PROTEINS COMPRISING DELTA-LIKE LIGAND 3 (DLL3) ANTIGEN BINDING DOMAINS AND THEIR USES

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “sequence listing JBI6411” and a creation date of Oct. 7, 2021 and having a size of 275 KB. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The application relates to a protein comprising an antigen binding region that binds Delta-like canonical Notch Ligand 3 (DLL3), and related compositions and methods.

BACKGROUND

Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer death in males, accounting for 14% (903,500) of the total new cancer cases and 6% (258,400) of the total cancer deaths in males worldwide. Metastatic prostate cancer is the second leading cause of cancer death in men in the United States. The course of prostate cancer from diagnosis to death is best categorized as a series of clinical stages based on the extent of disease, hormonal status, and absence or presence of detectable metastases: localized disease, rising levels of prostate-specific antigen (PSA) after radiation therapy or surgery with no detectable metastases, and clinical metastases in the non-castrate or castrate stage. Although surgery, radiation, or a combination of both can be curative for patients with localized disease, a significant proportion of these patients have recurrent disease as evidenced by a rising level of PSA, which can lead to the development of metastases, especially in the high-risk group—a transition to the lethal stage of the disease.

Androgen depletion therapy (ADT) is the standard treatment with a generally predictable outcome: decline in PSA, a period of stability in which the tumor does not proliferate, followed by rising PSA and regrowth as castration-resistant disease. Historically, ADT has been the standard of care for patients with metastatic prostate cancer.

However, recent clinical data suggests that androgen deprivation therapies can lead to the emergence of an androgen independent tumor phenotype known as neuroendocrine prostate cancer (NEPC) through the process of cellular trans-differentiation. Delta-like canonical Notch Ligand 3 (DLL3) has been shown to be enriched in NEPC tumors at both the RNA and protein level. Thus, strategies designed to target DLL3 can have clinical utility in NEPC/small cell carcinoma patient populations.

Small-cell lung cancer accounts for approximately 20% of all lung cancer incidence. The small-cell lung cancer rapidly progresses and is difficult to be surgically removed because lymph node metastasis or distant metastasis has already occurred at the time of diagnosis in many cases. This cancer exhibits high response rates to an anticancer agent in its early stage. Thus, chemotherapy is considered as the first choice for treating the cancer. The cancer, however, immediately becomes resistant to chemotherapy and recurs, resulting in a 3-year survival rate of 5% or lower.

Hence, there is a need for new therapy to treat cancers, such as NEPC, small cell carcinoma or small-cell lung cancer.

In normal cells, DLL3 regulates notch signaling intracellularly. In cancer cells, DLL3 is expressed extracellularly, e.g., with 618 amino acids and 8 extracellular domains including six EGF-like repeats in human. The human DLL3 is highly homologous to that of cynomolgus and mouse/rat sharing 96% and 83% amino acid sequence identity, respectively, while it only shares about <40% identity with DLL1 and DLL4. DLL3 has low to undetectable expression in normal tissues, but has been highly expressed on the cell surface of neuroendocrine tumors including small cell lung cancer, prostate, large cell carcinoma, and bladder, and has become a target in T-cell redirection for the treatment of neuroendocrine cancers.

SUMMARY OF THE INVENTION

In one general aspect, the disclosure relates to an isolated protein comprising an antigen binding region that binds delta-like protein 3 (DLL3), wherein the antigen binding region binds to an epitope within residues 429-618 of human DLL3 as set forth in SEQ ID NO:263.

In some embodiments, the isolated protein comprises an antigen binding region that competes for binding to DLL3 with a reference antibody comprising: a) a heavy chain variable region (VH) having the heavy chain complementarity determining region (HCDR)1, the HCDR2 and the HCDR3 of a VH having the amino acid sequence of SEQ ID NO:1, and a light chain variable region (VL) having the light chain complementarity determining region (LCDR)1, the LCDR2 and the LCDR3 of a VL having the amino acid sequence of SEQ ID NO:2; b) a VH having the HCDR1, the HCDR2 and the HCDR3 of a VH having the amino acid sequence of SEQ ID NO:3, and a VL having the LCDR1, the LCDR2 and the LCDR3 of a VL having the amino acid sequence of SEQ ID NO:4; c) a VH having the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:5 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:6; d) the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:7 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:8; e) the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:9 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO: 10; f) the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO: 11 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO: 12; or g) the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO: 13 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:14. Optionally, the reference antibody comprises the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:4.

Optionally, the isolated protein comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of: a) SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively; b) SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively; c) SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively; d) SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively; e) SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively; f) SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively; g) SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively; h) SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively; i) SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively; j) SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively; k) SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively; 1) SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; or m) SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively. Optionally, the isolated protein comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, and 35, respectively. Optionally, the antigen binding region that binds DLL3 is a scFv, a (scFv)₂, a Fv, a Fab, a F(ab′)₂, a Fd, a dAb or a VHH. Optionally, the antigen binding region that binds DLL3 is the Fab. Optionally, the antigen binding region that binds DLL3 is the scFv. Optionally, the scFv comprises, from the N- to C-terminus, a VH, a first linker (L1) and a VL (VH-L1-VL) or the VL, the L1 and the VH (VL-L1-VH). Optionally, the L1 comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids. Optionally, the L1 comprises an amino acid sequence of SEQ ID NOs:27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139. Optionally, the L1 comprises the amino acid sequence of SEQ ID NO: 120.

The disclosure also provides an antigen binding region that binds DLL3 comprising the VH of SEQ ID NOs:1, 3, 5, 7, 9, 11, or 13 and the VL of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14. Optionally, the antigen binding region that binds DLL3 comprises: a) the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2; b) the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4; c) the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6; d) the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8; e) the VH of SEQ ID NO:9 and the VL of SEQ ID NO: 10; f) the VH of SEQ ID NO:11 and the VL of SEQ ID NO:12; and/or g) the VH of SEQ ID NO:13 and the VL of SEQ ID NO:14. Optionally, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:4. Optionally, the antigen binding region that binds DLL3 comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NOs:63 or 64.

The disclosure provides an isolated protein that is a monospecific protein or a multispecific antigen-binding construct. Optionally, the isolated protein is a multispecific antigen-binding construct. Optionally, the multispecific antigen-binding construct is a bispecific protein. Optionally, the multispecific antigen-binding construct is a trispecific protein. Optionally, the multispecific antigen-binding construct comprises an antigen binding region that binds an antigen on a lymphocyte. Optionally, the lymphocyte is a T cell. Optionally, the T cell is a CD8+ T cell. Optionally, the lymphocyte is a natural killer (NK) cell. Optionally, in the multispecific antigen-binding construct, the antigen on the lymphocyte is CD3, CD3 epsilon (CD3E), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. Optionally, the antigen on the lymphocyte is CD3E.

In some embodiments, the multispecific antigen-binding construct comprises an antigen binding region that binds CD3ε comprising a) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO: 100, a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 106, a LCDR2 of SEQ ID NO:107 and a LCDR3 of SEQ ID NO: 108; or b) the VH of SEQ ID NO:84 and the VL of SEQ ID NO:85. In some embodiments, in the multispecific antigen-binding construct, the antigen binding region that binds CD3ε comprises a HCDR1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO:100, a LCDR1 of SEQ ID NO: 106, a LCDR2 of SEQ ID NO:107 and a LCDR3 of SEQ ID NO:108. In some embodiments, in the multispecific antigen-binding construct, the antigen binding region that binds CD3ε comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:84 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:85.

In some embodiments, the multispecific antigen-binding construct comprises the antigen binding region that binds CD3ε comprising a) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:95, a HCDR2 of SEQ ID NO:96, a HCDR3 of SEQ ID NO:97, a light chain complementarity determining region (LCDR)1 of SEQ ID NO: 101, a LCDR2 of SEQ ID NO:102 and a LCDR3 of SEQ ID NO: 104; or b) the VH of SEQ ID NO:77 and the VL of SEQ ID NO:80. In some embodiments, the multispecific antigen-binding construct comprises the antigen binding region that binds CD3ε comprising a HCDR1 of SEQ ID NO:95, a HCDR2 of SEQ ID NO:96, a HCDR3 of SEQ ID NO:97, a LCDR1 of SEQ ID NO:101, a LCDR2 of SEQ ID NO:102 and a LCDR3 of SEQ ID NO:104. In some embodiments, the multispecific antigen-binding construct comprises the antigen binding region that binds CD3E comprising a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:77 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:80.

The disclosure also provides an isolated multispecific antigen-binding construct comprising an antigen binding region that binds delta-like protein 3 (DLL3), wherein the antigen binding region that binds DLL3 comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a) SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively; b) SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively; c) SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively; d) SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively; e) SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively; f) SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively; g) SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively; h) SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively; i) SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively; j) SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively; k) SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively; 1) SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; m) SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively; n) the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2; o) the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4; p) the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6; q) the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8; r) the VH of SEQ ID NO:9 and the VL of SEQ ID NO: 10; s) the VH of SEQ ID NO:11 and the VL of SEQ ID NO:12; or t) the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:14. Optionally, the multispecific antigen-binding construct comprises the binding domain that binds DLL3 comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34 and 35, respectively.

In a particular embodiment, the disclosure provides an isolated multispecific antigen-binding construct comprising an antigen binding region that binds delta-like protein 3 (DLL3), wherein the antigen binding region that binds DLL3 comprises a heavy chain complementarity determining region (HCDR)1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO:3 and a light chain complementarity determining region (LCDR)1, a LCDR2 and a LCDR3 of a light chain variable region (VL) of SEQ ID NO:4.

The disclosure also provides an isolated multispecific antigen-binding construct comprising an antigen binding region that binds delta-like protein 3 (DLL3), wherein the antigen binding region that binds DLL3 comprises a heavy chain variable region (VH) of SEQ ID NO:3 and a light chain variable region (VL) of SEQ ID NO:4.

In some embodiments, the isolated protein is conjugated to a half-life extending moiety. Optionally, the half-life extending moiety is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, a Fc region, transferrin, albumin, an albumin binding domain or polyethylene glycol. Optionally, the fragment of the Ig constant region comprises a Fc region. Optionally, the antigen binding region that binds DLL3 is conjugated to the N-terminus of the Ig constant region or the fragment of the Ig constant region. Optionally, the antigen binding region that binds DLL3 is conjugated to the C-terminus of the Ig constant region or the fragment of the Ig constant region. Optionally, the antigen binding region that binds DLL3 is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2). Optionally, the L2 comprises the amino acid sequence of SEQ ID NOs:27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139. Optionally, the Ig constant region or the fragment of the Ig constant region is an IgG1, an IgG2, an IgG3 or an IgG4 isotype. Optionally, the Ig constant region or the fragment of the Ig constant region is an IgG1 isotype. Optionally, the Ig constant region or the fragment of the Ig constant region comprises at least one mutation that results in reduced binding of the protein to a Fcγ receptor (FcγR). Optionally, the at least one mutation that results in reduced binding of the protein to the FcγR is selected from the group consisting of F234A/L235A, L234A/L235A, L234A/L235A/D265 S, V234A/G237A/P238 S/H268A/V309L/A330 S/P33IS, F234A/L235A, S228P/F234A/L235A, N297A, V234A/G237A, K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D365E/L358M, H268Q/V309L/A330 S/P331 S, S267E/L328F, L234F/L235E/D265A, L234A/L235A/G237A/P238 S/H268A/A330 S/P331 S, S228P/F234A/L235A/G237A/P238 S and S228P/F234A/L235A/G236-deleted/G237A/P238 S, wherein residue numbering is according to the EU index. Optionally, the mutations that results in reduced binding of the protein to the FcγR are L234A_L235A_D265 S.

The disclosure provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively; b) a VH of SEQ ID NO: 1 and a VL of SEQ ID NO:2; c) a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4; d) a scFv of SEQ ID NO:63; and/or e) a scFv of SEQ ID NO:64. Optionally, the isolated protein comprises an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively; and/or b) a VH of SEQ ID NO:1 and a VL of SEQ ID NO:2. Optionally, the isolated protein comprises an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively; b) a VH of SEQ ID NO:5 and a VL of SEQ ID NO:6; and/or c) a scFv of SEQ ID NO:65. Optionally, the isolated protein comprises an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively; b) a VH of SEQ ID NO:7 and a VL of SEQ ID NO:8; and/or c) a scFv of SEQ ID NO:66. Optionally, the isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively; b) a VH of SEQ ID NO:9 and a VL of SEQ ID NO:10; and/or c) a scFv of SEQ ID NO:67. Optionally, the isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:27, 28, 29, 44, 45, 46, respectively; b) a VH of SEQ ID NO:11 and a VL of SEQ ID NO: 12; and/or c) a scFv of SEQ ID NO:68. Optionally, the isolated protein comprises an antigen binding region that binds DLL3, wherein the antigen binding region comprises a) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs: 30, 31, 32, 47, 48, 49, respectively; b) a VH of SEQ ID NO:13 and a VL of SEQ ID NO:14; and/or c) a scFv of SEQ ID NO:69.

Optionally, the isolated protein is a multispecific antigen-binding construct comprising an antigen binding region that binds CD3E. Optionally, the multispecific antigen-binding construct comprises an antigen binding region that binds CD3ε comprising a) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO: 100, a light chain complementarity determining region (LCDR)1 of SEQ ID NO:106, a LCDR2 of SEQ ID NO:107 and a LCDR3 of SEQ ID NO: 108; and/or b) the VH of SEQ ID NO:84 and the VL of SEQ ID NO:85. Optionally, the multispecific antigen-binding construct comprises an antigen binding region that binds CD3ε comprising a) a heavy chain complementarity determining region (HCDR)1 of SEQ ID NO:95, a HCDR2 of SEQ ID NO:96, a HCDR3 of SEQ ID NO:97, a light chain complementarity determining region (LCDR)1 of SEQ ID NO:101, a LCDR2 of SEQ ID NO: 102 and a LCDR3 of SEQ ID NO:104; and/or b) the VH of SEQ ID NO:77 and the VL of SEQ ID NO:80.

The disclosure provides an isolated anti-DLL3/anti-CD3 protein comprising a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen. Optionally, the lymphocyte antigen is a T cell antigen. Optionally, the T cell antigen is a CD8⁺ T cell antigen. Optionally, the lymphocyte antigen is a NK cell antigen. Optionally, the lymphocyte antigen is CD3, CD3 epsilon (CD3E), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C. Optionally, the lymphocyte antigen is CD3E.

Optionally, in an isolated anti-DLL3/anti-CD3 protein, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise a scFv, a (scFv)₂, a Fv, a Fab, a F(ab′)₂, a Fd, a dAb or a VHH. Optionally, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the Fab. Optionally, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the scFv. Optionally, the first antigen binding region that binds DLL3 comprises the scFv and the second antigen binding region that binds the lymphocyte antigen comprise the Fab. Optionally, the first antigen binding region that binds DLL3 comprises the Fab and the second antigen binding region that binds the lymphocyte antigen comprise the scFv. Optionally, the scFv comprises, from the N- to C-terminus, a VH, a first linker (L1) and a VL (VH-L1-VL) or the VL, the L1 and the VH (VL-L1-VH). Optionally, the L1 comprises a) about 5-50 amino acids; b) about 5-40 amino acids; c) about 10-30 amino acids; or d) about 10-20 amino acids. Optionally, the L1 comprises the amino acid sequence of SEQ ID NOs:27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139. Optionally, the L1 comprises the amino acid sequence of SEQ ID NO:120.

Optionally, in an isolated anti-DLL3/anti-CD3 protein, the first antigen binding region that binds DLL3 comprises a HCDR1 of SEQ ID NOs:15, 18, 21, 24, 27, 30, 50, 52, 53, 55, 57, 59, or 61, a HCDR2 of SEQ ID NOs:16, 19, 22, 25, 28, 31, 51, 54, 56, 58, 60, or 62, a HCDR3 of SEQ ID NOs:17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32, a LCDR1 of SEQ ID NOs:33, 36, 39, 41, 44, or 47, a LCDR2 of SEQ ID NOs:34, 37, 42, 45, or 48, and a LCDR3 of SEQ ID NOs:35, 38, 40, 43, 46, or 49. Optionally, the first antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of a. SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively; b. SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively; c. SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively; d. SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively; e. SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively; f. SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively; g. SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively; h. SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively; i. SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively; j. SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively; k. SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively; 1. SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; or m. SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively. Optionally, the first antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively.

In some embodiments, the first antigen binding region that binds DLL3 comprises a. the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2; b. the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4; c. the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6; d. the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8; e. the VH of SEQ ID NO:9 and the VL of SEQ ID NO:10; f. the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12; or g. the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:14. Optionally, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NOs:63 or 64. Optionally, the first antigen binding region that binds DLL3 comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the amino acid sequence of SEQ ID NO:64. Optionally, the first antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:4. Optionally, the second antigen binding region that binds CD3 comprises a HCDR1 of SEQ ID NOs:95 or 98, a HCDR2 of SEQ ID NOs:96 or 99, a HCDR3 of SEQ ID NOs:97 or 100, a LCDR1 of SEQ ID NOs:101 or 106, a LCDR2 of SEQ ID NOs:102 or 107, and a LCDR3 of SEQ ID NOs:104 or 108. Optionally, the second antigen binding region that binds CD3 comprises a HCDR1 of SEQ ID NO:95, a HCDR2 of SEQ ID NO:96, a HCDR3 of SEQ ID NO:97, a LCDR1 of SEQ ID NO: 101, a LCDR2 of SEQ ID NO: 102, and a LCDR3 of SEQ ID NO: 104. Optionally, the second antigen binding region that binds CD3 comprises a HCDR1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO: 100, a LCDR1 of SEQ ID NO: 106, a LCDR2 of SEQ ID NO: 107, and a LCDR3 of SEQ ID NO:108. Optionally, the second antigen binding region that binds CD3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:77 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:80. Optionally, the second antigen binding region that binds CD3 comprises a VH of SEQ ID NO:77 and a VL of SEQ ID NO:80. Optionally, the second antigen binding region that binds CD3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:84 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:85. Optionally, the second antigen binding region that binds the lymphocyte antigen comprises a VH of SEQ ID NO:84 and a VL of SEQ ID NO:85.

In some embodiments, the first antigen binding region that binds DLL3 is conjugated to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region and/or the second antigen binding region that binds the lymphocyte antigen is conjugated to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region. Optionally, the isolated anti-DLL3/anti-CD3 protein further comprises second linker (L2) between the first antigen binding region that binds DLL3 and the first Ig constant region or the fragment of the first Ig constant region and the second antigen binding region that binds the lymphocyte antigen and the second Ig constant region or the fragment of the second Ig constant region. Optionally, the L2 comprises the amino acid sequence of SEQ ID NOs:27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, or 138. Optionally, the fragment of the Ig constant region comprises a Fc region. Optionally, wherein the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgG1, an IgG2, and IgG3 or an IgG4 isotype. Optionally, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgG1. Optionally, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that results in reduced binding of the multispecific antigen-binding construct to a FcγR. Optionally, the at least one mutation that results in reduced binding of the multispecific antigen-binding construct to the FcγR is selected from the group consisting of F234A/L235A, L234A/L235A, L234A/L235A/D265 S, V234A/G237A/P238 S/H268A/V309L/A330 S/P331 S, F234A/L235A, S228P/F234A/L235A, N297A, V234A/G237A, K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D365E/L358M, H268Q/V309L/A330 S/P331 S, S267E/L328F, L234F/L235E/D265A, L234A/L235A/G237A/P238 S/H268A/A330 S/P331 S, S228P/F234A/L235A/G237A/P238 S and S228P/F234A/L235A/G236-deleted/G237A/P238 S, wherein residue numbering is according to the EU index. Optionally, the mutations that results in reduced binding of the multispecific antigen-binding construct to the FcγR are L234A_L235A_D265 S. Optionally, the protein comprises at least one mutation in a CH3 domain of the Ig constant region. Optionally, the at least one mutation in the CH3 domain of the Ig constant region is selected from the group consisting of T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394 S, Y407T, Y407A, T366 S/L368A/Y407V, L351Y/F405A/Y407V, T3661/K392M/T394W, F405A/Y407V, T366L/K392M/T394W, L351Y/Y407A, T366A/K409F, L351Y/Y407A, T366V/K409F, T366A/K409F, T350V/L351Y/F405A/Y407V and T350V/T366L/K392L/T394W, wherein residue numbering is according to the EU index.

In one general aspect, the application relates to a bispecific antigen-binding construct comprising:

- (1) a first antigen binding region that binds DLL3, wherein the first antigen binding region comprises a first VH having a HCDR1, a HCDR2 and a HCDR3, and a first VL having a LCDR1, a LCDR2 and a LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprise the amino acid sequences of
  - (a) SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively;
  - (b) SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively;
  - (c) SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively;
  - (d) SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively;
  - (e) SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively;
  - (f) SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively;
  - (g) SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively;
  - (h) SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively;
  - (i) SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively;
  - (j) SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively;
  - (k) SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively;
  - (l) SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; or
  - (m)SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively.
- (2) a second antigen binding region that binds CD3E, wherein the second antigen binding region comprises:
  - (a) a second VH having a HCDR1, a HCDR2 and a HCDR3 of the amino acid sequences of SEQ ID NOs: 95, 96 and 97, respectively, and a second VL having a LCDR1, a LCDR2 and a LCDR3 of the amino acid sequences of SEQ ID NOs: 101, 102 and 104, respectively; or
  - (b) a second VH having a HCDR1, a HCDR2 and a HCDR3 of the amino acid sequences of SEQ ID NOs: 98, 99 and 100, respectively, and a second VL having a LCDR1, a LCDR2 and a LCDR3 of the amino acid sequences of SEQ ID NOs: 106, 107 and 108, respectively.

The bispecific antigen-binding construct is referred to herein as an “anti-DLL3/anti-CD3 construct” or “an anti-DLL3/anti-CD3”.

In some embodiments, an isolated anti-DLL3/anti-CD3 protein comprising a first antigen binding region that binds DLL3 and a second antigen binding region that binds CD3, wherein a. The first antigen binding region that binds DLL3 comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively, and the second domain that binds CD3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 98, 99, 100, 106, 107, 108, respectively; and/or b. the first antigen binding region that binds DLL3 comprises a scFv which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the scFv of SEQ ID NO:64, and the second antigen binding region that binds CD3 comprises a Fab comprising a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:84 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:85; and/or c. the isolated anti-DLL3/anti-CD3 protein comprises a HC1 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC1 of SEQ ID NO:71, a HC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC2 of SEQ ID NO: 118, and a LC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the of SEQ ID NO:117.

In some embodiments, an isolated anti-DLL3/anti-CD3 protein comprising a first antigen binding region that binds DLL3 and a second antigen binding region that binds CD3, wherein a. the first antigen binding region that binds DLL3 comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2 and a LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively, and the second domain that binds CD3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 98, 99, 100, 106, 107, 108, respectively; and/or b. the first antigen binding region that binds DLL3 comprises a scFv which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the scFv of SEQ ID NO:64, and the second antigen binding region that binds CD3 comprises a Fab comprising a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VH of SEQ ID NO:84 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the VL of SEQ ID NO:85; and/or c. the isolated anti-DLL3/anti-CD3 protein comprises a HC1 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC1 of SEQ ID NO:229, a HC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the HC2 of SEQ ID NO:230, and a LC2 which is at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 99% or 100%) identical to the of SEQ ID NO:117.

The disclosure also provides an immunoconjugate comprising the isolated antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides an immunoconjugate comprising the isolated protein comprising the antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides an immunoconjugate comprising the isolated multispecific antigen-binding construct comprising the antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides a pharmaceutical composition comprising the isolated antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides a pharmaceutical composition comprising the isolated protein comprising the antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides a pharmaceutical composition comprising the isolated multispecific antigen-binding construct comprising the antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides an isolated polynucleotide encoding the isolated antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides an isolated polynucleotide encoding the isolated protein comprising the antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides an isolated polynucleotide encoding the isolated multispecific antigen-binding construct comprising the antigen binding region that binds DLL3 of the disclosure.

The disclosure also provides a vector comprising the polynucleotide of the disclosure.

The disclosure also provides a host cell comprising the polynucleotide or the vector of the disclosure.

The disclosure also provides a method of treating a DLL3 expressing cancer in a subject, comprising administering a therapeutically effective amount of the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure to the subject in need thereof for a time sufficient to treat the DLL3 expressing cancer.

The disclosure also provides a method of reducing the amount of DLL3 expressing tumor cells in a subject, comprising administering to the subject the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure for a time sufficient to reduce the amount of DLL3 expressing tumor cells.

The disclosure also provides a method of preventing establishment of a DLL3 expressing cancer in a subject, comprising administering the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure to the subject in need thereof to prevent establishment of the DLL3 expressing cancer in the subject.

The disclosure also provides a method of treating a noncancerous condition in a subject at risk of developing a DLL3 expressing cancerous condition, comprising administering the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure to the subject in need thereof to treat the noncancerous condition.

The disclosure also provides a method of treating prostate cancer in a subject, comprising administering a therapeutically effective amount of the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure to the subject in need thereof for a time sufficient to treat the prostate cancer.

The disclosure also provides a method of treating small cell lung cancer in a subject, comprising administering a therapeutically effective amount of the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure to the subject in need thereof for a time sufficient to treat the small cell lung cancer.

The disclosure also provides a method of detecting prostate cancer or small cell lung cancer in a subject, comprising administering to the subject the immunoconjugate of the disclosure, and detecting binding of the immunoconjugate to DLL3, thereby detecting prostate cancer or small cell lung cancer.

The disclosure also provides a kit comprising the antigen binding region that binds DLL3, the protein comprising the antigen binding region that binds DLL3, the multispecific antigen-binding construct comprising the antigen binding region that binds DLL3, the immunoconjugate of the disclosure or the pharmaceutical composition of the disclosure.

The disclosure also provides an anti-idiotypic antibody binding to the antigen binding region that binds DLL3 of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular descriptions of example embodiments, as illustrated in the accompanying drawings.

FIG. 1 shows the schematic view of a DLL3 extracellular domain including a DSL domain and 6 EGF domains. The amino acid sequence shown represents residues 176-215 of DSL domain (SEQ ID NO:246), residues 216-249 of EGF-1 domain (SEQ ID NO:247), residues 274-310 of EGF-2 domain (SEQ ID NO:248), residues 312-351 of EGF-3 domain (SEQ ID NO:249), residues 353-389 of EGF-4 domain (SEQ ID NO:250), residues 391-427 of EGF-5 domain (SEQ ID NO:251), residues 429-465 of EGF-6 domain (SEQ ID NO:252) and residues 429-618 of EGF-6 domain+C-terminal domain (SEQ ID NO:263).

FIG. 2A and FIG. 2B show cells binding of bispecific anti-DLL3×CD3 antibodies to DLL3+ tumor cell lines. FIG. 2A shows cells binding of bispecific anti-DLL3×CD3 antibodies to DLL3+ tumor cell lines, SHP77 cells. FIG. 2B shows cells binding of bispecific anti-DLL3×CD3 antibodies to DLL3+ tumor cell lines, HCC1833 cells.

FIG. 3 shows binding of bispecific anti-DLL3×CD3 antibodies on human pan T cells using FACS.

FIG. 4 shows tumor lysis of anti-DLL3×CD3 bispecific antibodies with and without optimized anti-DLL3 sequence evaluated in an IncuCyte-based cytotoxicity assay.

FIG. 5A and FIG. 5B show in vitro target cytotoxicity of bispecific anti-DLL3×CD3 antibodies measured by incuCyte imaging system in real-time for quantifying target cell death.

FIG. 5A shows in vitro target cytotoxicity of anti-DLL3×CD3 bispecific molecules measured by incuCyte imaging system in real-time for quantifying target cell death. Isolated pan-T cells were co-incubated with DLL3+ SHP77 cells in the presence of bispecific anti-DLL3×CD3 antibodies for 120 hours. FIG. 5B shows in vitro target cytotoxicity of anti-DLL3×CD3 bispecific molecules measured by incuCyte imaging system in real-time for quantifying target cell death. Isolated pan-T cells were co-incubated with DLL3⁻ HEK293 cells in the presence of bispecific anti-DLL3×CD3 antibodies for 120 hours.

FIG. 6 shows isolated pan-T cells were co-incubated with DLL3+ SHP77 cells in the presence of bispecific anti-DLL3/CD3 antibodies for 120 hours.

FIG. 7 shows in vitro T cell IFN-γ release by bispecific anti-DLL3×CD3 antibodies. IFN-γ concentration was measured from supernatants collected at the indicated time points.

FIGS. 8A-8C show the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by bispecific anti-DLL3×CD3 antibodies. FIG. 8A shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by bispecific anti-DLL3×CD3 antibodies with an E:T ratio of 10:1. FIG. 8B shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by bispecific anti-DLL3×CD3 antibodies with an E:T ratio of 5:1. FIG. 8C shows the cytotoxicity against DLL3+ target cell lines in PBMCs mediated by bispecific anti-DLL3×CD3 antibodies with an E:T ratio of 1:1.

FIG. 9 shows proliferation of CD3+ T cells in response to bispecific anti-DLL3×CD3 antibodies in whole PBMC cytotoxicity assay.

FIGS. 10A-10C show activation of T cells in response to bispecific anti-DLL3×CD3 antibodies. FIG. 10A shows activation of T cells in response to bispecific anti-DLL3×CD3 antibodies % CD25⁺ cells. FIG. 10B shows activation of T cells in response to bispecific anti-DLL3×CD3 antibodies % CD69⁺ cells. FIG. 10C shows activation of T cells in response to bispecific anti-DLL3×CD3 antibodies % CD71⁺ cells.

FIG. 11A shows dose response curves for IFNy concentrations at 48 hours.

FIG. 11B shows dose response curves for IFNy concentrations at 120 hours.

FIG. 12A shows dose response curves for CD8+CD25+ T-cells as a percentage of total CD8+ T-cells at 48 hours.

FIG. 12B shows dose response curves for CD8+CD25+ T-cells as a percentage of total CD8+ T-cells at 120 hours.

FIG. 13A shows dose response curves for CD8+ T-cells proliferation at 72 hours.

FIG. 13B shows dose response curves for CD8+ T-cells proliferation at 120 hours.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles, patents and patent applications are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present application, exemplary materials and methods are described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this application pertains. Otherwise, certain terms used herein have the meanings as set in the specification. All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth fully herein. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

Unless otherwise stated, any numerical value, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ±10% of the recited value. For example, a dosage of 10 mg includes 9 mg to 11 mg. As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”

The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.”

“About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in the Specification in the context of a particular assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

“Activation” or “stimulation” or “activated” or “stimulated” refers to induction of a change in the biologic state of a cell resulting in expression of activation markers, cytokine production, proliferation or mediating cytotoxicity of target cells. Cells may be activated by primary stimulatory signals.

“Alternative scaffold” refers to a single chain protein framework that contains a structured core associated with variable domains of high conformational tolerance. The variable domains tolerate variation to be introduced without compromising scaffold integrity, and hence the variable domains can be engineered and selected for binding to a specific antigen.

“Antibody-dependent cellular cytotoxicity”, “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to the mechanism of inducing cell death that depends upon the interaction of antibody-coated target cells with effector cells possessing lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils via Fc gamma receptors (FcγR) expressed on effector cells.

“Antibody-dependent cellular phagocytosis” or “ADCP” refers to the mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells.

“Antigen” refers to any molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of being bound by an antigen binding region or a T-cell receptor that is capable of mediating an immune response. Exemplary immune responses include antibody production and activation of immune cells, such as T cells, B cells or NK cells. Antigens may be expressed by genes, synthetized, or purified from biological samples such as a tissue sample, a tumor sample, a cell or a fluid with other biological components, organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates.

An “antigen binding region” or “antigen binding fragment” or “antigen binding domain” each refers to a portion of a full-length antibody that binds an antigen. An antigen binding region can be synthetic, enzymatically obtainable or genetically engineered polypeptides. An antigen binding region typically comprises one or more portions of at least the VH region. Antigen-binding fragments include multivalent molecules comprising one, two, three, or more antigen-binding portions of an antibody, and single-chain constructs wherein the VL and VH regions, or selected portions thereof, are joined by synthetic linkers or by recombinant methods to form a functional, antigen-binding molecule. Antigen-binding fragments can also be a single-domain antibody (sdAb), also known as a nanobody, which is an antibody fragment consisting of a single monomeric variable antibody domain (VHH). Examples of antigen-binding fragments include Fab, Fab′, F(ab)₂, F(ab′)₂, F(ab)₃, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv, see e.g., Bird et al., Science 1988; 242:423-426; and Huston et al. PNAS 1988; 85:5879-5883), dsFv, Fd (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VHH, and V-NAR domains; monovalent molecules comprising a single VH and a single VL chain; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10:949-57); camel IgG; IgNAR; as well as one or more isolated CDRs or a functional paratope, where the isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment, minimal recognition units consisting of the amino acid residues that mimic the CDRs of an antibody, such as FR3-CDR3-FR4 portions, the HCDR1, the HCDR2 and/or the HCDR3 and the LCDR1, the LCDR2 and/or the LCDR3, alternative scaffolds that bind an antigen, and multispecific antigen-binding constructs comprising the antigen binding regions. Various types of antibody fragments have been described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol 2005; 23:1126-1136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201. Antibody fragments can be obtained using conventional recombinant or protein engineering techniques, and the fragments can be screened for antigen-binding or other function in the same manner as are intact antibodies. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of full-length antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See WO 1993/16185; U.S. Pat. Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example. Such linear antibody fragments can be monospecific or bispecific. Antigen binding regions (such as VH and VL) cane be linked together via a synthetic linker to form various types of single antibody designs where the VH/VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chains, to form a monovalent antigen binding region, such as single chain Fv (scFv) or diabody. Antigen binding regions can also be conjugated to other antibodies, proteins, antigen binding regions or alternative scaffolds which may be monospecific or multispecific to engineer bispecific and multispecific antigen-binding constructs.

“Antibody” or “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, humanized, chimeric monoclonal antibodies, antigen binding regions, multispecific antibodies, such as bispecific, trispecific, tetraspecific etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (k), based on the amino acid sequences of their constant domains. General principles of antibody molecule structure and various techniques relevant to the production of antibodies are provided in, e.g., Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1988).

An isolated protein or construct of the application can also comprise an antibody derivative. The term “antibody derivative” as used herein refers to a molecule comprising a full-length antibody or an antigen-binding fragment thereof, wherein one or more amino acids are chemically modified or substituted. Chemical modifications that can be used in antibody derivative includes, e.g., alkylation, PEGylation, acylation, ester formation or amide formation or the like, e.g., for linking the antibody to a second molecule. Exemplary modifications include PEGylation (e.g., cysteine-PEGylation), biotinylation, radiolabeling, and conjugation with a second agent (such as a cytotoxic agent).

Antibodies herein include “amino acid sequence variants” with altered antigen-binding or biological activity. Examples of such amino acid alterations include antibodies with enhanced affinity for antigen (e.g. “affinity matured” antibodies), and antibodies with altered Fc region, if present, e.g. with altered (increased or diminished) antibody dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) (see, for example, WO 00/42072, Presta, L. and WO 99/51642, Iduosogie et al); and/or increased or diminished serum half-life (see, for example, WO00/42072, Presta, L.).

A “bispecific antigen-binding construct” or “bispecific construct” refers to a construct that specifically binds two distinct antigens or two distinct epitopes within the same antigen. A bispecific antigen-binding construct can be a protein, a protein complex, or an antibody. The bispecific construct can have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

A “bispecific anti-DLL3/anti-CD3 antibody,” “anti-DLL3×CD3,” “DLL3/CD3 antibody,” “DLL3×CD3 antibody,” “anti-DLL3/anti-CD3 protein”, and the like refer to a construct or antibody that binds DLL3 and CD3 and that comprises at least one binding domain specifically binding DLL3 and at least one binding domain specifically binding CD3. The domains specifically binding DLL3 and CD3 are typically V_H/V_Lpairs. The bispecific anti-DLL3×CD3 antibody can be monovalent in terms of its binding to either DLL3 or CD3.

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity-determining region” or “CDR” (residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; (Kabat 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/or those residues from a “hypervariable loop” (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987; 196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, “variable domain residue numbering as in Kabat” and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.

The term “administering” with respect to the methods of the invention, means a method for therapeutically or prophylactically preventing, treating or ameliorating a syndrome, disorder or disease as described herein by using a conjugate of the invention or a form, composition or medicament thereof. Such methods include administering an effective amount of said antibody, antigen-binding fragment thereof, or conjugate, or a form, composition or medicament thereof at different times during the course of a therapy or concurrently in a combination form. The methods of the invention are to be understood as embracing all known therapeutic treatment regimens.

The ability of a target antibody to “block” the binding of a target molecule to a natural target ligand, means that the antibody, in an assay using soluble or cell-surface associated target and ligand molecules, can detectably reduce the binding of a target molecule to the ligand in a dose-dependent fashion, where the target molecule detectably binds to the ligand in the absence of the antibody.

“Cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor.

“Complement-dependent cytotoxicity” or “CDC”, refers to the mechanism of inducing cell death in which the Fc effector domain of a target-bound protein binds and activates complement component C1q which in turn activates the complement cascade leading to target cell death. Activation of complement may also result in deposition of complement components on the target cell surface that facilitate CDC by binding complement receptors (e.g., CR3) on leukocytes.

“Complementarity determining regions” (CDR) are antibody regions that bind an antigen.

There are three CDRs in the VH (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (LCDR1, LCDR2, LCDR3). CDRs may be defined using various delineations such as Kabat (Wu et al. (1970) J Exp Med 132: 211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77) and AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996). The correspondence between the various delineations and variable region numbering is described (see e.g., Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun (2001), J Mol Biol 309:657-70; International ImMunoGeneTics (IMGT) database; Web resources, http://www_imgt_org). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” as used herein include CDRs defined by any of the methods described supra, Kabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia, supra; Martin, supra; Lefranc et al., supra).

TABLE 1

IMGT
Kabat
AbM
Chothia

V_HCDR1
27-38
31-35
26-35
26-32

V_HCDR2
56-65
50-65
50-58
53-55

V_HCDR3
105-117
95-102
95-102
96-101

V_LCDR1
27-38
24-34
24-34
26-32

V_LCDR2
56-65
50-56
50-56
50-52

V_LCDR3
105-117
89-97
89-97
91-96

“CD3” refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) complex and which consists of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta and CD3 gamma. Human CD3 epsilon comprises the amino acid sequence of SEQ ID NO:253. All references to proteins, polypeptides and protein fragments herein are intended to refer to the human version of the respective protein, polypeptide or protein fragment unless explicitly specified as being from a non-human species. Thus, “CD3” means human CD3 unless specified as being from a non-human species, e.g., “mouse CD3,” “monkey CD3,” etc.

Throughout the specification, “CD3-specific” or “specifically binds CD3” or “anti-CD3 antibody” refers to antibodies that bind specifically to the CD3-epsilon polypeptide (SEQ ID NO:253), including antibodies that bind specifically to the CD3-epsilon extracellular domain (ECD) (SEQ ID NO:254). CD3-epsilon, together with CD3-gamma, -delta and-zeta, and the T-cell receptor alpha/beta and gamma/delta heterodimers, forms the T-cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response.

A “conjugate” as used herein refer to a protein covalently linked to one or more heterologous molecule(s), including but not limited to a therapeutic peptide or protein, an antibody, a label, or a neurological disorder drug. When one protein is conjugated to another protein, it is also referred to the two proteins are fused together. By way of a non-limiting example, an antibody or antigen-binding fragment of the application can be conjugated to another polypeptide to form a fusion protein. In certain embodiments, an antibody or antigen-binding fragment of the application can be fused or conjugated to another polypeptide through a linker.

“Decrease,” “lower,” “lessen,” “reduce,” or “abate” refers generally to the ability of a test molecule to mediate a reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle. Exemplary responses are T cell expansion, T cell activation or T-cell mediated tumor cell killing or binding of a protein to its antigen or receptor, enhanced binding to a Fey or enhanced Fc effector functions such as enhanced ADCC, CDC and/or ADCP. Decrease may be a statistically significant difference in the measured response between the test molecule and the control (or the vehicle), or a decrease in the measured response, such as a decrease of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

“Delta-like protein 3” or “DLL3” refers to a known protein which is also called delta-like 3, delta 3, or drosophila Delta homolog 3. Unless specified, as used herein, DLL3 refers to human DLL3. All DLL3 isoforms and variants are encompassed in “DLL3”. The amino acid sequences of the various isoforms are retrievable from databases, such as NCBI accession numbers NP_058637.1 (isoform 1 precursor, 618 amino acids) and NP 982353.1(isoform 2 precursor, 587 amino acids). The amino acid sequence of a full length human DLL3 is shown in SEQ ID NO:255. The sequence of DLL3 includes the DSL domain (residues 176-215), EGF-1 domain (residues 216-249), EGF-2 domain (residues 274-310), EGF-3 domain (residues 312-351), EGF-4 domain (residues 353-389), EGF-5 domain (residues 391-427), EGF-6 domain (residues 429-465) and C-terminal domain (residues 466-618) (FIG. 1). The amino acid sequence of the DLL3 DSL domain is shown in SEQ ID NO: 246. The amino acid sequence of the DLL3 EGF-1 domain is shown in SEQ ID NO:247. The amino acid sequence of the DLL3 EGF-2 domain is shown in SEQ ID NO:248. The amino acid sequence of the DLL3 EGF-3 domain is shown in SEQ ID NO:249. The amino acid sequence of the DLL3 EGF-4 domain is shown in SEQ ID NO:250. The amino acid sequence of the DLL3 EGF-5 domain is shown in SEQ ID NO:251. The amino acid sequence of the DLL3 EGF-6 domain is shown in SEQ ID NO:252. The amino acid sequence of the DLL3 EGF-6+C-terminal domain is shown in SEQ ID NO:263.

“Differentiation” refers to a method of decreasing the potency or proliferation of a cell or moving the cell to a more developmentally restricted state.

“Encode” or “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

“Enhance,” “promote,” “increase,” “expand” or “improve” refers generally to the ability of a test molecule to mediate a greater response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle. Exemplary responses are T cell expansion, T cell activation or T-cell mediated tumor cell killing or binding of a protein to its antigen or receptor, enhanced binding to a Fcγ or enhanced Fc effector functions such as enhanced ADCC, CDC and/or ADCP. Enhance may be a statistically significant difference in the measured response between the test molecule and control (or vehicle), or an increase in the measured response, such as an increase of about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 30 fold or more, such as 500, 600, 700, 800, 900 or 1000 fold or more (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

“Epitope” refers to a portion of an antigen to which an antibody, or the antigen binding portion thereof, specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and/or discontinuous amino acids that form a conformational spatial unit. For a discontinuous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule. Antibody “epitope” depends on the methodology used to identify the epitope.

“Expansion” refers to the outcome of cell division and cell death.

“Express” and “expression” refers to the well-known transcription and translation occurring in cells or in vitro. The expression product, e.g., the protein, is thus expressed by the cell or in vitro and may be an intracellular, extracellular or a transmembrane protein.

“Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

“dAb” or “dAb fragment” refers to an antibody fragment composed of a VH domain (Ward et al. (1989), Nature 341:544 546).

“Fab” or “Fab fragment” refers to an antibody fragment composed of VH, CH1, VL and CL domains.

“F(ab′)₂” or “F(ab′)₂fragment” refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge in the hinge region.

“Fd” or “Fd fragment” refers to an antibody fragment composed of VH and CH1 domains.

“Fv” or “Fv fragment” refers to an antibody fragment composed of the VH and the VL domains from a single arm of the antibody. Fv fragments lack the constant regions of Fab (CH1 and CL) regions. The VH and VL in Fv fragments are held together by non-covalent interactions.

“Framework region” or “FR” residues are those VH or VL residues other than the CDRs as herein defined.

“Full length antibody” is comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain comprised of subdomains CH1, hinge, CH2 and CH3. Each light chain is comprised of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and the VL may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

“Genetic modification” refers to the introduction of a “foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences operably linked to polynucleotide encoding the chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “genetically engineered.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

“Heterologous” refers to two or more polynucleotides or two or more polypeptides that are not found in the same relationship to each other in nature.

“Heterologous polynucleotide” refers to a non-naturally occurring polynucleotide that encodes two or more neoantigens as described herein.

“Heterologous polypeptide” refers to a non-naturally occurring polypeptide comprising two or more neoantigen polypeptides as described herein.

“Host cell” refers to any cell that contains a heterologous nucleic acid. An exemplary heterologous nucleic acid is a vector (e.g., an expression vector).

“Human antibody” refers to an antibody that is optimized to have minimal immune response when administered to a human subject. Variable regions of human antibody are derived from human immunoglobulin sequences. If human antibody contains a constant region or a portion of the constant region, the constant region is also derived from human immunoglobulin sequences. Human antibody comprises heavy and light chain variable regions that are “derived from” sequences of human origin if the variable regions of the human antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice or rats carrying human immunoglobulin loci. “Human antibody” typically contains amino acid differences when compared to the immunoglobulins expressed in humans due to differences between the systems used to obtain the human antibody and human immunoglobulin loci, introduction of somatic mutations or intentional introduction of substitutions into the frameworks or CDRs, or both. Typically, “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical in amino acid sequence to an amino acid sequence encoded by human germline immunoglobulin or rearranged immunoglobulin genes. In some cases, “human antibody” may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al., (2000) J Mol Biol 296:57-86, or a synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et al., (2010) J Mol Biol 397:385-96, and in Int. Patent Publ. No. WO2009/085462. Antibodies in which at least one CDR is derived from a non-human species are not included in the definition of “human antibody”.

“Humanized antibody” refers to an antibody in which at least one CDR is derived from non-human species and at least one framework is derived from human immunoglobulin sequences. Humanized antibody may include substitutions in the frameworks so that the frameworks may not be exact copies of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

“In combination with” means that two or more therapeutic agents are to be administered to a subject together in a mixture, concurrently as single agents or sequentially as single agents in any order.

“Isolated” refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step.

“Isolated” refers to a molecule that is substantially free of other cellular material and/or chemicals and encompasses molecules that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity. An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessment of antibody purity, see, e.g., Flatman et al, J. Chromatogr. B 848:79-87 (2007).

A “linker” as used herein refers to a chemical linker or a single chain peptide linker that covalently connects two different entities. A linker can be used to connect any two of an antibody or a fragment thereof, a fusion protein and a conjugate of the present invention. The linker can connect, for example, the VH and VL in scFv, or the monoclonal antibody or antigen-binding fragment thereof with a therapeutic molecule, such as a second antibody. Single chain peptide linkers, comprised of from 1 to 25 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids, joined by peptide bonds, can be used. In certain embodiments, the amino acids are selected from the twenty naturally occurring amino acids. In certain other embodiments, one or more of the amino acids are selected from glycine, alanine, proline, asparagine, glutamine and lysine. Chemical linkers, such as a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a hybrid linker consisting of PEG and an embedded heterocycle, and a hydrocarbon chain can also be used.

“Modulate” refers to either enhanced or decreased ability of a test molecule to mediate an enhanced or a reduced response (i.e., downstream effect) when compared to the response mediated by a control or a vehicle.

“Monoclonal antibody” refers to an antibody obtained from a substantially homogenous population of antibody molecules, i.e., the individual antibodies comprising the population are identical except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain or post-translational modifications such as amino acid isomerization or deamidation, methionine oxidation or asparagine or glutamine deamidation. A bispecific monoclonal antibody binds two distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibodies may be monospecific or multispecific such as bispecific, monovalent, bivalent or multivalent.

A “multispecific antigen-binding construct” or “multispecific molecules” refers to a construct that specifically binds more than one distinct antigens or more than one distinct epitopes within the same antigen. A multispecific antigen-binding construct can be a protein, a protein complex, or an antibody. It comprises an antibody, or an antigen-binding fragment thereof, which is associated with or linked to at least one other functional molecule (e.g. another peptide or protein such as another antibody or ligand for a receptor) thereby forming a molecule that binds to at least two different binding sites or target molecules. Multispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens. Exemplary multispecific molecules include tr-specific or bi-specific antibodies and antibodies linked to soluble receptor fragments or ligands.

“Natural killer cell” and “NK cell” are used interchangeably and synonymously herein. NK cell refers to a differentiated lymphocyte with a CD16+ CD56+ and/or CD57⁺ TCR⁻ phenotype. NK cells are characterized by their ability to bind to and kill cells that fail to express “self” MHC/HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

“Operatively linked” and similar phrases, when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequences, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5′ and 3′ UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA) and in some instances to the production of a polypeptide (i.e., expression of the open reading frame). Operatively linked peptide refers to a peptide in which the functional domains of the peptide are placed with appropriate distance from each other to impart the intended function of each domain.

The term “paratope” refers to the area or region of an antibody molecule which is involved in binding of an antigen and comprise residues that interact with an antigen. A paratope may composed of continuous and/or discontinuous amino acids that form a conformational spatial unit. The paratope for a given antibody can be defined and characterized at different levels of details using a variety of experimental and computational methods. The experimental methods include hydrogen/deuterium exchange mass spectrometry (HX-MS). The paratope will be defined differently depending on the mapping method employed. A paratope can comprise amino acid residues directly involved in epitope binding, several of which are typically in CDRs, and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically bound antigen (in other words, the amino acid residue is within the “solvent-excluded surface” and/or “footprint” of the specifically bound antigen).

“Pharmaceutical combination” refers to a combination of two or more active ingredients administered either together or separately.

“Pharmaceutical composition” refers to a composition that results from combining an active ingredient and a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject. Exemplary pharmaceutically acceptable carriers are a buffer, stabilizer or preservative.

“Polynucleotide” or “nucleic acid” refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide. Polynucleotide may be a DNA or a RNA molecule.

“Prevent,” “preventing,” “prevention,” or “prophylaxis” of a disease or disorder means preventing that a disorder occurs in a subject.

“Proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells.

“Promoter” refers to the minimal sequences required to initiate transcription. Promoter may also include enhancers or repressor elements which enhance or suppress transcription, respectively.

“Protein” or “polypeptide” are used interchangeably herein and refers to a molecule that comprises one or more polypeptides each comprised of at least two amino acid residues linked by a peptide bond. Protein may be a monomer, or may be protein complex of two or more subunits, the subunits being identical or distinct. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Protein may be a heterologous fusion protein, a glycoprotein, or a protein modified by post-translational modifications such as phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation, ADP-ribosylation, pegylation or biotinylation. Protein may be recombinantly expressed.

“Recombinant” refers to polynucleotides, polypeptides, vectors, viruses and other macromolecules that are prepared, expressed, created or isolated by recombinant means.

“Regulatory element” refers to any cis- or trans acting genetic element that controls some aspect of the expression of nucleic acid sequences.

“Relapsed” refers to the return of a disease or the signs and symptoms of a disease after a period of improvement after prior treatment with a therapeutic.

“Refractory” refers to a disease that does not respond to a treatment. A refractory disease can be resistant to a treatment before or at the beginning of the treatment, or a refractory disease can become resistant during a treatment.

The phrases “sequence identity” or “percent (%) sequence identity” or “% identity” or “% identical to” when used with reference to an amino acid sequence describe the number of matches (“hits”) of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences. In other terms, using an alignment, for two or more sequences the percentage of amino acid residues that are the same (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity over the full-length of the amino acid sequences) may be determined, when the sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected. The sequences which are compared to determine sequence identity may thus differ by substitution(s), addition(s) or deletion(s) of amino acids. Suitable programs for aligning protein sequences are known to the skilled person. The percentage sequence identity of protein sequences can, for example, be determined with programs such as CLUSTALW, Clustal Omega, FASTA or BLAST, e.g. using the NCBI BLAST algorithm (Altschul S F, et al (1997), Nucleic Acids Res. 25:3389-3402).

“Single chain Fv” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and the VH are contiguously linked via a polypeptide linker, and capable of being expressed as a single chain polypeptide. Unless specified, as used herein, a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

“(scFv)₂” or “tandem scFv” or “bis-scFv” fragments refers to a fusion protein comprising two light chain variable regions (VL) and two heavy chain variable regions (VH), wherein the two VL and the two VH regions are contiguously linked via polypeptide linkers, and capable of being expressed as a single chain polypeptide. The two VL and two VH regions fused by peptide linkers form a bivalent molecule VL_A-linker-VH_A-linker-VL_B-linker-VH_Bto form two binding sites, capable of binding two different antigens or epitopes concurrently.

“Specifically binds,” “specific binding,” “specifically binding” or “binds” refer to a proteinaceous molecule binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the proteinaceous molecule binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K_D) of about 1×10⁻⁷M or less, for example about 5×10⁻⁸M or less, about 1×10⁻⁸M or less, about 1×10⁻⁹M or less, about 1×10⁻¹⁰M or less, about 1×10⁻¹¹M or less, or about 1×10⁻¹²M or less, typically with the K_Dthat is at least one hundred fold less than its K_Dfor binding to a non-specific antigen (e.g., BSA, casein).

“Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. The terms “subject” and “patient” can be used interchangeably herein.

“T cell” and “T lymphocyte” are interchangeable and used synonymously herein. T cell includes thymocytes, naïve T lymphocytes, memory T cells, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T cell can be a T helper (Th) cell, for example a T helper 1 (Th1) or a T helper 2 (Th2) cell. The T cell can be a helper T cell (HTL; CD⁴⁺ T cell) CD⁴⁺ T cell, a cytotoxic T cell (CTL; CD8⁺ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8⁺ T cell), CD⁴⁺ CD8⁺ T cell, or any other subset of T cells. Also included are “NKT cells”, which refer to a specialized population of T cells that express a semi-invariant αβ T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK1.1⁺ and NK1.1⁻, as well as CD⁴⁺, CD4⁻, CD8⁺ and CD8⁻ cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Also included are “gamma-delta T cells (γδ T cells),” which refer to a specialized population that to a small subset of T cells possessing a distinct TCR on their surface, and unlike the majority of T cells in which the TCR is composed of two glycoprotein chains designated α- and β-TCR chains, the TCR in γδ T cells is made up of a γ-chain and a δ-chain. γδ T cells can play a role in immunosurveillance and immunoregulation and were found to be an important source of IL-17 and to induce robust CD8⁺ cytotoxic T cell response. Also included are “regulatory T cells” or “Tregs” which refer to T cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs are typically transcription factor Foxp3-positive CD⁴⁺ T cells and can also include transcription factor Foxp3-negative regulatory T cells that are IL-10-producing CD⁴⁺ T cells.

“Therapeutically effective amount” or “effective amount” used interchangeably herein, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Example indicators of an effective therapeutic or combination of therapeutics that include, for example, improved well-being of the patient, reduction of a tumor burden, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.

“Transduction” refers to the introduction of a foreign nucleic acid into a cell using a viral vector.

“Treat,” “treating” or “treatment” of a disease or disorder such as cancer refers to accomplishing one or more of the following: reducing the severity and/or duration of the disorder, inhibiting worsening of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in subjects that have previously had the disorder, or limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder.

“Tumor cell” or a “cancer cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. Although transformation may arise from infection with a transforming virus and incorporation of new genomic nucleic acid, uptake of exogenous nucleic acid or it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene.

Transformation/cancer is exemplified by morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, modulation of tumor specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo.

“Variant,” “mutant” or “altered” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, for example one or more substitutions, insertions or deletions.

The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

“VHH” refers to a single-domain antibody or nanobody, exclusively composed of the antigen binding region of a heavy chain. A VHH single domain antibody lacks the light chain and the CH1 domain of the heavy chain of conventional Fab region.

Compositions of Matter

Antigen Binding Regions that Bind DLL3

The disclosure provides antigen binding regions that bind DLL3, monospecific and multispecific antigen-binding constructs comprising the antigen binding regions that bind DLL3, polynucleotides encoding the foregoing, vectors, host cells and methods of making and using the foregoing. The antigen binding regions that bind DLL3 identified herein demonstrated improved properties in terms of improved thermostability. The multispecific antigen-binding constructs disclosed herein can be particularly effective at mediating T cell mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy.

The disclosure provides an isolated protein comprising an antigen binding region that binds delta-like protein 3 (DLL3), wherein the antigen binding region that binds DLL3 binds to an epitope within the EGF-6+C-terminal domain of DLL3 set forth in SEQ ID NO:263 (residues 429-618 of DLL3). As shown in the examples, multispecific antigen-binding constructs targeting an epitope within the EGF-6 domain or closer to the C-terminus of DLL3 achieved potent levels of anti-tumor cytotoxicity.

Any method known in the art can be used to identify the region within DLL3 an antibody of the application binds in view of the present disclosure. For example, an ELISA assay can be used to identify the domain(s) within DLL3 to which an antibody binds. In a domain mapping ELISA assay, anti-DLL3 antibodies were evaluated for binding to recombinant DLL3 domain antigens spanning the N-terminal DSL fusion domain (DL3W44, SEQ ID NO: 189), EGF-1+2 fusion (DL3W42, SEQ ID NO:187), EGF-2 (DL3W41, SEQ ID NO:186), EGF-3 (DL3W40, SEQ ID NO:185), EGF-4 (DL3W39, SEQ ID NO:184), EGF-5 (DL3W38, SEQ ID NO:183), EGF-6 (DL3W37, SEQ ID NO: 182) and EGF-6+C-terminal domain fusion (DL3W36, SEQ ID NO:181). MesoScale Discovery high bind plates were coated overnight at 4° C. with 20 nM antigen. The plates were washed with PBS with 0.1% Tween and then blocked with Starting block solution for 30 minutes. The antibodies were added and incubated for 60 minutes at ambient temperature and then excess antibodies were removed by washing 3 times with PBS (Gibco, #14190-136). Antigen bound antibody was detected with sulfo-tagged anti-human antibody (Meso Scale Discovery, R32AJ) for 60 minutes at ambient temperature followed by another PBS wash. Signal acquisition was done in the presence of 1× MSD read buffer T (MSD, Cat #R92TC-1) on the MSD Sector 600 imager with appropriate plate settings. Data was analyzed for the highest binding signal per domain indicating the preferential domain binding.

An H/D exchange assay can be used to determine the residues within DLL3 to which an antibody binds. In an H/D exchange assay, recombinantly expressed soluble DLL3 is incubated in the presence or absence of the antibody in deuterated water for predetermined times resulting in deuterium incorporation at exchangeable hydrogen atoms which are unprotected by the antibody, followed by protease digestion of the protein and analyses of the peptide fragments using LC-MS. H/D exchange assay can be performed using known protocols. In some embodiments, the H/D exchange mixture is quenched by the addition of a quenching buffer (e.g., 8 M urea, 1M TCEP, pH 3.0) before being passed over an equilibrated immobilized pepsin/FPXIII column at room temperature (e.g., 600 μL/min). The peptic fragments are then loaded onto a reverse phase trap column (e.g., at 600 μL/min) and desalted (e.g., for 1 min at 600 μL), separated (e.g., on a C18 column) and analyzed by mass spectrometry (e.g., using an LTQ™ Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) with the capillary temperature at 275° C., resolution 150,000, and mass range (m/z) 300-1,800).

In some embodiments, the application provides an isolated protein, such as an antibody, comprising an antigen binding region, wherein the antigen binding region that binds DLL3 competes for binding to DLL3 with a reference antibody disclosed herein. In some embodiments, the reference antibody comprises a VH having a HCDR1, a HCDR2 and a HCDR3, and a VL having a LCDR1, a LCDR2 and a LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are:

- a. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:1 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:2;
- b. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:4;
- c. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:5 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:6;
- d. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:7 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:8;
- e. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:9 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO: 10;
- f. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:11 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO: 12; or
- g. the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:13 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO: 14.

In certain such embodiments, the reference antibody comprises a HCDR1, a HCDR2 and a HCDR3 of a VH of SEQ ID NO:3 and a LCDR1, a LCDR2 and a LCDR3 of a VL of SEQ ID NO:4.

Competition for binding of a test antibody that binds to SEQ ID NO:263 of soluble DLL3 with a reference antibody of the application can be assayed in vitro using well known methods in view of the present disclosure. For example, binding of labeled antibody to DLL3, e.g., the membrane proximal region of DLL3, in the presence of an unlabeled reference antibody can be assessed by ELISA. Bioacore analyses or flow cytometry can be used to demonstrate competition. The test antibody competes for binding to DLL3 with the reference antibody when the test antibody inhibits binding of the reference antibody to soluble DLL3 by 85% or more, for example 90% or more, or 95% or more.

In some embodiments, the application provides an isolated protein, such as an antibody, comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises a VH having a HCDR1, a HCDR2 and a HCDR3, and a VL having a LCDR1, a LCDR2 and a LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:1 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:2; or the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:4; or the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:5 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:6; or the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:7 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:8; or the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:9 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:10; or the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:11 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:12; or the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:13 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:14. In a particular embodiment, the isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:4.

- SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively;
- SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively;
- SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively;
- SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively;
- SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively;
- SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively;
- SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively;
- SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively;
- SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively;
- SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively;
- SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively;
- SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; or
- SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively.

In one embodiment, the disclosure provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 having the amino acid sequences of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively.

In another embodiment, the disclosure provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises a VH having the amino acid sequence of SEQ ID NOs:1, 3, 5, 7, 9, 11, or 13 and a VL having the amino acid sequence of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 14.

In one embodiment, the disclosure provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises: a VH of the amino acid sequence of SEQ ID NO:1 and a VL of the amino acid sequence of SEQ ID NO:2 (also referred to as a VH of SEQ ID NO:1 and a VL of SEQ ID NO:2);

- a VH of SEQ ID NO:1 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:1 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:1 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO:1 and a VL of SEQ ID NO:10;
- a VH of SEQ ID NO:1 and a VL of SEQ ID NO: 12;
- a VH of SEQ ID NO:1 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO: 12;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO:12;
- a VH of SEQ ID NO:3 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO: 12;
- a VH of SEQ ID NO:5 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO: 12;
- a VH of SEQ ID NO:7 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO:12;
- a VH of SEQ ID NO:9 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO:11 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:11 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO:11 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:11 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO:11 and a VL of SEQ ID NO:12;
- a VH of SEQ ID NO: 11 and a VL of SEQ ID NO: 14;
- a VH of SEQ ID NO: 13 and a VL of SEQ ID NO:2;
- a VH of SEQ ID NO:13 and a VL of SEQ ID NO:4;
- a VH of SEQ ID NO: 13 and a VL of SEQ ID NO:6;
- a VH of SEQ ID NO:13 and a VL of SEQ ID NO:8;
- a VH of SEQ ID NO: 13 and a VL of SEQ ID NO: 10;
- a VH of SEQ ID NO: 13 and a VL of SEQ ID NO: 12; or
- a VH of SEQ ID NO:13 and a VL of SEQ ID NO:14.

In a particular embodiment, the disclosure provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4, or a derivative thereof.

The disclosure also provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 95% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 95% identical to the VH of SEQ ID NO:3 and a VL which is at least 99% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 99% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

The disclosure provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NOs:63, 64, 65, 66, 67, 68, or 69.

The disclosure also provides an isolated protein comprising an antigen binding region that binds DLL3, wherein the antigen binding region that binds DLL3 comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of SEQ ID NO:63.

In some embodiments, the antigen binding region that binds DLL3 is a scFv.

In some embodiments, the antigen binding region that binds DLL3 is a (scFv)₂.

In some embodiments, the antigen binding region that binds DLL3 is a Fv.

In some embodiments, the antigen binding region that binds DLL3 is a Fab.

In some embodiments, the antigen binding region that binds DLL3 is a F(ab′)₂.

In some embodiments, the antigen binding region that binds DLL3 is a Fd.

In some embodiments, the antigen binding region that binds DLL3 is a dAb.

In some embodiments, the antigen binding region that binds DLL3 is a VHH.

In a particular embodiment, the antigen binding region that binds DLL3 is a scFv.

DLL3 Binding scFvs

Any of the VH and the VL or components thereof identified herein that bind DLL3 can be engineered into scFv format in either VH-linker-VL or VL-linker-VH orientation. Any of the VH and the VL identified herein can also be used to generate sc(Fv)₂structures, such as VH-linker-VL-linker-VL-linker-VH, VH-linker-VL-linker-VH-linker-VL, VH-linker-VH-linker-VL-linker-VL, VL-linker-VH-linker-VH-linker-VL, VL-linker-VH-linker-VL-linker-VH or VL-linker-VL-linker-VH-linker-VH.

The VH and the VL or components thereof identified herein can be incorporated into a scFv format and the binding and thermostability of the resulting scFv to DLL3 can be assessed using known methods in view of the present disclosure. Binding can be assessed using ProteOn XPR36, Biacore 3000 or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art. Binding can be evaluated using purified scFvs or Ecoli supernatants or lysed cells containing the expressed scFv. The measured affinity of a test scFv to DLL3 can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other binding parameters (e.g., K_D, K_on, K_off) are typically made with standardized conditions and standardized buffers. Thermostability may be evaluated by heating the test scFv at elevated temperatures, such as at 50° C., 55° C. or 60° C. for a period of time, such as 5 minutes (min), 10 min, 15 min, 20 min, 25 min or 30 min and measuring binding of the test scFv to DLL3. The scFvs retaining comparable binding to DLL3 when compared to a non-heated scFv sample are referred to as being thermostable.

In recombinant expression systems, the linker is a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and The. The linker should have a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to DLL3.

The linker can be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10-20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker is 6 amino acids long. In some embodiments, the linker is 7 amino acids long. In some embodiments, the linker is 8 amino acids long. In some embodiments, the linker is 9 amino acids long. In some embodiments, the linker is 10 amino acids long. In some embodiments, the linker is 11 amino acids long. In some embodiments, the linker is 12 amino acids long. In some embodiments, the linker is 13 amino acids long. In some embodiments, the linker is 14 amino acids long. In some embodiments, the linker is 15 amino acids long. In some embodiments, the linker is 16 amino acids long. In some embodiments, the linker is 17 amino acids long. In some embodiments, the linker is 18 amino acids long. In some embodiments, the linker is 19 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is 21 amino acids long. In some embodiments, the linker is 22 amino acids long. In some embodiments, the linker is 23 amino acids long. In some embodiments, the linker is 24 amino acids long. In some embodiments, the linker is 25 amino acids long. In some embodiments, the linker is 26 amino acids long. In some embodiments, the linker is 27 amino acids long. In some embodiments, the linker is 28 amino acids long. In some embodiments, the linker is 29 amino acids long. In some embodiments, the linker is 30 amino acids long. In some embodiments, the linker is 31 amino acids long. In some embodiments, the linker is 32 amino acids long. In some embodiments, the linker is 33 amino acids long. In some embodiments, the linker is 34 amino acids long. In some embodiments, the linker is 35 amino acids long. In some embodiments, the linker is 36 amino acids long. In some embodiments, the linker is 37 amino acids long. In some embodiments, the linker is 38 amino acids long. In some embodiments, the linker is 39 amino acids long. In some embodiments, the linker is 40 amino acids long. Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers.

Other linker sequences can include portions of immunoglobulin hinge area, CL or CH1 derived from any immunoglobulin heavy or light chain isotype. Alternatively, a variety of non-proteinaceous polymers, including polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers. Exemplary linkers that may be used are shown in Table 2. Additional linkers are described for example in Int. Pat. Publ. No. WO2019/060695.

In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (L1) and a VL (VH-L1-VL).

In some embodiments, the scFv comprises, from the N-to C-terminus, the VL, the L1 and the VH (VL-L1-VH). In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NO:120, SEQ ID NO:27, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO:138, or SEQ ID NO: 139.

TABLE 2

The amino acid sequences of linkers.

SEQ ID

Linker name
Amino acid sequence
NO:

Linker 1
GGSEGKSSGSGSESKSTGGS
120

Linker 2
GGGSGGGS
27

Linker 3
GGGSGGGSGGGS
72

Linker 4
GGGSGGGSGGGSGGGS
73

Linker 5
GGGSGGGSGGGSGGGSGGGS
74

Linker 6
GGGGSGGGGSGGGGS
75

Linker 7
GGGGSGGGGSGGGGSGGGGS
76

Linker 8
GGGGSGGGGSGGGGSGGGGSGGGGS
79

Linker 9
GSTSGSGKPGSGEGSTKG
81

Linker 10
IRPRAIGGSKPRVA
82

Linker 11
GKGGSGKGGSGKGGS
83

Linker 12
GGKGSGGKGSGGKGS
88

Linker 13
GGGKSGGGKSGGGKS
90

Linker 14
GKGKSGKGKSGKGKS
91

Linker 15
GGGKSGGKGSGKGGS
92

Linker 16
GKPGSGKPGSGKPGS
121

Linker 17
GKPGSGKPGSGKPGSGKPGS
122

Linker 18
GKGKSGKGKSGKGKSGKGKS
123

Linker 19
STAGDTHLGGEDFD
124

Linker 20
GEGGSGEGGSGEGGS
125

Linker 21
GGEGSGGEGSGGEGS
126

Linker 22
GEGESGEGESGEGES
127

Linker 23
GGGESGGEGSGEGGS
128

Linker 24
GEGESGEGESGEGESGEGES
129

Linker 25
GSTSGSGKPGSGEGSTKG
130

Linker 26
PRGASKSGSASQTGSAPGS
131

Linker 27
GTAAAGAGAAGGAAAGAAG
132

Linker 28
GTSGSSGSGSGGSGSGGGG
133

Linker 29
GKPGSGKPGSGKPGSGKPGS
134

Linker 30
GSGS
135

Linker 31
APAPAPAPAP
136

Linker 32
APAPAPAPAPAPAPAPAPAP
137

Linker 33
AEAAAKEAAAKEAAAAKEAAAAKEAA
138

AAKAAA

Linker 34
GTEGKSSGSGSESKST
139

In a particular embodiment, the L1 comprises or consists of the amino acid sequence of SEQ ID NO:120.

In some embodiments, the scFv comprises a heavy chain complementarity determining region (HCDR)1, a HCDR2 and a HCDR3 of a heavy chain variable region (VH) of SEQ ID NO:1 and a light chain complementarity determining region (LCDR)1, a LCDR2 and a LCDR3 of a light chain variable region (VL) of SEQ ID NO:2; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:4; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:5 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:6; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:7 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:8; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:9 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO: 10; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO: 11 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:12; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:13 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO: 14. In a particular embodiment, the scFv comprises the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH having a HCDR1, a HCDR2 and a HCDR3, and a VL having a LCDR1, a LCDR2 and a LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprises the amino acid sequences of:

- SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively;
- SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively;
- SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively;
- SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively;
- SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively;
- SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively;
- SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively;
- SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively;
- SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively;
- SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively;
- SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively;
- SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; or
- SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively.

In a particular embodiment, the scFv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:14.

In a particular embodiment, the scFv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:1 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:2.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:5 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:6.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:7 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:8.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:9 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:10.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO: 11 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:12.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO: 13 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:14.

In some embodiments, the scFv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL of SEQ ID NO: 4.

In some embodiments, the scFv comprises a VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH which is at least 95% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 99% identical to the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises a VH which is at least 95% identical to the VH of SEQ ID NO:3 and a VL which is at least 99% identical to the VL of SEQ ID NO:4.

In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NOs:63, 64, 65, 66, 67, 68, or 69.

In some embodiments, the scFv comprises an amino acid sequence which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of SEQ ID NOs:63, 64, 65, 66, 67, 68, or 69.

In a particular embodiment, the scFv comprises the amino acid sequence of SEQ ID NO:63.

In a particular embodiment, the scFv comprises the amino acid sequence of SEQ ID NO:64.

Other Antigen Binding Regions that Bind DLL3

Any of the VH and the VL or components thereof identified herein that bind DLL3 can also be engineered into Fab, F(ab′)₂, Fd or Fv format and their binding to DLL3 and thermostability can be assessed using the assays described herein.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises a VH having a HCDR1, a HCDR2 and a HCDR3, and a VL having a LCDR1, a LCDR2 and a LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 comprises: the HCDR1, the HCDR2 and the HCDR3 of a VH of SEQ ID NO:1 and the LCDR1, the LCDR2 and the LCDR3 of a VL of SEQ ID NO:2; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:4; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:5 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:6; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:7 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:8; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:9 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO: 10; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO: 11 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:12; or the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO: 13 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:14.

In a particular embodiment, a Fab, F(ab′)₂, Fd or Fv comprises the HCDR1, the HCDR2 and the HCDR3 of the VH of SEQ ID NO:3 and the LCDR1, the LCDR2 and the LCDR3 of the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of

- SEQ ID NOs:15, 16, 17, 33, 34, and 35, respectively;
- SEQ ID NOs:18, 19, 20, 36, 37, and 38, respectively;
- SEQ ID NOs:21, 22, 23, 39, 37, and 40, respectively;
- SEQ ID NOs:24, 25, 26, 41, 42, and 43, respectively;
- SEQ ID NOs:18, 28, 29, 44, 45, and 46, respectively;
- SEQ ID NOs:30, 31, 32, 47, 48, and 49, respectively;
- SEQ ID NOs:50, 51, 17, 33, 34, and 35, respectively;
- SEQ ID NOs:52, 51, 17, 33, 34, and 35, respectively;
- SEQ ID NOs:53, 54, 20, 36, 37, and 38, respectively;
- SEQ ID NOs:55, 56, 23, 39, 37, and 40, respectively;
- SEQ ID NOs:57, 58, 26, 41, 42, and 43, respectively;
- SEQ ID NOs:59, 60, 29, 44, 45, and 46, respectively; or
- SEQ ID NOs:61, 62, 32, 47, 48, and 49, respectively.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:1 and the VL of SEQ ID NO:14.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:14.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:5 and the VL of SEQ ID NO:14.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:7 and the VL of SEQ ID NO:14.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:9 and the VL of SEQ ID NO:14.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:11 and the VL of SEQ ID NO:14.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:2.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:4.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:6.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:8.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:10.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:12.

In some embodiments, a Fab, F(ab′)₂, Fd or Fv comprises the VH of SEQ ID NO:13 and the VL of SEQ ID NO:14.

In a particular embodiment, a Fab, F(ab′)₂, Fd or Fv comprises a VH of SEQ ID NO:1 and a VL of SEQ ID NO:2.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:1 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:2.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH of SEQ ID NO:1 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:2.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 95% identical to the VH of SEQ ID NO:1 and a VL which is at least 95% identical to the VL of SEQ ID NO:2.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:1 and a VL which is at least 95% identical to the VL of SEQ ID NO:2.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:1 and a VL which is at least 99% identical to the VL of SEQ ID NO:2.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:1 and a VL which is at least 95% identical to the VL of SEQ ID NO:2.

In a particular embodiment, the Fab, F(ab′)₂, Fd or Fv comprises a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 95% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 99% identical to the VL of SEQ ID NO:4.

In some embodiments, the Fab, F(ab′)₂, Fd or Fv comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

The VH and VL of the Fab comprising the antigen binding region that binds DLL3 can be engineered into Fab-Fc HC (VH-CH1-hinge-CH2-CH3) and Fab-Fc LC (VL-CL) formats respectively. In certain such embodiments, the Fab-Fc HC comprises an amino acid sequence which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to SEQ ID NO: 109. In a particular embodiment, the Fab-Fc HC comprises an amino acid sequence which is identical to SEQ ID NO:109.

In some embodiments, the Fab-Fc LC comprises an amino acid sequence which is at least 80% (e.g. at least 85%, at least 90%, at least 95% or at least 99%) identical to SEQ ID NO: 110.

In a particular embodiment, the Fab-Fc LC comprises an amino acid sequence which is identical to SEQ ID NO:110.

As shown in the examples, a particularly suitable antigen binding region that binds DLL3 for incorporating into a multispecific construct comprises a Fab-Fc HC having the amino acid sequence of SEQ ID NO: 109 and a Fab-Fc LC having the amino acid sequence of SEQ ID NO:110.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:63.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:64.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:65.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:66.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:67.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:68.

In some embodiments, the F(ab′)₂comprises the amino acid sequence of SEQ ID NO:69.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:63.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:64.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:65.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:66.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:67.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:68.

In some embodiments, the Fv comprises the amino acid sequence of SEQ ID NO:69.

Homologous Antigen Binding Regions and Antigen Binding Regions with Conservative Substitutions

Variants of the antigen binding regions that bind DLL3 are within the scope of the disclosure. For example, variants may comprise 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 or 29 amino acid substitutions in the antigen binding region that bind DLL3 as long as they retain or have improved functional properties when compared to the parent antigen binding regions. In some embodiments, the sequence identity may be about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the antigen binding regions that bind DLL3 of the disclosure. In some embodiments, the variation is in the framework regions. In some embodiments, variants are generated by conservative substitutions.

In some embodiments, an isolated protein comprising an antigen binding region that binds DLL3 comprises a VH and a VL which are at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH and VL, respectively, of an antigen binding region that binds DLL3 disclosed herein.

Also provided are antigen binding regions that bind DLL3 comprising the VH and the VL which are at least 80% identical to the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2;

- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:11 and the VL of SEQ ID NO: 12; or
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 14.

In some embodiments, the identity is 85%. In some embodiments, the identity is 90%. In some embodiments, the identity is 91%. In some embodiments, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. In some embodiments, the identity is 94%. In some embodiments, the identity is 94%. In some embodiments, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. In some embodiments, the identity is 98%. In some embodiments, the identity is 99%.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:1 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:2.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:5 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:6.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:7 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:8.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:9 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO: 10.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO: 11 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:12.

In some embodiments, the antigen binding regions that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO: 13 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:14.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 85% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 90% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 91% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 92% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 93% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 94% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 95% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 96% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 97% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 98% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH which is at least 99% identical to the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 85% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 90% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 91% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 92% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 93% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 94% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 95% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 96% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 97% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 98% identical to the VL of SEQ ID NO:4.

In some embodiments, the antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 99% identical to the VL of SEQ ID NO:4.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical positions/total number of positions ×100), 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 percent identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci 4:11-17 (1988)) 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. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch (J Mol Biol 48:444-453 (1970)) 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 some embodiments, variant antigen binding regions that bind DLL3 comprise one or two conservative substitutions in any of the CDR regions, while retaining desired functional properties of the parent antigen binding regions that bind DLL3.

“Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid modifications. Conservative modifications include amino acid substitutions, additions and deletions. Conservative amino acid substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis (MacLennan et al., (1988) Acta Physiol Scand Suppl 643:55-67; Sasaki et al., (1988) Adv Biophys 35:1-24). Amino acid substitutions to the antibodies of the application may be made by known methods for example by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting variants may be tested for their characteristics using assays described herein.

Methods of Generating Antigen Binding Region that Bind DLL3

Antigen binding regions that bind DLL3 provided in the disclosure may be generated using various technologies. For example, the hybridoma method of Kohler and Milstein may be used to identify VH/VL pairs that bind DLL3. In the hybridoma method, a mouse or other host animal, such as a hamster, rat or chicken is immunized with human and/or cyno DLL3, followed by fusion of spleen cells from immunized animals with myeloma cells using standard methods to form hybridoma cells. Colonies arising from single immortalized hybridoma cells may be screened for production of the antibodies containing the antigen binding regions that bind DLL3 with desired properties, such as specificity of binding, cross-reactivity or lack thereof, affinity for the antigen, and any desired functionality.

Antigen binding regions that bind DLL3 generated by immunizing non-human animals may be humanized. Exemplary humanization techniques including selection of human acceptor frameworks include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing (Padlan, (1991) Mol Immunol 28:489-499), Specificity Determining Residues Resurfacing (U.S. Patent Publ. No. 2010/0261620), human framework adaptation (U.S. Pat. No. 8,748,356) or superhumanization (U.S. Pat. No. 7,709,226). In these methods, CDRs or a subset of CDR residues of parental antibodies are transferred onto human frameworks that may be selected based on their overall homology to the parental frameworks, based on similarity in CDR length, or canonical structure identity, or a combination thereof.

Humanized antigen binding regions may be further optimized to improve their selectivity or affinity to a desired antigen by incorporating altered framework support residues to preserve binding affinity (backmutations) by techniques such as those described in Int. Patent Publ. Nos. WO1090/007861 and WO1992/22653, or by introducing variation at any of the CDRs for example to improve affinity of the antigen binding region.

Transgenic animals, such as mice, rat or chicken carrying human immunoglobulin (Ig) loci in their genome may be used to generate antigen binding regions that bind DLL3, and are described in for example U.S. Pat. No. 6,150,584, Int. Patent Publ. No. WO1999/45962, Int. Patent Publ. Nos. WO2002/066630, WO2002/43478, WO2002/043478 and WO1990/04036. The endogenous immunoglobulin loci in such animal may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the animal using homologous or non-homologous recombination, using transchromosomes, or using minigenes. Companies such as Regeneron (http://_www_regeneron_com), Harbour Antibodies (http://_www_harbourantibodies_com), Open Monoclonal Technology, Inc. (OMT) (http://_www_omtinc_net), KyMab (http://_www_kymab_com), Trianni (http://_www.trianni_com) and Ablexis (http://_www_ablexis_com) may be engaged to provide human antibodies directed against a selected antigen using technologies as described above. In some embodiments, Ablexis mice were immunized with soluble full length DLL3 protein.

Antigen binding regions that bind DLL3 may be selected from a phage display library, where the phage is engineered to express human immunoglobulins or portions thereof such as Fabs, single chain antibodies (scFv), or unpaired or paired antibody variable regions. The antigen binding regions that bind DLL3 may be isolated for example from phage display library expressing antibody heavy and light chain variable regions as fusion proteins with bacteriophage pIX coat protein as described in Shi et al., (2010) JMol Biol 397:385-96, and Int. Patent Publ. No. WO09/085462). The libraries may be screened for phage binding to human and/or cyno DLL3 and the obtained positive clones may be further characterized, the Fabs isolated from the clone lysates, and converted to scFvs or other configurations of antigen binding regions.

Preparation of immunogenic antigens and expression and production of antigen binding regions of the disclosure may be performed using any suitable technique, such as recombinant protein production. The immunogenic antigens may be administered to an animal in the form of purified protein, or protein mixtures including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the animal's body from nucleic acids encoding said antigen or a portion thereof.

Fusions or Conjugations to Half-Life Extending Moieties

The antigen binding regions that bind DLL3 of the disclosure can be fused or conjugated to a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, immunoglobulins (Ig) or fragments thereof, such as Fc regions. Amino acid sequences of the aforementioned half-life extending moieties are known. Ig or fragments thereof include all isotypes, i.e., IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE.

Additional half-life extending moieties that can be conjugated to the antigen binding regions that bind DLL3 of the disclosure include polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the antigen binding regions that bind DLL3 of the disclosure and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced antigen binding regions that bind DLL3 of the disclosure.

A pegyl moiety can for example be conjugated to the antigen binding region that bind DLL3 of the disclosure by incorporating a cysteine residue to the C-terminus of the antigen binding region that bind DLL3 of the disclosure, or engineering cysteines into residue positions that face away from the DLL3 binding site and attaching a pegyl group to the cysteine using well known methods.

In some embodiments, the antigen binding region that binds DLL3 is fused or conjugated to a half-life extending moiety.

In some embodiments, the half-life extending moiety is an immunoglobulin (Ig), a fragment of the Ig, an Ig constant region, a fragment of the Ig constant region, a Fc region, transferrin, albumin, an albumin binding domain or polyethylene glycol. In some embodiments, the half-life extending moiety is an Ig constant region.

In some embodiments, the half-life extending moiety is the Ig.

In some embodiments, the half-life extending moiety is the fragment of the Ig.

In some embodiments, the half-life extending moiety is the Ig constant region.

In some embodiments, the half-life extending moiety is the fragment of the Ig constant region.

In some embodiments, the half-life extending moiety is the Fc region.

In some embodiments, the half-life extending moiety is albumin.

In some embodiments, the half-life extending moiety is the albumin binding domain.

In some embodiments, the half-life extending moiety is transferrin.

In some embodiments, the half-life extending moiety is polyethylene glycol.

The antigen binding regions that bind DLL3 fused or conjugated to a half-life extending moiety can be evaluated for their pharmacokinetic properties utilizing known in vivo models in view of the present disclosure.

Fusion to Immunoglobulin (Ig) Constant Regions or Fragments of the Ig Constant Regions

The antigen binding regions that bind DLL3 of the disclosure can be fused to an Ig constant region or a fragment of the Ig constant region to impart antibody-like properties, including Fc effector functions C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis or down regulation of cell surface receptors (e.g., B cell receptor; BCR). The Ig constant region or the fragment of the Ig constant region functions also as a half-life extending moiety as discussed herein. The antigen binding regions that bind DLL3 of the disclosure may be engineered into conventional full-length antibodies using standard methods. The full-length antibodies comprising the antigen binding region that binds DLL3 may further be engineered as described herein.

Immunoglobulin heavy chain constant region comprised of subdomains CH1, hinge, CH2 and CH3. The CH1 domain spans residues A118-V215, the CH2 domain residues A231-K340 and the CH3 domain residues G341-K447 on the heavy chain, residue numbering according to the EU Index. In some instances, G341 is referred as a CH2 domain residue. Hinge is generally defined as including E216 and terminating at P230 of human IgG1. Ig Fc region comprises at least the CH2 and the CH3 domains of the Ig constant region, and therefore comprises at least a region from about A231 to K447 of Ig heavy chain constant region.

The application also provides an antigen binding region that binds DLL3 conjugated to an immunoglobulin (Ig) constant region or a fragment of the Ig constant region.

In some embodiments, the Ig constant region is a heavy chain constant region In some embodiments, the Ig constant region is a light chain constant region.

In some embodiments, the fragment of the Ig constant region comprises a Fc region.

In some embodiments, the fragment of the Ig constant region comprises a CH2 domain.

In some embodiments, the fragment of the Ig constant region comprises a CH3 domain.

In some embodiments, the fragment of the Ig constant region comprises the CH2 domain and the CH3 domain.

In some embodiments, the fragment of the Ig constant region comprises at least portion of a hinge, the CH2 domain and the CH3 domain. Portion of the hinge refers to one or more amino acid residues of the Ig hinge.

In some embodiments, the fragment of the Ig constant region comprises the hinge, the CH2 domain and the CH3 domain.

In a particular embodiment, the fragment of the Ig constant region comprises the hinge, the CH2 domain and the CH3 domain.

In some embodiments, the antigen binding region that binds DLL3 is conjugated to the N-terminus of the Ig constant region or the fragment of the Ig constant region.

In some embodiments, the antigen binding region that binds DLL3 is conjugated to the C-terminus of the Ig constant region or the fragment of the Ig constant region.

In some embodiments, the antigen binding region that binds DLL3 is conjugated to the Ig constant region or the fragment of the Ig constant region via a second linker (L2).

In some embodiments, the L2 comprises the amino acid sequence of SEQ ID NOs:27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139.

In a particular embodiment, the L2 comprises the amino acid sequence of SEQ ID NO:120.

The antigen binding regions that bind DLL3 of the disclosure conjugated to Ig constant region or the fragment of the Ig constant region may be assessed for their functionality using several known assays. Binding to DLL3 may be assessed using methods described herein. Altered properties imparted by the Ig constant domain or the fragment of the Ig constant region such as Fc region may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcγRI, FcγRII, FcγRIII or FcRn receptors, or using cell-based assays measuring for example ADCC, CDC or ADCP.

ADCC can be assessed using an in vitro assay using DLL3 expressing cells as target cells and NK cells as effector cells. Cytolysis may be detected by the release of label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells. In an exemplary assay, target cells are used with a ratio of 1 target cell to 4 effector cells. Target cells are pre-labeled with BATDA and combined with effector cells and the test antibody. The samples are incubated for 2 hours and cell lysis measured by measuring released BATDA into the supernatant. Data is normalized to maximal cytotoxicity with 0.67% Triton X-100 (Sigma Aldrich) and minimal control determined by spontaneous release of BATDA from target cells in the absence of any antibody.

ADCP can be evaluated by using monocyte-derived macrophages as effector cells and any DLL3 expressing cells as target cells which are engineered to express GFP or other labeled molecule. In an exemplary assay, effector:target cell ratio may be for example 4:1. Effector cells may be incubated with target cells for 4 hours with or without the antibody of the application. After incubation, cells may be detached using accutase. Macrophages can be identified with anti-CD11b and anti-CD14 antibodies coupled to a fluorescent label, and percent phagocytosis may be determined based on % GFP fluorescence in the CD11⁺ CD14⁺ macrophages using standard methods.

CDC of cells can be measured for example by plating Daudi cells at 1×10⁵cells/well (50 μL/well) in RPMI-B (RPMI supplemented with 1% BSA), adding 50 μL of test protein to the wells at final concentration between 0-100 μg/mL, incubating the reaction for 15 min at room temperature, adding 11 μL of pooled human serum to the wells, and incubation the reaction for 45 min at 37° C. Percentage (%) lysed cells may be detected as % propidium iodide stained cells in FACS assay using standard methods.

In some embodiments, the first antigen binding region that binds DLL3 is fused to a first immunoglobulin (Ig) constant region or a fragment of the first Ig constant region and/or the second antigen binding region that binds the lymphocyte antigen is fused to a second immunoglobulin (Ig) constant region or a fragment of the second Ig constant region.

In some embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises a Fc region.

In some embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises a CH2 domain.

In some embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises a CH3 domain.

In some embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises the CH2 domain and the CH3 domain.

In some embodiments, the fragment of the first Ig constant region and/or the fragment of the second Ig constant region comprises at least portion of a hinge, the CH2 domain and the CH3 domain.

In some embodiments, the fragment of the Ig constant region comprises the hinge, the CH2 domain and the CH3 domain.

In some embodiments, the multispecific antigen-binding construct further comprises a second linker (L2) between the first antigen binding region that binds DLL3 and the first Ig constant region or the fragment of the first Ig constant region and the second antigen binding region that binds the lymphocyte antigen and the second Ig constant region or the fragment of the second Ig constant region.

In a particular embodiment, the L2 comprises the amino acid sequence of SEQ ID NO:120.

In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgG1, an IgG2, and IgG3 or an IgG4 isotype.

In a particular embodiment, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region is an IgG1 isotype.

The first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region can further be engineered as described herein.

In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that results in reduced binding of the multispecific antigen-binding construct to a FcγR.

In some embodiments, the at least one mutation that results in reduced binding of the multispecific antigen-binding construct to the FcγR is selected from the group consisting of F234A/L235A, L234A/L235A, L234A/L235A/D265 S, V234A/G237A/P238 S/H268A/V309L/A330 S/P331 S, F234A/L235A, S228P/F234A/L235A, N297A, V234A/G237A, K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D365E/L358M, H268Q/V309L/A330 S/P331 S, S267E/L328F, L234F/L235E/D265A, L234A/L235A/G237A/P238 S/H268A/A330 S/P331 S, S228P/F234A/L235A/G237A/P238 S and S228P/F234A/L235A/G236-deleted/G237A/P238 S, wherein residue numbering is according to the EU index. In a particular embodiment, the first Ig constant region or the fragment of the first Ig constant region and/or the second Ig constant region or the fragment of the second Ig constant region comprise the following mutations: L234A_L235A_D265 S.

In some embodiments, the FcγR is FcγRI, FcγRIIA, FcγRIIB or FcγRIII, or any combination thereof.

In some embodiments, the first Ig constant region or the fragment of the first Ig constant region and the second Ig constant region or the fragment of the second Ig constant region comprises at least one mutation that modulates a half-life of the multispecific antigen-binding construct.

In some embodiments, the multispecific antigen-binding construct comprises at least one mutation in a CH3 domain of the first Ig constant region or in a CH3 domain of the fragment of the first Ig constant region and/or at least one mutation in a CH3 domain of the second Ig constant region or in a CH3 domain of the fragment of the second Ig constant region.

- L351Y_F405A_Y407V/T394W, T3661_K392M_T394W/F405A_Y407V,
- T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,
- L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or
- T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in US2012/0149876 or US2013/0195849 (Zymeworks).

In some embodiments, the at least one mutation in a CH3 domain of the first Ig constant region or in a CH3 domain of the fragment of the first Ig constant region and/or at least one mutation in a CH3 domain of the second Ig constant region or in a CH3 domain of the fragment of the second Ig constant region is selected from the group consisting of T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394 S/Y407A, T366W/T394 S, F405W/T394 S and T366W/T366 S_L368A_Y407V as described in WO1996/027011.

In some embodiment, a protein or multispecific antigen-binding construct of the application can comprise one or more amino acid modifications that reduces or eliminates the effector function, such as the ADCC or CDC, such as mutations that reduce or abolish the binding to Fc gamma receptor. Such mutations can be at positions L234, L235, D270, N297, E318, K320, K322, P331, and P329, such as one, two or three mutations of L234A, L235A and P331 S, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.

Proteins Comprising the Antigen Binding Regions that Bind DLL3 of the Disclosure

The antigen binding regions that bind DLL3 of the disclosure can be engineered into monospecific or multispecific antigen-binding constructs of various designs using standard methods.

The disclosure also provides a monospecific protein comprising the antigen binding region that binds DLL3 of the disclosure.

In some embodiments, the monospecific protein is an antibody.

The disclosure also provides a multispecific antigen-binding construct comprising the antigen binding region that binds DLL3 of the disclosure.

In some embodiments, the multispecific antigen-binding construct is bispecific.

In some embodiments, the multispecific antigen-binding construct is trispecific.

In some embodiments, the multispecific antigen-binding construct is tetraspecific.

In some embodiments, the multispecific antigen-binding construct is monovalent for binding to DLL3.

In some embodiments, the multispecific antigen-binding construct is bivalent for binding to DLL3.

The disclosure also provides an isolated multispecific antigen-binding construct comprising a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (such as CD3).

In some embodiments, the lymphocyte antigen is a T cell antigen.

In some embodiments, the T cell antigen is a CD8⁺ T cell antigen.

In some embodiments, the lymphocyte antigen is a NK cell antigen.

In some embodiments, the lymphocyte antigen is CD3, CD3 epsilon (CD3E), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C.

In some embodiments, the lymphocyte antigen is CD3ε.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise a scFv, a (scFv)₂, a Fv, a Fab, a F(ab′)₂, a Fd, a dAb or a VHH.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the Fab.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the F(ab′)₂.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the VHH.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the Fv.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the Fd.

In some embodiments, the first antigen binding region that binds DLL3 and/or the second antigen binding region that binds the lymphocyte antigen comprise the scFv.

In a particular embodiment, the multispecific antigen-binding construct is bispecific, wherein the first antigen binding region that binds DLL3 comprises a scFv and the second antigen binding region that binds the lymphocyte antigen (e.g., CD3) comprises a Fab.

In a particular embodiment, the multispecific antigen-binding construct is bispecific, wherein the first antigen binding region that binds DLL3 comprises a Fab and the second antigen binding region that binds the lymphocyte antigen (e.g., CD3) comprises a scFv.

In some embodiments, the scFv comprises, from the N- to C-terminus, a VH, a first linker (L1) and a VL (VH-L1-VL) or the VL, the L1 and the VH (VL-L1-VH).

In some embodiments, the L1 comprises about 5-50 amino acids.

In some embodiments, the L1 comprises about 5-40 amino acids.

In some embodiments, the L1 comprises about 10-30 amino acids.

In some embodiments, the L1 comprises about 10-20 amino acids.

In some embodiments, the L1 comprises the amino acid sequence of SEQ ID NOs: 27, 72, 73, 74, 75, 76, 79, 81, 82, 83, 88, 90, 91, 92, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, or 139.

In a particular embodiment, the L1 comprises the amino acid sequence of SEQ ID NO:120.

In some embodiments, the first antigen binding region that binds DLL3 comprises the HCDR1 of SEQ ID NOs:15, 18, 21, 24, 18, 30, 50, 52, 53, 55, 57, 59, or 61, a HCDR2 of SEQ ID NOs:16, 19, 22, 25, 28, 31, 51, 54, 56, 58, 60, or 62, a HCDR3 of SEQ ID NOs:17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32, a LCDR1 of SEQ ID NOs:33, 36, 39, 41, 44, or 47, a LCDR2 of SEQ ID NOs:34, 37, 42, 45, or 48, and a LCDR3 of SEQ ID NOs:35, 38, 40, 43, 46, or 49.

In a particular embodiment, the first antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34 and 35, respectively. In some embodiments, the multispecific antigen-binding construct mediates T cell mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy.

In some embodiments, the multispecific antigen-binding construct potently mediates the expansion of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69 and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct displays increased tumor killing.

In some embodiments, the first antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4, and the second antigen binding region that binds a lymphocyte antigen, optionally which is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

In some embodiments, the isolated multispecific antigen-binding construct mediates T cell mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy. In some embodiments, the multispecific antigen-binding construct potently mediates the expansion of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69 and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct displays increased tumor killing.

In some embodiments, the bispecific anti-DLL3×CD3 antibody achieves >90% (e.g., 95%) tumor lysis by 5 days in a T cell cytotoxicity assay.

In some embodiments, the first antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL of SEQ ID NO:4, and the second antigen binding region that binds a lymphocyte antigen, optionally which is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

In some embodiments, the first antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4, and the second antigen binding region that binds a lymphocyte antigen, optionally which is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein may be particularly effective at mediating T cell mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy.

In a particular embodiment, the first antigen binding region that binds DLL3 comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4.

In a particular embodiment, the first antigen binding region that binds DLL3 comprises the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4, and the second antigen binding region that binds a lymphocyte antigen, optionally which is CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, such as CD3.

In some embodiments, the multispecific antigen-binding construct mediates T cell mediated cytotoxicity. In some embodiments, the multispecific antigen-binding construct potently mediates the expansion of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69 and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct displays increased tumor killing. In some embodiments, the bispecific anti-DLL3×CD3 antibody achieves >90% (e.g., 95%) tumor lysis by 5 days in a T cell cytotoxicity assay.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NOs:63, 64, 65, 66, 67, 68, or 69.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:63.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:64.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:65.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:66.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:67.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:68.

In some embodiments, the first antigen binding region that binds DLL3 comprises the amino acid sequence of SEQ ID NO:69.

In some embodiments, the first antigen binding region that binds DLL3 comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of SEQ ID NO:63.

In a particular embodiment, the first antigen binding region that binds DLL3 comprises an amino acid sequence of SEQ ID NOs:63 or 64.

The disclosure also provides a second antigen binding region that binds lymphocyte antigen (such as CD3), wherein the antigen binding region that binds lymphocyte comprises the heavy chain variable region (VH) of SEQ ID NO:77 and the light chain variable region (VL) of SEQ ID NO:80 or the VH of SEQ ID NO:84 and the VL of SEQ ID NO:85.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen (such as CD3) comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:77 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:80.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:77 and a VL of SEQ ID NO:80.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen comprises a VH of SEQ ID NO:77 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:80.

In a particular embodiment, the second antigen binding region that binds a lymphocyte antigen comprises a VH of SEQ ID NO:77 and a VL of SEQ ID NO:80.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen (such as CD3) comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:84 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:85.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen comprises a VH of SEQ ID NO:84 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:85.

In a particular embodiment, the second antigen binding region that binds a lymphocyte antigen comprises a VH of SEQ ID NO:84 and a VL of SEQ ID NO:85.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen comprises:

- a HCDR1 of SEQ ID NO:95, a HCDR2 of SEQ ID NO:96, a HCDR3 of SEQ ID NO:97, a LCDR1 of SEQ ID NO:101, a LCDR2 of SEQ ID NO: 102 and a LCDR3 of SEQ ID NO:104; or the VH of SEQ ID NO:77 and the VL of SEQ ID NO:80.

In some embodiments, the second antigen binding region that binds a lymphocyte antigen comprises:

- a HCDR1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO:100, a LCDR1 of SEQ ID NO: 106, a LCDR2 of SEQ ID NO:107 and a LCDR3 of SEQ ID NO:108; or the VH of SEQ ID NO:84 and the VL of SEQ ID NO:85.

In a particular embodiment, the second antigen binding region that binds a lymphocyte antigen (such as CD3) comprises the HCDR1 of SEQ ID NO:95, the HCDR2 of SEQ ID NO:96, the HCDR3 of SEQ ID NO:97, the LCDR1 of SEQ ID NO:101, the LCDR2 of SEQ ID NO:102 and the LCDR3 of SEQ ID NO:104.

In a particular embodiment, the first antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, and 35, respectively and the second antigen binding region that binds a lymphocyte antigen (such as CD3) comprises a VH of SEQ ID NO:84 and a VL of SEQ ID NO:85. In some embodiments, the multispecific antigen-binding construct mediates T cell mediated cytotoxicity, promoting T cell activation, proliferation, and expansion, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy. In some embodiments, the multispecific antigen-binding construct potently mediates the expansion of cytotoxic CD8 T cells. In some embodiments, the multispecific antigen-binding construct upregulates CD25, CD69 and CD71 expression on the surface of CD8 T cells. In some embodiments, the multispecific antigen-binding construct displays increased tumor killing. In some embodiments, the bispecific anti-DLL3×CD3 antibody achieves >90% (e.g., 95%) tumor lysis by 5 days in a T cell cytotoxicity assay.

In a particular embodiment, the first antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL of SEQ ID NO:4 and the second antigen binding region that binds a lymphocyte antigen (such as CD3) comprises the HCDR1 of SEQ ID NO:95, the HCDR2 of SEQ ID NO:96, the HCDR3 of SEQ ID NO:97, the LCDR1 of SEQ ID NO:101, the LCDR2 of SEQ ID NO:102 and the LCDR3 of SEQ ID NO: 104.

Generation of Multispecific Antigen-Binding Constructs that Comprise Antigen Binding Regions that Bind DLL3

The antigen binding regions that bind DLL3 of the disclosure may be engineered into multispecific antibodies which are also encompassed within the scope of the application.

The antigen binding regions that bind DLL3 may be engineered into full length multispecific antibodies which are generated using Fab arm exchange, in which substitutions are introduced into two monospecific bivalent antibodies within the Ig constant region CH3 domain which promote Fab arm exchange in vitro. In the methods, two monospecific bivalent antibodies are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization; thereby generating the bispecific antibody by Fab arm exchange. The incubation conditions may optimally be restored to non-reducing. Exemplary reducing agents that may be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. For example, incubation for at least 90 min at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pH of 7.4 may be used.

CH3 mutations that may be used include technologies such as Knob-in-Hole mutations (Genentech), electrostatically-matched mutations (Chugai, Amgen, NovoNordisk, Oncomed), the Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Duobody® mutations (Genmab), and other asymmetric mutations (e.g., Zymeworks).

Knob-in-hole mutations are disclosed for example in WO1996/027011 and include mutations on the interface of CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in preferential interaction between the first CH3 region and the second CH3 region. Exemplary CH3 region mutations forming a knob and a hole are T366Y/F405A, T366W/F405W, F405W/Y407A, T394W/Y407T, T394 S/Y407A, T366W/T394 S, F405W/T394 S and T366W/T366 S_L368A_Y407V.

Heavy chain heterodimer formation may be promoted by using electrostatic interactions by substituting positively charged residues on the first CH3 region and negatively charged residues on the second CH3 region as described in US2010/0015133, US2009/0182127, US2010/028637 or US2011/0123532.

Other asymmetric mutations that can be used to promote heavy chain heterodimerization are L351Y_F405A_Y407V/T394W, T3661_K392M_T394W/F405A_Y407V,

- T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,
- L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or
- T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in US2012/0149876 or US2013/0195849 (Zymeworks).

SEEDbody mutations involve substituting select IgG residues with IgA residues to promote heavy chai heterodimerization as described in US20070287170.

Other exemplary mutations that may be used are R409D_K370E/D399K_E357K, S354C_T366W/Y349C_T366 S_L368A_Y407V, Y349C_T366W/S354C_T366 S_L368A_Y407V, T366K/L351D, L351K/Y349E, L351K/Y349D, L351K/L368E, L351Y_Y407A/T366A_K409F, L351Y_Y407A/T366V_K409F, K392D/D399K, K392D/E356K, K253E_D282K_K322D/D239K_E240K_K292D, K392D_K409D/D356K_D399K as described in WO2007/147901, WO 2011/143545, WO2013157954, WO2013096291 and US2018/0118849.

Duobody® mutations (Genmab) are disclosed for example in U.S. Pat. No. 9,150,663 and US2014/0303356 and include mutations F405L/K409R, wild-type/F405L_R409K, T350I_K370T_F405L/K409R, K370W/K409R, D399AFGHILMNRSTVWY/K409R, T366ADEFGHILMQVY/K409R, L368ADEGHNRSTVQ/K409AGRH, D399FHKRQ/K409AGRH, F405IKLSTVW/K409AGRH and Y407LWQ/K409AGRH.

Additional bispecific or multispecific structures into which the antigen binding regions that bind DLL3 can be incorporated include Dual Variable Domain Immunoglobulins (DVD) (Int. Pat. Publ. No. WO2009/134776; DVDs are full length antibodies comprising the heavy chain having a structure VH1-linker-VH2-CH and the light chain having the structure VL1-linker-VL2-CL; linker being optional), structures that include various dimerization domains to connect the two antibody arms with different specificity, such as leucine zipper or collagen dimerization domains (Int. Pat. Publ. No. WO2012/022811; U.S. Pat. Nos. 5,932,448; 6,833,441), two or more domain antibodies (dAbs) conjugated together, diabodies, heavy chain only antibodies such as camelid antibodies and engineered camelid antibodies, Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer), IgG-like Bispecific (InnClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche), ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics) and Dual(ScFv)2-Fab (National Research Center for Antibody Medicine—China), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). ScFv-, diabody-based, and domain antibodies, include but are not limited to, Bispecific T Cell Engager (BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies.

The antigen binding regions that bind DLL3 of the disclosure may also be engineered into multispecific antigen-binding constructs which comprise three polypeptide chains. In such designs, at least one antigen binding region is in the form of a scFv. Exemplary designs include (in which “1” indicates the first antigen binding region, “2” indicates the second antigen binding region and “3” indicates the third antigen binding region):

- Design 1: Chain A) scFv1-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3
- Design 2: Chain A) scFv1-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3
- Design 3: Chain A) scFv1-CH1-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3
- Design 4: Chain A) CH2-CH3-scFv1; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3
- CH3 engineering may be incorporated to the Designs 1-4, such as mutations
- L351Y_F405A_Y407V/T394W, T3661_K392M_T394W/F405A_Y407V,
- T366L_K392M_T394W/F405A_Y407V, L351Y_Y407A/T366A_K409F,
- L351Y_Y407A/T366V_K409F, Y407A/T366A_K409F, or
- T350V_L351Y_F405A_Y407V/T350V_T366L_K392L_T394W as described in US2012/0149876 or US2013/0195849 (Zymeworks).

In a particular embodiment, the design is Chain A) scFv1-hinge-CH2-CH3; Chain B) VL2-CL; Chain C) VH2-CH1-hinge-CH2-CH3.

In some embodiment, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (such as CD3), wherein the first antigen binding region that binds DLL3 comprises a HCDR1 of SEQ ID NOs:15, 18, 21, 24, 18, 30, 50, 52, 53, 55, 57, 59, or 61, a HCDR2 of SEQ ID NOs:16, 19, 22, 25, 28, 31, 51, 54, 56, 58, 60, or 62, a HCDR3 of SEQ ID NOs:17, 20, 23, 26, 29, 32, 17, 20, 23, 26, 29, or 32, a LCDR1 of SEQ ID NOs:33, 36, 39, 41, 44, or 47, a LCDR2 of SEQ ID NOs:34, 37, 42, 45, or 48, and a LCDR3 of SEQ ID NOs:35, 38, 40, 43, 46, or 49.

- SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively;
- SEQ ID NOs:18, 19, 20, 36, 37, 38, respectively;
- SEQ ID NOs:21, 22, 23, 39, 37, 40, respectively;
- SEQ ID NOs:24, 25, 26, 41, 42, 43, respectively;
- SEQ ID NOs:18, 28, 29, 44, 45, 46, respectively;
- SEQ ID NOs:30, 31, 32, 47, 48, 49, respectively;
- SEQ ID NOs:50, 51, 17, 33, 34, 35, respectively;
- SEQ ID NOs:52, 51, 17, 33, 34, 35, respectively;
- SEQ ID NOs:53, 54, 20, 36, 37, 38, respectively;
- SEQ ID NOs:55, 56, 23, 39, 37, 40, respectively;
- SEQ ID NOs:57, 58, 26, 41, 42, 43, respectively;
- SEQ ID NOs:59, 60, 29, 44, 45, 46, respectively; or
- SEQ ID NOs:61, 62, 32, 47, 48, 49, respectively.

In a particular embodiment, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding region that binds DLL3 comprises the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 of SEQ ID NOs:15, 16, 17, 33, 34, 35, respectively. In some embodiments, the isolated multispecific antigen-binding construct mediates T cell mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy. In some embodiments, the isolated multispecific antigen-binding construct potently mediates the expansion of cytotoxic CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct upregulates CD25, CD69 and CD71 expression on the surface of CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct displays increased tumor killing. In some embodiments, the bispecific anti-DLL3×CD3 antibody achieves >90% (e.g., 95%) tumor lysis by 5 days in a T cell cytotoxicity assay.

- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:12;
- the VH of SEQ ID NO:1 and the VL of SEQ ID NO:14;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:12;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:14;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:12;
- the VH of SEQ ID NO:3 and the VL of SEQ ID NO:14;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:12;
- the VH of SEQ ID NO:5 and the VL of SEQ ID NO:14;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:12;
- the VH of SEQ ID NO:7 and the VL of SEQ ID NO:14;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:10;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:12;
- the VH of SEQ ID NO:9 and the VL of SEQ ID NO:14;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 10;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 12;
- the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 14;
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:2;
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:4;
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:6;
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO:8;
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 10;
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 12; or
- the VH of SEQ ID NO: 13 and the VL of SEQ ID NO: 14.

In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4.

In a particular embodiment, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding region that binds DLL3 comprises a VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4. In some embodiments, the isolated multispecific antigen-binding constructs disclosed herein may be particularly effective at mediating T cell mediated cytotoxicity, promoting T cell activation and proliferation, increasing T cell cytokine release and/or displaying increased anti-tumor efficacy. In a particular embodiment, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL of SEQ ID NO:4.

In a particular embodiment, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding region that binds DLL3 comprises a VH which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VH of SEQ ID NO:3 and a VL which is at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the VL of SEQ ID NO:4. In some embodiments, the isolated multispecific antigen-binding construct mediates T cell mediated cytotoxicity. In some embodiments, the isolated multispecific antigen-binding construct potently mediates the expansion of cytotoxic CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct upregulates CD25, CD69 and CD71 expression on the surface of CD8 T cells. In some embodiments, the isolated multispecific antigen-binding construct displays increased tumor killing. In some embodiments, the bispecific anti-DLL3×CD3 antibody achieves >90% (e.g., 95%) tumor lysis by 5 days in a T cell cytotoxicity assay.

In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the first antigen binding region that binds DLL3 comprises an amino acid sequence at least 80% (e.g., at least 85%, at least 90%, at least 95% or at least 99%) identical to the amino acid sequence of SEQ ID NOs:63 or 64.

In some embodiments, the isolated multispecific antigen-binding construct comprises a first antigen binding region that binds DLL3 and a second antigen binding region that binds a lymphocyte antigen (e.g., CD3), wherein the second antigen binding region that binds the lymphocyte antigen comprises a HCDR1 of SEQ ID NOs:95 or 98, a HCDR2 of SEQ ID NOs:96 or 99, a HCDR3 of SEQ ID NOs:97 or 100, a LCDR1 of SEQ ID NO:101 or 106, a LCDR2 of SEQ ID NOs:102 or 107, and a LCDR3 of SEQ ID NOs:103, 104, or 108.

- a HCDR1 of SEQ ID NO:95, a HCDR2 of SEQ ID NO:96, a HCDR3 of SEQ ID NO:97, a LCDR1 of SEQ ID NO:101, a LCDR2 of SEQ ID NO: 102 and a LCDR3 of SEQ ID NO:104; or
- a HCDR1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO:100, a LCDR1 of SEQ ID NO: 106, a LCDR2 of SEQ ID NO:107 and a LCDR3 of SEQ ID NO:108.

- a HCDR1 of SEQ ID NO:98, a HCDR2 of SEQ ID NO:99, a HCDR3 of SEQ ID NO:100, a LCDR1 of SEQ ID NO: 106, a LCDR2 of SEQ ID NO:107 and a LCDR3 of SEQ ID NO:108; or the VH of SEQ ID NO:84 and the VL of SEQ ID NO:85.