the immune cell engager comprises a B cell, macrophage, and/or dendritic cell engager chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to OX40; an OX40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., as described herein, e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4), or a TLR9 agonists); a 41BB; a CD2; a CD47; or a STING agonist, or a combination thereof.

In some embodiments, the B cell engager is a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to OX40, CD40 or CD70.

In some embodiments, the macrophage engager is a CD2 agonist. In some embodiments, the macrophage engager is an antigen binding domain that binds to: CD40L or antigen binding domain or ligand that binds CD40, a Toll like receptor (TLR) agonist (e.g., as described herein), e.g., a TLR9 or TLR4 (e.g., caTLR4 (constitutively active TLR4), CD47, or a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). In some embodiments, the STING agonist is biotinylated.

In some embodiments, the dendritic cell engager is a CD2 agonist. In some embodiments, the dendritic cell engager is a ligand, a receptor agonist, or an antibody molecule that binds to one or more of: OX40L, 41BB, a TLR agonist (e.g., as described herein) (e.g., TLR9 agonist, TLR4 (e.g., caTLR4 (constitutively active TLR4)), CD47, or and a STING agonist. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., cyclic di-GMP (cdGMP) or cyclic di-AMP (cdAMP). In some embodiments, the STING agonist is biotinylated.

In other embodiments, the immune cell engager mediates binding to, or activation of, one or more of a B cell, a macrophage, and/or a dendritic cell. Exemplary B cell, macrophage, and/or dendritic cell engagers can be chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to OX40; an OX40 ligand (OX40L); a Toll-like receptor agonist (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); a 41BB agonist; a CD2; a CD47; or a STING agonist, or a combination thereof.

In some embodiments, the B cell engager is chosen from one or more of a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to OX40, CD40 or CD70.

In other embodiments, the macrophage cell engager is chosen from one or more of a CD2 agonist; a CD40L; an OX40L; an antibody molecule that binds to OX40, CD40 or CD70; a Toll-like receptor agonist or a fragment thereof (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4)); a CD47 agonist; or a STING agonist.

In other embodiments, the dendritic cell engager is chosen from one or more of a CD2 agonist, an OX40 antibody, an OX40L, 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4)), CD47 agonist, or a STING agonist.

In one embodiment, the OX40L comprises the amino acid sequence:

(SEQ ID NO: 6210)

QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDG

FYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDK

VYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL,

In another embodiment the CD40L comprises the amino acid sequence:

(SEQ ID NO: 6211)

MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQL

TVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAAN

THSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLL

KL,

In yet other embodiments, the STING agonist comprises a cyclic dinucleotide, e.g., a cyclic di-GMP (cdGMP), a cyclic di-AMP (cdAMP), or a combination thereof, optionally with 2′,5′ or 3′,5′ phosphate linkages.

In one embodiment, the immune cell engager includes 41BB ligand, e.g., comprising the amino acid sequence:

(SEQ ID NO: 6212)

ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQ

NVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQL

ELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSA

FGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP

AGLPSPRSE,

Toll-Like Receptors

Toll-Like Receptors (TLRs) are evolutionarily conserved receptors are homologues of the Drosophila Toll protein, and recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by microbial pathogens, or danger-associated molecular patterns (DAMPs) that are endogenous molecules released from necrotic or dying cells. PAMPs include various bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN) and lipopeptides, as well as flagellin, bacterial DNA and viral double-stranded RNA. DAMPs include intracellular proteins such as heat shock proteins as well as protein fragments from the extracellular matrix. Stimulation of TLRs by the corresponding PAMPs or DAMPs initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB and interferon regulatory factors (IRFs). Signaling by TLRs results in a variety of cellular responses, including the production of interferons (IFNs), pro-inflammatory cytokines and effector cytokines that direct the adaptive immune response. TLRs are implicated in a number of inflammatory and immune disorders and play a role in cancer (Rakoff-Nahoum S. & Medzhitov R., 2009. Toll-like receptors and cancer. Nat Revs Cancer 9:57-63.)

TLRs are type I transmembrane proteins characterized by an extracellular domain containing leucine-rich repeats (LRRs) and a cytoplasmic tail that contains a conserved region called the Toll/IL-1 receptor (TIR) domain. Ten human and twelve murine TLRs have been characterized, TLR1 to TLR10 in humans, and TLR1 to TLR9, TLR11, TLR12 and TLR13 in mice, the homolog of TLR10 being a pseudogene. TLR2 is essential for the recognition of a variety of PAMPs from Gram-positive bacteria, including bacterial lipoproteins, lipomannans and lipoteichoic acids. TLR3 is implicated in virus-derived double-stranded RNA. TLR4 is predominantly activated by lipopolysaccharide. TLR5 detects bacterial flagellin and TLR9 is required for response to unmethylated CpG DNA. Finally, TLR7 and TLR8 recognize small synthetic antiviral molecules, and single-stranded RNA was reported to be their natural ligand. TLR11 has been reported to recognize uropathogenic E. coli and a profilin-like protein from Toxoplasma gondii. The repertoire of specificities of the TLRs is apparently extended by the ability of TLRs to heterodimerize with one another. For example, dimers of TLR2 and TLR6 are required for responses to diacylated lipoproteins while TLR2 and TLR1 interact to recognize triacylated lipoproteins. Specificities of the TLRs are also influenced by various adapter and accessory molecules, such as MD-2 and CD14 that form a complex with TLR4 in response to LPS.

TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a MyD88-independent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells. The MyD88-dependent pathway is common to all TLRs, except TLR3 (Adachi O. et al., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 9(1):143-50). Upon activation by PAMPs or DAMPs, TLRs hetero- or homodimerize inducing the recruitment of adaptor proteins via the cytoplasmic TIR domain. Individual TLRs induce different signaling responses by usage of the different adaptor molecules. TLR4 and TLR2 signaling requires the adaptor TIRAP/Mal, which is involved in the MyD88-dependent pathway. TLR3 triggers the production of IFN-β in response to double-stranded RNA, in a MyD88-independent manner, through the adaptor TRIF/TICAM-1. TRAM/TICAM-2 is another adaptor molecule involved in the MyD88-independent pathway which function is restricted to the TLR4 pathway.

TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFNs. The signaling mechanisms leading to the induction of type I IFNs differ depending on the TLR activated. They involve the interferon regulatory factors, IRFs, a family of transcription factors known to play a critical role in antiviral defense, cell growth and immune regulation. Three IRFs (IRF3, IRF5 and IRF7) function as direct transducers of virus-mediated TLR signaling. TLR3 and TLR4 activate IRF3 and IRF7, while TLR7 and TLR8 activate IRF5 and IRF7 (Doyle S. et al., 2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity. 17(3):251-63). Furthermore, type I IFN production stimulated by TLR9 ligand CpG-A has been shown to be mediated by PI(3)K and mTOR (Costa-Mattioli M. & Sonenberg N. 2008. RAPping production of type I interferon in pDCs through mTOR. Nature Immunol. 9: 1097-1099).

TLR-9

TLR9 recognizes unmethylated CpG sequences in DNA molecules. CpG sites are relatively rare (˜1%) on vertebrate genomes in comparison to bacterial genomes or viral DNA. TLR9 is expressed by numerous cells of the immune system such as B lymphocytes, monocytes, natural killer (NK) cells, and plasmacytoid dendritic cells. TLR9 is expressed intracellularly, within the endosomal compartments and functions to alert the immune system of viral and bacterial infections by binding to DNA rich in CpG motifs. TLR9 signals leads to activation of the cells initiating pro-inflammatory reactions that result in the production of cytokines such as type-I interferon and IL-12.

TLR Agonists

A TLR agonist can agonize one or more TLR, e.g., one or more of human TLR-1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, an adjunctive agent described herein is a TLR agonist. In some embodiments, the TLR agonist specifically agonizes human TLR-9. In some embodiments, the TLR-9 agonist is a CpG moiety. As used herein, a CpG moiety, is a linear dinucleotide having the sequence: 5′-C-phosphate-G-3′, that is, cytosine and guanine separated by only one phosphate.

In some embodiments, the CpG moiety comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more CpG dinucleotides. In some embodiments, the CpG moiety consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 CpG dinucleotides. In some embodiments, the CpG moiety has 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 10-20, 10-30, 10-40, or 10-50 CpG dinucleotides.

In some embodiments, the TLR-9 agonist is a synthetic ODN (oligodeoxynucleotides). CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs). CpG ODNs possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO) backbone found in genomic bacterial DNA. There are three major classes of CpG ODNs: classes A, B and C, which differ in their immunostimulatory activities. CpG-A ODNs are characterized by a PO central CpG-containing palindromic motif and a PS-modified 3′ poly-G string. They induce high IFN-α production from pDCs but are weak stimulators of TLR9-dependent NF-κB signaling and pro-inflammatory cytokine (e.g. IL-6) production. CpG-B ODNs contain a full PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion. CpG-C ODNs combine features of both classes A and B. They contain a complete PS backbone and a CpG-containing palindromic motif C-Class CpG ODNs induce strong IFN-α production from pDC as well as B cell stimulation.

Cytokine Molecules

Cytokines are generally polypeptides that influence cellular activity, for example, through signal transduction pathways. Accordingly, a cytokine of the multispecific or multifunctional polypeptide is useful and can be associated with receptor-mediated signaling that transmits a signal from outside the cell membrane to modulate a response within the cell. Cytokines are proteinaceous signaling compounds that are mediators of the immune response. They control many different cellular functions including proliferation, differentiation and cell survival/apoptosis; cytokines are also involved in several pathophysiological processes including viral infections and autoimmune diseases. Cytokines are synthesized under various stimuli by a variety of cells of both the innate (monocytes, macrophages, dendritic cells) and adaptive (T- and B-cells) immune systems. Cytokines can be classified into two groups: pro- and anti-inflammatory. Pro-inflammatory cytokines, including IFNγ, IL-1, IL-6 and TNF-alpha, are predominantly derived from the innate immune cells and Th1 cells. Anti-inflammatory cytokines, including IL-10, IL-4, IL-13 and IL-5, are synthesized from Th2 immune cells.

The present disclosure provides, inter alia, multispecific (e.g., bi-, tri-, quad-specific) or multifunctional molecules, that include, e.g., are engineered to contain, one or more cytokine molecules, e.g., immunomodulatory (e.g., proinflammatory) cytokines and variants, e.g., functional variants, thereof. Accordingly, in some embodiments, the cytokine molecule is an interleukin or a variant, e.g., a functional variant thereof. In some embodiments the interleukin is a proinflammatory interleukin. In some embodiments the interleukin is chosen from interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-7 (IL-7), or interferon gamma. In some embodiments, the cytokine molecule is a proinflammatory cytokine.

In certain embodiments, the cytokine is a single chain cytokine. In certain embodiments, the cytokine is a multichain cytokine (e.g., the cytokine comprises 2 or more (e.g., 2) polypeptide chains. An exemplary multichain cytokine is IL-12.

Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-21, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNFβ. In one embodiment the cytokine of the multispecific or multifunctional polypeptide is a cytokine selected from the group of GM-CSF, IL-2, IL-7, IL-8, IL-10, IL-12, IL-15, IL-21, IFN-α, IFN-γ, MIP-1α, MIP-1p and TGF-β. In one embodiment the cytokine of the multispecific or multifunctional polypeptide is a cytokine selected from the group of IL-2, IL-7, IL-10, IL-12, IL-15, IFN-α, and IFN-γ. In certain embodiments the cytokine is mutated to remove N- and/or O-glycosylation sites. Elimination of glycosylation increases homogeneity of the product obtainable in recombinant production. In certain embodiments, the cytokine is TGF-β. In certain embodiments, the multispecific or multifunctional polypeptide comprises a TGF-β inhibitor.

In one embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-2. In a specific embodiment, the IL-2 cytokine can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity. In another particular embodiment the IL-2 cytokine is a mutant IL-2 cytokine having reduced binding affinity to the .alpha.subunit of the IL-2 receptor. Together with the .beta. and .gamma.subunits (also known as CD122 and CD132, respectively), the .alpha.subunit (also known as CD25) forms the heterotrimeric high-affinity IL-2 receptor, while the dimeric receptor consisting only of the β- and γ-subunits is termed the intermediate-affinity IL-2 receptor. As described in PCT patent application number PCT/EP2012/051991, which is incorporated herein by reference in its entirety, a mutant IL-2 polypeptide with reduced binding to the .alpha.subunit of the IL-2 receptor has a reduced ability to induce IL-2 signaling in regulatory T cells, induces less activation-induced cell death (AICD) in T cells, and has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. The use of such an cytokine with reduced toxicity is particularly advantageous in a multispecific or multifunctional polypeptide according to the invention, having a long serum half-life due to the presence of an Fc domain. In one embodiment, the mutant IL-2 cytokine of the multispecific or multifunctional polypeptide according to the invention comprises at least one amino acid mutation that reduces or abolishes the affinity of the mutant IL-2 cytokine to the .alpha.subunit of the IL-2 receptor (CD25) but preserves the affinity of the mutant IL-2 cytokine to the intermediate-affinity IL-2 receptor (consisting of the β and γ subunits of the IL-2 receptor), compared to the non-mutated IL-2 cytokine. In one embodiment the one or more amino acid mutations are amino acid substitutions. In a specific embodiment, the mutant IL-2 cytokine comprises one, two or three amino acid substitutions at one, two or three position(s) selected from the positions corresponding to residue 42, 45, and 72 of human IL-2. In a more specific embodiment, the mutant IL-2 cytokine comprises three amino acid substitutions at the positions corresponding to residue 42, 45 and 72 of human IL-2. In an even more specific embodiment, the mutant IL-2 cytokine is human IL-2 comprising the amino acid substitutions F42A, Y45A and L72G. In one embodiment the mutant IL-2 cytokine additionally comprises an amino acid mutation at a position corresponding to position 3 of human IL-2, which eliminates the O-glycosylation site of IL-2. Particularly, said additional amino acid mutation is an amino acid substitution replacing a threonine residue by an alanine residue. A particular mutant IL-2 cytokine useful in the invention comprises four amino acid substitutions at positions corresponding to residues 3, 42, 45 and 72 of human IL-2. Specific amino acid substitutions are T3A, F42A, Y45A and L72G. As demonstrated in PCT patent application number PCT/EP2012/051991 and in the appended Examples, said quadruple mutant IL-2 polypeptide (IL-2 qm) exhibits no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in T.sub.reg cells, and a reduced toxicity profile in vivo. However, it retains ability to activate IL-2 signaling in effector cells, to induce proliferation of effector cells, and to generate IFN-γ as a secondary cytokine by NK cells.

The IL-2 or mutant IL-2 cytokine according to any of the above embodiments may comprise additional mutations that provide further advantages such as increased expression or stability. For example, the cysteine at position 125 may be replaced with a neutral amino acid such as alanine, to avoid the formation of disulfide-bridged IL-2 dimers. Thus, in certain embodiments the IL-2 or mutant IL-2 cytokine of the multispecific or multifunctional polypeptide according to the invention comprises an additional amino acid mutation at a position corresponding to residue 125 of human IL-2. In one embodiment said additional amino acid mutation is the amino acid substitution C125A.

In a specific embodiment, the IL-2 cytokine of the multispecific or multifunctional polypeptide comprises the polypeptide sequence of SEQ ID NO: 6364

[APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPK

KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK

GSETTFMCEYADETATIVEFLNRWITFAQSIISTLT].

In another specific embodiment the IL-2 cytokine of the multispecific or multifunctional polypeptide comprises the polypeptide sequence of SEQ ID NO: 6365

[APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPK

KATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELK

GSETTFMCEYADETATIVEFLNRWITFAQSIISTLT].

In another embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-12. In a specific embodiment, said IL-12 cytokine is a single chain IL-12 cytokine. In an even more specific embodiment, the single chain IL-12 cytokine comprises the polypeptide sequence of SEQ ID NO: 6366

[IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLG

SGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKD

QKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGV

TCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHK

LKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPH

SYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYY

SSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQN

LLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTK

NESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTM

NAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYK

TKIKLCILLHAFRIRAVTIDRVMSYLNAS].

In one embodiment, the IL-12 cytokine can elicit one or more of the cellular responses selected from the group consisting of: proliferation in a NK cell, differentiation in a NK cell, proliferation in a T cell, and differentiation in a T cell.

In another embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-10. In a specific embodiment, said IL-10 cytokine is a single chain IL-10 cytokine. In an even more specific embodiment, the single chain IL-10 cytokine comprises the polypeptide sequence of SEQ ID NO: 6367

[SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLL

KESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGEN

LKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIF

INYIEAYMTMKIRNGGGGSGGGGSGGGGSGGGGSSPGQGTQSENSCTHF

PGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA

LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLP

CENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN].

In another specific embodiment, the IL-10 cytokine is a monomeric IL-10 cytokine. In a more specific embodiment, the monomeric IL-10 cytokine comprises the polypeptide sequence of SEQ ID NO: 6368

[SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLL

KESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGEN

LKTLRLRLRRCHRFLPCENGGGSGGKSKAVEQVKNAFNKLQEKGIYKAM

SEFDIFINYIEAYMTMKIRN].

In one embodiment, the IL-10 cytokine can elicit one or more of the cellular responses selected from the group consisting of: inhibition of cytokine secretion, inhibition of antigen presentation by antigen presenting cells, reduction of oxygen radical release, and inhibition of T cell proliferation. A multispecific or multifunctional polypeptide according to the invention wherein the cytokine is IL-10 is particularly useful for downregulation of inflammation, e.g. in the treatment of an inflammatory disorder.

In another embodiment, the cytokine of the multispecific or multifunctional polypeptide is IL-15. In a specific embodiment said IL-15 cytokine is a mutant IL-15 cytokine having reduced binding affinity to the α-subunit of the IL-15 receptor. Without wishing to be bound by theory, a mutant IL-15 polypeptide with reduced binding to the .alpha.subunit of the IL-15 receptor has a reduced ability to bind to fibroblasts throughout the body, resulting in improved pharmacokinetics and toxicity profile, compared to a wild-type IL-15 polypeptide. The use of an cytokine with reduced toxicity, such as the described mutant IL-2 and mutant IL-15 effector moieties, is particularly advantageous in a multispecific or multifunctional polypeptide according to the invention, having a long serum half-life due to the presence of an Fc domain. In one embodiment the mutant IL-15 cytokine of the multispecific or multifunctional polypeptide according to the invention comprises at least one amino acid mutation that reduces or abolishes the affinity of the mutant IL-15 cytokine to the .alpha.subunit of the IL-15 receptor but preserves the affinity of the mutant IL-15 cytokine to the intermediate-affinity IL-15/IL-2 receptor (consisting of the .beta. and .gamma.subunits of the IL-15/IL-2 receptor), compared to the non-mutated IL-15 cytokine. In one embodiment the amino acid mutation is an amino acid substitution. In a specific embodiment, the mutant IL-15 cytokine comprises an amino acid substitution at the position corresponding to residue 53 of human IL-15. In a more specific embodiment, the mutant IL-15 cytokine is human IL-15 comprising the amino acid substitution E53A. In one embodiment the mutant IL-15 cytokine additionally comprises an amino acid mutation at a position corresponding to position 79 of human IL-15, which eliminates the N-glycosylation site of IL-15. Particularly, said additional amino acid mutation is an amino acid substitution replacing an asparagine residue by an alanine residue. In an even more specific embodiment the IL-15 cytokine comprises the polypeptide sequence of SEQ ID NO: 6370 [NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGDASIHDTVEN LIILANNSLSSNGAVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS]. In one embodiment, the IL-15 cytokine can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity.

Mutant cytokine molecules useful as effector moieties in the multispecific or multifunctional polypeptide can be prepared by deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing. Substitution or insertion may involve natural as well as non-natural amino acid residues. Amino acid modification includes well known methods of chemical modification such as the addition or removal of glycosylation sites or carbohydrate attachments, and the like.

In one embodiment, the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is GM-CSF. In a specific embodiment, the GM-CSF cytokine can elicit proliferation and/or differentiation in a granulocyte, a monocyte or a dendritic cell. In one embodiment, the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is IFN-α. In a specific embodiment, the IFN-α cytokine can elicit one or more of the cellular responses selected from the group consisting of: inhibiting viral replication in a virus-infected cell, and upregulating the expression of major histocompatibility complex I (MHC I). In another specific embodiment, the IFN-α cytokine can inhibit proliferation in a tumor cell. In one embodiment the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is IFNγ. In a specific embodiment, the IFN-γ cytokine can elicit one or more of the cellular responses selected from the group of: increased macrophage activity, increased expression of MHC molecules, and increased NK cell activity. In one embodiment the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is IL-7. In a specific embodiment, the IL-7 cytokine can elicit proliferation of T and/or B lymphocytes. In one embodiment, the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is IL-8. In a specific embodiment, the IL-8 cytokine can elicit chemotaxis in neutrophils. In one embodiment, the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide, is MIP-1α. In a specific embodiment, the MIP-1α cytokine can elicit chemotaxis in monocytes and T lymphocyte cells. In one embodiment, the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is MIP-1β. In a specific embodiment, the MIP-1β cytokine can elicit chemotaxis in monocytes and T lymphocyte cells. In one embodiment, the cytokine, particularly a single-chain cytokine, of the multispecific or multifunctional polypeptide is TGF-β. In a specific embodiment, the TGF-β cytokine can elicit one or more of the cellular responses selected from the group consisting of: chemotaxis in monocytes, chemotaxis in macrophages, upregulation of IL-1 expression in activated macrophages, and upregulation of IgA expression in activated B cells.

In one embodiment, the multispecific or multifunctional polypeptide of the invention binds to an cytokine receptor with a dissociation constant (K_D) that is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times greater than that for a control cytokine. In another embodiment, the multispecific or multifunctional polypeptide binds to an cytokine receptor with a K_Dthat is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than that for a corresponding multispecific or multifunctional polypeptide comprising two or more effector moieties. In another embodiment, the multispecific or multifunctional polypeptide binds to an cytokine receptor with a dissociation constant K_Dthat is about 10 times greater than that for a corresponding the multispecific or multifunctional polypeptide comprising two or more cytokines.

In some embodiments, the multispecific molecules disclosed herein include a cytokine molecule. In some embodiments, the cytokine molecule includes a full length, a fragment or a variant of a cytokine; a cytokine receptor domain, e.g., a cytokine receptor dimerizing domain; or an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor.

In some embodiments the cytokine molecule is chosen from IL-2, IL-12, IL-15, IL-18, IL-7, IL-21, or interferon gamma, or a fragment or variant thereof, or a combination of any of the aforesaid cytokines. The cytokine molecule can be a monomer or a dimer. In some embodiments, the cytokine molecule can further include a cytokine receptor dimerizing domain.

In other embodiments, the cytokine molecule is an agonist of a cytokine receptor, e.g., an antibody molecule (e.g., an agonistic antibody) to a cytokine receptor chosen from an IL-15Ra or IL-21R.

In one embodiment, the cytokine molecule is IL-15, e.g., human IL-15 (e.g., comprising the amino acid sequence.

(SEQ ID NO: 6191)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQV

ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKE

FLQSFVHIVQMFINTS,

In some embodiments, the cytokine molecule comprises a receptor dimerizing domain, e.g., an IL15Ralpha dimerizing domain. In one embodiment, the IL15Ralpha dimerizing domain comprises the amino acid sequence:

(SEQ ID NO: 6192)

MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSY

SLYSRERYICNSGFKRKAGTSSLTECVL,

a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 6192. In some embodiments, the cytokine molecule (e.g., IL-15) and the receptor dimerizing domain (e.g., an IL15Ralpha dimerizing domain) of the multispecific molecule are covalently linked, e.g., via a linker (e.g., a Gly-Ser linker, e.g., a linker comprising the amino acid sequence SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 6193). In other embodiments, the cytokine molecule (e.g., IL-15) and the receptor dimerizing domain (e.g., an IL15Ralpha dimerizing domain) of the multispecific molecule are not covalently linked, e.g., are non-covalently associated.

In other embodiments, the cytokine molecule is IL-2, e.g., human IL-2 (e.g., comprising the amino acid sequence:

(SEQ ID NO: 6194)

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKK

ATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKG

SETTFMCEYADETATIVEFLNRWITFCQSIISTLT,

In other embodiments, the cytokine molecule is IL-18, e.g., human IL-18 (e.g., comprising the amino acid sequence:

(SEQ ID NO: 6195)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFII

SMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSD

IIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDR

SIMFTVQNED,

In other embodiments, the cytokine molecule is IL-21, e.g., human IL-21 (e.g., comprising the amino acid sequence:

(SEQ ID NO: 6196)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSC

FQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDS

YEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS,

In yet other embodiments, the cytokine molecule is interferon gamma, e.g., human interferon gamma (e.g., comprising the amino acid sequence:

(SEQ ID NO: 6197)

QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQI

VSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNY

SVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG,

TGF-β Inhibitor

In one aspect, provided herein is a multispecific or multifunctional polypeptide (e.g., antibody molecule) comprising a modulator of TGF-β (e.g., a TGF-β inhibitor). In some embodiments, the TGF-β inhibitor binds to and inhibits TGF-β, e.g., reduces the activity of TGF-β. In some embodiments, the TGF-β inhibitor inhibits (e.g., reduces the activity of) TGF-β 1. In some embodiments, the TGF-β inhibitor inhibits (e.g., reduces the activity of) TGF-β 2. In some embodiments, the TGF-β inhibitor inhibits (e.g., reduces the activity of) TGF-β 3. In some embodiments, the TGF-β inhibitor inhibits (e.g., reduces the activity of) TGF-β 1 and TGF-β 3. In some embodiments, the TGF-β inhibitor inhibits (e.g., reduces the activity of) TGF-β 1, TGF-β 2, and TGF-β 3.

In some embodiments, the TGF-β inhibitor comprises a portion of a TGF-β receptor (e.g., an extracellular domain of a TGF-β receptor) that is capable of inhibiting (e.g., reducing the activity of) TGF-β, or functional fragment or variant thereof. In some embodiments, the TGF-β inhibitor comprises a TGFBR1 polypeptide (e.g., an extracellular domain of TGFBR1 or functional variant thereof). In some embodiments, the TGF-β inhibitor comprises a TGFBR2 polypeptide (e.g., an extracellular domain of TGFBR2 or functional variant thereof). In some embodiments, the TGF-β inhibitor comprises a TGFBR3 polypeptide (e.g., an extracellular domain of TGFBR3 or functional variant thereof). In some embodiments, the TGF-β inhibitor comprises a TGFBR1 polypeptide (e.g., an extracellular domain of TGFBR1 or functional variant thereof) and a TGFBR2 polypeptide (e.g., an extracellular domain of TGFBR2 or functional variant thereof). In some embodiments, the TGF-β inhibitor comprises a TGFBR1 polypeptide (e.g., an extracellular domain of TGFBR1 or functional variant thereof) and a TGFBR3 polypeptide (e.g., an extracellular domain of TGFBR3 or functional variant thereof). In some embodiments, the TGF-β inhibitor comprises a TGFBR2 polypeptide (e.g., an extracellular domain of TGFBR2 or functional variant thereof) and a TGFBR3 polypeptide (e.g., an extracellular domain of TGFBR3 or functional variant thereof).

Exemplary TGF-β receptor polypeptides that can be used as TGF-β inhibitors have been disclosed in U.S. Pat. Nos. 8,993,524, 9,676,863, 8,658,135, US20150056199, US20070184052, and WO2017037634, all of which are herein incorporated by reference in their entirety.

In some embodiments, the TGF-β inhibitor comprises an extracellular domain of TGFBR1 or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6381, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6382, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6383, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6390, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6391, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto).

In some embodiments, the TGF-β inhibitor comprises an extracellular domain of TGFBR2 or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6384, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6385, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6386, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6387, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6388, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6389, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto).

In some embodiments, the TGF-β inhibitor comprises an extracellular domain of TGFBR3 or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6392, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises an extracellular domain of SEQ ID NO: 6393, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto). In some embodiments, the TGF-β inhibitor comprises the amino acid sequence of SEQ ID NO: 6394, or a sequence substantially identical thereto (e.g., a sequence that is at least 80%, 85%, 90%, or 95% identical thereto).

In some embodiments, the TGF-β inhibitor comprises no more than one TGF-β receptor extracellular domain. In some embodiments, the TGF-β inhibitor comprises two or more (e.g., two, three, four, five, or more) TGF-β receptor extracellular domains, linked together, e.g., via a linker.

In some embodiments, the multispecific molecule comprises a configuration shown in FIGS. 24A-24D. In some embodiments, the TGFRβ inhibitor comprises a TGF-beta receptor ECD homodimer. In some embodiments, the TGFRβ inhibitor comprises a TGF-beta receptor ECD heterodimer. In some embodiments, the two TGFBR ECD domains are linked to two Fc regions, e.g., the C-terminus of two Fc regions. In some embodiments, the two TGFBR ECD domains are linked to CH1 and CL, respectively.

TABLE 37

Exemplary amino acid sequences of TGF-β polypeptides or TGF-β receptor polypeptides

SEQ ID

NO
Description
Amino acid sequence

SEQ ID
Immature
MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIE

NO:
human
AIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEP

6378
TGF-ß 1
EPEPEADYYAKEVTRVLMVETHNEIYDKFKQSTHSIYMFFNTSELRE

(P01137-1)
AVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWRYLSNRLLA

PSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQV

DINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRAL

DTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPC

PYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGR

KPKVEQLSNMIVRSCKCS

SEQ ID
Human
LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVL

NO:
TGF-ß 1
ALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEIYDKF

6395
(P01137-1)
KQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELY

QKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGF

RLSAHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMAT

PLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWK

WIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPC

CVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

SEQ ID
Immature
MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSK

NO:
human
LKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSD

6379
TGF-ß 2
EEYYAKEVYKIDMPPFFPSENAIPPTFYRPYFRIVRFDVSAMEKNASN

(P61812-1)
LVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVV

KTRAEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNN

YIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLM

LLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDL

GWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASA

SPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS

SEQ ID
Human
LSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVIS

NO:
TGF-ß 2
IYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPFFPSEN

6396
(P61812-1)
AIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPE

QRIELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHE

WLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTS

TYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRA

LDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAG

ACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGK

TPKIEQLSNMIVKSCKCS

SEQ ID
Immature
MKMHLQRALVVLALLNFATVSLSLSTCTTLDFGHIKKKRVEAIRGQI

NO:
human
LSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQE

6380
TGF-β 3
NTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEK

(P10600-1)
NRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGK

NLPTRGTAEWLSFDVTDTVREWLLRRESNLGLEISIHCPCHTFQPNG

DILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILM

MIPPHRLDNPGQGGQRKKRALDTNYCFRNLEENCCVRPLYIDFRQD

LGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEA

SASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS

SEQ ID
Human
LSTCTTLDFGHIKKKRVEAIRGQILSKLRLTSPPEPTVMTHVPYQVLA

NO:
TGF-ß 3
LYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEH

6397
(P10600-1)
NELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRN

EQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTDTVREW

LLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDH

GRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDT

NYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCP

YLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTP

KVEQLSNMVVKSCKCS

SEQ ID
Immature
MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDN

NO:
human
FTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTG

6381
TGFBR1
SVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCFVCISL

isoform 1
MLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSG

(P36897-1)
SGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAVKIFS

SREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWLVSD

YHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQGKP

AIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVG

TKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSIGGIH

EDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRV

MAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM

SEQ ID
Human
LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRD

NO:
TGFBR1
RPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAA

6398
isoform 1
VIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTT

(P36897-1)
LKDLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGK

WRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDN

GTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAH

LHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSA

TDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLV

FWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNI

PNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQE

GIKM

SEQ ID
Immature
MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDN

NO:
human
FTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTG

6382
TGFBR1
SVTTTYCCNQDHCNKIELPTTGPFSVKSSPGLGPVELAAVIAGPVCF

isoform 2
VCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDM

(P36897-2)
TTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVWRGKWRGEEVAV

KIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGTWTQLWL

VSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQ

GKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRHDSATDTIDIAPNH

RVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVFWEIARRCSI

GGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEA

LRVMAKIMRECWYANGAARLTALRIKKTLSQLSQQEGIKM

SEQ ID
Human
LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRD

NO:
TGFBR1
RPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTGPFSVKSSPGLGPVE

6399
isoform 2
LAAVIAGPVCFVCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISE

(P36897-2)
GTTLKDLIYDMTTSGSGSGLPLLVQRTIARTIVLQESIGKGRFGEVW

RGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADN

KDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASG

LAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVRH

DSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAM

GLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLR

PNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLSQLSQ

QEGIKM

SEQ ID
Immature
MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDN

NO:
human
FTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTG

6383
TGFBR1
SVTTTYCCNQDHCNKIELPTTGLPLLVQRTIARTIVLQESIGKGRFGE

isoform 3
VWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIA

(P36897-3)
ADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALST

ASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLA

VRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIY

AMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQ

KLRPNIPNRWQSCEALRVMAKIMRECWYANGAARLTALRIKKTLS

QLSQQEGIKM

SEQ ID
Human
LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRD

NO:
TGFBR1
RPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTGLPLLVQRTIARTIV

6400
isoform 3
LQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTV

(P36897-3)
MLRHENILGFIAADNKDNGTWTQLWLVSDYHEHGSLFDYLNRYTV

TVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNILVKK

NGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSIN

MKHFESFKRADIYAMGLVFWEIARRCSIGGIHEDYQLPYYDLVPSDP

SVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWYANGA

ARLTALRIKKTLSQLSQQEGIKM

SEQ ID
Human
LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRD

NO:
TGFBR1
RPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVEL

6390
fragment 1

SEQ ID
Human
ALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPR

NO:
TGFBR1
DRPFVCAPSSKTGSVTTTYCCNQDHCNKIEL

6391
fragment 2

SEQ ID
Immature
MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVK

NO:
human
FPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKND

6384
TGFBR2
ENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSS

isoform B
DECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRV

(short
NRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHN

isoform)
TELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYA

(P37173-1)
SWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGN

LQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIVHRD

LKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTARYM

APEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKD

YEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCE

TLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPEDGS

LNTTK

SEQ ID
Human
TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCM

NO:
TGFBR2
SNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6401
isoform B
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIF

(short
QVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFS

isoform)
EHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKA

(P37173-1)
KLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLT

AEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLA

RGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSL

RLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDV

YSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVL

RDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERF

SELEHLDRLSGRSCSEEKIPEDGSLNTTK

SEQ ID
Immature
MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSDVEMEAQKDEIICPSC

NO:
human
NRTAHPLRHINNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCM

6385
TGFBR2
SNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

isoform A
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIF

(long
QVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFS

isoform)
EHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKA

(P37173-2)
KLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLT

AEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLA

RGIAHLHSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSL

RLDPTLSVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDV

YSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREHPCVESMKDNVL

RDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERF

SELEHLDRLSGRSCSEEKIPEDGSLNTTK

SEQ ID
Human
TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGA

NO:
TGFBR2
VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

6402
isoform A
NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCS

(long
CSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYC

isoform)
YRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNI

(P37173-2)
NHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYE

EYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHA

KGNLQEYLTRHVISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMPIV

HRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGTA

RYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGE

VKDYEPPFGSKVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQM

VCETLTECWDHDPEARLTAQCVAERFSELEHLDRLSGRSCSEEKIPE

DGSLNTTK

SEQ ID
Human
TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCM

NO:
TGFBR2
SNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6386
fragment 1
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(ECD of

human

TGFBR2

isoform B)

SEQ ID
Human
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS

NO:
TGFBR2
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6387
fragment 2
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

SEQ ID
Human
TIPPHVQKSDVEMEAQKDEIICPSCNRTAHPLRHINNDMIVTDNNGA

NO:
TGFBR2
VKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRK

6388
fragment 3
NDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCS

(ECD of
CSSDECNDNIIFSEEYNTSNPD

human

TGFBR2

isoform A)

SEQ ID
Human
QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI

NO:
TGFBR2
TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE

6389
fragment 4
CNDNIIF

SEQ ID
Immature
MTSHYVIAIFALMSSCLATAGPEPGALCELSPVSASHPVQALMESFT

NO:
human
VLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPISSV

6392
TGFBR3
HIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSA

isoform 1
NFSLTAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKV

(Q03167-1)
GEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCVMSSQPQNEEVH

IIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVI

KSFDVKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWA

LDNGYSPITSYTMAPVANRFHLRLENNAEEMGDEEVHTIPPELRILL

DPGALPALQNPPIRGGEGQNGGLPFPFPDISRRVWNEEGEDGLPRPK

DPVIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQAS

GYSGMDVTLLDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVV

YYNSIVIQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTR

PEIVVFNCSLQQVRNPSSFQEQPHGNITFNMELYNTDLFLVPSQGVFS

VPENGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENIC

PKDESVKFYSPKRVHFPIPQADMDKKRFSFVFKPVFNTSLLFLQCEL

TLCTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPLA

VIHHEAESKEKGPSMKEPNPISPPIFHGLDTLTVMGIAFAAFVIGALL

TGALWYIYSHTGETAGRQQVPTSPPASENSSAAHSIGSTQSTPCSSSS

TA

SEQ ID
Human
GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVL

NO:
TGFBR3
NLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHL

6403
isoform 1
KTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLL

(Q03167-1)
NWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNY

LAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIR

PSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGFGKES

ERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFH

LRLENNAEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNG

GLPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSV

DIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNG

THFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDG

YEDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQ

PHGNITFNMELYNTDLFLVPSQGVFSVPENGHVYVEVSVTKAEQEL

GFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQA

DMDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPD

EACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKEPNPI

SPPIFHGLDTLTVMGIAFAAFVIGALLTGALWYIYSHTGETAGRQQV

PTSPPASENSSAAHSIGSTQSTPCSSSSTA

SEQ ID
Immature
MTSHYVIAIFALMSSCLATAGPEPGALCELSPVSASHPVQALMESFT

NO:
human
VLSGCASRGTTGLPQEVHVLNLRTAGQGPGQLQREVTLHLNPISSV

6393
TGFBR3
HIHHKSVVFLLNSPHPLVWHLKTERLATGVSRLFLVSEGSVVQFSSA

isoform 2
NFSLTAETEERNFPHGNEHLLNWARKEYGAVTSFTELKIARNIYIKV

(Q03167-2)
GEDQVFPPKCNIGKNFLSLNYLAEYLQPKAAEGCVMSSQPQNEEVH

IIELITPNSNPYSAFQVDITIDIRPSQEDLEVVKNLILILKCKKSVNWVI

KSFDVKGSLKIIAPNSIGFGKESERSMTMTKSIRDDIPSTQGNLVKWA

LDNGYSPITSYTMAPVANRFHLRLENNEEMGDEEVHTIPPELRILLDP

GALPALQNPPIRGGEGQNGGLPFPFPDISRRVWNEEGEDGLPRPKDP

VIPSIQLFPGLREPEEVQGSVDIALSVKCDNEKMIVAVEKDSFQASGY

SGMDVTLLDPTCKAKMNGTHFVLESPLNGCGTRPRWSALDGVVYY

NSIVIQVPALGDSSGWPDGYEDLESGDNGFPGDMDEGDASLFTRPEI

VVFNCSLQQVRNPSSFQEQPHGNITFNMELYNTDLFLVPSQGVFSVP

ENGHVYVEVSVTKAEQELGFAIQTCFISPYSNPDRMSHYTIIENICPK

DESVKFYSPKRVHFPIPQADMDKKRFSFVFKPVFNTSLLFLQCELTL

CTKMEKHPQKLPKCVPPDEACTSLDASIIWAMMQNKKTFTKPLAVI

HHEAESKEKGPSMKEPNPISPPIFHGLDTLTVMGIAFAAFVIGALLTG

ALWYIYSHTGETAGRQQVPTSPPASENSSAAHSIGSTQSTPCSSSSTA

SEQ ID
Human
GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVL

NO:
TGFBR3
NLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHL

6404
isoform 2
KTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLL

(Q03167-2)
NWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNY

LAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIR

PSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGFGKES

ERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFH

LRLENNEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNGG

LPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSVD

IALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNGT

HFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDGY

EDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQP

HGNITFNMELYNTDLFLVPSQGVFSVPENGHVYVEVSVTKAEQELG

FAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQAD

MDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPDE

ACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKEPNPIS

PPIFHGLDTLTVMGIAFAAFVIGALLTGALWYIYSHTGETAGRQQVP

TSPPASENSSAAHSIGSTQSTPCSSSSTA

SEQ ID
Human
GPEPGALCELSPVSASHPVQALMESFTVLSGCASRGTTGLPQEVHVL

NO:
TGFBR3
NLRTAGQGPGQLQREVTLHLNPISSVHIHHKSVVFLLNSPHPLVWHL

6394
fragment 1
KTERLATGVSRLFLVSEGSVVQFSSANFSLTAETEERNFPHGNEHLL

NWARKEYGAVTSFTELKIARNIYIKVGEDQVFPPKCNIGKNFLSLNY

LAEYLQPKAAEGCVMSSQPQNEEVHIIELITPNSNPYSAFQVDITIDIR

PSQEDLEVVKNLILILKCKKSVNWVIKSFDVKGSLKIIAPNSIGFGKES

ERSMTMTKSIRDDIPSTQGNLVKWALDNGYSPITSYTMAPVANRFH

LRLENNAEEMGDEEVHTIPPELRILLDPGALPALQNPPIRGGEGQNG

GLPFPFPDISRRVWNEEGEDGLPRPKDPVIPSIQLFPGLREPEEVQGSV

DIALSVKCDNEKMIVAVEKDSFQASGYSGMDVTLLDPTCKAKMNG

THFVLESPLNGCGTRPRWSALDGVVYYNSIVIQVPALGDSSGWPDG

YEDLESGDNGFPGDMDEGDASLFTRPEIVVFNCSLQQVRNPSSFQEQ

PHGNITFNMELYNTDLFLVPSQGVFSVPENGHVYVEVSVTKAEQEL

GFAIQTCFISPYSNPDRMSHYTIIENICPKDESVKFYSPKRVHFPIPQA

DMDKKRFSFVFKPVFNTSLLFLQCELTLCTKMEKHPQKLPKCVPPD

EACTSLDASIIWAMMQNKKTFTKPLAVIHHEAESKEKGPSMKEPNPI

SPPIFHGLDTLTV

SEQ ID
hCH1-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

NO:
hFc_Hole-
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

6405
3x4GS-
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TGFbR2
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCT

LPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGXGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFP

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI

TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE

CNDNIIFSEEYNTSNPD, wherein X is K or absent

SEQ ID
hCH1-
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT

NO:
hFc_Knob-
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV

6406
3x4GS-
DKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV

TGFbR2
TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT

LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP

VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

SLSPGXGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFP

QLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENI

TLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDE

CNDNIIFSEEYNTSNPD, wherein X is K or absent

SEQ ID
hFc_Hole-
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

NO:
3x4GS-
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

6407
TGFbR2
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMT

KNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGXGGGG

SGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRF

STCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPK

LPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEY

NTSNPD, wherein X is K or absent

SEQ ID
hFc_Knob-
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

NO:
3x4GS-
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD

6408
TGFbR2
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEM

TKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGXGG

GGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCD

VRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCH

DPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFS

EEYNTSNPD, wherein X is K or absent

SEQ ID
TGFbR2-
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS

NO:
3x4GS-
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6409
hCH1-
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGS

hFc_Hole
GGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

KGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGX, wherein X is K or absent

SEQ ID
TGFbR2-
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS

NO:
3x4GS-
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6410
hCH1-
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGS

hFc_Knob
GGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA

KGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNG

QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA

LHNHYTQKSLSLSPGX, wherein X is K or absent

SEQ ID
TGFbR2-
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS

NO:
3x4GS-
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6411
hCLIg_vl
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGS

GGGGSGGGGSGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGA

VTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSH

RSYSCQVTHEGSTVEKTVAPTECS

SEQ ID
TGFβR2-
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMS

NO:
3x4GS-
NCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDA

6412
hCLIg_vk
ASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGS

GGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC

Stromal Modifying Moieties

Solid tumors have a distinct structure that mimics that of normal tissues and comprises two distinct but interdependent compartments: the parenchyma (neoplastic cells) and the stroma that the neoplastic cells induce and in which they are dispersed. All tumors have stroma and require stroma for nutritional support and for the removal of waste products. In the case of tumors which grow as cell suspensions (e.g., leukemias, ascites tumors), the blood plasma serves as stroma (Connolly J L et al. Tumor Structure and Tumor Stroma Generation. In: Kufe D W et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton: BC Decker; 2003). The stroma includes a variety of cell types, including fibroblasts/myofibroblasts, glial, epithelial, fat, vascular, smooth muscle, and immune cells along with extracellular matrix (ECM) and extracellular molecules (Li Hanchen et al. Tumor Microenvironment: The Role of the Tumor Stroma in Cancer. J of Cellular Biochemistry 101: 805-815 (2007)).

Stromal modifying moieties described herein include moieties (e.g., proteins, e.g., enzymes) capable of degrading a component of the stroma, e.g., an ECM component, e.g., a glycosaminoglycan, e.g., hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin sulfate, heparin, entactin, tenascin, aggrecan and keratin sulfate; or an extracellular protein, e.g., collagen, laminin, elastin, fibrinogen, fibronectin, and vitronectin.

Stromal Modifying Enzymes

In some embodiments, the stromal modifying moiety is an enzyme. For example, the stromal modifying moiety can include, but is not limited to a hyaluronidase, a collagenase, a chondroitinase, a matrix metalloproteinase (e.g., macrophage metalloelastase).

Hyaluronidases

Hyaluronidases are a group of neutral- and acid-active enzymes found throughout the animal kingdom. Hyaluronidases vary with respect to substrate specificity, and mechanism of action. There are three general classes of hyaluronidases: (1) Mammalian-type hyaluronidases, (EC 3.2.1.35) which are endo-beta-N-acetylhexosaminidases with tetrasaccharides and hexasaccharides as the major end products. They have both hydrolytic and transglycosidase activities, and can degrade hyaluronan and chondroitin sulfates; (2) Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, and to various extents, chondroitin sulfate and dermatan sulfate. They are endo-beta-N-acetylhexosaminidases that operate by a beta elimination reaction that yields primarily disaccharide end products; (3) Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-beta-glucuronidases that generate tetrasaccharide and hexasaccharide end products through hydrolysis of the beta 1-3 linkage.

Mammalian hyaluronidases can be further divided into two groups: (1) neutral active and (2) acid active enzymes. There are six hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3 HYAL4 HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown to possess enzyme activity toward any known substrates. HYAL4 is a chondroitinase and lacks activity towards hyaluronan. HYAL1 is the prototypical acid-active enzyme and PH20 is the prototypical neutral-active enzyme. Acid active hyaluronidases, such as HYAL1 and HYAL2 lack catalytic activity at neutral pH. For example, HYAL1 has no catalytic activity in vitro over pH 4.5 (Frost and Stem, “A Microtiter-Based Assay for Hyaluronidase Activity Not Requiring Specialized Reagents”, Analytical Biochemistry, vol. 251, pp. 263-269 (1997). HYAL2 is an acid active enzyme with a very low specific activity in vitro.

In some embodiments the hyaluronidase is a mammalian hyaluronidase. In some embodiments the hyaluronidase is a recombinant human hyaluronidase. In some embodiments, the hyaluronidase is a neutral active hyaluronidase. In some embodiments, the hyaluronidase is a neutral active soluble hyaluronidase. In some embodiments, the hyaluronidase is a recombinant PH20 neutral-active enzyme. In some embodiments, the hyaluronidase is a recombinant PH20 neutral-active soluble enzyme. In some embodiments the hyaluronidase is glycosylated. In some embodiments, the hyaluronidase possesses at least one N-linked glycan. A recombinant hyaluronidase can be produced using conventional methods known to those of skill in the art, e.g., U.S. Pat. No. 7,767,429, the entire contents of which are incorporated by reference herein.

In some embodiments the hyaluronidase is rHuPH20 (also referred to as Hylenex®; presently manufactured by Halozyme; approved by the FDA in 2005 (see e.g., Scodeller P (2014) Hyaluronidase and other Extracellular Matrix Degrading Enzymes for Cancer Therapy: New Uses and Nano-Formulations. J Carcinog Mutage 5:178; U.S. Pat. Nos. 7,767,429; 8,202,517; 7,431,380; 8,450,470; 8,772,246; 8,580,252, the entire contents of each of which is incorporated by reference herein). rHuPH20 is produced by genetically engineered CHO cells containing a DNA plasmid encoding for a soluble fragment of human hyaluronidase PH20. In some embodiments the hyaluronidase is glycosylated. In some embodiments, the hyaluronidase possesses at least one N-linked glycan. A recombinant hyaluronidase can be produced using conventional methods known to those of skill in the art, e.g., U.S. Pat. No. 7,767,429, the entire contents of which are incorporated by reference herein. In some embodiments, rHuPH20 has a sequence at least 95% (e.g., at least 96° 98% 99%, 100%) identical to the amino acid sequence of

(SEQ ID NO: 6213)

LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINAT

GQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITF

YMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLT

EATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKK

PGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRN

RVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETV

ALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQ

VLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLE

QFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETE

EPQIFYNASPSTLS.

In any of the methods provided herein, the anti-hyaluronan agent can be an agent that degrades hyaluronan or can be an agent that inhibits the synthesis of hyaluronan. For example, the anti-hyaluronan agent can be a hyaluronan degrading enzyme. In another example, the anti-hyaluronan agent or is an agent that inhibits hyaluronan synthesis. For example, the anti-hyaluronan agent is an agent that inhibits hyaluronan synthesis such as a sense or antisense nucleic acid molecule against an HA synthase or is a small molecule drug. For example, an anti-hyaluronan agent is 4-methylumbelliferone (MU) or a derivative thereof, or leflunomide or a derivative thereof. Such derivatives include, for example, a derivative of 4-methylumbelliferone (MU) that is 6,7-dihydroxy-4-methyl coumarin or 5,7-dihydroxy-4-methyl coumarin.

In further examples of the methods provided herein, the hyaluronan degrading enzyme is a hyaluronidase. In some examples, the hyaluronan-degrading enzyme is a PH20 hyaluronidase or truncated form thereof to lacking a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In specific examples, the hyaluronidase is a PH20 selected from a human, monkey, bovine, ovine, rat, mouse or guinea pig PH20. For example, the hyaluronan-degrading enzyme is a human PH20 hyaluronidase that is neutral active and N-glycosylated and is selected from among (a) a hyaluronidase polypeptide that is a full-length PH20 or is a C-terminal truncated form of the PH20, wherein the truncated form includes at least amino acid residues 36-464 of SEQ ID NO: 6213, such as 36-481, 36-482, 36-483, where the full-length PH20 has the sequence of amino acids set forth in SEQ ID NO: 6213; or (b) a hyaluronidase polypeptide comprising a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the polypeptide or truncated form of sequence of amino acids set forth in SEQ ID NO: 6213; or (c) a hyaluronidase polypeptide of (a) or (b) comprising amino acid substitutions, whereby the hyaluronidase polypeptide has a sequence of amino acids having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the polypeptide set forth in SEQ ID NO: 6213 or the with the corresponding truncated forms thereof. In exemplary examples, the hyaluronan-degrading enzyme is a PH20 that comprises a composition designated rHuPH20.

In other examples, the anti-hyaluronan agent is a hyaluronan degrading enzyme that is modified by conjugation to a polymer. The polymer can be a PEG and the anti-hyaluronan agent a PEGylated hyaluronan degrading enzyme. Hence, in some examples of the methods provided herein the hyaluronan-degrading enzyme is modified by conjugation to a polymer. For example, the hyaluronan-degrading enzyme is conjugated to a PEG, thus the hyaluronan degrading enzyme is PEGylated. In an exemplary example, the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). In the methods provided herein, the corticosteroid can be a glucocorticoid that is selected from among cortisones, dexamethasones, hydrocortisones, methylprednisolones, prednisolones and prednisones.

Chondroitinases

Chondroitinases are enzymes found throughout the animal kingdom which degrade glycosaminoglycans, specifically chondroitins and chondroitin sulfates, through an endoglycosidase reaction. In some embodiments the chondroitinase is a mammalian chondroitinase. In some embodiments the chondroitinase is a recombinant human chondroitinase. In some embodiments the chondroitinase is HYAL4. Other exemplary chondroitinases include chondroitinase ABC (derived from Proteus vulgaris; Japanese Patent Application Laid-open No 6-153947, T. Yamagata et al. J. Biol. Chem., 243, 1523 (1968), S. Suzuki et al, J. Biol. Chem., 243, 1543 (1968)), chondroitinase AC (derived from Flavobacterium heparinum; T. Yamagata et al., J. Biol. Chem., 243, 1523 (1968)), chondroitinase AC II (derived from Arthrobacter aurescens; K. Hiyama, and S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. (Tokyo), 80, 1201 (1976)), Hyaluronidase ACIII (derived from Flavobacterium sp. Hp102; Hirofumi Miyazono et al., Seikagaku, 61, 1023 (1989)), chondroitinase B (derived from Flavobacterium heparinum; Y. M. Michelacci and C. P. Dietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), Y. M. Michelacci and C. P. Dietrich, Biochem. J., 151, 121 (1975), Kenichi Maeyama et al, Seikagaku, 57, 1189 (1985)), chondroitinase C (derived from Flavobacterium sp. Hp102; Hirofumi Miyazono et al, Seikagaku, 61, 1023 (1939)), and the like.

Matrix Metalloproteinases

Matrix metalloproteases (MMPs) are zinc-dependent endopeptidases that are the major proteases involved in extracellular matrix (ECM) degradation. MMPs are capable of degrading a wide range of extracellular molecules and a number of bioactive molecules. Twenty-four MMP genes have been identified in humans, which can be organized into six groups based on domain organization and substrate preference: Collagenases (MMP-1, -8 and -13), Gelatinases (MMP-2 and MMP-9), Stromelysins (MMP-3, -10 and -11), Matrilysin (MMP-7 and MMP-26), Membrane-type (MT)-MMPs (MMP-14, -15, -16, -17, -24 and -25) and others (MMP-12, -19, -20, -21, -23, -27 and -28). In some embodiments, the stromal modifying moiety is a human recombinant MMP (e.g., MMP-1, -2, -3, -4, -5, -6, -7, -8, -9, 10, -11, -12, -13, -14, 15, -15, -17, -18, -19, 20, -21, -22, -23, or -24).

Collagenases

The three mammalian collagenases (MMP-1, -8, and -13) are the principal secreted endopeptidases capable of cleaving collagenous extracellular matrix. In addition to fibrillar collagens, collagenases can cleave several other matrix and non-matrix proteins including growth factors. Collagenases are synthesized as inactive pro-forms, and once activated, their activity is inhibited by specific tissue inhibitors of metalloproteinases, TIMPs, as well as by non-specific proteinase inhibitors (Ala-aho R et al. Biochimie. Collagenases in cancer. 2005 Mar-Apr; 87(3-4):273-86). In some embodiments, the stromal modifying moiety is a collagenase. In some embodiments, the collagenase is a human recombinant collagenase. In some embodiments, the collagenase is MMP-1. In some embodiments, the collagenase is MMP-8. In some embodiments, the collagenase is MMP-13.

Macrophage Metalloelastase

Macrophage metalloelastase (MME), also known as MMP-12, is a member of the stromelysin subgroup of MMPs and catalyzes the hydrolysis of soluble and insoluble elastin and a broad selection of matrix and nonmatrix substrates including type IV collagen, fibronectin, laminin, vitronectin, entactin, heparan, and chondroitin sulfates (Erja Kerkelä et al. Journal of Investigative Dermatology (2000) 114, 1113-1119; doi: 10.1046/j.1523-1747.2000.00993). In some embodiments, the stromal modifying moiety is a MME. In some embodiments, the MME is a human recombinant MME. In some embodiments, the MME is MMP-12.

Additional Stromal Modifying Moieties

In some embodiments, the stromal modifying moiety causes one or more of: decreases the level or production of a stromal or extracellular matrix (ECM) component; decreases tumor fibrosis; increases interstitial tumor transport; improves tumor perfusion; expands the tumor microvasculature; decreases interstitial fluid pressure (IFP) in a tumor; or decreases or enhances penetration or diffusion of an agent, e.g., a cancer therapeutic or a cellular therapy, into a tumor or tumor vasculature.

In some embodiments, the stromal or ECM component decreased is chosen from a glycosaminoglycan or an extracellular protein, or a combination thereof. In some embodiments, the glycosaminoglycan is chosen from hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparin, heparin sulfate, entactin, tenascin, aggrecan and keratin sulfate. In some embodiments, the extracellular protein is chosen from collagen, laminin, elastin, fibrinogen, fibronectin, or vitronectin. In some embodiments, the stromal modifying moiety includes an enzyme molecule that degrades a tumor stroma or extracellular matrix (ECM). In some embodiments, the enzyme molecule is chosen from a hyaluronidase molecule, a collagenase molecule, a chondroitinase molecule, a matrix metalloproteinase molecule (e.g., macrophage metalloelastase), or a variant (e.g., a fragment) of any of the aforesaid. The term “enzyme molecule” includes a full length, a fragment or a variant of the enzyme, e.g., an enzyme variant that retains at least one functional property of the naturally-occurring enzyme.

In some embodiments, the stromal modifying moiety decreases the level or production of hyaluronic acid. In other embodiments, the stromal modifying moiety comprises a hyaluronan degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid.

In some embodiments, the hyaluronan degrading enzyme is a hyaluronidase molecule, e.g., a full length or a variant (e.g., fragment thereof) thereof. In some embodiments, the hyaluronan degrading enzyme is active in neutral or acidic pH, e.g., pH of about 4-5. In some embodiments, the hyaluronidase molecule is a mammalian hyaluronidase molecule, e.g., a recombinant human hyaluronidase molecule, e.g., a full length or a variant (e.g., fragment thereof, e.g., a truncated form) thereof. In some embodiments, the hyaluronidase molecule is chosen from HYAL1, HYAL2, or PH-20/SPAM1, or a variant thereof (e.g., a truncated form thereof). In some embodiments, the truncated form lacks a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In some embodiments, the hyaluronidase molecule is glycosylated, e.g., comprises at least one N-linked glycan.

In some embodiments, the hyaluronidase molecule comprises the amino acid sequence:

or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 6213.

In some embodiments, the hyaluronidase molecule comprises:

- (i) the amino acid sequence of 36-464 of SEQ ID NO: 6213;
- (ii) the amino acid sequence of 36481, 36482, or 36-483 of PH20, wherein PH20 has the sequence of amino acids set forth in SEQ ID NO: 6213; or
- (iii) an amino acid sequence having at least 95% to 100% sequence identity to the polypeptide or truncated form of sequence of amino acids set forth in SEQ ID NO: 6213; or
- (iv) an amino acid sequence having 30, 20, 10, 5 or fewer amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises an amino acid sequence at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule is encoded by a nucleotide sequence at least 95% (e.g., at least 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence of SEQ ID NO: 6213.

In some embodiments, the hyaluronidase molecule is PH20, e.g., rHuPH20. In some embodiments, the hyaluronidase molecule is HYAL1 and comprises the amino acid sequence:

(SEQ ID NO: 6218)

FRGPLLPNRPFTTVWNANTQWCLERHGVDVDVSVFDVVANPGQTFRGPD

MTIFYSSQGTYPYYTPTGEPVFGGLPQNASLIAHLARTFQDILAAIPAP

RSRALVQAQHPDWPAPQVEAVAQDQFQGAARAWMAGTLQLGRALRPRGL

WDFSGLAVIDWEAWRPRWAFNWDTKDIYRQGFYGFPDCYNYDFLSPNYT

GQCPSGIRAQNDQLGWLWGQSRALYPSIYMPAVLEGTGKSQMYVQHRVA

EAFRVAVAAGDPNLPVLPYVQIFYDTTNHFLPLDELEHSLGESAAQGAA

GVVLWVSWENTRTKESCQAIKEYMDTTLGPFILNVTSGALLCSQALCSG

HGRCVRRTSHPKALLLLNPASFSIQLTPGGGPLSLRGALSLEDQAQMAV

EFKCRCYPGWQAPWCERKSMW,

In some embodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule, further comprises a polymer, e.g., is conjugated to a polymer, e.g., PEG. In some embodiments, the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). In some embodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule, further comprises an immunoglobulin chain constant region (e.g., Fc region) chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, more particularly, the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin constant region (e.g., the Fc region) is linked, e.g., covalently linked to, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule. In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fe receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function. In some embodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule forms a dimer.

In some embodiments, the stromal modifying moiety comprises an inhibitor of the synthesis of hyaluronan, e.g., an HA synthase. In some embodiments, the inhibitor comprises a sense or an antisense nucleic acid molecule against an HA synthase or is a small molecule drug. In some embodiments, the inhibitor is 4-methylumbelliferone (MU) or a derivative thereof (e.g., 6,7-dihydroxy-4-methyl coumarin or 5,7-dihydroxy-4-methyl coumarin), or leflunomide or a derivative thereof.

In some embodiments, the stromal modifying moiety comprises antibody molecule against hyaluronic acid.

In some embodiments, the stromal modifying moiety comprises a collagenase molecule, e.g., a mammalian collagenase molecule, or a variant (e.g., fragment) thereof. In some embodiments, the collagenase molecule is collagenase molecule IV, e.g., comprising the amino acid sequence of:

(SEQ ID NO: 6219)

YNFFPRKPKWDKNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLR

FSRIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTGVGGDSHF

DDDELWTLGEGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGF

LWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSC

TTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFT

FLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLVAAH

EFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKGIQELYGASPDID

LGTGPTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTVTPRD

KPMGPLLVATFWPELPEKIDAVYEAPQEEKAVFFAGNEYWIYSASTLER

GYPKPLTSLGLPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMD

PGFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKLENQSLKSV

KFGSIKSDWLGC,

Targeting Moieties

In some embodiments, the multispecific and/or multifunctional molecules disclosed herein comprise a tumor-targeting moiety. In some embodiments, the tumor-targeting moiety targets (e.g., binds to) a tumor antigen selected from: G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1. In some embodiments, the tumor-targeting moiety targets (e.g., binds to) G6B.

G6B refers to MPIG6B, also known as megakaryocyte and platelet inhibitory receptor G6b or C6orf25. Swiss-Prot accession number 095866 provides exemplary human G6B amino acid sequences. In some embodiments, G6B or G6B molecule is a naturally-existing G6B or a functional variant or fragment thereof.

CD34 refers to hematopoietic progenitor cell antigen CD34. Swiss-Prot accession number P28906 provides exemplary human CD34 amino acid sequences. In some embodiments, CD34 or CD34 molecule is a naturally-existing CD34 or a functional variant or fragment thereof.

CD41 refers to ITGA2B, also known as Integrin alpha-IIb. Swiss-Prot accession number P08514 provides exemplary human CD41 amino acid sequences. In some embodiments, CD41 or CD41 molecule is a naturally-existing CD41 or a functional variant or fragment thereof.

P-selectin refers to SELP, also known as CD62P, GMP-140 or LECAM3. Swiss-Prot accession number P16109 provides exemplary human P-selectin amino acid sequences. In some embodiments, P-selectin or P-selectin molecule is a naturally-existing P-selectin or a functional variant or fragment thereof.

Clec2 refers to CLECIB, also known as C-type lectin domain family 1 member B. Swiss-Prot accession number Q9P126 provides exemplary human Clec2 amino acid sequences. In some embodiments, Clec2 or Clec2 molecule is a naturally-existing Clec2 or a functional variant or fragment thereof.

cKIT refers to mast/stem cell growth factor receptor kit, also known as CD117. Swiss-Prot accession number P10721 provides exemplary human cKIT amino acid sequences. In some embodiments, cKIT or cKIT molecule is a naturally-existing cKIT or a functional variant or fragment thereof.

FLT3 refers to receptor-type tyrosine-protein kinase FLT3, also known as CD135. Swiss-Prot accession number P36888 provides exemplary human FLT3 amino acid sequences. In some embodiments, FLT3 or FLT3 molecule is a naturally-existing FLT3 or a functional variant or fragment thereof.

MPL refers to thrombopoietin receptor, also known as C7403. Swiss-Prot accession number P40238 provides exemplary human MPL amino acid sequences. In some embodiments, MPL or MPL molecule is a naturally-existing MPL or a functional variant or fragment thereof.

ITGB3 refers to Integrin beta-3, also known as CD61. Swiss-Prot accession number P05106 provides exemplary human ITGB3 amino acid sequences. In some embodiments, ITGB3 or ITGB3 molecule is a naturally-existing ITGB3 or a functional variant or fragment thereof.

ITGB2 refers to Integrin beta-2, also known as CD18. Swiss-Prot accession number P05107 provides exemplary human ITGB2 amino acid sequences. In some embodiments, ITGB2 or ITGB2 molecule is a naturally-existing ITGB2 or a functional variant or fragment thereof.

GP5 refers to platelet glycoprotein V, also known as CD42d. Swiss-Prot accession number P40197 provides exemplary human GP5 amino acid sequences. In some embodiments, GP5 or GP5 molecule is a naturally-existing GP5 or a functional variant or fragment thereof.

GP6 refers to platelet glycoprotein VI. Swiss-Prot accession number Q9HCN6 provides exemplary human GP6 amino acid sequences. In some embodiments, GP6 or GP6 molecule is a naturally-existing GP6 or a functional variant or fragment thereof.

GP9 refers to platelet glycoprotein IX, also known as CD42a. Swiss-Prot accession number P14770 provides exemplary human GP9 amino acid sequences. In some embodiments, GP9 or GP9 molecule is a naturally-existing GP9 or a functional variant or fragment thereof.

GP1BA refers to platelet glycoprotein Ib alpha chain, also known as CD42b. Swiss-Prot accession number P07359 provides exemplary human GP1BA amino acid sequences. In some embodiments, GP1BA or GP1BA molecule is a naturally-existing GP1BA or a functional variant or fragment thereof.

DSC2 refers to desmocollin-2, also known as cadherin family member 2. Swiss-Prot accession number Q02487 provides exemplary human DSC2 amino acid sequences. In some embodiments, DSC2 or DSC2 molecule is a naturally-existing DSC2 or a functional variant or fragment thereof.

FCGR2A refers to Fc-gamma-RIIa, also known as CD32. Swiss-Prot accession number P12318 provides exemplary human FCGR2A amino acid sequences. In some embodiments, FCGR2A or FCGR2A molecule is a naturally-existing FCGR2A or a functional variant or fragment thereof.

TNFRSF10A refers to Tumor necrosis factor receptor superfamily member 10A, also known as Death receptor 4, TNF-related apoptosis-inducing ligand receptor 1, TRAIL-R1, or CD261. Swiss-Prot accession number 000220 provides exemplary human TNFRSF10A amino acid sequences. In some embodiments, TNFRSF10A or TNFRSF10A molecule is a naturally-existing TNFRSF10A or a functional variant or fragment thereof.

TNFRSF10B refers to Tumor necrosis factor receptor superfamily member 10B, also known as Death receptor 5, TNF-related apoptosis-inducing ligand receptor 2, TRAIL-R2, or CD262. Swiss-Prot accession number 014763 provides exemplary human TNFRSF10B amino acid sequences. In some embodiments, TNFRSF10B or TNFRSF10B molecule is a naturally-existing TNFRSF10B or a functional variant or fragment thereof.

TM4SF1 refers to transmembrane 4 L6 family member 1. Swiss-Prot accession number P30408 provides exemplary human TM4SF1 amino acid sequences. In some embodiments, TM4SF1 or TM4SF1 molecule is a naturally-existing TM4SF1 or a functional variant or fragment thereof.

In some embodiments, the multispecific and/or multifunctional molecule comprises one or more additional tumor-targeting moieties. In some embodiments, the one or more additional tumor-targeting moieties target (e.g., bind to) the same tumor antigen as the first tumor-targeting moiety. In some embodiments, the one or more additional tumor-targeting moieties target (e.g., bind to) a different tumor antigen from the first tumor-targeting moiety. In some embodiments, the multispecific and/or multifunctional molecule comprises a plurality of tumor-targeting moieties targeting different tumor antigens present on the same cell (e.g., a tumor cell). In some embodiments, the multispecific and/or multifunctional molecule comprises a plurality of tumor-targeting moieties targeting different tumor antigens present on different cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different tumor cells). In some embodiments, each of the tumor antigens is selected from: G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In some embodiments, the multispecific and/or multifunctional molecule comprises a first tumor-targeting moiety (e.g., targeting a first tumor antigen) and a second tumor-targeting moiety (e.g., targeting a second tumor antigen). In some embodiments, the first and second tumor antigens are present on the same tumor cell. In some embodiments, the first and third tumor antigens are present on the same tumor cell. In some embodiments, the second and third tumor antigens are present on the same tumor cell. In some embodiments, the first, second, and third tumor antigens are present on the same tumor cell. In some embodiments, the first and second tumor antigens are present on different tumor cells. In some embodiments, the first and third tumor antigens are present on different tumor cells. In some embodiments, the second and third tumor antigens are present on different tumor cells. In some embodiments, the first, second, and third tumor antigens are present on different tumor cells.

In some embodiments, the first, second, and/or third tumor antigens show higher expression in a tumor cell, e.g., a myeloproliferative neoplasm cell, than a non-tumor cell. In some embodiments, the expression of the first, second, and/or third tumor antigens in a tumor cell, e.g., a myeloproliferative neoplasm cell, is at least 1.5, 2, 4, 6, 8, or 10-fold higher than the expression of the first, second, and/or third tumor antigens in a non-tumor cell. In some embodiments, the multifunctional molecule preferentially binds to a tumor cell, e.g., a myeloproliferative neoplasm cell, over a non-tumor cell. In some embodiments, the binding between the multifunctional molecule and the tumor cell, e.g., a myeloproliferative neoplasm cell, is more than 10, 20, 30, 40, 50-fold greater than the binding between the multifunctional molecule and a non-tumor cell. In some embodiments, the affinity, e.g., the combined affinity, of the first and second tumor-targeting moieties for a tumor cell, e.g., a myeloproliferative neoplasm cell, is greater than the affinity of a similar multifunctional molecule having only one of the first tumor-targeting moiety or the second tumor-targeting moiety. In some embodiments, the affinity, e.g., the combined affinity, of the first and second tumor-targeting moieties for a tumor cell, e.g., a myeloproliferative neoplasm cell, is at least 2, 5, 10, 20, 30, 40, 50, 75 or 100 times greater than the affinity of a similar multifunctional molecule having only one of the first tumor-targeting moiety or the second tumor-targeting moiety.

In some embodiments, the affinity, e.g., the combined affinity, of the first, second, and third tumor-targeting moieties for a tumor cell, e.g., a myeloproliferative neoplasm cell, is greater than the affinity of a similar multifunctional molecule having only one of the first tumor-targeting moiety, the second tumor-targeting moiety, or the third tumor-targeting moiety, or a similar multifunctional molecule having only two of the first tumor-targeting moiety, the second tumor-targeting moiety, or the third tumor-targeting moiety. In some embodiments, the affinity, e.g., the combined affinity, of the first, second, and third tumor-targeting moieties for a tumor cell, e.g., a myeloproliferative neoplasm cell, is at least 2, 5, 10, 20, 30, 40, 50, 75 or 100 times greater than the affinity of a similar multifunctional molecule having only one of the first tumor-targeting moiety, the second tumor-targeting moiety, or the third tumor-targeting moiety, or a similar multifunctional molecule having only two of the first tumor-targeting moiety, the second tumor-targeting moiety, or the third tumor-targeting moiety.

In some embodiments, the affinity, e.g., the combined affinity, for the first and second tumor antigens of the first tumor-targeting moiety and the second tumor-targeting moiety is equal to or greater than the affinity of (iii), (iv) or (v), either alone or as part of the multifunctional molecule, for its corresponding binding member. In some embodiments, the affinity, e.g., the combined affinity, for the first and second tumor antigens of the first tumor-targeting moiety and the second tumor-targeting moiety is at least 2, 5, 10, 20, 30, 40, 50, 75 or 100 times greater than the affinity of (iii), (iv) or (v), either alone or as part of the multifunctional molecule, for its corresponding binding member.

In some embodiments, the affinity, e.g., the combined affinity, for the first, second, and third tumor antigens of the first tumor-targeting moiety, the second tumor-targeting moiety, and the third tumor-targeting moiety is equal to or greater than the affinity of (iii), (iv) or (v), either alone or as part of the multifunctional molecule, for its corresponding binding member. In some embodiments, the affinity, e.g., the combined affinity, for the first, second, and third tumor antigens of the first tumor-targeting moiety, the second tumor-targeting moiety, and the third tumor-targeting moiety is at least 2, 5, 10, 20, 30, 40, 50, 75 or 100 times greater than the affinity of (iii), (iv) or (v), either alone or as part of the multifunctional molecule, for its corresponding binding member.

In some embodiments of the aforementioned aspects, the first tumor antigen is CD34 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is CD34, the second tumor antigen is CD41, and the third tumor antigen is G6B.

In some embodiments of the aforementioned aspects, the first tumor antigen is P-selectin and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD34, the second tumor antigen is CD41, and the third tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD34, the second tumor antigen is G6B, and the third tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD41, the second tumor antigen is G6B, and the third tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD34, the second tumor antigen is CD41, and the third tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD34, the second tumor antigen is G6B, and the third tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD41, the second tumor antigen is G6B, and the third tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD34, the second tumor antigen is P-selectin, and the third tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD41, the second tumor antigen is P-selectin, and the third tumor antigen is Clec2. In some embodiments, the first tumor antigen is G6B, the second tumor antigen is P-selectin, and the third tumor antigen is Clec2.

In some embodiments of the aforementioned aspects, the first tumor antigen is CD34 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is CD34 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is CD41 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is CD41 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is G6B and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is G6B and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is P-selectin and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is P-selectin and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is Clec2 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is Clec2 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is cKIT and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is cKIT and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is FLT3 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is FLT3 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is MPL and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is MPL and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is ITGB3 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is ITGB3 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is ITGB2 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is ITGB2 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is GP5 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is GP5 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is GP6 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is GP6 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is GP9 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is GP9 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is GP1BA and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is GP1BA and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is DSC2 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is DSC2 and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is FCGR2A and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is FCGR2A and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is TNFRSF10A and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is TNFRSF10A and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is TNFRSF10B and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is TNFRSF10B and the second tumor antigen is TM4SF1.

In some embodiments of the aforementioned aspects, the first tumor antigen is TM4SF1 and the second tumor antigen is CD34. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is CD41. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is G6B. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is P-selectin. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is Clec2. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is cKIT. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is FLT3. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is MPL. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is ITGB3. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is ITGB2. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is GP5. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is GP6. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is GP9. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is GP1BA. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is DSC2. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is FCGR2A. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is TNFRSF10A. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is TNFRSF10B. In some embodiments, the first tumor antigen is TM4SF1 and the second tumor antigen is TM4SF1.

Antibody Molecules Targeting Tumor Antigens

In some embodiments, the tumor-targeting moiety comprises a CDR, a framework region, or a variable region sequence shown in Table 38 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

TABLE 38

Sequences for exemplary antibodies capable of binding to exemplary target molecules

Target
Description
SEQ ID NO
Sequence

CD34
Exemplary
SEQ ID
EVQLQQSGPELVKPGASVKISCKASGYSFIGYFMNWV

anti-CD34
NO: 2001
MQSHGRSLEWIGRINPYNGYTFYNQKFKGKATLTVD

VH

KSSSTAHMELRSLASEDSAVYYCARHFRYDGVFYYA

MDYWGQGTSVTVSS

Exemplary
SEQ ID
QLVLTQSSSASFSLGASAKLTCTLSSQHSTFTIEWYQQ

anti-CD34
NO: 2002
QPLKPPKYVMDLKKDGSHSTGDGVPDRFSGSSSGAD

VL

RYLSISNIQPEDEATYICGVGDTIKEQFVYVFGGGTKV

TVL

cKIT
Exemplary
SEQ ID
EVQLVESGGGLVQPGGSLRLSCAASGFAFSGYYMAW

(CD117)
anti-cKIT
NO: 2003
VRQAPGKGLEWVANINYPGSSTYYLDSVKGRFTISRD

VH

NAKNSLYLQMNSLRAEDTAVYYCARGDYYGTTYWY

FDVWGQGTTVTVSS

Exemplary
SEQ ID
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQ

anti-cKIT
NO: 2004
KPGKAPKLLIYYTSRLQSGVPSRFSGSGSGTDFTLTISS

VL

LQPEDFATYYCQQGRRLWSFGGGTKVEIK

FLT3
Exemplary
SEQ ID
QVQLQQPGAELVKPGASLKLSCKSSGYTFTSYWMHW

anti-FLT3
NO: 2005
VRQRPGHGLEWIGEIDPSDSYKDYNQKFKDKATLTVD

VH

RSSNTAYMHLSSLTSDDSAVYYCARAITTTPFDFWGQ

GTTLTVSS

Exemplary
SEQ ID
DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQ

anti-FLT3
NO: 2006
KSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSV

VL

ETEDFGVYFCQQSNTWPYTFGGGTKLEIKR

CD41
Exemplary
SEQ ID
EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYVHW

(ITGA2B)
anti-CD41
NO: 2007
VKQRPEQGLEWIGRIDPANGYTKYDPKFQGKATITAD

VH

TSSNTAYLQLSSLTSEDTAVYYCVRPLYDYYAMDYW

GQGTSVTVSS

Exemplary
SEQ ID
DILMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQ

anti-CD41
NO: 2008
KPGKSFMGLIYYGTNLVDGVPSRFSGSGSGADYSLTIS

VL

SLDSEDFADYYCVQYAQLPYTFGGGTKLEIK

MPL
1.75 VH
SEQ ID
EVLVESGGGLVQPKGSLKLSCAASGFSFNTYAMNW

NO: 2009
VRQAPGKGLEWIAHIRSKSNNFATYYADSVKDRFSIS

RDASENILFLQMNNLKTEDTAMYYCVRQGGDFPMDY

WGQGTSVTVSS

1.75 VL
SEQ ID
QIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQK

NO: 2010
PGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISN

MEAEDAAAYYCQQYHSYPTTFGGGTKLEVK

1.78 VH
SEQ ID
QVQLQQSGPELVKPGASVKMSCKASGYAFSSSWLNW

NO: 2011
VRQRPGKGLEWIGRIYPGDGENHYNGKFKGKATLTA

DKSSSTGYMQLSSLTSEDSAVYFCASYYEGGYWGQG

TLITVSA

1.78 VL
SEQ ID
DIVMTQAAPSIPVTPGESVSISCRSDKSLLHSNGNTYLF

NO: 2012
WFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAF

TLRISGVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK

P-
Exemplary
SEQ ID
EVLVESGGGLVRPGGSLRLSCAASGFTFSNYDMHW

Selectin
anti-P-
NO: 2013
VRQATGKGLEWVSAITAAGDIYYPGSVKGRFTISREN

(SELP)
Selectin VH

AKNSLYLQMNSLRAGDTAVYYCARGRYSGSGSYYN

DWFDPWGQGTLVTVSS

Exemplary
SEQ ID
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQ

anti-P-
NO: 2014
KPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISS

Selectin VL

LEPEDFAVYYCQQRSNWPLTFGGGTKVEIK

DSC2
Exemplary
SEQ ID
MDSRLNLVFLVLILKGVQCDVQLVESGGGLVQPGGS

anti-DSC2
NO: 2015
RKLSCAASGFTFSSFGMHWVRQAPEKGLEWVAYISSG

#1 VH

SSTIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTA

MYYCARVHYYYFDYWGQGTTLTVSS

Exemplary
SEQ ID
MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGK

anti-DSC2
NO: 2016
VTITCKASQDINKYIAWYQHKPGKGPRLLIHYTSTLQP

#1 VL

GIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLW

TFGGGTKL

Exemplary
SEQ ID
MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGET

anti-DSC2
NO: 2017
VKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWIN

#2 VH

TETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNED

TATYFCARWLLFDYWGQGTTLTVSS

Exemplary
SEQ ID
MESQTQVLMFLLLWVSGACADIVMTQSPSSLAMSVG

anti-DSC2
NO: 2018
QKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKL

#2 VL

LVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLA

DYFCQQHYSTPLTFGAGTKL

FCGR2A
AT-10 VH
SEQ ID
EVKLEESGGGLVQPGGSMKLSCVASGFTFSYYWMN

(CD32a)

NO: 2019
WVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTI

SRDDSKNNVYLQMNNLRAEDTGIYYCNRRDEYYAM

DYWGQGTSVSVSS

AT-10 VL
SEQ ID
DIVLTQSPGSLAVSLGQRATISCRASESVDNFGISFMN

NO: 2020
WFQQKPGQPPRLLIYGASNQGSGVPARFSGSGSGTDF

SLNIHPVEEDDAAMYFCQQSKEVPWTFGGGTKLEIK

IV.3 VH
SEQ ID
QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWV

NO: 2021
KQAPGKGLKWMGWLNTYTGESIYPDDFKGRFAFSSE

TSASTAYLQINNLKNEDMATYFCARGDYGYDDPLDY

WGQGTSVTVSS

IV.3 VL
SEQ ID
DIVMTQAAPSVPVTPGESVSISCRSSKSLLHTNGNTYL

NO: 2022
HWFLQRPGQSPQLLIYRMSVLASGVPDRFSGSGSGTA

FTLSISRVEAEDVGVFYCMQHLEYPLTFGAGTKLELK

MDE-8 VH
SEQ ID
QVHLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHW

NO: 2023
VRQAPGKGLEWVAVIWYDGSNYYYTDSVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCARDLGAAASDY

WGQGTLVTVSS

MDE-8 VL
SEQ ID
AIQLTQSPSSLSASVGDRVTITCRASQGINSALAWYQQ

NO: 2024
KPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISS

LQPEDFATYYCQQFNSYPHTFGQGTKLEIK

TNFRS
E-11-13 VH
SEQ ID
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGP

F10A

NO: 2025
GLVKPSETLSLTCTVSGGSIISKSSYWGWIRQPPGKGL

or

EWIGSIYYSGSTFYNPSLKSRVTISVDTSKNQFSLKLSS

TNFRS

VTAADTAVYYCARLTVAEFDYWGQGTLVTVSSAS

F10B
E-11-13 VL
SEQ ID
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGER

NO: 2026
ATLSCRASQSVSSFLAWYQQKPGQAPRLLIYDASNRA

TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN

WPLTFGPGTKVDIKRT

L-30-10 VH
SEQ ID
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGP

NO: 2027
GLVKPSETLSLTCTVSGGSISSRSNYWGWIRQPPGKGL

EWIGNVYYRGSTYYNSSLKSRVTISVDTSKNQFSLKLS

SVTVADTAVYYCARLSVAEFDYWGQGILVTVSSAS

L-30-10 VL
SEQ ID
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGER

NO: 2028
ATLSCRASQSVSSFLAWYQQKPGQAPRLLIYDASNRA

TGSPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSD

WPLTFGPGTKVDIKRT

H-48-2 VH
SEQ ID
MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGP

NO: 2029
GLVKPSETLSLTCTVSGGSISSSSYYWGWVRQPPGKG

LEWIGSIHYSGSTFYNPSLKSRVTISVDTSKNQFSLKLS

SVTAADTTVYYCARQGSTVVRGVYYYGMDVWGQG

TTVTVSSAS

H-48-2 VL
SEQ ID
METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGER

NO: 2030
ATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSR

ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYG

SSPLYTFGQGTKLEIKRT

0304 VH
SEQ ID
MDWTWRILFLVAAATSAHSQVQLVQSGAEMKKPGA

NO: 2031
SVKVSCKTSGYTFTNYKINWVRQAPGQGLEWMGWM

NPDTDSTGYPQKFQGRVTMTRNTSISTAYMELSSLRS

EDTAVYYCARSYGSGSYYRDYYYGMDVWGQGTTVT

VSS

0304 VL
SEQ ID
MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGER

NO: 2032
ATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRA

TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN

WPLTFGGGTKVEIKR

KMTR1 VH
SEQ ID
MEFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGRSL

NO: 2033
RLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSG

GSRYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA

VYYCAKESSGWFGAFDYWGQGTLVTVSS

KMTRI VL
SEQ ID
MSPSQLIGFLLLWVPASRGEIVLTQSPDFQSVTPKEKV

NO: 2034
TITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSG

VPSRFSGSGSGTDFTLTINSLEAEDAAAYYCHQSSSLPI

TFGQGTRLEIKR

TM4SF1
Exemplary
SEQ ID
EVILVESGGGLVKPGGSLKLSCAASGFTFSSFAMSWV

anti-
NO: 2035
RQTPEKRLEWVATISSGSIYIYYTDGVKGRFTISRDNA

TM4SF1

KNTVHLQMSSLRSEDTAMYYCARRGIYYGYDGYAM

VH

DYWGQGTSVTVSS

Exemplary
SEQ ID
AVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYL

anti-
NO: 2036
HWYMQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTD

TM4SF1

FTLKISRVEADDLGIYFCSQSTHIPLAFGAGTKLELK

VL

In some embodiments, the first, second, or third tumor antigen is CD34. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) a VH of SEQ ID NO: 2001 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (iii) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2002 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (iv) a VL of SEQ ID NO: 2002 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, wherein the first, second, or third tumor antigen is CD41. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2007 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) a VH of SEQ ID NO: 2007 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (iii) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2008 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (iv) a VL of SEQ ID NO: 2008 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is P-selectin. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2013 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) a VH of SEQ ID NO: 2013 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (iii) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2014 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (iv) a VL of SEQ ID NO: 2014 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is cKIT. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2003 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) a VH of SEQ ID NO: 2003 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (iii) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2004 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (iv) a VL of SEQ ID NO: 2004 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is FLT3. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2005 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) a VH of SEQ ID NO: 2005 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (iii) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2006 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (iv) a VL of SEQ ID NO: 2006 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is MPL. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2009 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2009 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2010 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2010 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); or
- (ii) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2011 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2011 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2012 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2012 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is DSC2. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2015 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2015 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2016 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2016 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); or
- (ii) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2017 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2017 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2018 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2018 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is FCGR2A. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2019 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2019 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2020 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2020 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (ii) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2021 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO:20 21 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2022 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2022 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or
- (iii) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2023 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2023 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2024 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2024 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is TNFRSF10A or TNFRSF10B. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2025 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2025 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2026 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2026 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); or
- (ii) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2027 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2027 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2028 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2028 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (iii) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2029 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2029 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2030 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2030 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (iv) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2031 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2031 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2032 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2032 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); or
- (v) (a) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2033 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (b) a VH of SEQ ID NO: 2033 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (c) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2034 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (d) a VL of SEQ ID NO: 2034 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the first, second, or third tumor antigen is TM4SF1. In some embodiments, the first, second, or third tumor-targeting moiety comprises:

- (i) a HCDR1, HCDR2, and/or HCDR3 from SEQ ID NO: 2035 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) a VH of SEQ ID NO: 2035 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- (iii) a LCDR1, LCDR2, and/or LCDR3 from SEQ ID NO: 2036 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (iv) a VL of SEQ ID NO: 2036 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

Exemplary Anti-CD34 Antibody Sequences

In one aspect, provided herein is a multispecific or multifunctional molecule comprising a tumor targeting moiety that comprises a CD34-targeting moiety. In another aspect, provided herein is an anti-CD34 antibody molecule (e.g., a monoclonal anti-CD34 antibody molecule).

In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises an antibody, or an antigen-binding fragment thereof, disclosed in Table 20 or Table 21. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a CDR, a framework region, or a variable region sequence disclosed in Table 20 or Table 21 (or a sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6239 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6241 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6243 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6239, a VHCDR2 amino acid sequence of SEQ ID NO: 6241, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6243. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VL comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6245 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 1236 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6246 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6245, a VLCDR2 amino acid sequence of SEQ ID NO: 1236, and a VLCDR3 amino acid sequence of SEQ ID NO: 6246.

In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79, 6225, 6227, or 6229, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO: 6231, 6233, 6235, or 6237, or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6231 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 6231. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6231 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 and a VL comprising the amino acid sequence of SEQ ID NO: 6231. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6231 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 and a VL comprising the amino acid sequence of SEQ ID NO: 6231. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6231 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 and a VL comprising the amino acid sequence of SEQ ID NO: 6231. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6233 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 6233. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6233 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 and a VL comprising the amino acid sequence of SEQ ID NO: 6233. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6233 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 and a VL comprising the amino acid sequence of SEQ ID NO: 6233. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6233 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 and a VL comprising the amino acid sequence of SEQ ID NO: 6233. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 6235. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 and a VL comprising the amino acid sequence of SEQ ID NO: 6235. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 and a VL comprising the amino acid sequence of SEQ ID NO: 6235. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6235 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 and a VL comprising the amino acid sequence of SEQ ID NO: 6235. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 6237. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6225 and a VL comprising the amino acid sequence of SEQ ID NO: 6237. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6227 and a VL comprising the amino acid sequence of SEQ ID NO: 6237. In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto) and a VL comprising the amino acid sequence of SEQ ID NO: 6237 (or an amino acid sequence having at least about 80%, 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 6229 and a VL comprising the amino acid sequence of SEQ ID NO: 6237.

In some embodiments, the CD34-targeting moiety or anti-CD34 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence of SEQ ID NO: 6233.

TABLE 20

Exemplary variable region sequences of anti-CD34 antibodies

SEQ ID

NO
Description
Sequence

SEQ ID
Mouse VH
EIQLQQSGPELMKPGASLKISCKTSGYSFTSYYMHWVKQSHGQ

NO: 6222

SLEWIGFIDPFKVITGYNHNFRGKATLTVDRSSTTAYMHLRSLT

SEDSAVYYCARRYYSDYDGYALDYWGQGTSVTVSS

SEQ ID
Mouse VL
DVVMTQTPLSLPVSLGDQASIFCRSSQSLVHSDGNTYLHWYLQ

NO: 6223

KPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDL

GVYFCSQSTHVPPYTFGGGTKLEIK

SEQ ID
Humanization
QIQLQESGPGLVKPSETLSLTCTTSGYSFTSYYMHWIRQPPGKG

NO: 79
variant VH1
LEWIGFIDPFKVITGYNHNFRGRVTISVDRSKTQASLKLSSVTAA

DTAVYYCARRYYSDYDGYALDYWGQGTLVTVSS

SEQ ID
Humanization
EIQLVQSGAEVKKPGATVKISCKTSGYSFTSYYMHWVQQAPGK

NO: 6225
variant VH2
GLEWMGFIDPFKVITGYNHNFRGRVTITVDRSTTTAYMELSSLR

SEDTAVYYCARRYYSDYDGYALDYWGQGTLVTVSS

SEQ ID
Humanization
QIQLVQSGAEVKKTGSSVKVSCKTSGYSFTSYYMHWVRQAPG

NO: 6227
variant VH3
QALEWMGFIDPFKVITGYNHNFRGRVTITVDRSMTTAYMELSS

LRSEDTAMYYCARRYYSDYDGYALDYWGQGTLVTVSS

SEQ ID
Humanization
QIQLVQSGAEVKKPGASVKVSCKTSGYSFTSYYMHWVRQAPG

NO: 6229
variant VH4
QGLEWMGFIDPFKVITGYNHNFRGRVTSTVDRSITTAYMELSRL

RSDDTVVYYCARRYYSDYDGYALDYWGQGTLVTVSS

SEQ ID
Humanization
EVVMTQSPGTLSLSPGERATLSCRSSQSLVHSDGNTYLHWYQQ

NO: 6231
variant VL1
KPGQAPRLLIYKVSNRFSGIPDRFSGSGSGTDFTLTISRLEPEDFA

VYFCSQSTHVPPYTFGGGTKVEIK

SEQ ID
Humanization
EVVMTQSPATLSLSPGERATLSCRSSQSLVHSDGNTYLHWYQQ

NO: 6233
variant VL2
KPGQAPRLLIYKVSNRFSGIPARFSGSGSGTDFTLTISSLEPEDFA

VYFCSQSTHVPPYTFGGGTKVEIK

SEQ ID
Humanization
EVVMTQSPATLSVSPGERATLSCRSSQSLVHSDGNTYLHWYQQ

NO: 6235
variant VL3
KPGQAPRLLIYKVSNRFSGIPARFSGSGSGTEFTLTISSLQSEDFA

VYFCSQSTHVPPYTFGGGTKVEIK

SEQ ID
Humanization
VVWMTQSPSLLSASTGDRVTISCRSSQSLVHSDGNTYLHWYQQ

NO: 6237
variant VL4
KPGKAPELLIYKVSNRFSGVPSRFSGSGSGTDFTLTISCLQSEDF

ATYFCSQSTHVPPYTFGGGTKVEIK

TABLE 21

Exemplary CDRs of anti-CD34 antibodies

SEQ ID NO
Description
Sequence

SEQ ID NO: 6239
VH CDR1
FTSYYMH

SEQ ID NO: 6241
VH CDR2
FIDPFKVITGYNHNFRG

SEQ ID NO: 6243
VH CDR3
RYYSDYDGYALDY

SEQ ID NO: 6245
VL CDR1
RSSQSLVHSDGNTYLH

SEQ ID NO: 1236
VL CDR2
KVSNRFS

SEQ ID NO: 6246
VL CDR3
SQSTHVPPYT

Linkers

The multispecific or multifunctional molecule disclosed herein can further include a linker, e.g., a linker between one or more of: the antigen binding domain and the cytokine molecule, the antigen binding domain and the immune cell engager, the antigen binding domain and the stromal modifying moiety, the cytokine molecule and the immune cell engager, the cytokine molecule and the stromal modifying moiety, the immune cell engager and the stromal modifying moiety, the antigen binding domain and the immunoglobulin chain constant region, the cytokine molecule and the immunoglobulin chain constant region, the immune cell engager and the immunoglobulin chain constant region, or the stromal modifying moiety and the immunoglobulin chain constant region. In some embodiments, the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker, or a combination thereof.

In one embodiment, the multispecific molecule can include one, two, three or four linkers, e.g., a peptide linker. In one embodiment, the peptide linker includes Gly and Ser. In some embodiments, the peptide linker is selected from GGGGS (SEQ ID NO: 6214); GGGGSGGGGS (SEQ ID NO: 6215); GGGGSGGGGSGGGGS (SEQ ID NO: 6216); and DVPSGPGGGGGSGGGGS (SEQ ID NO: 6217). In some embodiments, the peptide linker is a A(EAAAK)nA (SEQ ID NO: 6413) family of linkers (e.g., as described in Protein Eng. (2001) 14 (8): 529-532). These are stiff helical linkers with n ranging from 2-5. In some embodiments, the peptide linker is selected from AEAAAKEAAAKAAA (SEQ ID NO: 6220); AEAAAKEAAAKEAAAKAAA (SEQ ID NO: 6221); AEAAAKEAAAKEAAAKEAAAKAAA (SEQ ID NO: 77); and AEAAAKEAAAKEAAAKEAAAKEAAAKAAA(SEQ ID NO: 78).

Nucleic Acids

Nucleic acids encoding the aforementioned multispecific or multifunctional molecules are also disclosed.

In certain embodiments, the invention features nucleic acids comprising nucleotide sequences that encode heavy and light chain variable regions and CDRs or hypervariable loops of the antibody molecules, as described herein. For example, the invention features a first and second nucleic acid encoding heavy and light chain variable regions, respectively, of an antibody molecule chosen from one or more of the antibody molecules disclosed herein. The nucleic acid can comprise a nucleotide sequence as set forth in the tables herein, or a sequence substantially identical thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which differs by no more than 3, 6, 15, 30, or 45 nucleotides from the sequences shown in the tables herein.

In certain embodiments, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs or hypervariable loops from a heavy chain variable region having an amino acid sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or having one or more substitutions, e.g., conserved substitutions). In other embodiments, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs or hypervariable loops from a light chain variable region having an amino acid sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or having one or more substitutions, e.g., conserved substitutions). In yet another embodiment, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs or hypervariable loops from heavy and light chain variable regions having an amino acid sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or having one or more substitutions, e.g., conserved substitutions).

In certain embodiments, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs or hypervariable loops from a heavy chain variable region having the nucleotide sequence as set forth in the tables herein, a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or capable of hybridizing under the stringency conditions described herein). In another embodiment, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, or three CDRs or hypervariable loops from a light chain variable region having the nucleotide sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or capable of hybridizing under the stringency conditions described herein). In yet another embodiment, the nucleic acid can comprise a nucleotide sequence encoding at least one, two, three, four, five, or six CDRs or hypervariable loops from heavy and light chain variable regions having the nucleotide sequence as set forth in the tables herein, or a sequence substantially homologous thereto (e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or capable of hybridizing under the stringency conditions described herein).

In certain embodiments, the nucleic acid can comprise a nucleotide sequence encoding a cytokine molecule, an immune cell engager, or a stromal modifying moiety disclosed herein.

Vectors

Further provided herein are vectors comprising the nucleotide sequences encoding a multispecific or multifunctional molecule described herein. In one embodiment, the vectors comprise nucleotides encoding a multispecific or multifunctional molecule described herein. In one embodiment, the vectors comprise the nucleotide sequences described herein. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, for example, bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (Rous Sarcoma Virus, MMTV or MOMLV) or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semliki Forest virus, Eastern Equine Encephalitis virus and Flaviviruses.

Additionally, cells which have stably integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow for the selection of transfected host cells. The marker may provide, for example, prototropy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper, or the like. The selectable marker gene can be either directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcriptional promoters, enhancers, and termination signals.

Once the expression vector or DNA sequence containing the constructs has been prepared for expression, the expression vectors may be transfected or introduced into an appropriate host cell. Various techniques may be employed to achieve this, such as, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid based transfection or other conventional techniques. In the case of protoplast fusion, the cells are grown in media and screened for the appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the antibody molecule produced are known to those skilled in the art, and may be varied or optimized depending upon the specific expression vector and mammalian host cell employed, based upon the present description.

Cells

In another aspect, the application features host cells and vectors containing the nucleic acids described herein. The nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell. The host cell can be a eukaryotic cell, e.g., a mammalian cell, an insect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. For example, the mammalian cell can be a cultured cell or a cell line. Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO), Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cells from a transgenic animal, e.g., mammary epithelial cell.

The invention also provides host cells comprising a nucleic acid encoding an antibody molecule as described herein.

In one embodiment, the host cells are genetically engineered to comprise nucleic acids encoding the antibody molecule.

In one embodiment, the host cells are genetically engineered by using an expression cassette. The phrase “expression cassette,” refers to nucleotide sequences, which are capable of affecting expression of a gene in hosts compatible with such sequences. Such cassettes may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or helpful in effecting expression may also be used, such as, for example, an inducible promoter.

The invention also provides host cells comprising the vectors described herein.

The cell can be, but is not limited to, a eukaryotic cell, a bacterial cell, an insect cell, or a human cell. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

Uses and Combination Therapies

Methods described herein include treating a cancer in a subject by using a multispecific or multifunctional molecule described herein, e.g., using a pharmaceutical composition described herein. Also provided are methods for reducing or ameliorating a symptom of a cancer in a subject, as well as methods for inhibiting the growth of a cancer and/or killing one or more cancer cells. In some embodiments, the methods described herein decrease the size of a tumor and/or decrease the number of cancer cells in a subject administered with a described herein or a pharmaceutical composition described herein.

In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is a leukemia or a lymphoma. As used herein, a “hematologic cancer” refers to a tumor of the hematopoietic or lymphoid tissues, e.g., a tumor that affects blood, bone marrow, or lymph nodes. Exemplary hematologic malignancies include, but are not limited to, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, acute monocytic leukemia (AMoL), chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), or large granular lymphocytic leukemia), lymphoma (e.g., AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma (e.g., classical Hodgkin lymphoma or nodular lymphocyte-predominant Hodgkin lymphoma), mycosis fungoides, non-Hodgkin lymphoma (e.g., B-cell non-Hodgkin lymphoma (e.g., Burkitt lymphoma, small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, or mantle cell lymphoma) or T-cell non-Hodgkin lymphoma (mycosis fungoides, anaplastic large cell lymphoma, or precursor T-lymphoblastic lymphoma)), primary central nervous system lymphoma, Sézary syndrome, Waldenstrom macroglobulinemia), chronic myeloproliferative neoplasm, Langerhans cell histiocytosis, multiple myeloma/plasma cell neoplasm, myelodysplastic syndrome, or myelodysplastic/myeloproliferative neoplasm.

In some embodiments, the cancer is a myeloproliferative neoplasm, e.g., primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML). In some embodiments, the cancer is myelofibrosis. In some embodiments, the subject has myelofibrosis. In some embodiments, the subject has a calreticulin mutation, e.g., a calreticulin mutation disclosed herein. In some embodiments, the subject does not have the JAK2-V617F mutation. In some embodiments, the subject has the JAK2-V617F mutation. In some embodiments, the subject has a MPL mutation. In some embodiments, the subject does not have a MPL mutation.

In some embodiments, the cancer is a solid cancer. Exemplary solid cancers include, but are not limited to, ovarian cancer, rectal cancer, stomach cancer, testicular cancer, cancer of the anal region, uterine cancer, colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, Kaposi's sarcoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, brain stem glioma, pituitary adenoma, epidermoid cancer, carcinoma of the cervix squamous cell cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the vagina, sarcoma of soft tissue, cancer of the urethra, carcinoma of the vulva, cancer of the penis, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, spinal axis tumor, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, metastatic lesions of said cancers, or combinations thereof.

In some embodiments, the multispecific or multifunctional molecules (or pharmaceutical composition) are administered in a manner appropriate to the disease to be treated or prevented. The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease. Appropriate dosages may be determined by clinical trials. For example, when “an effective amount” or “a therapeutic amount” is indicated, the precise amount of the pharmaceutical composition (or multispecific or multifunctional molecules) to be administered can be determined by a physician with consideration of individual differences in tumor size, extent of infection or metastasis, age, weight, and condition of the subject. In some embodiments, the pharmaceutical composition described herein can be administered at a dosage of 10⁴to 10⁹cells/kg body weight, e.g., 10⁵to 10⁶cells/kg body weight, including all integer values within those ranges. In some embodiments, the pharmaceutical composition described herein can be administered multiple times at these dosages. In some embodiments, the pharmaceutical composition described herein can be administered using infusion techniques described in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

In some embodiments, the multispecific or multifunctional molecules or pharmaceutical composition is administered to the subject parenterally. In some embodiments, the cells are administered to the subject intravenously, subcutaneously, intratumorally, intranodally, intramuscularly, intradermally, or intraperitoneally. In some embodiments, the cells are administered, e.g., injected, directly into a tumor or lymph node. In some embodiments, the cells are administered as an infusion (e.g., as described in Rosenberg et al., New Eng. J. of Med. 319:1676, 1988) or an intravenous push. In some embodiments, the cells are administered as an injectable depot formulation.

In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, monkey, pig, dog, cat, cow, sheep, goat, rabbit, rat, or mouse. In embodiments, the subject is a human. In some embodiments, the subject is a pediatric subject, e.g., less than 18 years of age, e.g., less than 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or less years of age. In some embodiments, the subject is an adult, e.g., at least 18 years of age, e.g., at least 19, 20, 21, 22, 23, 24, 25, 25-30, 30-35, 35-40, 40-50, 50-60, 60-70, 70-80, or 80-90 years of age.

Combination Therapies

The multispecific or multifunctional molecules disclosed herein can be used in combination with a second therapeutic agent or procedure.

In some embodiments, the multispecific or multifunctional molecule and the second therapeutic agent or procedure are administered/performed after a subject has been diagnosed with a cancer, e.g., before the cancer has been eliminated from the subject. In some embodiments, the multispecific or multifunctional molecule and the second therapeutic agent or procedure are administered/performed simultaneously or concurrently. For example, the delivery of one treatment is still occurring when the delivery of the second commences, e.g., there is an overlap in administration of the treatments. In other embodiments, the multispecific or multifunctional molecule and the second therapeutic agent or procedure are administered/performed sequentially. For example, the delivery of one treatment ceases before the delivery of the other treatment begins.

In some embodiments, combination therapy can lead to more effective treatment than monotherapy with either agent alone. In some embodiments, the combination of the first and second treatment is more effective (e.g., leads to a greater reduction in symptoms and/or cancer cells) than the first or second treatment alone. In some embodiments, the combination therapy permits use of a lower dose of the first or the second treatment compared to the dose of the first or second treatment normally required to achieve similar effects when administered as a monotherapy. In some embodiments, the combination therapy has a partially additive effect, wholly additive effect, or greater than additive effect.

In one embodiment, the multispecific or multifunctional molecule is administered in combination with a therapy, e.g., a cancer therapy (e.g., one or more of anti-cancer agents, immunotherapy, photodynamic therapy (PDT), surgery and/or radiation). The terms “chemotherapeutic,” “chemotherapeutic agent,” and “anti-cancer agent” are used interchangeably herein. The administration of the multispecific or multifunctional molecule and the therapy, e.g., the cancer therapy, can be sequential (with or without overlap) or simultaneous. Administration of the multispecific or multifunctional molecule can be continuous or intermittent during the course of therapy (e.g., cancer therapy). Certain therapies described herein can be used to treat cancers and non-cancerous diseases. For example, PDT efficacy can be enhanced in cancerous and non-cancerous conditions (e.g., tuberculosis) using the methods and compositions described herein (reviewed in, e.g., Agostinis, P. et al. (2011) CA Cancer J. Clin. 61:250-281).

Anti-Cancer Therapies

In other embodiments, the multispecific or multifunctional molecule is administered in combination with a low or small molecular weight chemotherapeutic agent. Exemplary low or small molecular weight chemotherapeutic agents include, but not limited to, 13-cis-retinoic acid (isotretinoin, ACCUTANE®), 2-CdA (2-chlorodeoxyadenosine, cladribine, LEUSTATIN™), 5-azacitidine (azacitidine, VIDAZA®), 5-fluorouracil (5-FU, fluorouracil, ADRUCIL®), 6-mercaptopurine (6-MP, mercaptopurine, PURINETHOL®), 6-TG (6-thioguanine, thioguanine, THIOGUANINE TABLOID®), abraxane (paclitaxel protein-bound), actinomycin-D (dactinomycin, COSMEGEN®), alitretinoin (PANRETIN®), all-transretinoic acid (ATRA, tretinoin, VESANOID®), altretamine (hexamethylmelamine, HMM, HEXALEN®), amethopterin (methotrexate, methotrexate sodium, MTX, TREXALL™ RHEUMATREX®), amifostine (ETHYOL®), arabinosylcytosine (Ara-C, cytarabine, CYTOSAR-U®), arsenic trioxide (TRISENOX®), asparaginase (Erwinia L-asparaginase, L-asparaginase, ELSPAR®, KIDROLASE®), BCNU (carmustine, BiCNUo), bendamustine (TREANDA®), bexarotene (TARGRETIN®), bleomycin (BLENOXANE®), busulfan (BUSULFEX®, MYLERAN®), calcium leucovorin (Citrovorum Factor, folinic acid, leucovorin), camptothecin-11 (CPT-11, irinotecan, CAMPTOSAR®), capecitabine (XELODA®), carboplatin (PARAPLATIN®), carmustine wafer (prolifeprospan 20 with carmustine implant, GLIADEL® wafer), CCI-779 (temsirolimus, TORISEL®), CCNU (lomustine, CeeNU), CDDP (cisplatin, PLATINOL®, PLATINOL-AQ®), chlorambucil (leukeran), cyclophosphamide (CYTOXAN®, NEOSAR®), dacarbazine (DIC, DTIC, imidazole carboxamide, DTIC-DOME®), daunomycin (daunorubicin, daunorubicin hydrochloride, rubidomycin hydrochloride, CERUBIDINE®), decitabine (DACOGEN®), dexrazoxane (ZINECARD®), DHAD (mitoxantrone, NOVANTRONE®), docetaxel (TAXOTERE®), doxorubicin (ADRIAMYCIN®, RUBEX®), epirubicin (ELLENCE™), estramustine (EMCYT®), etoposide (VP-16, etoposide phosphate, TOPOSAR®, VEPESID®, ETOPOPHOS®), floxuridine (FUDR®), fludarabine (FLUDARA®), fluorouracil (cream) (CARAC™, EFUDEX®, FLUOROPLEX®), gemcitabine (GEMZAR®), hydroxyurea (HYDREA®, DROXIA™, MYLOCEL™), idarubicin (IDAMYCIN®), ifosfamide (IFEX®), ixabepilone (IXEMPRA™), LCR (leurocristine, vincristine, VCR, ONCOVIN®, VINCASAR PFS®), L-PAM (L-sarcolysin, melphalan, phenylalanine mustard, ALKERAN®), mechlorethamine (mechlorethamine hydrochloride, mustine, nitrogen mustard, MUSTARGEN®), mesna (MESNEX™) mitomycin (mitomycin-C, MTC, MUTAMYCIN®), nelarabine (ARRANON®), oxaliplatin (ELOXATIN™), paclitaxel (TAXOL®, ONXAL™), pegaspargase (PEG-L-asparaginase, ONCOSPAR®), PEMETREXED (ALIMTA®), pentostatin (NIPENT®), procarbazine (MATULANE®), streptozocin (ZANOSAR®), temozolomide (TEMODAR®), teniposide (VM-26, VUMON®), TESPA (thiophosphoamide, thiotepa, TSPA, THIOPLEX®), topotecan (HYCAMTIN®), vinblastine (vinblastine sulfate, vincaleukoblastine, VLB, ALKABAN-AQ®, VELBAN®), vinorelbine (vinorelbine tartrate, NAVELBINE®), and vorinostat (ZOLINZA®).

In another embodiment, the multispecific or multifunctional molecule is administered in conjunction with a biologic. Biologics useful in the treatment of cancers are known in the art and a binding molecule of the invention may be administered, for example, in conjunction with such known biologics. For example, the FDA has approved the following biologics for the treatment of breast cancer: HERCEPTIN® (trastuzumab, Genentech Inc., South San Francisco, Calif.; a humanized monoclonal antibody that has anti-tumor activity in HER2-positive breast cancer); FASLODEX® (fulvestrant, AstraZeneca Pharmaceuticals, LP, Wilmington, Del.; an estrogen-receptor antagonist used to treat breast cancer); ARIMIDEX® (anastrozole, AstraZeneca Pharmaceuticals, LP; a nonsteroidal aromatase inhibitor which blocks aromatase, an enzyme needed to make estrogen); Aromasin® (exemestane, Pfizer Inc., New York, N.Y.; an irreversible, steroidal aromatase inactivator used in the treatment of breast cancer); FEMARA® (letrozole, Novartis Pharmaceuticals, East Hanover, N.J.; a nonsteroidal aromatase inhibitor approved by the FDA to treat breast cancer); and NOLVADEX® (tamoxifen, AstraZeneca Pharmaceuticals, LP; a nonsteroidal antiestrogen approved by the FDA to treat breast cancer). Other biologics with which the binding molecules of the invention may be combined include: AVASTIN® (bevacizumab, Genentech Inc.; the first FDA-approved therapy designed to inhibit angiogenesis); and ZEVALIN® (ibritumomab tiuxetan, Biogen Idec, Cambridge, Mass.; a radiolabeled monoclonal antibody currently approved for the treatment of B-cell lymphomas).

In addition, the FDA has approved the following biologics for the treatment of colorectal cancer: AVASTIN®; ERBITUX® (cetuximab, ImClone Systems Inc., New York, N.Y., and Bristol-Myers Squibb, New York, N.Y.; is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR)); GLEEVEC® (imatinib mesylate; a protein kinase inhibitor); and ERGAMISOL® (levamisole hydrochloride, Janssen Pharmaceutica Products, LP, Titusville, N.J.; an immunomodulator approved by the FDA in 1990 as an adjuvant treatment in combination with 5-fluorouracil after surgical resection in patients with Dukes' Stage C colon cancer).

For the treatment of lung cancer, exemplary biologics include TARCEVA® (erlotinib HCL, OSI Pharmaceuticals Inc., Melville, N.Y.; a small molecule designed to target the human epidermal growth factor receptor 1 (HER1) pathway).

For the treatment of multiple myeloma, exemplary biologics include VELCADE® Velcade (bortezomib, Millennium Pharmaceuticals, Cambridge Mass.; a proteasome inhibitor). Additional biologics include THALIDOMID® (thalidomide, Clegene Corporation, Warren, N.J.; an immunomodulatory agent and appears to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells and anti-angiogenesis).

Additional exemplary cancer therapeutic antibodies include, but are not limited to, 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab pegol, alemtuzumab (CAMPATH®, MABCAMPATH®), altumomab pentetate (HYBRI-CEAKER®), anatumomab mafenatox, anrukinzumab (IMA-638), apolizumab, arcitumomab (CEA-SCAN®), bavituximab, bectumomab (LYMPHOSCAN®), belimumab (BENLYSTA®, LYMPHOSTAT-B®), besilesomab (SCINTIMUN®), bevacizumab (AVASTIN®), bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, capromab pendetide (PROSTASCINT®), catumaxomab (REMOVAB®), CC49, cetuximab (C225, ERBITUX®), citatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, denosumab (PROLIA®), detumomab, ecromeximab, edrecolomab (PANOREX®), elotuzumab, epitumomab cituxetan, epratuzumab, ertumaxomab (REXOMUN®), etaracizumab, farletuzumab, figitumumab, fresolimumab, galiximab, gemtuzumab ozogamicin (MYLOTARG®), girentuximab, glembatumumab vedotin, ibritumomab (ibritumomab tiuxetan, ZEVALIN®), igovomab (INDIMACIS-125®), intetumumab, inotuzumab ozogamicin, ipilimumab, iratumumab, labetuzumab (CEA-CIDE®), lexatumumab, lintuzumab, lucatumumab, lumiliximab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, nacolomab tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab (THERACIM®, THERALOC®), nofetumomab merpentan (VERLUMA®), ofatumumab (ARZERRA®), olaratumab, oportuzumab monatox, oregovomab (OVAREX®), panitumumab (VECTIBIX®), pemtumomab (THERAGYN®), pertuzumab (OMNITARG®), pintumomab, pritumumab, ramucirumab, ranibizumab (LUCENTIS®), rilotumumab, rituximab (MABTHERA®, RITUXAN®), robatumumab, satumomab pendetide, sibrotuzumab, siltuximab, sontuzumab, tacatuzumab tetraxetan (AFP-CIDE®), taplitumomab paptox, tenatumomab, TGN1412, ticilimumab (tremelimumab), tigatuzumab, TNX-650, tositumomab (BEXXAR®), trastuzumab (HERCEPTIN®), tremelimumab, tucotuzumab celmoleukin, veltuzumab, volociximab, votumumab (HUMASPECT®), zalutumumab (HUMAX-EGFR®), and zanolimumab (HUMAX-CD4®).

In other embodiments, the multispecific or multifunctional molecule is administered in combination with a viral cancer therapeutic agent. Exemplary viral cancer therapeutic agents include, but not limited to, vaccinia virus (vvDD-CDSR), carcinoembryonic antigen-expressing measles virus, recombinant vaccinia virus (TK-deletion plus GM-CSF), Seneca Valley virus-001, Newcastle virus, coxsackie virus A21, GL-ONC1, EBNA1 C-terminal/LMP2 chimeric protein-expressing recombinant modified vaccinia Ankara vaccine, carcinoembryonic antigen-expressing measles virus, G207 oncolytic virus, modified vaccinia virus Ankara vaccine expressing p53, OncoVEX GM-CSF modified herpes-simplex 1 virus, fowlpox virus vaccine vector, recombinant vaccinia prostate-specific antigen vaccine, human papillomavirus 16/18 L1 virus-like particle/AS04 vaccine, MVA-EBNA1/LMP2 Inj. vaccine, quadrivalent HPV vaccine, quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine (GARDASIL®), recombinant fowlpox-CEA(6D)/TRICOM vaccine; recombinant vaccinia-CEA(6D)-TRICOM vaccine, recombinant modified vaccinia Ankara-5T4 vaccine, recombinant fowlpox-TRICOM vaccine, oncolytic herpes virus NV1020, HPV L1 VLP vaccine V504, human papillomavirus bivalent (types 16 and 18) vaccine (CERVARIX®), herpes simplex virus HF10, Ad5CMV-p53 gene, recombinant vaccinia DF3/MUC1 vaccine, recombinant vaccinia-MUC-1 vaccine, recombinant vaccinia-TRICOM vaccine, ALVAC MART-1 vaccine, replication-defective herpes simplex virus type I (HSV-1) vector expressing human Preproenkephalin (NP2), wild-type reovirus, reovirus type 3 Dearing (REOLYSIN®), oncolytic virus HSV1716, recombinant modified vaccinia Ankara (MVA)-based vaccine encoding Epstein-Barr virus target antigens, recombinant fowlpox-prostate specific antigen vaccine, recombinant vaccinia prostate-specific antigen vaccine, recombinant vaccinia-B7.1 vaccine, rAd-p53 gene, Ad5-delta24RGD, HPV vaccine 580299, JX-594 (thymidine kinase-deleted vaccinia virus plus GM-CSF), HPV-16/18 L1/AS04, fowlpox virus vaccine vector, vaccinia-tyrosinase vaccine, MEDI-517 HPV-16/18 VLP AS04 vaccine, adenoviral vector containing the thymidine kinase of herpes simplex virus TK99UN, HspE7, FP253/Fludarabine, ALVAC(2) melanoma multi-antigen therapeutic vaccine, ALVAC-hB7.1, canarypox-hIL-12 melanoma vaccine, Ad-REIC/Dkk-3, rAd-IFN SCH 721015, TIL-Ad-INFg, Ad-ISF35, and coxsackievirus A21 (CVA21, CAVATAK®).

In other embodiments, the multispecific or multifunctional molecule is administered in combination with a nanopharmaceutical. Exemplary cancer nanopharmaceuticals include, but not limited to, ABRAXANE® (paclitaxel bound albumin nanoparticles), CRLX101 (CPT conjugated to a linear cyclodextrin-based polymer), CRLX288 (conjugating docetaxel to the biodegradable polymer poly (lactic-co-glycolic acid)), cytarabine liposomal (liposomal Ara-C, DEPOCYT^M), daunorubicin liposomal (DAUNOXOME®), doxorubicin liposomal (DOXIL®, CAELYX®), encapsulated-daunorubicin citrate liposome (DAUNOXOME®), and PEG anti-VEGF aptamer (MACUGEN®).

In some embodiments, the multispecific or multifunctional molecule is administered in combination with paclitaxel or a paclitaxel formulation, e.g., TAXOL®, protein-bound paclitaxel (e.g., ABRAXANE®). Exemplary paclitaxel formulations include, but are not limited to, nanoparticle albumin-bound paclitaxel (ABRAXANE®, marketed by Abraxis Bioscience), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin, marketed by Protarga), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX, marketed by Cell Therapeutic), the tumor-activated prodrug (TAP), ANG105 (Angiopep-2 bound to three molecules of paclitaxel, marketed by ImmunoGen), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1; see Li et al., Biopolymers (2007) 87:225-230), and glucose-conjugated paclitaxel (e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate, see Liu et al., Bioorganic & Medicinal Chemistry Letters (2007) 17:617-620).

Exemplary RNAi and antisense RNA agents for treating cancer include, but not limited to, CALAA-01, siG12D LODER (Local Drug EluteR), and ALN-VSP02.

Other cancer therapeutic agents include, but not limited to, cytokines (e.g., aldesleukin (IL-2, Interleukin-2, PROLEUKIN®), alpha Interferon (IFN-alpha, Interferon alfa, INTRON® A (Interferon alfa-2b), ROFERON-A® (Interferon alfa-2a)), Epoetin alfa (PROCRIT®), filgrastim (G-CSF, Granulocyte-Colony Stimulating Factor, NEUPOGEN®), GM-CSF (Granulocyte Macrophage Colony Stimulating Factor, sargramostim, LEUKINE™), IL-11(Interleukin-11, oprelvekin, NEUMEGA®), Interferon alfa-2b (PEG conjugate) (PEG interferon, PEG-INTRON™), and pegfilgrastim (NEULASTA™)), hormone therapy agents (e.g., aminoglutethimide (CYTADREN®), anastrozole (ARIMIDEX®), bicalutamide (CASODEX®), exemestane (AROMASIN®), fluoxymesterone (HALOTESTIN®), flutamide (EULEXIN®), fulvestrant (FASLODEX®), goserelin (ZOLADEX®), letrozole (FEMARA®), leuprolide (ELIGARD™, LUPRON®, LUPRON DEPOT®, VIADUR™) megestrol (megestrol acetate, MEGACE®), nilutamide (ANANDRON®, NILANDRON®), octreotide (octreotide acetate, SANDOSTATIN®, SANDOSTATIN LAR®), raloxifene (EVISTA®), romiplostim (NPLATE®), tamoxifen (NOVALDEX®), and toremifene (FARESTON®)), phospholipase A2 inhibitors (e.g., anagrelide (AGRYLIN®)), biologic response modifiers (e.g., BCG (THERACYS®, TICE®), and Darbepoetin alfa (ARANESP®)), target therapy agents (e.g., bortezomib (VELCADE®), dasatinib (SPRYCEL™), denileukin diftitox (ONTAK®), erlotinib (TARCEVA®), everolimus (AFINITOR®), gefitinib (IRESSA®), imatinib mesylate (STI-571, GLEEVEC™), lapatinib (TYKERB®), sorafenib (NEXAVAR®), and SU11248 (sunitinib, SUTENT®)), immunomodulatory and antiangiogenic agents (e.g., CC-5013 (lenalidomide, REVLIMID®), and thalidomide (THALOMID®)), glucocorticosteroids (e.g., cortisone (hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, ALA-CORT®, HYDROCORT ACETATE®, hydrocortone phosphate LANACORT®, SOLU-CORTEF®), decadron (dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, DEXASONE®, DIODEX®, HEXADROL®, MAXIDEX®), methylprednisolone (6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, DURALONE®, MEDRALONE®, MEDROL®, M-PREDNISOL®, SOLU-MEDROL®), prednisolone (DELTA-CORTEF®, ORAPRED®, PEDIAPRED®, PRELONE®), and prednisone (DELTASONE®, LIQUID PRED®, METICORTEN®, ORASONE®)), and bisphosphonates (e.g., pamidronate (AREDIA®), and zoledronic acid (ZOMETA®))

In some embodiments, the multispecific or multifunctional molecule is used in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor). Exemplary tyrosine kinase inhibitor include, but are not limited to, an epidermal growth factor (EGF) pathway inhibitor (e.g., an epidermal growth factor receptor (EGFR) inhibitor), a vascular endothelial growth factor (VEGF) pathway inhibitor (e.g., an antibody against VEGF, a VEGF trap, a vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, a VEGFR-3 inhibitor)), a platelet derived growth factor (PDGF) pathway inhibitor (e.g., a platelet derived growth factor receptor (PDGFR) inhibitor (e.g., a PDGFR-β inhibitor)), a RAF-1 inhibitor, a KIT inhibitor and a RET inhibitor. In some embodiments, the anti-cancer agent used in combination with the AHCM agent is selected from the group consisting of: axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™ AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, AEE788, AG-490, AST-6, BMS-599626, CUDC-101, PD153035, pelitinib (EKB-569), vandetanib (zactima), WZ3146, WZ4002, WZ8040, ABT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951 (tivozanib), axitinib, BAY 73-4506 (regorafenib), brivanib alaninate (BMS-582664), brivanib (BMS-540215), cediranib (AZD2171), CHIR-258 (dovitinib), CP 673451, CYC116, E7080, Ki8751, masitinib (AB1010), MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, Pazopanib Hydrochloride, PD173074, Sorafenib Tosylate (Bay 43-9006), SU 5402, TSU-68 (SU6668), vatalanib, XL880 (GSK1363089, EXEL-2880). Selected tyrosine kinase inhibitors are chosen from sunitinib, erlotinib, gefitinib, or sorafenib. In one embodiment, the tyrosine kinase inhibitor is sunitinib.

In one embodiment, the multispecific or multifunctional molecule is administered in combination with one of more of: an anti-angiogenic agent, or a vascular targeting agent or a vascular disrupting agent. Exemplary anti-angiogenic agents include, but are not limited to, VEGF inhibitors (e.g., anti-VEGF antibodies (e.g., bevacizumab); VEGF receptor inhibitors (e.g., itraconazole); inhibitors of cell proliferatin and/or migration of endothelial cells (e.g., carboxyamidotriazole, TNP-470); inhibitors of angiogenesis stimulators (e.g., suramin), among others. A vascular-targeting agent (VTA) or vascular disrupting agent (VDA) is designed to damage the vasculature (blood vessels) of cancer tumors causing central necrosis (reviewed in, e.g., Thorpe, P. E. (2004) Clin. Cancer Res. Vol. 10:415-427). VTAs can be small-molecule. Exemplary small-molecule VTAs include, but are not limited to, microtubule destabilizing drugs (e.g., combretastatin A-4 disodium phosphate (CA4P), ZD6126, AVE8062, Oxi 4503); and vadimezan (ASA404).

Immune Checkpoint Inhibitors

In other embodiments, methods described herein comprise use of an immune checkpoint inhibitor in combination with the multispecific or multifunctional molecule. The methods can be used in a therapeutic protocol in vivo.

In some embodiments, an immune checkpoint inhibitor inhibits a checkpoint molecule. Exemplary checkpoint molecules include but are not limited to CTLA4, PD1, PD-L1, PD-L2, TIM3, LAG3, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), BTLA, KIR, MHC class I, MHC class II, GAL9, VISTA, BTLA, TIGIT, LAIRI, and A2aR. See, e.g., Pardoll. Nat. Rev. Cancer 12.4(2012):252-64, incorporated herein by reference.

In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, e.g., an anti-PD-1 antibody such as Nivolumab, Pembrolizumab or Pidilizumab. Nivolumab (also called MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558) is a fully human IgG4 monoclonal antibody that specifically inhibits PD1. See, e.g., U.S. Pat. No. 8,008,449 and WO2006/121168. Pembrolizumab (also called Lambrolizumab, MK-3475, MK03475, SCH-900475 or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. See, e.g., Hamid, O. et al. (2013) New England Journal of Medicine 369 (2): 134-44, U.S. Pat. No. 8,354,509 and WO2009/114335. Pidilizumab (also called CT-011 or Cure Tech) is a humanized IgGik monoclonal antibody that binds to PDi. See, e.g., WO2009/101611. In one embodiment, the inhibitor of PD-1 is an antibody molecule having a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence of Nivolumab, Pembrolizumab or Pidilizumab. Additional anti-PD1 antibodies, e.g., AMP 514 (Amplimmune), are described, e.g., in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649.

In some embodiments, the PD-1 inhibitor is an immunoadhesin, e.g., an immunoadhesin comprising an extracellular/PD-1 binding portion of a PD-1 ligand (e.g., PD-L1 or PD-L2) that is fused to a constant region (e.g., an Fc region of an immunoglobulin). In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg, e.g., described in WO2011/066342 and WO2010/027827), a PD-L2 Fc fusion soluble receptor that blocks the interaction between B7-H1 and PD-1.

In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, e.g., an antibody molecule. In some embodiments, the PD-L1 inhibitor is YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105. In some embodiments, the anti-PD-L1 antibody is MSB0010718C (also called A09-246-2; Merck Serono), which is a monoclonal antibody that binds to PD-L1. Exemplary humanized anti-PD-L1 antibodies are described, e.g., in WO2013/079174. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody, e.g., YW243.55.S70. The YW243.55.S70 antibody is described, e.g., in WO 2010/077634. In one embodiment, the PD-L1 inhibitor is MDX-1105 (also called BMS-936559), which is described, e.g., in WO2007/005874. In one embodiment, the PD-L1 inhibitor is MDPL3280A (Genentech/Roche), which is a human Fc-optimized IgG1 monoclonal antibody against PD-L1. See, e.g., U.S. Pat. No. 7,943,743 and U.S Publication No.: 20120039906. In one embodiment, the inhibitor of PD-L1 is an antibody molecule having a sequence substantially identical or similar thereto, e.g., a sequence at least 85%, 90%, 95% identical or higher to the sequence of YW243.55.S70, MPDL3280A, MEDI-4736, MSB-0010718C, or MDX-1105.

In some embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor, e.g., AMP-224 (which is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD1 and B7-H1. See, e.g., WO2010/027827 and WO2011/066342.

In one embodiment, the immune checkpoint inhibitor is a LAG-3 inhibitor, e.g., an anti LAG-3 antibody molecule. In some embodiments, the anti-LAG-3 antibody is BMS-986016 (also called BMS986016; Bristol-Myers Squibb). BMS-986016 and other humanized anti-LAG-3 antibodies are described, e.g., in US 2011/0150892, WO2010/019570, and WO2014/008218.

In some embodiments, the immune checkpoint inhibitor is a TIM-3 inhibitor, e.g., anti-TIM3 antibody molecule, e.g., described in U.S. Pat. No. 8,552,156, WO 2011/155607, EP 2581113 and U.S Publication No.: 2014/044728.

In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, e.g., anti-CTLA-4 antibody molecule. Exemplary anti-CTLA4 antibodies include Tremelimumab (IgG2 monoclonal antibody from Pfizer, formerly known as ticilimumab, CP-675,206); and Ipilimumab (also called MDX-010, CAS No. 477202-00-9). Other exemplary anti-CTLA-4 antibodies are described, e.g., in U.S. Pat. No. 5,811,097.

CRS Grading

In some embodiments, the compositions described herein may induce lower levels of cytokine release syndrome (CRS) and/or may have a lower chance of causing (e.g., may not cause) CRS compared to other compositions. In some embodiments, CRS can be graded in severity from 1-5 as follows. Grades 1-3 are less than severe CRS. Grades 4-5 are severe CRS. For Grade 1 CRS, only symptomatic treatment is needed (e.g., nausea, fever, fatigue, myalgias, malaise, headache) and symptoms are not life threatening. For Grade 2 CRS, the symptoms require moderate intervention and generally respond to moderate intervention. Subjects having Grade 2 CRS develop hypotension that is responsive to either fluids or one low-dose vasopressor; or they develop grade 2 organ toxicity or mild respiratory symptoms that are responsive to low flow oxygen (<40% oxygen). In Grade 3 CRS subjects, hypotension generally cannot be reversed by fluid therapy or one low-dose vasopressor. These subjects generally require more than low flow oxygen and have grade 3 organ toxicity (e.g., renal or cardiac dysfunction or coagulopathy) and/or grade 4 transaminitis. Grade 3 CRS subjects require more aggressive intervention, e.g., oxygen of 40% or higher, high dose vasopressor(s), and/or multiple vasopressors. Grade 4 CRS subjects suffer from immediately life-threatening symptoms, including grade 4 organ toxicity or a need for mechanical ventilation. Grade 4 CRS subjects generally do not have transaminitis. In Grade 5 CRS subjects, the toxicity causes death. Sets of criteria for grading CRS are provided herein as Table 39, Table 40, and Table 41. Unless otherwise specified, CRS as used herein refers to CRS according to the criteria of Table 40.

In some embodiments, CRS is graded according to Table 39:

TABLE 39

CRS grading

Gr1
Supportive care only

Gr2
IV therapies +/− hospitalization.

Gr3
Hypotension requiring IV fluids or low-dose vasoactives or

hypoxemia requiring oxygen, CPAP, or BIPAP.

Gr4
Hypotension requiring high-dose vasoactives or hypoxemia

requiring mechanical ventilation.

Gr 5
Death

TABLE 40

CTCAE v 4.0 CRS grading scale

CRS

grade
Characteristics

Grade 1
Mild; No infusion interruption; No intervention

Grade 2
Infusion interruption indicated but responds promptly to

symptomatic treatment (e.g., antihistamines, NSAIDS, narcotics,

IV fluids); prophylactic medications indicated for <=24 hrs

Grade 3
Prolonged (e.g., not rapidly responsive to symptomatic

medications and/or brief interruption of infusion); recurrence

of symptoms following initial improvement; hospitalization

indicated for clinical sequelae (e.g., renal impairment,

pulmonary infiltrates)

Grade 4
Life threatening consequences; pressor or ventilator support

TABLE 41

NCI CRS grading scale

CRS

grade
Characteristics

Grade
Symptoms are not life threatening and require symptomatic

1
treatment only; e.g., fever, nausea, fatigue, headache, myalgias,

malaise

Grade
Symptoms require and respond to moderate intervention;

2
Oxygen requirement <40% or hypotension responsive to fluids

or low dose pressors or Grade 2 organ toxicity

Grade
Symptoms require and respond to aggressive intervention;

3
Oxygen requirement >=40% or Hypotension requiring high dose

or multiple pressors or grade 3 organ toxicity or grade 4

transaminitis

Grade
Life threatening symptoms Requirement for ventilator support or

4
Grade 4; organ toxicity (excluding transaminitis)

Example 1. Humanization of α-TRBV6-5 Antibody Clone Antibody A

The germline for the mouse α-TCRβ antibody clone Antibody A VH and VL were assigned using IMGT nomenclature, with CDR regions defined by a combined Kabat and Chothia classification. SEQ ID NO: 1 and SEQ ID NO: 2 are the Antibody A VH and VL sequences respectively where the VH germline is mouse IGHV1S12*01 and the VL germline is mouse IGKV6-15*01. SEQ ID NOs: 3-5 are the Antibody A VH CDR regions 1-3 respectively and SEQ ID NOs: 6-8 correspond to the VL CDR regions 1-3 (as described in TABLE 30).

Humanization of the Antibody A VH and VL sequences was done separately using similar methodology. Amino acids positions were identified in the framework regions which were important for the success of CDR grafting. Human germline sequences were identified which preserved the necessary residues and contained a high amount of overall identity. When the human germline framework sequence did not contain a matching important amino acid, it was back mutated to match the mouse sequence. CDR regions were grafted onto the human germline unchanged. The Antibody A VH was humanized into human IGHV1-69*01 and the Antibody A VL was humanized into IGKV1-17*01 and IGKV1-27*01. All 3 humanized sequences were confirmed to contain no introduced potential negative post translational modification sites such as NG, DG, NS, NN, DS, NT, NXS, or NXT as a result of the humanization process. SEQ ID NO: 9 is the humanized Antibody A-H.1 VH and SEQ ID NOs: 10 and 11 are the humanized VL IGKV1-17*01 and IGKV1-27*01 germlines respectively (as described in TABLE 30). FIGS. 1A and 1B show the murine and humanized sequences with annotations depicting the CDR and framework regions (FR).

Example 2: Humanization of α-TRBV12-3 and TRBV12-4 Antibody Clone Antibody B

The germline for the mouse α-TCRβ antibody clone Antibody B VH and VL were assigned using IMGT nomenclature, with CDR regions defined by a combined Kabat and Chothia classification. SEQ ID NO: 15 and SEQ ID NO: 16 are the Antibody B VH and VL sequences respectively where the VH germline is mouse IGHV5-17*02 and the VL germline is mouse IGKV4-50*01. SEQ ID NOs: 17-19 are the B-H VH CDR regions 1-3 respectively and SEQ ID NOs: 20-22 are the B-H VL CDR regions 1-3 (as described in Table 31).

The method applied to humanize Antibody A described in Example 1 was used to humanize Antibody B. The Antibody B VH was humanized into human IGHV3-30*01, IGHV3-48*01, and IGHV3-66*01 and the Antibody B VL was humanized into human IGKV1-9*01, IGKV1-39*01, IGKV3-15*01, IGLV1-47*01 and IGLV3-10*01. SEQ ID NOs: 23-25 are the B-H.1A, B-H.1B, and B-H.1C humanized heavy chains and SEQ ID NOs: 26-30 are the B-H.1D, B-H.1E, B-H.1F, B-H.1G and B-H.1H humanized light chains (as described in Table 31). FIGS. 2A and 2B show the murine and humanized sequences with annotations depicting the CDR and framework regions (FR).

Example 3: Characteristics of Anti-TCRβV Antibodies

Introduction

Current bispecific constructs designed to redirect T cells to promote tumor cell lysis for cancer immunotherapy typically utilize single chain variable fragments (scFvs) that are derived from monoclonal antibodies (mAb) directed against the CD3e subunit of the T cell receptor (TCR). However, there are limitations to this approach which may prevent the full realization of the therapeutic potential for such bispecific constructs. Previous studies have shown that, e.g., low “activating” doses of anti-CD3e mAb can cause long-term T cell dysfunction and exert immunosuppressive effects. In addition, anti-CD3e mAbs bind to all T cells and thus activate equally all T cells, which has been associated with the first dose side effects of anti-CD3e mAbs that result from massive T cell activation. These large number of activated T cells secrete substantial amounts of cytokines, the most important of which is Interferon gamma (IFNg). This excess amount of IFNg in turn, e.g., activates macrophages which then can overproduce proinflammatory cytokines such as IL-1, IL-6 and TNF-alpha, causing a “cytokine storm” known as the cytokine release syndrome (CRS). Thus, it might be advantageous to develop antibodies that are capable of binding and activating only a subset of necessary effector T cells to reduce the CRS.

Results

To that end, antibodies directed to the variable chain of the beta subunit of TCR (TCR Vb) were identified. These anti-TCR Vb antibodies bind and activate a subset of T cells, but with, e.g., no or markedly reduced CRS. Using plate-bound anti-TCR Vb13.1 mAbs (A-H.1 and A-H.2) it was shown that a population of T cells, defined by positive staining with A-H.1, can be expanded (from ˜5% of T cells on day 0 to almost 60% of total T cells on day 6 of cell culture) (FIGS. 4A-4C). For this experiment, human CD3+ T cells were isolated using magnetic-bead separation (negative selection) and activated with immobilized (plate-coated) A-H.1 or OKT3 (anti-CD3e) antibodies at 100 nM for 6 days. The expanded Vb13.1+ T cells display cytolytic activity against transformed cell line RPMI-8226 when co-cultured with purified CD3+ T cells (FIGS. 5A-5B).

Next, the ability of PBMCs activated by anti-TCR VB antibodies to produce cytokines was assessed. The cytokine production of PBMCs activated with anti-TCR VB antibodies was compared to the cytokine production of PBMCs activated with: (i) anti-CD3e antibodies (OKT3 or SP34-2); (ii) anti-TCR V alpha (TCR VA) antibodies including anti-TCR VA 12.1 antibody 6D6.6, anti-TCR VA24JA18 antibody 6B11; (iii) anti-TCR alpha beta antibody T10B9; and/or (iv) isotype control (BGM0109). The anti-TCR VB antibodies tested include: humanized anti-TCRVB 13.1 antibodies (A-H.1, or A-H.2), murine anti-TCR VB5 antibody E, murine anti-TCR VB8.1 antibody B, and murine anti-TCR VB12 antibody D. BGM0109 comprises the amino acid sequence of

(SEQ ID NO: 3282)

METDTLLLWVLLLWVPGSTGGLNDIFEAQKIEWHEGGGGSEPRTDTDTC

PNPPDPCPTCPTPDLLGGPSVFIFPPKPKDVLMISLTPKITCVVVDVSE

EEPDVQFNWYVNNVEDKTAQTETRQRQYNSTYRVVSVLPIKHQDWMSGK

VFKCKVNNNALPSPIEKTISKPRGQVRVPQIYTFPPPIEQTVKKDVSVT

CLVTGFLPQDIHVEWESNGQPQPEQNYKNTQPVLDSDGSYFLYSKLNVP

KSRWDQGDSFTCSVIHEALHNHHMTKTISRSLGNGGGGS.

As shown in FIG. 6A, when plate-bound A-H.1 or A-H.2, or anti-CD3e antibodies (OKT3 or SP34-2) were used to activate human PBMCs, the T cell cytokine IFNg was induced (FIG. 6A). All anti-TCR VB antibodies tested had a similar effect on the production of IFNg (FIG. 6B). The anti-TCR VA antibodies did not induce similar IFNg production.

With respect to IL-2 production, PBMCs activated with A-H.1 and A-H.2 resulted in increased IL-2 production (FIG. 7A) with delayed kinetics (FIG. 7B) as compared to PBMCs activated with anti-CD3e antibodies (OKT3 or SP34-2). FIG. 7B shows that anti-TCR VB antibody activated PBMCs demonstrate peak production of IL-2 at Day 5 or Day 6 post-activation (incubation with plate-coated antibodies). In contrast, IL-2 production in PBMCs activated with OKT3 peaked at day 2 post-activation. As with IFNG, the IL-2 effect (e.g., enhanced production of IL-2 and delayed kinetics) was similar across all anti-TCR VB antibodies tested (FIG. 7B).

The production of cytokines IL-6, IL-1β and TNF-alpha which are associated with “cytokine storms” (and accordingly CRS) was also assessed under similar conditions. FIGS. 8A, 9A and 10A shows that while PBMCs activated with anti-CD3e antibodies demonstrate production of IL-6 (FIG. 8A), TNF-alpha (FIG. 9A) and IL-1β (FIG. 10A), no or little induction of these cytokines was observed with PBMCs activated with A-H.1 or A-H.2. As shown in FIGS. 9B and 10B, TNF-alpha and IL-1β production was not induced by activation of PBMCs with any of the anti-TCR VB antibodies.

It was further noted that the kinetics of IFNg production by A-H.1-activated CD3+ T cells was delayed relative to those produced by CD3+ T cells activated by anti-CD3e mAbs (OKT3 and SP34-2) (FIGS. 11A and 11B).

Finally, it was observed that the subset of memory effector T cells known as T_EMRA was preferentially expanded in CD8+ T cells activated by A-H.1 or A-H.2 (FIG. 12). Isolated human PBMCs were activated with immobilized (plate-coated) anti-CD3e or anti-TCR Vβ13.1 at 100 nM for 6-days. After a 6-day incubation, T-cell subsets were identified by FACS staining for surface markers for Naive T cell (CD8+, CD95-, CD45RA+, CCR7+), T stem cell memory (TSCM; CD8+, CD95+, CD45RA+, CCR7+), T central memory (Tcm; CD8+, CD95+, CD45RA-, CCR7+), T effector memory (Tem; CD8+, CD95+, CD45RA-, CCR7-), and T effector memory re-expressing CD45RA (Temra; CD8+, CD95+, CD45RA+, CCR7-). Human PBMCs activated by anti-TCR Vβ 13.1 antibodies (A-H.1 or A-H.2) increased CD8+TSCM and Temra T cell subsets when compared to PBMCs activated by anti-CD3e antibodies (OKT3 or SP34-2). Similar expansion was observed with CD4+ T cells.

CONCLUSION

The data provided in this Example show that antibodies directed against TCR Vb can, e.g., preferentially activate a subset of T cells, leading to an expansion of T_EMRA, which can, e.g., promote tumor cell lysis but not CRS. Thus, bispecific constructs utilizing either a Fab or scFv or a peptide directed to the TCR Vb can, e.g., be used to activate and redirect T cells to promote tumor cell lysis for cancer immunotherapy, without, e.g., the harmful side-effects of CRS associated with anti-CD3e targeting.

Example 4: On-Target T Cell Mediated Cytotoxicity of Multiple Myeloma (MM) Cells with a Dual-Targeting Antibody Molecule Against BCMA and a T Cell Engager

This example shows on-target T cell mediated cytotoxicity of multiple myeloma (MM) cells with dual-targeting antibody molecules that recognize a T cell engager, e.g., TCRVb, on T cells and BCMA on MM cells.

As shown in FIG. 13A, purified human T cells activated with plate-bound anti-TCRVb antibody for 5 days proliferate at a higher rate than purified human T cells activated with plate-bound anti-CD3 (OKT3) antibody. Anti-TCRVb antibody stimulation of T cells resulted in selective expansion of CD45RA+ effector memory CD8+ and CD4+ T cells (TEMRA) cells (FIG. 13B). Both CD8+ and CD4+ Temra cell populations expanded more when stimulated with an anti-TCRVb antibody, compared to unstimulated cells or cells stimulated with an anti-CD3 (SP34) antibody. Anti-TCRVb antibodies resulted in delayed secretion of IFN-g by PBMCs stimulated with an anti-TCRVb antibody compared to PBMCs stimulated with anti-CD3 antibodies (FIG. 13C). Additionally, T cells stimulated with anti-TCRVb antibody or anti-CD3 antibodies resulted in comparable lysis of multiple myeloma target cells, as shown in FIG. 13D. As shown in FIGS. 13E-13F, T cells stimulated for 5 days with 100 ng/ml plate-bound an anti-TCRVb antibody, or an anti-CD3 antibody secreted perforin and Granzyme B.

Activation of PBMCs with anti-TCRVb antibody resulted in higher production and/or secretion of IL-2 and/or IL-15 compared to PBMCs activated with an anti-OKT3 antibody (FIG. 14A). Anti-TCRVb antibody activated of PBMCs also resulted in expansion and/or survival, e.g., proliferation of Natural Killer (NK) cells (FIG. 14B). In comparison, PBMCS activated with an anti-OKT3 antibody did not result in NK cell expansion. Further, as described in Example 3, PBMCs activated with an anti-TCRVb antibody did not result in the production of cytokines IL-6, IL-1β and TNF-alpha which are associated with CRS (FIG. 15). These in vitro characterization studies show that in some embodiments, anti-TCRVb antibodies, e.g., activate and/or stimulate, T cells to promote T cell killing as evidenced by target cell lysis, perforin secretion and granzyme B secretion, and secretion of IFN-g with, e.g., delayed kinetics.

Next, the ability of a dual-targeting antibody molecule, which targets BCMA on one arm and TCRVb on the other arm, to target and kill multiple myeloma (MM) cells was tested. Healthy donor PBMCs were co-incubated with the RMPI8226 MM cell line and one of the following dual-targeting antibody molecules: BCMA-TCRVb, BCMA-CD3, or Control-TCRVb; or an isotype control Target cell lysis was then assessed using flow cytometry. As shown in FIG. 16A, the dual-targeting BCMA-TCRVb antibody molecule resulted in killing of MM cells in vitro.

The dual-targeting BCMA-TCRVb antibody molecule was further tested in vivo for its ability to inhibit MM tumor growth in a MM mouse model. The NCI-H929 cell line was injected in NOD-scid IL2rγnull (NSG)recipient mice on Day 0 followed by delivery of PBMCs on Day 9. On Days 12, 15, 18 and 21, the dual-targeting BCMA-TCRVb antibody molecule was administered via intraperitoneal injection at a dose of 0.5 mg/kg. FIG. 16B shows prevention, e.g., inhibition, of MM tumor growth in vivo with the dual-targeting BCMA-TCRVb antibody molecule. These results demonstrate that in some embodiments the dual-targeting BCMA-TCRVb antibody molecule, e.g., can kill tumor cells, e.g., MM tumor cells, in vitro and in vivo. Accordingly, in some embodiments, a dual-targeting BCMA-TCRVb antibody molecule can be used, e.g., as a therapy for cancer, e.g., a hematological cancer, e.g., MM.

Example 5: In Vitro Cytotoxicity of a Dual-Targeting Antibody Molecule Against FcRH5 and a T Cell Engager

This example shows in vitro cytotoxicity on multiple myeloma (MM) cells with a dual-targeting antibody molecule that recognizes a T cell engager, e.g., TCRVb, on T cells and FcRH5 on MM cells. Healthy donor PBMCs or purified T cells were co-incubated with the MOL8M MM cell line and a dual-targeting antibody molecule which targets FcRH5 on one arm and TCRVb on the other arm, or with an isotype control antibody. Target cell lysis was then assessed using flow cytometry. As shown in FIG. 17, the dual targeting FcRH5-TCRVb molecule resulted in killing of MM cells by both purified T cells or PBMCs. This shows that the dual targeting FcRH5-TCRVb molecule can target and promote killing of MM cells by immune cells, e.g., in PBMCs, including T cells.

Example 6: Immunization of Armenian Hamster to Generate Anti-NKp30 Antibodies

Briefly, Armenian hamsters were immunized with the extracellular domain of human NKp30 protein in complete Freund's adjuvant and boosted twice on day 14 and day 28 with NKp30 in incomplete Freund's adjuvant (IFA). On day 56 one more boost in IFA was given and the animals harvested three days later. Spleens were collected and fused with P3X63Ag8.653 murine myeloma cell line. 0.9×10{circumflex over ( )}5 cells/well in 125 μl were seated in 96 well plate and feed with 125 μl of I-20+2ME+ HAT (IMDM (4 g/L glucose) supplemented with 20% fetal bovine serum, 4 mM L-glutamine, 1 mM sodium pyruvate, 50 U penicillin, 50 μg streptomycin and 50 μM 2-ME in the absence or presence of HAT or HT for selection, and Hybridoma Cloning Factor (1% final) on days 7, 11 and thereafter as needed. At approximately 2 weeks after fusion (cells are about 50% confluent) supernatant was collected and assayed for binding.

Example 7: Hybridoma Screen for NKp30 mAbs

Expi293 cells were transfected with BG160 (hNKp30 cell antigen) 18 hours prior to screening. The day of screening, transfected cells were diluted to 0.05×10{circumflex over ( )}⁶/mL and anti-Armenian hamster Fc Alexa Fluor 488 added to a final concentration of 0.4 ug/mL. 50 uL (2,500 cells) of this mixture was added to each well of a 384 well plate. The same density of untransfected 293 cells with secondary were used as a negative control. 5 uL of hybridoma supernatant was added to the cell mixture and the plate incubated for 1 hour at 37° C. The plates were then imaged on Mirrorball. Positive clones were identified and subcloned by serial dilution to obtain clonal selected hybridoma. After reconfirmation using the same protocols the hybridoma cells were harvested and the corresponding heavy and light chain sequences recovered. The DNA was subcloned into pcDNA3.4 for subsequent expression of the corresponding antibodies and further validation.

Example 8: Binding of NKp30 Antibodies to NK92 Cells

NK-92 cells were washed with PBS containing 0.5% BSA and 0.1% sodium azide (staining buffer) and added to 96-well V-bottom plates with 200,000 cells/well. Hamster NKp30 antibodies were added to the cells in 2.0 fold serial dilutions and incubated for 1 hour at room temperature. The plates were washed twice with staining buffer. The secondary antibody against hamster Fc conjugated to AF647 (Jackson, 127-605-160) was added at 1:100 dilution (1.4 mg/ml stock) and incubated with the cells for 30 minutes at 4° C. followed by washing with staining buffer. Cells were subsequently were fixed for 10 minutes with 4% paraformaldehyde at room temperature. The plates were read on CytoFLEX LS (Beckman Coulter). Data was calculated as the percent-AF747 positive population (FIG. 22).

Example 9: Bioassay to Measure Activity of NKp30 Antibodies Using NK92 Cell Line

NKp30 antibodies were three-fold serially diluted in PBS and incubated at 2-8° C. overnight in flat bottom 96 well plates. Plates were washed twice in PBS and 40,000 NK-92 cells were added in growth medium containing IL-2. Plates were incubated at 37° C., 5% CO2, humidified incubator for 16-24 hours before supernatants were collected. IFNγ levels in supernatants was measured following MSD assay instructions (FIG. 23). Supernatant collected from cells incubated with hamster isotype IgG was used as negative control and supernatants from cells incubated with NKp30 monoclonal antibody (R&D, clone 210847) was utilized as a positive control. Data were generated using hamster anti-NKp30 mABs.

Example 10: Characterization of Anti-Calreticulin Antibodies

A murine anti-calreticulin antibody AbM-1 (also referred to as BIM0031) which comprises a VH of SEQ ID NO: 6250 and a VL of SEQ ID NO: 6252 was humanized. Five humanized VHs (SEQ ID NOs: 6372 and 234-237 shown in Table 16) and five humanized VLs (SEQ ID NOs: 238-242 shown in Table 16) were generated. All the humanized VHs comprise a cysteine to alanine substitution in HCDR2. Antibodies BJM0040-BJM0064, as disclosed in Table 19, were synthesized and characterized for their biochemical and functional activities.

Briefly, expression level of purified proteins was measured after protein A elution. Proteins were analyzed by analytical SEC to assess aggregation and tested by differential scanning fluorimetry (DSF) to identify more stable cantidates. Binding affinity of the cantidates was measured in ELISA assay against mutant calreticulin C-terminal peptide fused to a human Fc. The results were summarized in Table 22. Humanized antibodies comprising the cysteine to alanine substitution in HCDR2 demonstrated reduced aggregation compared to the parental murine antibody.

TABLE 22

Summary of characterization of anti-calreticulin antibodies

yield
% aggregation
Tm
ELISA

(mg/L)
after ProA
(C.)
IC50

BJM0040
95.7
0
75
12.34

BJM0041
193.6
5.3
75
18.79

BJM0042
106.7
0
75
10.52

BJM0043
181.5
3
75
8.279

BJM0044
161.7
5.6
75
16.19

BJM0045
42.9
8
73
~836101

BJM0046
116.6
7.5
76
~362165

BJM0047
93.5
7
76
802.9

BJM0048
111.1
6
75
430.3

BJM0049
103.4
6.8
76
943.6

BJM0050
261.8
10.3
—
597627

BJM0051
112.2
7.4
77
780.7

BJM0052
123.2
12.4
77
776.2

BJM0053
132
10.3
76
357.2

BJM0054
128.7
12.3
77
657.2

BJM0055
72.6
17.1
69
1E+06

BJM0056
113.3
11.6
69
889.5

BJM0057
67.1
12.1
69
4E+06

BJM0058
92.4
9.1
69
498.4

BJM0059
136.4
12
68
83.11

BJM0060
134.2
7.5
72
~4347

BJM0061
140.8
8.5
73
356

BJM0062
91.3
8.5
73
351.8

BJM0063
145.2
8.8
73
988.6

BJM0064
139.7
10
73
637.4

BIM0031

24.32

Example 11: Generation and Characterization of Humanized Anti-NKp30 Antibodies

A series of hamster anti-NKp30 antibodies were selected. These antibodies were shown to bind to human NKp30 and cynomolgus NKp30 and induce IFNγ production from NK-90 cells (data not shown). The VH and VL sequences of exemplary hamster anti-NKp30 antibodies 15E1, 9G1, 151H6, 9D9, 3A12, and 12D10 are disclosed in Table 9. The VH and VL sequences of exemplary humanized anti-NKp30 antibodies based on 15E1, 9G1, and 151H6 are also disclosed in Table 9. The Kabat CDRs of these antibodies are disclosed in Table 34 and Table 8.

Two humanized constructs based on 15E1 were selected. The first construct BJM0407 is a Fab comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7302 and a lambda light chain variable region comprising the amino acid sequence of SEQ ID NO: 7305. Its corresponding scFv construct BJM0859 comprises the amino acid sequence of SEQ ID NO: 7310. The second construct BJM0411 is a Fab comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7302 and a kappa light chain variable region comprising the amino acid sequence of SEQ ID NO: 7309. Its corresponding scFv construct BJM0860 comprises the amino acid sequence of SEQ ID NO: 7311. BJM0407 and BJM0411 showed comparable biophysical characteristics, e.g., binding affinity to NKp30 and thermal stability. The scFv constructs BJM0859 and BJM0860 also showed comparable biophysical properties.

Example 12: Binding of an Exemplary Anti-Calreticulin Antibody Molecule to Wild-Type or Mutant Calreticulin

In this example, an exemplary anti-calreticulin antibody molecule, BKM0106 (parental IgG form of antibody 6C10), was tested for binding to wild-type calreticulin (CALR WT) compared to two calreticulin mutants (CALR ins and CALR del, the sequences of which are listed in Table 2 as SEQ ID Nos: 1002 and 1003, respectively). Two ELISA tests were run, one in which the antigen was coated in the plate, and one in which the antibody was coated on the plate.

For the experiment in which antigen was coated on the plate, a Maxisorp plate was coated with CALR WT, CALR in, or CALR del protein. The plate was blocked with 3% BSA. BKM0106 was diluted to 100 nM and added to the wells. Anti-Human HRP (Jackson Immunoresearch 309-035-008) 1:20,000 was added to each well. 1-Step Turbo TMB-ELISA Substrate solution was added and the reaction was stopped by adding 1M HCl. The plate was read at 450 nm.

For the experiment in which antibody was coated on the plate, a Maxisorp plate was coated with BKM0106. The plate was blocked with 3% BSA. CALR WT, CALR ins, and CALR del were diluted to 100 nM and added to separate wells. Anti-His-Tag-HRP (Southern Biotech 4603-05) 1:5,000 was added to each well. 1-Step Turbo TMB-ELISA Substrate solution was added and the reaction was stopped by adding 1M HCl. The plate was read at 450 nm.

As shown in FIGS. 25A-25B, the exemplary antibody BKM0106 bound to the CALR ins and CALR del mutants, but did not bind to wild-type calreticulin.

Example 13: Binding of an Exemplary Anti-Calreticulin Antibody Molecule to Cells Expressing Mutant Calreticulin

In this example, an exemplary anti-calreticulin antibody molecule, BKM0106 (parental IgG form of antibody 6C10), was tested for binding to cells expressing mutant calreticulin (CALR ins and CALR del, the sequences of which are listed in Table 2 as SEQ ID Nos: 1002 and 1003, respectively). Briefly, Expi293F cells (Thermo Fisher A14527) were triple transfected with BH470 (human TpoR), BH472 (human JAK2) and either human CALR ins, human CALR del, or BH800 (human CALR WT) cell antigens. BKM0106 and the hIgG1 isotype control at 0.78, 1.56, 3.125, 6.25, 12.5, 25, and 50 nM were added to the wells. Cells were incubated with secondary antibody Alexa Fluor 488 Anti-Human IgG (Jackson ImmunoResearch 109-545-088) 1:200. Zombie Violet BV412 (BioLegend 423114) viability dye was added to the cells 1:100.

As shown in FIGS. 26A-26B, BKM0106 bound to both CALR ins and CALR del with similar responses.

Example 14: Targeting mtCALR+ Cells Via Bridging mtCALR+ Cells to Immune Effector Cells Via Bispecific Antibodies

In this example, a series of antibody molecules was tested for therapeutic efficacy in a syngeneic murine cancer model. Briefly, surrogate molecules with a mIgG2a backbone were generated from the exemplary anti-calreticulin antibody molecule 6C10 and each of the following effector arms:

- BKM0201-mutCALR-6C10 monoclonal-ADCC enabled
- BKM0202-mutCALR-6C10×TCRvB (2×2) bispecific-LALAPG
- BKM0204-mutCALR-6C10×CD3-2C11 (1×1) bispecific-LALAPG

The therapeutic efficacy of the above molecules were tested in a systemic murine model generated using Ba/F3 cells engineered cells expressing mtCALR, hMPL, and hJAK2. On day 1 of the study, 2×10{circumflex over ( )}6 engineered Ba/F3 mtCALR, hMPL, hJAK2 cells, suspended in PBS, were intravenously injected in to female Balb/C mice. Mice were treated a dose of 1 mg/kg or 5 mg/kg of CALR×/TCRvB and control bispecific molecules every three days for a total of 4 doses (day 4, 7, 10 and 13) by intravenous bolus injection. Animals were monitored for humane endpoints and Body weights were monitored twice weekly. All animals were euthanized on day 15 and spleens were excised, and spleen weights were monitored as a surrogate for disease burden. Data indicates that vehicle treated mice in Ba/F3 mtCALR, hMPL, hJAK2 cell engrafted mice show nearly three-fold increase in spleen weight as compared to the naïve mice suggesting increased disease burden.

As shown in FIG. 27, all three antibody treated mice showed a significant decrease in spleen weights as compared to vehicle control.

Example 15: Biacore Analysis of Exemplary Anti-NKp30 Antibody Molecules

In this example, a series of exemplary anti-NKp30 antibody molecules were analyzed for their binding affinity for NKp30. Briefly, surface plasmon resonance (SPR) measurements were performed by using the BIAcore T200. Human NKp30 (BKM0179) was immobilized on a CM5 chip via anti-mouse Fc antibody to a response of 50 RU. Each exemplary antibody construct were injected at concentrations of 3.9, 7.8, 15.6, 31.2, 62.5, and 125 nM, and at a flow rate of 20 μl/min, over the surface on which the human NKp30 was immobilized. The data was fit using a 1:1 binding model.

As shown in Table 23, most of the exemplary antibodies showed preserved affinity to human NKp30 compared to the parental antibody.

TABLE 23

Biacore results

Human Nkp30

Construct
Description
(BKM0179)

BJM1078
BJM0407 Parental
1.48
nM

BJM1079
BJM0411 Parental
1.26
nM

BKM0138
BJM0411 VL-N95A
3.2
nM

BKM0139
BJM0411 VL-D92A
3.2
nM

BKM0140
BJM0407 VL-D92A
3.3
nM

BKM0141
BJM0407 VL-N95A
3.0
nM

BKM0142
BJM0411 VH-N60A
1.28
nM

BKM0143
BJM0407 VH-N60A
1.45
nM

BKM0144
BJM0411 VH-N60A-VL-D92A-N95A
6.4
nM

BKM0145
BJM0407 VH-N60A-VL-D92A-N95A
4.2
nM

Example 16: Production and Assessment of Exemplary Anti-CD3 Antibody Molecules

Production of Anti-CD3 Antibody Molecules

Four C57/BL6 mice were immunized with KLH conjugated CD3e peptide bearing the sequence QDGNEEMGGITQTPYKVSISGTTVILTC (SEQ ID NO: 7487). Animals were bled three days after fourth immunization to check for titers. After the fourth immunization, animals were boosted twice with the antigen. Three to four days after final boost, animals are sacrificed for spleen or lymph node tissue harvest. Spleen was fused using standard fusion methods which produced 20 plates of hybridoma cells. The primary screen was performed using ELISA by checking binding to the peptide followed by CD3e protein. One clone, 4D4, was selected for expansion and following subcloning protocol, generated monoclonal hybridoma.

Binding of Anti-CD3 Antibody Molecules to CD3 In Vitro

In this example, exemplary humanized anti-CD3 antibody molecules BKM0020, BKM0025, BKM0028, BKM0038 (as described herein) were tested for their binding affinity to human or cynomolgus CD3e using surface plasmon resonance (SPR). SPR measurements were performed by using the BIAcore T200. Each construct was immobilized on a CM5 chip via Anti-human Fc antibody to a response of 200 RU. Human CD3e (Acro Biosystems CDE-H5223) was diluted to 500 nM and then diluted two-fold. Each analyte concentration was injected at a flow rate of 20 μl/min over the surface on which each antibody was immobilized. The data was fit using a 1:1 binding model. Binding affinity results are shown in FIG. 28. Affinity to human CD3e was preserved compared to the parental and affinity to cyno CD3e was two to three-fold lower compared to the parental antibody.

Binding of Anti-CD3 Antibody Molecules to CD3 Expressed on Cells

Binding of the exemplary humanized anti-CD3 antibodies to CD3-expressing Jurkat cells was performed using FACS. Antibodies were tested in a series of 3-fold dilutions starting with 10 ug/ml concentration and detected using anti-human IgG secondary antibodies conjugated to AF647. As shown in FIG. 29, humanized anti-CD3 mAb showed strong binding to Jurkat cells expressing CD3.

Example 17: Generation of Anti-Calreticulin (CALR) Antibodies

Two Armenian Hamsters (86 and 87) were immunized with mutCALR 5 bp ins (BJ028) antigen. Hamster #86 was chosen to perform fusion using standard procedures. Fusion produced 12 hybridoma plates that were screened by ELISA for binding to BJ028 (mut CALR ins), BJ027 (wild-type CALR) and BIM0167 (CALR mutant peptide fused to a human Fc). 15 clones were further selected for expansion and subcloning. Two clones were selected for further characterization and sequencing to obtain V gene sequences. The hamster sequences were further humanized by grafting CDRs on to human frameworks. The resulting humanized mAbs were tested for binding using Biacore and FACS, e.g., as described above.

Example 18: Optimization of α-TRBV6-5 Antibody

The anti TRBV6-5 antibody was optimized to improve affinity for the human and cyno antigen, improve thermal stability, and remove sequence motifs that might pose chemical stability liabilities. ScFv libraries were built using random mutagenesis (Caldwell et al. (1992) Randomization of genes by PCR mutagenesis. PCR Meth. Appl. 2:28) or a modified version of Kunkel mutagenesis (Kunkel T A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. PNAS 82(2): 488-92). For affinity improvement, library selections vs human and cyno antigens were performed using standard phage display (Lee, C M et al. (2007) Selection of human antibody fragments by phage display. Nature protocols 2, 3001) and yeast display techniques (Chao G, et al. (2006) Isolating and engineering human antibodies using yeast surface display. Nature Protocols. 1(2):755-69). Thermal challenge of phage or yeast populations was used to select for clones with improved thermal stability. Selections were followed by standard screening methods such as ELISA and flow cytometry to identify individual clones with improved properties. Following hit sequencing and analysis of mutation-activity correlation, second-generation libraries were constructed using the same methods above. Library selections and individual clone screening were repeated as above with the modification that more stringent conditions were applied to select for clones with maximized activity. Following hit sequencing, scFv genes were reformatted into the biologically relevant antibody format for expression, purification, and triaging.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

EQUIVALENTS

EXEMPLARY EMBODIMENTS

Additional features of any of the aforesaid multifunctional molecules, nucleic acids, vectors, host cells, or methods include one or more of the following exemplary embodiments.

Exemplary Embodiment 1

The disclosure relates, inter alia, to novel multispecific or multifunctional molecules that include (i) an antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein); and one, two or all of: (ii) an immune cell engager (e.g., chosen from an NK cell engager, a T cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager); (iii) a cytokine molecule; and/or (iv) a stromal modifying moiety. The terms “multispecific” or “multifunctional” are used interchangeably herein.

Without wishing to be bound by theory, the multispecific or multifunctional molecules disclosed herein are expected to target (e.g., localize, bridge and/or activate) an immune cell (e.g., an immune effector cell chosen form an NK cell, a T cell, a B cell, a dendritic cell or a macrophage), at a target cell, e.g., a cancer cell, expressing a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and/or alter the tumor stroma, e.g., alter the tumor microenvironment near the cancer site. Increasing the proximity and/or activity of the immune cell using the multispecific molecules described herein is expected to enhance an immune response against the target cell (e.g., the cancer cell), thereby providing a more effective therapy (e.g., a more effective cancer therapy). Without being bound by theory, a targeted, localized immune response against the target cell (e.g., the cancer cell) is believed to reduce the effects of systemic toxicity of the multispecific molecules described herein.

Accordingly, provided herein are, inter alia, multispecific molecules (e.g., multispecific or multifunctional antibody molecules) that include the aforesaid moieties, nucleic acids encoding the same, methods of producing the aforesaid molecules, and methods of treating a cancer using the aforesaid molecules.

In an aspect, the disclosure features a method of detecting calreticulin (e.g., wild-type or mutant calreticulin) in a sample or subject, comprising: contacting the sample or subject with an anti-calreticulin antibody molecule described herein; and detecting formation of a complex between the antibody molecule and the sample or subject, thereby detecting calreticulin (e.g., wild-type or mutant calreticulin).

In some embodiments, calreticulin (e.g., wild-type or mutant calreticulin) is detected in vitro or in vivo.

In some embodiments, the method further comprises contacting a reference sample or subject with the antibody molecule; and detecting formation of a complex between the antibody molecule and the reference sample or subject, wherein a change, e.g., a statistically significant change, in the formation of the complex in the sample or subject, relative to the reference sample or subject is indicative of the presence of calreticulin (e.g., wild-type or mutant calreticulin) in the sample or subject.

In some embodiments, the method further comprises obtaining a sample from a subject.

In some embodiments, the sample comprises one or more of plasma, tissue (e.g., cancerous tissue), biopsy, blood (e.g., whole blood), PBMCs, bone marrow, and/or lymphatic tissue, e.g., lymph node. In some embodiments, the sample has not been frozen and/or fixed. In some embodiments, the sample has been formalin-fixed (e.g., formalin-fixed, paraffin-embedded (FFPE). In some embodiments, the sample has been stained (e.g., for analysis by immunohistochemistry). In some embodiments, the sample has been frozen and/or fixed.

In some embodiments, the subject has, or is at risk of having, a disease or disorder described herein (e.g., cancer, e.g., a myelofibrosis).

In some embodiments, the method further comprises performing a flow cytometry analysis, e.g., using a multi-panel method. In some embodiments, the method further comprises performing an immunohistochemical (IHC) analysis, e.g. monochrome or in a multiplexed format. In some embodiments, the IHC method comprises brightfield chromogenic IHC. In embodiments, the brightfield chromogenic IHC method comprises direct detection of antigens by primary antibodies, e.g., which are directly labeled with different chromogens. In some embodiments, the IHC method comprises fluorescent IHC. In embodiments, the fluorescent IHC method comprises direct detection of antigens by primary antibodies, e.g., which are directly labeled with different fluorophores. In some embodiments, the method further comprises performing immunohistochemistry on a sample, e.g., a fixed sample, e.g., an FFPE sample. In some embodiments, the method further comprises measuring the level of calreticulin+(e.g., wild-type calreticulin+ or mutant calreticulin+) cells from the biological sample (e.g., determining if calreticulin+(e.g., wild-type calreticulin+ or mutant calreticulin+) cells are depleted, e.g., relative to a reference sample or subject. In some embodiments, the method further comprises measuring the intracellular level of calreticulin (e.g., wild-type or mutant calreticulin). In some embodiments, the method further comprises measuring the membrane level of calreticulin (e.g., wild-type or mutant calreticulin).

In some embodiments, the method comprises combining two or more of the detection methods described herein. In embodiments, the method comprises a nucleic acid-based method and an antibody-based method.

In some embodiments, the method further comprises evaluating the subject for a change in prognosis, severity, or presence or absence of a disease or disorder (e.g., cancer, e.g., myelofibrosis), e.g., after treatment (e.g., with an antibody molecule described herein).

In some embodiments, the antibody molecule is detectably labeled. In some embodiments, the antibody molecule is an anti-calreticulin (e.g., wild-type or mutant calreticulin) antibody molecule.

In an aspect, the disclosure features a method of evaluating a subject, comprising: contacting a sample (e.g., a sample described herein) from the subject with an anti-calreticulin (e.g., wild-type or mutant calreticulin) antibody molecule described herein; and

- detecting formation of a complex between the antibody molecule and the sample, thereby evaluating the subject.

In some embodiments, the subject has, or is at risk of having, a disease or disorder described herein (e.g., cancer, e.g., myelofibrosis). In some embodiments, the subject has not been treated with an antibody molecule described herein. In some embodiments, the subject has been treated with an antibody molecule described herein.

In an aspect, the disclosure features a kit comprising an anti-calreticulin (e.g., wild-type or mutant calreticulin) antibody molecule described herein and instructions for use in a method of detecting calreticulin (e.g., wild-type or mutant calreticulin) in a sample or subject, e.g., in accordance with a method described herein.

Accordingly, in one aspect, the disclosure features a multifunctional molecule that includes:

- (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), e.g., a calreticulin-targeting antigen binding domain disclosed in any one of Table 4, Table 5, Table 6, Table 24, Table 25, Table 16, Table 17, Table 18, or Table 19,
- and
- (ii) a second antigen binding domain that binds to TCRβV, e.g., an anti-TCRβV antigen binding domain disclosed in any one of Table 30, Table 31, Table 32, Table 33, Table 11, Table 12, or Table 13, or a second antigen binding domain that binds to NKp30, e.g., an anti-NKp30 antigen binding domain disclosed in Tables 7, Table 8, Table 35, Table 36, Table 9, Table 10, or Table 34.

In some embodiments, the second antigen binding domain binds to TCRβV.

In some embodiments, the second antigen binding domain activates a T cell or the second antigen binding domain does not activate a T cell.

In some embodiments, the second antigen binding domain binds to TCRβ V12 or TCRβ V6 (e.g., comprising the amino acid sequence of SEQ ID NO: 1044).

In some embodiments, the second antigen binding domain comprises one or more amino acid sequences as listed in Table 30, Table 31, Table 32, Table 33, Table 11, Table 12, or Table 13.

In some embodiments, the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 3 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 4 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 5 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 7 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 8 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 45 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 46 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 47 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 51 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 52 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 53 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or
- (d) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 48 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 49 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 50 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 54 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 55 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 56 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of a VH in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of a VL in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto)
- (iii) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (iv) the VL comprises the amino acid sequence of SEQ ID NO: 10 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of SEQ ID NO: 11 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or
- (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of SEQ ID NO: 1312 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of SEQ ID NO: 1314 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 63 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or
- (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 66 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 67 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 68 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of SEQ ID NO: 15 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of SEQ ID NO: 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises: the amino acid sequence of SEQ ID NO: 23 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 24 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 25 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or
- (ii) the VL comprises: the amino acid sequence of SEQ ID NO: 26 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 27 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 28 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 30 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

In some embodiments, the multifunctional molecule comprises: a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to TCR (e.g., TCRVβ) (e.g., a first scFv that binds to TCR (e.g., TCRVβ)),

- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to TCR (e.g., TCRVβ) (e.g., a second scFv that binds to TCR (e.g., TCRVβ)),
- a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH form a third antigen binding domain that binds to a second calreticulin protein,
- optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286,
- optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In some embodiments, the second antigen binding domain binds to NKp30.

In some embodiments, the second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 7313 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions; and/or
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 7326 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of any of SEQ ID NOs: 7298 or 7300-7304 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of SEQ ID NOs: 7298 or 7300-7304); and/or
- (ii) a VL comprising the amino acid sequence of any of SEQ ID NOs: 7299 or 7305-7309 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to any of SEQ ID NOs: 7299 or 7305-7309).

In some embodiments, the second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7305 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7305); or
- (ii) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7309 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7309).

In some embodiments, the second antigen binding domain comprises:

- (i) an amino acid sequence of SEQ ID NO: 7310 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7310); or
- (ii) an amino acid sequence of SEQ ID NO: 7311 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7311).

In some embodiments, the second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

In some embodiments, the second antigen binding domain comprises:

- (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) having an amino acid sequence of a VHFWR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 having an amino acid sequence of a VHFWR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 having an amino acid sequence of a VHFWR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 having an amino acid sequence of a VHFWR4 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), and/or
- (2) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) having an amino acid sequence of a VLFWR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 having an amino acid sequence of a VLFWR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 having an amino acid sequence of a VLFWR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 having an amino acid sequence of a VLFWR4 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

In some embodiments, the second antigen binding domain comprises:

- (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, a VHFWR2 amino acid sequence of SEQ ID NO: 6004, a VHFWR3 amino acid sequence of SEQ ID NO: 6005, or a VHFWR4 amino acid sequence of SEQ ID NO: 6006, and
- (3) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292, or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

In some embodiments, the second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of a VH of Table 7, Table 35, Table 9, Table 10, or Table 34 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) a VL comprising the amino acid sequence of a VL of Table 8, Table 36, Table 9, Table 10, or Table 34 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto).

In some embodiments, the multispecific molecule comprises:

- a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30),
- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH from a third antigen binding domain that binds to a second calreticulin protein,
- optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286,
- optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

In some embodiments, the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

In some embodiments, the multispecific molecule further comprises: a third antigen binding domain that binds to a second calreticulin protein, e.g., wherein the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein:

- (i) the third antigen binding domain is different from the first antigen binding domain, or
- (ii) the third antigen binding domain is the same as the first antigen binding domain.

In some embodiments, the second calreticulin molecule is the same as the calreticulin molecule bound by the first antigen binding domain.

In some embodiments, the second calreticulin molecule is different from the calreticulin molecule bound by the first antigen binding domain.

In some embodiments, the first antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VLCDR1 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VLCDR2 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VLCDR3 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (iii) a VH comprising the amino acid sequence of a VH in Table 24, Table 25, or Table 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (iv) a VL comprising the amino acid sequence of a VL in Table 24, Table 25, or Table 16 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto);
- (v) a VH comprising a heavy chain framework region 1 (VHFWR1) having an amino acid sequence of a VHFWR1 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 having an amino acid sequence of a VHFWR2 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 having an amino acid sequence of a VHFWR3 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 having an amino acid sequence of a VHFWR4 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or
- (vi) a VL comprising a light chain framework region 1 (VLFWR1) having an amino acid sequence of a VLFWR1 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 having an amino acid sequence of a VLFWR2 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VLFWR3 having an amino acid sequence of a VLFWR3 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or a VLFWR4 having an amino acid sequence of a VLFWR4 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the multifunctional molecule further comprises a tumor-targeting moiety.

In some embodiments, the tumor-targeting moiety binds to a tumor antigen.

In some embodiments, the tumor antigen is selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In some embodiments, the tumor-targeting moiety comprises a VH and/or VL sequence, e.g., as listed in Table 38 or Table 20.

In some embodiments, the multispecific molecule further comprises a linker, e.g., a linker between the first antigen binding domain and the second antigen binding domain.

In some embodiments, the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.

In some embodiments, the linker is a peptide linker.

In some embodiments, the peptide linker comprises Gly and Ser.

In some embodiments, the peptide linker comprises an amino acid sequence chosen from SEQ ID NOs: 6214-6217 or 6220-6221 and 77-78.

In another aspect, the disclosure provides a nucleic acid molecule encoding the multifunctional molecule as described herein.

In another aspect, the disclosure provides a vector, e.g., an expression vector, comprising the nucleic acid molecule as described herein.

In another aspect, the disclosure provides a host cell comprising the nucleic acid molecule or a vector as described herein.

In another aspect, the disclosure provides a method of making, e.g., producing, the multifunctional molecule as described herein, comprising culturing the host cell described herein, under suitable conditions, e.g., conditions suitable for gene expression and/or homo- or heterodimerization.

In another aspect, the disclosure provides a pharmaceutical composition comprising the multifunctional molecule as described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.

In another aspect, the disclosure provides a method of treating a cancer, comprising administering to a subject in need thereof the multifunctional molecule as disclosed herein, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

In another aspect, the disclosure provides a use of the multifunctional molecule as described herein in treating a cancer. In another aspect, the disclosure provides a multifunctional molecule disclosed herein for use in treating a cancer.

In some embodiments, the subject has cancer cells that express the first and/or second calreticulin protein.

In some embodiments, wherein the subject has the JAK2 V617F mutation.

In some embodiments, the subject does not have the JAK2 V617F mutation.

In some embodiments, the subject has an MPL mutation.

In some embodiments, the subject does not have an MPL mutation.

In some embodiments, the cancer is a solid tumor cancer.

In some embodiments, the method or use further comprises administering a second therapeutic treatment.

In some embodiments, the second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormonal therapy), radiation, or surgery.

In some embodiments, the therapeutic agent is selected from: a chemotherapeutic agent, or a biologic agent.

In another aspect, the disclosure features a multifunctional molecule (e.g., polypeptide or nucleic acid encoding the same) that includes:

- (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and
- (ii) one, two, or all of:
- (a) an immune cell engager chosen from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager;
- (b) a cytokine molecule;
- (c) a stromal modifying moiety; or
- (d) a tumor-targeting moiety that binds to a tumor antigen, e.g., chosen from: G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In an aspect, the disclosure features a multifunctional molecule (e.g., polypeptide or nucleic acid encoding the same) that includes:

- (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and
- (ii) a second antigen binding domain comprising an immune cell engager (e.g., a T cell engager, e.g., an antigen binding domain that binds to TCRβV, e.g., as described herein).

In an aspect, the disclosure features a multifunctional molecule (e.g., polypeptide or nucleic acid encoding the same) that includes:

- (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and
- (ii) a second antigen binding domain comprising a tumor-targeting moiety, e.g., that binds to a tumor antigen chosen from: G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In an aspect, the disclosure features a multifunctional molecule (e.g., polypeptide or nucleic acid encoding the same) that includes:

- (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein),
- (ii) a second antigen binding domain comprising an immune cell engager (e.g., a T cell engager, e.g., an antigen binding domain that binds to TCRβV, e.g., as described herein, e.g., an anti-TCRβV antibody molecule described herein), and
- (iii) a third antigen binding domain comprising a tumor-targeting moiety, e.g., that binds to a tumor antigen chosen from: G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

In some embodiments, the multifunctional molecule further comprises a cytokine molecule or a modulator of a cytokine molecule, e.g., a TGF-β inhibitor, e.g., as described herein.

In some embodiments, the multifunctional molecule further comprises an NK cell engager, e.g., an antigen binding domain that binds to Nkp30, e.g., as described herein.

In some embodiments, the calreticulin protein (e.g., the wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286. In some embodiments, the wild type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001. In some embodiments, the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286.

In some embodiments, the first antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, a VHFWR2 amino acid sequence of SEQ ID NO: 6226, a VHFWR3 amino acid sequence of SEQ ID NO: 6228, or a VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6232, a VHFWR2 amino acid sequence of SEQ ID NO: 6234, a VHFWR3 amino acid sequence of SEQ ID NO: 6236, or a VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first antigen binding domain comprises a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, a VLFWR2 amino acid sequence of SEQ ID NO: 6240, a VLFWR3 amino acid sequence of SEQ ID NO: 6242, or a VLFWR4 amino acid sequence of SEQ ID NO: 6244.

In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises an amino acid sequence chosen from SEQ ID NOs: 6285-6312 or 1001. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises an amino acid sequence chosen from SEQ ID NOs: 6313-6346 or 1002-1003. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is a calreticulin protein (e.g., a wild-type or mutant calreticulin protein) disclosed in Table 2 or 3. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6287. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6313. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6314.

In some embodiments, the multifunctional molecule further comprising a second antigen binding domain that preferentially binds to a second calreticulin protein (e.g., a wild-type or mutant calreticulin protein). In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the second antigen binding domain is different from the first antigen binding domain. In some embodiments, the second antigen binding domain is the same as the first antigen binding domain. In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises an amino acid sequence chosen from SEQ ID NOs: 6287-6312. In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) comprises an amino acid sequence chosen from SEQ ID NOs: 6313-6346 or 1002-1003. In some embodiments, the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is a calreticulin protein (e.g., a wild-type or mutant calreticulin protein) disclosed in Table 2 or 3. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6287. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6313. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6314.

In some embodiments, the first calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is a Type 1 calreticulin protein (e.g., a wild-type or mutant calreticulin protein), and the second calreticulin protein (e.g., a wild-type or mutant calreticulin protein) is a Type 2 calreticulin protein (e.g., a wild-type or mutant calreticulin protein). In some embodiments, the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6287, and the second calreticulin protein the amino acid sequence of SEQ ID NO: 6288. In some embodiments, the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6313, and the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6314.

In some embodiments, the wild type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

In some embodiments, the first antigen binding domain has about the same affinity (e.g., equal affinity) for the first calreticulin protein (e.g., a mutant calreticulin protein) and for a wild-type calreticulin protein.

In some embodiments, the second antigen binding domain has about the same affinity (e.g., equal affinity) for the second calreticulin protein (e.g., a mutant calreticulin protein) and for a wild-type calreticulin protein.

In some embodiments, the first antigen binding domain has a higher affinity for a first calreticulin mutant protein than for the wild type calreticulin protein. In some embodiments, the K_Dfor the binding between the first antigen binding domain and the first calreticulin mutant protein is no more than 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% of the K_Dfor the binding between the first antigen binding domain and the wild type calreticulin protein. In some embodiments, the first antigen binding domain binds to an epitope located within the C-terminus of the first calreticulin mutant protein. In some embodiments, the first antigen binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the first antigen binding domain does not bind to the wild type calreticulin protein. In some embodiments, the wild type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

In some embodiments, the second antigen binding domain has a higher affinity for a second calreticulin mutant protein than for the wild type calreticulin protein. In some embodiments, the K_Dfor the binding between the second antigen binding domain and the second calreticulin mutant protein is no more than 40%, 30%, 20%, 10%, 1%, 0.1%, or 0.01% of the K_Dfor the binding between the second antigen binding domain and the wild type calreticulin protein. In some embodiments, the second antigen binding domain binds to an epitope located within the C-terminus of the second calreticulin mutant protein. In some embodiments, the second antigen binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6286. In some embodiments, the second antigen binding domain does not bind to the wild type calreticulin protein. In some embodiments, the wild type calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

In some embodiments, the multifunctional molecule preferentially binds to a myeloproliferative neoplasm cell over a non-tumor cell. In some embodiments, the binding between the multifunctional molecule and the myeloproliferative neoplasm cell is more than 10, 20, 30, 40, 50-fold greater than the binding between the multifunctional molecule and a non-tumor cell. In some embodiments, the myeloproliferative neoplasm cell is chosen from a myelofibrosis cell, an essential thrombocythemia cell, a polycythemia vera cell, or a chronic myeloid cancer cell. In some embodiments, the myeloproliferative neoplasm cell does not comprise a JAK2 V617F mutation. In some embodiments, the myeloproliferative neoplasm cell does not comprise an MPL mutation.

In some embodiments, the first and/or second antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6253 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6254 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the first and/or second antigen binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6253 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6254 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6255 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6253, a VHCDR2 amino acid sequence of SEQ ID NO: 6254, and a VHCDR3 amino acid sequence of SEQ ID NO: 6255. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6259, a VLCDR2 amino acid sequence of SEQ ID NO: 6260, and a VLCDR3 amino acid sequence of SEQ ID NO: 6261.

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising a VHCDR1 amino acid sequence of SEQ ID NO: 6253, a VHCDR2 amino acid sequence of SEQ ID NO: 6254, and a VHCDR3 amino acid sequence of SEQ ID NO: 6255, and
- (ii) a VL comprising a VLCDR1 amino acid sequence of SEQ ID NO: 6259, a VLCDR2 amino acid sequence of SEQ ID NO: 6260, and a VLCDR3 amino acid sequence of SEQ ID NO: 6261.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, a VHFWR2 amino acid sequence of SEQ ID NO: 6226, a VHFWR3 amino acid sequence of SEQ ID NO: 6228, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, a VLFWR2 amino acid sequence of SEQ ID NO: 6240, a VLFWR3 amino acid sequence of SEQ ID NO: 6242, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6244.

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6224, a VHFWR2 amino acid sequence of SEQ ID NO: 6226, a VHFWR3 amino acid sequence of SEQ ID NO: 6228, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6230, and
- (ii) a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, a VLFWR2 amino acid sequence of SEQ ID NO: 6240, a VLFWR3 amino acid sequence of SEQ ID NO: 6242, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6244.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6263 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 amino acid sequence of SEQ ID NO: 6264 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 amino acid sequence of SEQ ID NO: 6265 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228, and
- (ii) a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6263, a VHFWR2 amino acid sequence of SEQ ID NO: 6264, a VHFWR3 amino acid sequence of SEQ ID NO: 6265, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277, a VLFWR2 amino acid sequence of SEQ ID NO: 6278, a VLFWR3 amino acid sequence of SEQ ID NO: 6279, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising a VHFWR1 amino acid sequence of SEQ ID NO: 6263, a VHFWR2 amino acid sequence of SEQ ID NO: 6264, a VHFWR3 amino acid sequence of SEQ ID NO: 6265, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 228, and
- (ii) a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277, a VLFWR2 amino acid sequence of SEQ ID NO: 6278, a VLFWR3 amino acid sequence of SEQ ID NO: 6279, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6247). In some embodiments, the first and/or second antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249).

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of SEQ ID NO: 6247 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6247), and
- (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249).

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6247. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the first and/or second antigen binding domain comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 6247, and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising an amino acid sequence of at least 70% or 75% sequence identity to SEQ ID NO: 6250. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising an amino acid sequence of at least 85% or 90% sequence identity to SEQ ID NO: 6252. In some embodiments, the first and/or second antigen binding domain comprises (i) a VH comprising an amino acid sequence of at least 70% or 75% sequence identity to SEQ ID NO: 6250, and (ii) a VL comprising an amino acid sequence of at least 85% or 90% sequence identity to SEQ ID NO: 6252.

In some embodiments, the first and/or second antigen binding domain comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6256 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6257 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6258 or 116 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions). In some embodiments, the first and/or second antigen binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6256 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6257 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6258 or 116 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6259 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 6260 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 6261 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6232, a VHFWR2 amino acid sequence of SEQ ID NO: 6234, a VHFWR3 amino acid sequence of SEQ ID NO: 6236, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6230. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, a VLFWR2 amino acid sequence of SEQ ID NO: 6240, a VLFWR3 amino acid sequence of SEQ ID NO: 6242, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6244.

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6232, a VHFWR2 amino acid sequence of SEQ ID NO: 6234, a VHFWR3 amino acid sequence of SEQ ID NO: 6236, and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6230, and
- (ii) a VL comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6238, a VLFWR2 amino acid sequence of SEQ ID NO: 6240, a VLFWR3 amino acid sequence of SEQ ID NO: 6242, and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6244.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising a heavy chain framework 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6266 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 amino acid sequence of SEQ ID NO: 6267 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 amino acid sequence of SEQ ID NO: 6268 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6269. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the first and/or second antigen binding domain comprises:

- (i) a VH comprising a heavy chain framework 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6266 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 amino acid sequence of SEQ ID NO: 6267 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 amino acid sequence of SEQ ID NO: 6268 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 amino acid sequence of SEQ ID NO: 6269, and
- (ii) a VL comprising a VLFWR1 amino acid sequence of SEQ ID NO: 6277 (or a sequence with no more than 1, 2, or 3 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 amino acid sequence of SEQ ID NO: 6278 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), a VLFWR3 amino acid sequence of SEQ ID NO: 6279 (or a sequence with no more than 1 mutation, e.g., substitution, addition, or deletion), and/or a VLFWR4 amino acid sequence of SEQ ID NO: 6280.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6248 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6248). In some embodiments, the first and/or second antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249).

In some embodiments, the first and/or second antigen binding domain comprises

- (i) a VH comprising the amino acid sequence of SEQ ID NO: 6248 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 6248), and
- (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to SEQ ID NO: 6249).

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 6248. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 6249. In some embodiments, the first and/or second antigen binding domain comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 6248, and (ii) a VL comprising the amino acid sequence of SEQ ID NO: 6249.

In some embodiments, the first and/or second antigen binding domain comprises a VH comprising an amino acid sequence of at least 70% or 74% sequence identity to SEQ ID NO: 6251. In some embodiments, the first and/or second antigen binding domain comprises a VL comprising an amino acid sequence of at least 85% or 90% sequence identity to SEQ ID NO: 6252. In some embodiments, the first and/or second antigen binding domain comprises (i) a VH comprising an amino acid sequence of at least 70% or 74% sequence identity to SEQ ID NO: 6251, and/or (ii) a VL comprising an amino acid sequence of at least 85% or 90% sequence identity to SEQ ID NO: 6252.

In some embodiments, the multifunctional molecule comprises an immune cell engager chosen from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager. In some embodiments, the immune cell engager binds to and activates an immune cell, e.g., an effector cell. In some embodiments, the immune cell engager binds to, but does not activate, an immune cell, e.g., an effector cell.

In some embodiments, the immune cell engager is a T cell engager, e.g., a T cell engager that mediates binding to and activation of a T cell, or a T cell engager that mediates binding to but not activation of a T cell. In some embodiments, the T cell engager binds to CD3, TCRα, TCRβ, TCRγ, TCRξ, ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In some embodiments, the T cell engager is an anti-CD3 antibody molecule. In some embodiments, the T cell engager is an anti-TCRβ antibody molecule, e.g., an anti-TCRβV antibody molecule described herein.

In some embodiments, the immune cell engager is an NK cell engager, e.g., an NK cell engager that mediates binding to and activation of an NK cell, or an NK cell engager that mediates binding to but not activation of an NK cell. In some embodiments, the NK cell engager is chosen from an antibody molecule, e.g., an antigen binding domain, or ligand that binds to (e.g., activates): NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16 (e.g., CD16a, CD16b, or both), CRTAM, CD27, PSGL1, CD96, CD100 (SEMA4D), NKp80, CD244 (also known as SLAMF4 or 2B4), SLAMF6, SLAMF7, KIR2DS2, KIR2DS4, KIR3DS1, KIR2DS3, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, or CD160. In some embodiments, the NK cell engager is an antibody molecule or ligand that binds to (e.g., activates) NKp30. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain. In some embodiments, the NK cell engager is an antibody molecule, e.g., an antigen binding domain, that binds to NKp30 or NKp46. In some embodiments, the NK cell engager is a ligand, optionally, the ligand further comprises an immunoglobulin constant region, e.g., an Fc region. In some embodiments, the NK cell engager is a ligand of NKp44 or NKp46, e.g., a viral HA. In some embodiments, the NK cell engager is a ligand of DAP10, e.g., a coreceptor for NKG2D. In some embodiments, the NK cell engager is a ligand of CD16, e.g., a CD16a/b ligand, e.g., a CD16a/b ligand further comprising an antibody Fc region. In some embodiments, the immune cell engager mediates binding to, or activation of, or both of, one or more of a B cell, a macrophage, and/or a dendritic cell.

In some embodiments, the immune cell engager comprises a B cell, macrophage, and/or dendritic cell engager chosen from one or more of CD40 ligand (CD40L) or a CD70 ligand; an antibody molecule that binds to CD40 or CD70; an antibody molecule to OX40; an OX40 ligand (OX40L); an agonist of a Toll-like receptor (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); a 41BB; a CD2 agonist; a CD47; or a STING agonist, or a combination thereof. In some embodiments, the immune cell engager is a B cell engager, e.g., a CD40L, an OX40L, or a CD70 ligand, or an antibody molecule that binds to OX40, CD40 or CD70. In some embodiments, the immune cell engager is a macrophage cell engager, e.g., a CD2 agonist; a CD40L; an OX40L; an antibody molecule that binds to OX40, CD40 or CD70; an agonist of a Toll-like receptor (TLR) (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4) or a TLR9 agonist); CD47; or a STING agonist. In some embodiments, the immune cell engager is a dendritic cell engager, e.g., a CD2 agonist, an OX40 antibody, an OX40L, 41BB agonist, a Toll-like receptor agonist or a fragment thereof (e.g., a TLR4, e.g., a constitutively active TLR4 (caTLR4)), CD47 agonist, or a STING agonist. In some embodiments, the STING agonist comprises a cyclic dinucleotide, e.g., a cyclic di-GMP (cdGMP), a cyclic di-AMP (cdAMP), or a combination thereof, optionally with 2′,5′ or 3′,5′ phosphate linkages, e.g., wherein the STING agonist is covalently coupled to the multifunctional molecule.

In some embodiments, the multifunctional molecule comprises a cytokine molecule or a modulator thereof. In some embodiments, the cytokine molecule is chosen from TGF-β, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), or interferon gamma, or a fragment or variant thereof, or a combination of any of the aforesaid cytokines. In some embodiments, the cytokine molecule is a monomer or a dimer. In some embodiments, the cytokine molecule further comprises a receptor dimerizing domain, e.g., an IL15Ralpha dimerizing domain. In some embodiments, the cytokine molecule (e.g., IL-15) and the receptor dimerizing domain (e.g., an IL15Ralpha dimerizing domain) are not covalently linked, e.g., are non-covalently associated.

In some embodiments, the modulator of the cytokine molecule comprises a TGF-β inhibitor.

In some embodiments, the multifunctional molecule comprises a stromal modifying moiety. In some embodiments, the stromal modifying moiety causes one or more of: decreases the level or production of a stromal or extracellular matrix (ECM) component; decreases tumor fibrosis; increases interstitial tumor transport; improves tumor perfusion; expands the tumor microvasculature; decreases interstitial fluid pressure (IFP) in a tumor; or decreases or enhances penetration or diffusion of an agent, e.g., a cancer therapeutic or a cellular therapy, into a tumor or tumor vasculature. In some embodiments, the stromal or ECM component decreased is chosen from a glycosaminoglycan or an extracellular protein, or a combination thereof. In some embodiments, the glycosaminoglycan is chosen from hyaluronan (also known as hyaluronic acid or HA), chondroitin sulfate, chondroitin, dermatan sulfate, heparan sulfate, heparin, entactin, tenascin, aggrecan or keratin sulfate. In some embodiments, the extracellular protein is chosen from collagen, laminin, elastin, fibrinogen, fibronectin, or vitronectin. In some embodiments, the stromal modifying moiety comprises an enzyme molecule that degrades a tumor stroma or extracellular matrix (ECM). In some embodiments, the enzyme molecule is chosen from a hyaluronidase molecule, a collagenase molecule, a chondroitinase molecule, a matrix metalloproteinase molecule (e.g., macrophage metalloelastase), or a variant (e.g., a fragment) of any of the aforesaid. In some embodiments, the stromal modifying moiety decreases the level or production of hyaluronic acid. In some embodiments, the stromal modifying moiety comprises a hyaluronan degrading enzyme, an agent that inhibits hyaluronan synthesis, or an antibody molecule against hyaluronic acid. In some embodiments, the hyaluronan degrading enzyme is a hyaluronidase molecule or a variant (e.g., fragment thereof) thereof. In some embodiments, the hyaluronan degrading enzyme is active in neutral or acidic pH, e.g., pH of about 4-5. In some embodiments, the hyaluronidase molecule is a mammalian hyaluronidase molecule, e.g., a recombinant human hyaluronidase molecule, or a variant thereof (e.g., a truncated form thereof). In some embodiments, the hyaluronidase molecule is chosen from HYAL1, HYAL2, or PH-20/SPAM1, or a variant thereof (e.g., a truncated form thereof). In some embodiments, the truncated form lacks a C-terminal glycosylphosphatidylinositol (GPI) attachment site or a portion of the GPI attachment site. In some embodiments, the hyaluronidase molecule is glycosylated, e.g., comprises at least one N-linked glycan. In some embodiments, the hyaluronidase molecule comprises the amino acid sequence of SEQ ID NO: 6213, or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 6213). In some embodiments, the hyaluronidase molecule comprises the amino acid residues 36-464 of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises the amino acid residues 36-481, 36-482, or 36-483 of PH20, wherein PH20 has the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises an amino acid sequence having at least 95% to 100% sequence identity to the polypeptide or truncated form of the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises an amino acid sequence having 30, 20, 10, 5 or fewer amino acid substitutions to the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule comprises an amino acid sequence at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%, 100%) identical to the amino acid sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule is encoded by a nucleotide sequence at least 95% (e.g., at least 96%, 97%, 98%, 99%, 100%) identical to the nucleotide sequence of SEQ ID NO: 6213. In some embodiments, the hyaluronidase molecule is PH20, e.g., rHuPH20. In some embodiments, the hyaluronidase molecule is HYAL1 and comprises the amino acid sequence of SEQ ID NO: 6218, or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 6218). In some embodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule, further comprises a polymer, e.g., is conjugated to a polymer, e.g., PEG. In some embodiments, the hyaluronan-degrading enzyme is a PEGylated PH20 enzyme (PEGPH20). In some embodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule, further comprises an immunoglobulin chain constant region (e.g., Fc region) chosen from, e.g., the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4, more particularly, the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin constant region (e.g., the Fc region) is linked, e.g., covalently linked to, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule. In some embodiments, the immunoglobulin chain constant region (e.g., Fc region) is altered, e.g., mutated, to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function. In some embodiments, the hyaluronan degrading enzyme, e.g., the hyaluronidase molecule, forms a dimer. In some embodiments, the stromal modifying moiety comprises an inhibitor of the synthesis of hyaluronan, e.g., an HA synthase. In some embodiments, the inhibitor comprises a sense or an antisense nucleic acid molecule against an HA synthase or is a small molecule drug. In some embodiments, the inhibitor is 4-methylumbelliferone (MU) or a derivative thereof (e.g., 6,7-dihydroxy-4-methyl coumarin or 5,7-dihydroxy-4-methyl coumarin), or leflunomide or a derivative thereof. In some embodiments, the stromal modifying moiety comprises a collagenase molecule, e.g., a mammalian collagenase molecule, or a variant (e.g., fragment) thereof. In some embodiments, the collagenase molecule is collagenase molecule IV, e.g., comprising the amino acid sequence of SEQ ID NO: 6219, or a fragment thereof, or an amino acid sequence substantially identical thereto (e.g., 95% to 99.9% identical thereto, or having at least one amino acid alteration, but not more than five, ten or fifteen alterations (e.g., substitutions, deletions, or insertions, e.g., conservative substitutions) to the amino acid sequence of SEQ ID NO: 6219.

In some embodiments, the multifunctional molecule comprises an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a cytokine molecule. In some embodiments, the multifunctional molecule comprises an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager) and a stromal modifying moiety. In some embodiments, the multifunctional molecule comprises a cytokine molecule and a stromal modifying moiety. In some embodiments, the multifunctional molecule comprises an immune cell engager (e.g., a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager), a cytokine molecule, and a stromal modifying moiety.

In some embodiments, the multifunctional molecule comprises at least two non-contiguous polypeptide chains.

In some embodiments, the multifunctional molecule comprises the following configuration:

- A, B-[dimerization module]-C, -D
  
  e.g., the configuration shown in FIGS. 1A, 1B, and 1C, wherein:
  
  (1) the dimerization module comprises an immunoglobulin constant domain, e.g., a heavy chain constant domain (e.g., a homodimeric or heterodimeric heavy chain constant region, e.g., an Fc region), or a constant domain of an immunoglobulin variable region (e.g., a Fab region); and
  
  (2) A, B, C, and D are independently absent; (i) an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a mutant calreticulin protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286; (ii) an immune cell engager chosen from a T cell engager, an NK cell engager, a B cell engager, a dendritic cell engager, or a macrophage cell engager; (iii) a cytokine molecule; or (iv) a stromal modifying moiety, provided that: at least one, two, or three of A, B, C, and D comprises an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and any of the remaining A, B, C, and D is absent or comprises one of an immune cell engager, a cytokine molecule, or a stromal modifying moiety.

In some embodiments,

- (i) A comprises an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule;
- (ii) A comprises an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises a cytokine molecule;
- (iii) A comprises an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises a stromal modifying moiety;
- (iv) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule;
- (v) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises a cytokine molecule;
- (vi) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises a stromal modifying moiety;
- (vii) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule;
- (viii) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises a cytokine molecule;
- (ix) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises a stromal modifying moiety;
- (x) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a cytokine molecule;
- (xi) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a stromal modifying moiety;
- (xii) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises (a) a cytokine molecule and (b) a stromal modifying moiety;
- (xiii) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a cytokine molecule;
- (xiv) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a stromal modifying moiety;
- (xv) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises (a) a cytokine molecule and (b) a stromal modifying moiety;
- (xvi) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a cytokine molecule;
- (xvii) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule and (b) a stromal modifying moiety;
- (xviii) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises (a) a cytokine molecule and (b) a stromal modifying moiety;
- (xix) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B, C, or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, (b) a cytokine molecule, and (c) a stromal modifying moiety;
- (xx) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, B comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and C or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, (b) a cytokine molecule, and (c) a stromal modifying moiety; or
- (xxi) A comprises a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the first calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, C comprises a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), wherein the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and B or D comprises (a) an immune cell engager, e.g., a T cell engager, e.g., an anti-CD3 antibody molecule, (b) a cytokine molecule, and (c) a stromal modifying moiety.

In some embodiments, the dimerization module comprises one or more immunoglobulin chain constant regions (e.g., Fc regions) comprising one or more of: a paired cavity-protuberance (“knob-in-a hole”), an electrostatic interaction, or a strand-exchange. In some embodiments, the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid substitution at a position chosen from one or more of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 397, 398, 399, 405, 407, or 409, e.g., of the Fc region of human IgG1. In some embodiments, the one or more immunoglobulin chain constant regions (e.g., Fc regions) comprise an amino acid substitution chosen from: T366S, L368A, or Y407V (e.g., corresponding to a cavity or hole), or T366W (e.g., corresponding to a protuberance or knob), or a combination thereof.

In some embodiments, the multifunctional molecule further comprises a linker, e.g., a linker between one or more of: the antigen binding domain and the immune cell engager, the antigen binding domain and the cytokine molecule, the antigen binding domain and the stromal modifying moiety, the immune cell engager and the cytokine molecule, the immune cell engager and the stromal modifying moiety, the cytokine molecule and the stromal modifying moiety, the antigen binding domain and the dimerization module, the immune cell engager and the dimerization module, the cytokine molecule and the dimerization module, or the stromal modifying moiety and the dimerization module. In some embodiments, the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises Gly and Ser. In some embodiments, the peptide linker comprises an amino acid sequence chosen from SEQ ID NOs: 6214-6217 or 6220-6221 and 77-78.

In one aspect, the invention provides a multifunctional molecule, comprising:

- (i) an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), e.g., wherein the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286, and
- (ii) a moiety that binds to CD3, e.g., an antibody molecule that binds to CD3.

- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to CD3 (e.g., a second scFv that binds to CD3),
- a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), and the second VL and the second VH form a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6286, optionally wherein the first and second calreticulin proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314.

In some embodiments, the multifunctional molecule comprises the configuration of FIG. 2A or 2B.

In one aspect, the invention provides a multifunctional molecule, comprising:

- (i) an antigen binding domain that binds to a calreticulin protein (e.g., a wild type calreticulin protein or a calreticulin mutant protein), e.g., wherein the calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6286, and
- (ii) a moiety that binds to TCR (e.g., TCRβ), e.g., an antibody molecule that binds to TCR (e.g., TCRβ).

In some embodiments, the multifunctional molecule comprises: a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to TCR (e.g., TCRβ) (e.g., a first scFv that binds to TCR (e.g., TCRβ)),

- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to TCR (e.g., TCRβ) (e.g., a second scFv that binds to TCR (e.g., TCRβ)), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), and the second VL and the second VH form a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6286, optionally wherein the first and second calreticulin proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314.

In some embodiments, the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

In one aspect, the invention provides a multifunctional molecule, comprising:

- (i) an antigen binding domain that binds to a calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), e.g., wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286, and
- (ii) a moiety that binds to NKp30, e.g., an antibody molecule or ligand that binds to (e.g., activates) NKp30.

In some embodiments, the multifunctional molecule comprises:

- a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30),
- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30), a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), and the second VL and the second VH form a second antigen binding domain that binds to a second calreticulin protein (e.g., a wild-type calreticulin protein or a calreticulin mutant protein), wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6286, optionally wherein the first and second calreticulin proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314.

In some embodiments, the multifunctional molecule comprises the configuration of FIG. 4A or 4B.

In another aspect, the disclosure provides an isolated nucleic acid molecule encoding any multispecific or multifunctional molecule described herein. In another aspect, the disclosure provides an isolated nucleic acid molecule, which comprises the nucleotide sequence encoding any of the multispecific or multifunctional molecules described herein, or a nucleotide sequence substantially homologous thereto (e.g., at least 80%, 90%, 95%, or 99.9% identical thereto). In another aspect, the disclosure provides a host cell comprising a nucleic acid molecule or a vector described herein.

In another aspect, the disclosure provides a method of making, e.g., producing, a multispecific or multifunctional molecule polypeptide described herein, comprising culturing a host cell described herein, under suitable conditions, e.g., conditions suitable for gene expression and/or homo- or heterodimerization.

In another aspect, the disclosure provides a pharmaceutical composition comprising a multispecific or multifunctional molecule polypeptide described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer.

In another aspect, the disclosure provides a method of treating a cancer, comprising administering to a subject in need thereof a multispecific or multifunctional molecule polypeptide described herein, wherein the multispecific antibody is administered in an amount effective to treat the cancer. In some embodiments, the subject has cancer cells that express the first and/or second calreticulin mutant. In some embodiments, the subject has tumor cells that express the first, second, or third tumor antigen, e.g., the subject has tumor cells that express a tumor antigen chosen from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1. In some embodiments, the subject has the JAK2 V617F mutation. In some embodiments, the subject does not have the JAK2 V617F mutation. In some embodiments, the subject has a MPL mutation. In some embodiments, the subject does not have a MPL mutation. In some embodiments, the cancer is a hematological cancer, optionally wherein the cancer is a myeloproliferative neoplasm, e.g., primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML). In some embodiments, the cancer is myelofibrosis. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is one or more of pancreatic (e.g., pancreatic adenocarcinoma), breast, colorectal, lung (e.g., small or non-small cell lung cancer), skin, ovarian, or liver cancer.

In some embodiments, the cancer cell comprises a myeloproliferative neoplasm cell. In some embodiments, the myeloproliferative neoplasm cell is chosen from a myelofibrosis cell, an essential thrombocythemia cell, a polycythemia vera cell, or a chronic myeloid cancer cell. In some embodiments, the myeloproliferative neoplasm cell is a myelofibrosis cell. In some embodiments, the myeloproliferative neoplasm cell is an essential thrombocythemia cell. In some embodiments, the myeloproliferative neoplasm cell is a polycythemia vera cell. In some embodiments, the myeloproliferative neoplasm cell is a chronic myeloid cancer cell. In some embodiments, the myeloproliferative neoplasm cell comprises a JAK2 mutation (e.g., a JAK2 V617F mutation). In some embodiments, the myeloproliferative neoplasm cell comprises a calreticulin mutation. In some embodiments, the myeloproliferative neoplasm cell comprises a MPL mutation.

In some embodiments, the method further comprises administering a second therapeutic treatment. In some embodiments, second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormonal therapy), radiation, or surgery. In some embodiments, therapeutic agent is selected from: a chemotherapeutic agent, or a biologic agent.

Exemplary Embodiment 2

1. A multifunctional molecule comprising:

- (i) a first antigen binding domain that binds to a calreticulin protein (e.g., a wild-type or mutant calreticulin protein), e.g., a calreticulin-targeting antigen binding domain disclosed in any one of Table 4, Table 5, Table 6, Table 24, Table 25, Table 16, Table 17, Table 18, or Table 19, and
- (ii) a second antigen binding domain that binds to TCRβV, e.g., an anti-TCRβV antigen binding domain disclosed in any one of Table 30, Table 31, Table 32, Table 33, Table 11, Table 12, or Table 13, or a second antigen binding domain that binds to NKp30, e.g., an anti-NKp30 antigen binding domain disclosed in Table 7, Table 8, Table 35, Table 36, Table 9, Table 10, or Table 34.

2. The multifunctional molecule of embodiment 1, wherein the second antigen binding domain binds to TCRβV.

3. The multifunctional molecule of embodiment 2, wherein the second antigen binding domain activates a T cell or the second antigen binding domain does not activate a T cell.

4. The multifunctional molecule of embodiment 2 or 3, wherein the second antigen binding domain binds to TCRβ V12 or TCRβ V6 (e.g., comprising the amino acid sequence of SEQ ID NO: 1044).

5. The multifunctional molecule of any of embodiments 2-4, wherein the second antigen binding domain comprises one or more amino acid sequences as listed in Table 30, Table 31, Table 32, Table 33, Table 11, Table 12, or Table 13.

6. The multifunctional molecule of any of embodiments 2-5, wherein the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions),
- (ii) the VL comprises a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 3 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 4 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 5 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 7 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 8 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 45 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 46 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 47 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 51 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 52 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 53 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or
- (d) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 48 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 49 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 50 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 54 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 55 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 56 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

7. The multifunctional molecule of any of embodiments 2-5, wherein the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of a VH in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of a VL in Table 30, Table 31, Table 33, Table 11, Table 12, or Table 13 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto)
- (iii) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (iv) the VL comprises the amino acid sequence of SEQ ID NO: 10 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of SEQ ID NO: 11 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or
- (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of SEQ ID NO: 1312 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of SEQ ID NO: 1314 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

8. The multifunctional molecule of any of embodiments 2-5, wherein the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 19 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 20 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 21 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 22 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 57 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 58 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 59 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 63 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 64 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 65 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions); and/or
- (c) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 60 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 61 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 62 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) the VL comprises a light chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 66 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 67 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 amino acid sequence of SEQ ID NO: 68 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

9. The multifunctional molecule of any of embodiments 2-5, wherein the second antigen binding domain comprises:

- (a) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises the amino acid sequence of SEQ ID NO: 15 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) the VL comprises the amino acid sequence of SEQ ID NO: 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or
- (b) a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
- (i) the VH comprises: the amino acid sequence of SEQ ID NO: 23 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), the amino acid sequence of SEQ ID NO: 24 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or the amino acid sequence of SEQ ID NO: 25 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto); and/or
- (ii) the VL comprises:
- the amino acid sequence of SEQ ID NO: 26 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- the amino acid sequence of SEQ ID NO: 27 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- the amino acid sequence of SEQ ID NO: 28 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto),
- the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), or
- the amino acid sequence of SEQ ID NO: 30 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

10. The multifunctional molecule of any of embodiments 2-9, comprising:

- a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to TCR (e.g., TCRVβ) (e.g., a first scFv that binds to TCR (e.g., TCRVβ)),
- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to TCR (e.g., TCRVβ) (e.g., a second scFv that binds to TCR (e.g., TCRVβ)),
- a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH form a third antigen binding domain that binds to a second calreticulin protein,
- optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286,
- optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

11. The multifunctional molecule of embodiment 1, wherein the second antigen binding domain binds to NKp30.

12. The multifunctional molecule of embodiment 11, wherein the second antigen binding domain is chosen from an antibody molecule, e.g., an antigen binding domain, or ligand that binds to (e.g., activates) NKp30, e.g., the second antigen binding domain is an antibody molecule or ligand that binds to (e.g., activates) NKp30.

13. The multifunctional molecule of embodiment 11 or 12, wherein the second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) having an amino acid sequence of a VLCDR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 having an amino acid sequence of a VLCDR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 having an amino acid sequence of a VLCDR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

14. The multifunctional molecule of embodiment 13, wherein the second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 7313 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 amino acid sequence of SEQ ID NO: 6001 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 7315 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions; and/or
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 7326 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VLCDR2 amino acid sequence of SEQ ID NO: 7327 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7329 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions).

15. The multifunctional molecule of embodiment 13 or 14, wherein the second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of any of SEQ ID NOs: 7298 or 7300-7304 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any of SEQ ID NOs: 7298 or 7300-7304); and/or
- (ii) a VL comprising the amino acid sequence of any of SEQ ID NOs: 7299 or 7305-7309 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity to any of SEQ ID NOs: 7299 or 7305-7309).

16. The multifunctional molecule of any of embodiments 13-15, wherein the second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7305 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7305); or
- (ii) a VH comprising the amino acid sequence of SEQ ID NO: 7302 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7302), and a VL comprising the amino acid sequence of SEQ ID NO: 7309 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7309).

17. The multifunctional molecule of any of embodiments 13-16, wherein the second antigen binding domain comprises:

- (i) an amino acid sequence of SEQ ID NO: 7310 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7310); or
- (ii) an amino acid sequence of SEQ ID NO: 7311 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to 7311).

18. The multifunctional molecule of embodiment 11 or 12, wherein the second antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO: 6000, a VHCDR2 amino acid sequence of SEQ ID NO: 6001, and/or a VHCDR3 amino acid sequence of SEQ ID NO: 6002, and
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VLCDR1) amino acid sequence of SEQ ID NO: 6063, a VLCDR2 amino acid sequence of SEQ ID NO: 6064, and/or a VLCDR3 amino acid sequence of SEQ ID NO: 7293.

19. The multifunctional molecule of any of embodiments 11, 12, or 18, wherein the second antigen binding domain comprises:

- (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) having an amino acid sequence of a VHFWR1 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR2 having an amino acid sequence of a VHFWR2 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VHFWR3 having an amino acid sequence of a VHFWR3 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VHFWR4 having an amino acid sequence of a VHFWR4 of Table 7, Table 35, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), and/or
- (2) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) having an amino acid sequence of a VLFWR1 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR2 having an amino acid sequence of a VLFWR2 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), a VLFWR3 having an amino acid sequence of a VLFWR3 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom), or a VLFWR4 having an amino acid sequence of a VLFWR4 of Table 8, Table 36, Table 9, Table 10, or Table 34 (or a sequence with no more than 1, 2, 3, 4, 5, or 6 mutations, e.g., substitutions, additions, or deletions, therefrom).

20. The multifunctional molecule of embodiment 19, wherein the second antigen binding domain comprises:

- (1) a heavy chain variable region (VH) comprising a heavy chain framework region 1 (VHFWR1) amino acid sequence of SEQ ID NO: 6003, a VHFWR2 amino acid sequence of SEQ ID NO: 6004, a VHFWR3 amino acid sequence of SEQ ID NO: 6005, or a VHFWR4 amino acid sequence of SEQ ID NO: 6006, and
- (3) a light chain variable region (VL) comprising a light chain framework region 1 (VLFWR1) amino acid sequence of SEQ ID NO: 6066, a VLFWR2 amino acid sequence of SEQ ID NO: 6067, a VLFWR3 amino acid sequence of SEQ ID NO: 7292, or a VLFWR4 amino acid sequence of SEQ ID NO: 6069.

21. The multifunctional molecule of any one of embodiments 11, 12, or 18-20, wherein the second antigen binding domain comprises:

- (i) a VH comprising the amino acid sequence of a VH of Table 7, Table 35, Table 9, Table 10, or Table 34 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and/or
- (ii) a VL comprising the amino acid sequence of a VL of Table 8, Table 36, Table 9, Table 10, or Table 34 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto).

22. The multifunctional molecule of either of embodiments 11, 12, or 18-21, wherein the second antigen binding domain comprises a heavy chain comprising the amino acid sequence of a heavy chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

23. The multifunctional molecule of either of embodiments 11, 12, or 18-22, wherein the second antigen binding domain comprises a light chain comprising the amino acid sequence of a light chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

24. The multifunctional molecule of either of embodiments 11, 12, or 18-23, wherein the second antigen binding domain comprises a heavy chain comprising the amino acid sequence of a heavy chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto), and a light chain comprising the amino acid sequence of a light chain of Table 10 (or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto).

25. The multifunctional molecule of any of embodiments 11-24, comprising:

- a first polypeptide comprising, e.g., from N-terminus to C-terminus, a first VL and a first CL, a second polypeptide comprising, e.g., from N-terminus to C-terminus, a first VH, a first CH1, a first dimerization domain (e.g., a first Fc), and a first moiety that binds to NKp30 (e.g., a first antibody molecule or ligand that binds to NKp30),
- a third polypeptide comprising, e.g., from N-terminus to C-terminus, a second VH, a second CH1, a second dimerization domain (e.g., a second Fc), and optionally a second moiety that binds to NKp30 (e.g., a second antibody molecule or ligand that binds to NKp30),
- a fourth polypeptide comprising, e.g., from N-terminus to C-terminus, a second VL and a second CL, wherein:
- the first VL and the first VH form a first antigen binding domain that binds to a first calreticulin protein, and the second VL and the second VH from a third antigen binding domain that binds to a second calreticulin protein,
- optionally wherein the first and second calreticulin proteins comprise the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286,
- optionally wherein the first and second calreticulin mutant proteins are each independently chosen from: a molecule comprising the amino acid sequence of SEQ ID NO: 6313, or a molecule comprising the amino acid sequence of SEQ ID NO: 6314, optionally wherein the multifunctional molecule comprises the configuration of FIG. 3A or 3B.

26. The multifunctional molecule of any of the preceding embodiments, wherein the calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6285-6312 or 1001, optionally wherein the calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6313-6346 or 1002-1003.

27. The multifunctional molecule of any of the preceding embodiments, wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6285 or 1001.

28. The multifunctional molecule of any of the preceding embodiments, wherein the calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

29. The multifunctional molecule of any of the preceding embodiments, wherein the first antigen binding domain binds to an epitope located within the C-terminus of the calreticulin protein, optionally wherein the first antigen binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286.

30. The multifunctional molecule of any of the preceding embodiments, further comprising a third antigen binding domain that binds to a second calreticulin protein, e.g., wherein the second calreticulin mutant protein comprises the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286, optionally wherein:

- (i) the third antigen binding domain is different from the first antigen binding domain, or
- (ii) the third antigen binding domain is the same as the first antigen binding domain.

31. The multifunctional molecule of embodiment 30, wherein the second calreticulin molecule is the same as the calreticulin molecule bound by the first antigen binding domain.

32. The multifunctional molecule of embodiment 30, wherein the second calreticulin molecule is different from the calreticulin molecule bound by the first antigen binding domain.

33. The multifunctional molecule of any of embodiments 30-32, wherein the second calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6285-6312 or 1001, optionally wherein the second calreticulin protein comprises an amino acid sequence chosen from SEQ ID NOs: 6313-6346 or 1002-1003.

34. The multifunctional molecule of embodiment 33, wherein the calreticulin protein bound by the first antigen binding domain comprises the amino acid sequence of SEQ ID NO: 6285 or 1001, and the second calreticulin protein comprises the amino acid sequence of SEQ ID NO: 6286.

35. The multifunctional molecule of any of embodiments 30-34, wherein the third antigen binding domain binds to an epitope located within the C-terminus of the second calreticulin protein, optionally wherein the third antigen binding domain binds to an epitope located within the amino acid sequence of SEQ ID NO: 6285, 1001, or 6286.

36. The multifunctional molecule of any of the preceding embodiments, wherein the first antigen binding domain comprises:

- (i) a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VHCDR1 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VHCDR2 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VHCDR3 in Table 4, Table 24, Table 25, or Table 17 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (ii) a light chain variable region (VL) comprising a light chain complementarity determining region 1 (VHCDR1) having an amino acid sequence of a VLCDR1 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), a VHCDR2 having an amino acid sequence of a VLCDR2 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions), and/or a VHCDR3 having an amino acid sequence of a VLCDR3 in Table 5, Table 24, Table 25, or Table 18 (or a sequence with no more than 1, 2, 3, or 4 mutations, e.g., substitutions, additions, or deletions);
- (iii) a VH comprising the amino acid sequence of a VH in Table 24, Table 25, or Table 16 (or an amino acid sequence having at least about 77%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto);
- (iv) a VL comprising the amino acid sequence of a VL in Table 24, Table 25, or Table 16 (or an amino acid sequence having at least about 93%, 95%, or 99% sequence identity thereto);
- (v) a VH comprising a heavy chain framework region 1 (VHFWR1) having an amino acid sequence of a VHFWR1 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR2 having an amino acid sequence of a VHFWR2 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VHFWR3 having an amino acid sequence of a VHFWR3 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or a VHFWR4 having an amino acid sequence of a VHFWR4 in Table 4 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or
- (vi) a VL comprising a light chain framework region 1 (VLFWR1) having an amino acid sequence of a VLFWR1 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VLFWR2 having an amino acid sequence of a VLFWR2 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), a VLFWR3 having an amino acid sequence of a VLFWR3 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions), and/or a VLFWR4 having an amino acid sequence of a VLFWR4 in Table 5 or Table 6 (or a sequence with no more than 1, 2, 3, 4, 5, 6, 7, 8, or 9 mutations, e.g., substitutions, additions, or deletions).

37. The multifunctional molecule of any of the preceding embodiments, wherein the multifunctional molecule further comprises a tumor-targeting moiety.

38. The multifunctional molecule of embodiment 37, wherein the tumor-targeting moiety binds to a tumor antigen.

39. The multifunctional molecule of embodiment 38, wherein the tumor antigen is selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

40. The multifunctional molecule of embodiment 37, wherein the tumor-targeting moiety comprises an antibody molecule, e.g., that binds to a tumor antigen selected from G6B, CD34, CD41, P-selectin, Clec2, cKIT, FLT3, MPL, ITGB3, ITGB2, GP5, GP6, GP9, GP1BA, DSC2, FCGR2A, TNFRSF10A, TNFRSF10B, or TM4SF1.

41. The multifunctional molecule of embodiment 40, wherein the tumor-targeting moiety comprises a VH and/or VL sequence, e.g., as listed in Table 38 or Table 20.

42. The multifunctional molecule of any one of the preceding embodiments, wherein the multifunctional molecule preferentially binds to a myeloproliferative neoplasm cell over a non-tumor cell, optionally wherein the binding between the multifunctional molecule and the myeloproliferative neoplasm cell is more than 10, 20, 30, 40, 50-fold greater than the binding between the multifunctional molecule and a non-tumor cell.

43. The multifunctional molecule of embodiment 42, wherein the myeloproliferative neoplasm cell is chosen from a myelofibrosis cell, an essential thrombocythemia cell, a polycythemia vera cell, or a chronic myeloid cancer cell, optionally wherein: the myeloproliferative neoplasm cell does not comprise a JAK2 V617F mutation, or the myeloproliferative neoplasm cell does not comprise a MPL mutation.

44. The multifunctional molecule of any one of the preceding embodiments, further comprising a linker, e.g., a linker between the first antigen binding domain and the second antigen binding domain.

45. The multifunctional molecule of embodiment 44, wherein the linker is chosen from: a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker.

46. The multifunctional molecule of embodiment 44 or 45, wherein the linker is a peptide linker.

47. The multifunctional molecule of 46, wherein the peptide linker comprises Gly and Ser.

48. The multifunctional molecule of 46, wherein the peptide linker comprises an amino acid sequence chosen from SEQ ID NOs: 6214-6217 or 6220-6221 and 77-78.

49. A nucleic acid molecule encoding the multifunctional molecule of any of the preceding embodiments.

50. A vector, e.g., an expression vector, comprising the nucleic acid molecule of embodiment 49.

51. A cell comprising the nucleic acid molecule of embodiment 49 or the vector of embodiment 50.

52. A method of making, e.g., producing, the multifunctional molecule of any one of embodiments 1-48, comprising culturing the cell of embodiment 51, under suitable conditions, e.g., conditions suitable for gene expression and/or homo- or heterodimerization.

53. A pharmaceutical composition comprising the multifunctional molecule of any one of embodiments 1-48 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

54. A method of treating a cancer, comprising administering to a subject in need thereof the multifunctional molecule of any one of embodiments 1-48, wherein the multifunctional molecule is administered in an amount effective to treat the cancer.

55. Use of the multifunctional molecule of any one of embodiments 1-48 for the manufacture of a medicament for treating a cancer.

56. The method of embodiment 54 or the use of embodiment 55, wherein the subject has cancer cells that express the first and/or second calreticulin protein.

57. The method of embodiment 54 or 56 or the use of embodiment 55 or 56, wherein the subject has the JAK2 V617F mutation.

58. The method of embodiment 54 or 56 or the use of embodiment 55 or 56, wherein the subject does not have the JAK2 V617F mutation.

59. The method of any one of embodiments 54 or 56-58 or the use of any one of embodiments 55-58, wherein the subject has a MPL mutation.

60. The method of any one of embodiments 54 or 56-58 or the use of any one of embodiments 55-58, wherein the subject does not have a MPL mutation.

61. The method of any one of embodiments 54 or 56-60 or the use of any one of embodiments 55-60, wherein the cancer is a hematological cancer, optionally wherein the cancer is a myeloproliferative neoplasm, e.g., primary or idiopathic myelofibrosis (MF), essential thrombocytosis (ET), polycythemia vera (PV), or chronic myelogenous leukemia (CML), optionally wherein the cancer is myelofibrosis.

62. The method of any one of embodiments 54 or 56-60 or the use of any one of embodiments 55-60, the cancer is a solid tumor cancer.

63. The method of any of embodiments 54 or 56-62 or the use of any one of embodiments 55-62, further comprising administering a second therapeutic treatment.

64. The method of embodiment 63 or the use of embodiment 63, wherein the second therapeutic treatment comprises a therapeutic agent (e.g., a chemotherapeutic agent, a biologic agent, hormonal therapy), radiation, or surgery.

65. The method of embodiment 64 or the use of embodiment 64, wherein the therapeutic agent is selected from: a chemotherapeutic agent, or a biologic agent.

66. A method of detecting calreticulin (e.g., wild-type and/or mutant calreticulin) in a sample or subject, comprising: contacting the sample or subject with an anti-calreticulin (e.g., wild-type and/or mutant calreticulin) antibody molecule described herein; and detecting formation of a complex between the antibody molecule and the sample or subject, thereby detecting calreticulin (e.g., wild-type and/or mutant calreticulin).

67. The method of embodiment 66, wherein calreticulin (e.g., wild-type and/or mutant calreticulin) is detected in vitro or in vivo.

68. The method of embodiment 66 or 67, further comprising contacting a reference sample or subject with the antibody molecule; and detecting formation of a complex between the antibody molecule and the reference sample or subject, wherein a change, e.g., a statistically significant change, in the formation of the complex in the sample or subject, relative to the reference sample or subject is indicative of the presence of calreticulin (e.g., wild-type and/or mutant calreticulin) in the sample or subject.

69. The method of any of embodiments 66-68, further comprising obtaining a sample from a subject.

70. The method of any of embodiment 66-69, wherein sample comprises one or more of plasma, tissue (e.g., cancerous tissue), biopsy, blood (e.g., whole blood), PBMCs, bone marrow, and/or lymphatic tissue, e.g., lymph node.

71. The method of any of embodiments 66-70, wherein the sample has not been frozen and/or fixed.

72. The method of any of embodiments 66-70, wherein the sample has been frozen (e.g., snap frozen) and/or fixed (e.g., formalin-fixed paraffin-embedded (FFPE)).

73. The method of any of embodiments 66-72, wherein the subject has, or is at risk of having, a disease or disorder described herein (e.g., cancer, e.g., myelofibrosis).

74. The method of any of embodiments 66-73, further comprising performing a flow analysis, e.g., using a multi-panel method.

75. The method of any of embodiments 66-74, further comprising assessing T-cell clonality, e.g., to determine the presence and/or level of T cell malignancy.

76. The method of any of embodiments 66-75, further comprising measuring the level of calreticulin+(e.g., wild-type calreticulin+ and/or mutant calreticulin+) cells from the biological sample (e.g., determining if the calreticulin+ cells are depleted, e.g., relative to a reference sample or subject).

77. The method of any of embodiments 66-76, further comprising measuring the intracellular level of calreticulin (e.g., wild-type and/or mutant calreticulin).

78. The method of any of embodiments 66-77, further comprising measuring the membrane level of calreticulin (e.g., wild-type and/or mutant calreticulin).

79. The method of any of embodiments 66-78, further comprising evaluating the subject for a change in prognosis, severity, or presence or absence of a disease or disorder (e.g., cancer, e.g., myelofibrosis), e.g., after treatment (e.g., with an antibody molecule described herein).

80. The method of any of embodiments 66-79, wherein the antibody molecule is detectably labeled.

81. A method of evaluating a subject, comprising: contacting a sample (e.g., a sample described herein) from the subject with an anti-calreticulin (e.g., wild-type and/or mutant calreticulin) antibody molecule described herein; and detecting formation of a complex between the antibody molecule and the sample, thereby evaluating the subject.

82. The method of embodiment 81, wherein the subject has, or is at risk of having, a disease or disorder described herein (e.g., cancer, e.g., myelofibrosis).

83. The method of embodiment 81 or 82, wherein the subject has not been treated with an antibody molecule described herein.

84. The method of embodiment 81 or 82, wherein the subject has been treated with an antibody molecule described herein.

85. A kit comprising an anti-calreticulin (e.g., wild-type and/or mutant calreticulin) antibody molecule described herein and instructions for use in a method of detecting calreticulin (e.g., wild-type and/or mutant calreticulin) in a sample or subject.

	Number	Date	Country
Parent	PCT/US2021/047571	Aug 2021	US
Child	18173995		US

MULTIFUNCTIONAL MOLECULES THAT BIND TO CALRETICULIN AND USES THEREOF

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