CD3 antigen binding fragment and application thereof

Information

  • Patent Grant
  • 11639388
  • Patent Number
    11,639,388
  • Date Filed
    Friday, February 22, 2019
    5 years ago
  • Date Issued
    Tuesday, May 2, 2023
    a year ago
Abstract
Provided in the present application is a humanized and multifunctional CD3 antibody; a heavy chain variable region of the CD3 antibody comprised herein contains the amino acid sequence of any one of SEQ ID NOs: 1-3 or a variant sequence thereof; and a light chain variable region thereof contains the amino acid sequence of any one of SEQ ID NOs: 26-28 or a variant sequence thereof. Further provided in the present application is a multifunctional antibody comprising (a) a light chain-heavy chain pair that has specificity for tumor cells or microorganisms; and (b) a fusion peptide that comprises a single-chain variable fragment and an Fc fragment having a CH2 domain and/or a CH3 domain, the fusion peptide having specificity for immune cells. The antibodies provided in the present application have improved biological activity, thermal stability and/or acid resistance.
Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application under 35 U.S.C. 371 of International Application No. PCT/CN2019/075901, filed Feb. 22, 2019, the content of which is incorporated by reference in its entirety into the present disclosure.


SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 20, 2021, is named YZY022_SEQLT.txt and is 188 kb size.


FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering, and in particular to the field of antibody engineering. Specifically, the present application relates to antibodies, such as multi-functional antibodies, such as antibodies against CD3, in particular humanized antibodies, antigen-binding fragments of the antibodies, and related uses.


BACKGROUND ART

CD3 is a T cell surface molecule that can bind to T cell receptors on the surface of T cells to form a TCR-CD3 complex to activate T cells, and play an important role in antigen recognition and immune signal transduction. Anti-CD3 antibodies are widely used in the treatment of transplant rejection and autoimmune diseases. It is known that murine anti-CD3 antibodies, such as OKT3, may cause a significant human anti-mouse antibody (HAMA) response, which is not conducive to use in humans. Therefore, it is necessary to subject these mouse antibodies to humanization or other treatments to reduce adverse reactions. At present, the main way to avoid or reduce the HAMA response is to humanize murine monoclonal antibodies or develop fully humanized antibodies. For example, the HAMA response can be reduced by introducing sequence fragments identical to the human antibody protein into the murine antibody. However, there may not be structurally similar proteins in humans, and such treatment may not be possible. Moreover, bottlenecks such as decreased antibody affinity, low activity, poor stability or low yield of humanized antibodies often occur and active therapeutic proteins cannot be obtained.


SUMMARY OF THE INVENTION

The present invention provides an antibody, such as a multi-functional antibody, such as an antibody for CD3, in particular a humanized antibody, an antigen-binding fragment of the antibody, and a related use.


In some embodiments, the invention provides an antibody or an antigen binding fragment thereof, in particular a humanized antibody or an antigen binding fragment thereof, the antibody specifically binding to CD3 of primates, e.g., humans and/or monkeys, the antibody comprising framework regions, which are FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3 and FR-L4, respectively, and complementarity-determining regions (CDRs), wherein CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of heavy chain variable regions are amino acid sequences shown in SEQ ID NOs:1, 2 and 3, respectively, or variant sequences thereof, such as any one of sequences shown in the CDR3 variant sequences SEQ ID Nos: 4-14 and 190-191; CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of light chain variable regions are amino acid sequences shown in SEQ ID NOs:26, 27 and 28, respectively, or variant sequences thereof, wherein the framework regions of the humanized antibody comprise one or more of the following sequences:

    • a) FR-H1 of SEQ ID No: 15 or 16;
    • b) FR-H2 of SEQ ID No: 17;
    • c) FR-H3 of any one of SEQ ID Nos: 18-24;
    • d) FR-H4 of SEQ ID No: 25;
    • e) FR-L1 of any one of SEQ ID Nos: 29-31;
    • f) FR-L2 of any one of SEQ ID Nos: 32-38;
    • g) FR-L3 of any one of SEQ ID Nos: 39-42; and/or
    • h) FR-L4 of any one of SEQ ID Nos: 43-44.


In some embodiments, the invention provides an antibody or an antigen binding fragment thereof, in particular a humanized antibody or an antigen binding fragment thereof, the antibody specifically binding to CD3 of primates, e.g., humans and/or monkeys, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises any one of the following sequences:

    • a) amino acid sequences of SEQ ID NOs: 45-62;
    • b) amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence of SEQ ID NOs: 45-62;
    • c) amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence of SEQ ID NOs: 45-62; and the light chain variable region comprises any one of the following sequences:
    • d) amino acid sequences of SEQ ID NOs: 63-73;
    • e) amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence of SEQ ID NOs: 63-73;
    • f) amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence of SEQ ID NOs: 63-73.


In some embodiments, the invention provides an antibody or an antigen binding fragment thereof, in particular a humanized antibody or an antigen binding fragment thereof, the antibody specifically binding to CD3 of primates, e.g., humans and/or monkeys, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise amino acid sequences selected from the group consisting of:

    • a) SEQ ID Nos: 46, 63; SEQ ID Nos: 47, 63; SEQ ID Nos: 49, 63; SEQ ID Nos: 50, 63; SEQ ID Nos: 51, 63; SEQ ID Nos: 46, 71; SEQ ID Nos: 47, 71; SEQ ID Nos: 49, 71; SEQ ID Nos: 51, 71; SEQ ID Nos: 52, 72; SEQ ID Nos: 53, 72; SEQ ID Nos: 54, 72; SEQ ID Nos: 55, 72; SEQ ID Nos: 56, 72; SEQ ID Nos: 57, 72; SEQ ID Nos: 58, 72; SEQ ID Nos: 62, 72; SEQ ID Nos: 52, 73; SEQ ID Nos: 53, 73; SEQ ID Nos: 54, 73; SEQ ID Nos: 55, 73; SEQ ID Nos: 56, 73; SEQ ID Nos: 57, 73; SEQ ID Nos: 58, 73; SEQ ID Nos: 61, 73; SEQ ID Nos: 62, 73; SEQ ID Nos: 45, 63; SEQ ID Nos: 48, 63; SEQ ID Nos: 45, 64; SEQ ID Nos: 45, 67; SEQ ID Nos: 48, 64; SEQ ID Nos: 48, 67; SEQ ID Nos: 45, 71; SEQ ID Nos: 48, 71; SEQ ID Nos: 50, 71; SEQ ID Nos: 61, 72; SEQ ID Nos: 60, 73; SEQ ID Nos: 60, 72; SEQ ID Nos: 59, 72;
    • b) amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence in a);
    • c) amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence in a).


In some embodiments, the invention provides a polyspecific antibody, preferably bispecific antibody, comprising the antibody or an antigen binding fragment thereof according to any one of claims 1-3, and an antibody against another antigen and/or antigenic epitope, or an antigen binding fragment thereof, for example, a protein over-expressed in tumor cells compared to corresponding non-tumor cells; tumor antigen, such as CD38, BCMA, PD-L1, SLAMF7, Claudin18.2 or CEA; viruses; bacteria; and/or endotoxins.


In some embodiments, the invention provides a polypeptide, comprising an amino acid sequence selected from SEQ ID NOs: 45-62, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence of SEQ ID NOs: 45-62, or an amino acid sequence having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence of SEQ ID NOs: 45-62.


In some embodiments, the invention provides a polypeptide, comprising an amino acid sequence selected from SEQ ID NOs: 63-73, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence of SEQ ID NOs: 63-73, or an amino acid sequence having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence of SEQ ID NOs: 63-73.


In some embodiments, the humanized antibody has comparable affinity and improved biological activity, thermal stability, and/or acid resistance compared to a control antibody.


In some embodiments, the invention provides a polynucleotide, which codes the polypeptide of the invention.


In some embodiments, the invention provides an antibody, comprising (a) a light chain-heavy chain pair that is specific for tumor cells or microorganisms; and (b) fusion peptide, comprising a single-chain variable fragment and a single-chain Fc fragment, and the fusion peptide is specific for immune cells. In some embodiments, the single-chain Fc fragment comprises the CH2 and/or CH3 sequence described herein, for example, CH2 having a sequence selected from any one of SEQ ID Nos: 155-161 and 192 and/or CH3 having a sequence selected from any one of SEQ ID Nos:162-183. In some embodiments, the fusion peptide comprises the corresponding sequence or a partial sequence thereof of the antibody described herein, for example, the fusion peptide comprises the light chain and/or heavy chain variable region and/or framework region sequences of the humanized antibody described herein. In some embodiments, the single-chain variable fragment (scFv) of the fusion peptide comprises the scFV of the humanized antibody described herein.


In some embodiments, the fusion peptide in the antibody of the present invention comprises VHs-linker1-VLs-hinge 1-CH2-CH3-b, the heavy chain comprises VHm-CH1-hinge 2-CH2-CH3-a, and the light chain comprises VLm-CL.


In some embodiments, the light chain-heavy chain pair in the antibody of the present invention specifically binds to

    • a) a protein over-expressed in tumor cells compared to corresponding non-tumor cells;
    • b) tumor antigen, such as CD38, BCMA, PD-L1, SLAMF7, Claudin18.2 or CEA;
    • c) viruses;
    • d) bacteria; and/or
    • e) endotoxins.


In some embodiments, the fusion peptide in the antibody of the present invention specifically binds to immune cell antigens, for example, the fusion peptide comprises an antigen binding site that specifically binds to CD3 of primates, e.g., humans and/or monkeys, such as the fusion peptide comprises the variable regions of the light and heavy chains of the antibodies described herein.


In some embodiments, the VH of the fusion peptide of the antibody of the present invention comprises a sequence selected from any one of SEQ ID Nos: 45-62, 74, 76, 78, 80, 82, 84, 86, and 88; VL of the fusion peptide comprises a sequence selected from any one of SEQ ID Nos: 63-73, 75, 77, 79, 81, 83, 85, 87, and 89; linker1 of the fusion peptide comprises a sequence selected from any one of SEQ ID Nos: 120-138; hinge 1 of the fusion peptide and hinge 2 of the heavy chain comprise a sequence selected from any one of SEQ ID Nos: 139-147; CH2 of the fusion peptide and CH2 of the heavy chain comprise a sequence selected from any one of SEQ ID Nos: 155-161 and 192; CH3-b of the fusion peptide comprises a sequence selected from any one of SEQ ID Nos: 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, and 183; CH3-a of the heavy chain comprises a sequence selected from any one of SEQ ID Nos: 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, and 182; VHm of the heavy chain comprises a sequence selected from any one of SEQ ID Nos: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 193; CH1 of the heavy chain comprises the sequence of SEQ ID Nos: 154; VLm of the light chain comprises a sequence selected from any one of SEQ ID Nos: 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, and 194; and/or CL of the light chain comprises a sequence selected from any one of SEQ ID Nos: 148-153.


In some embodiments, the VH of the fusion peptide and VL of the fusion peptide of the antibody of the present invention respectively comprise amino acid sequences selected from the group consisting of: a) SEQ ID Nos: 45, 63; SEQ ID Nos: 48, 63; SEQ ID Nos: 48, 71; SEQ ID Nos: 49, 63; SEQ ID Nos: 49, 71; SEQ ID Nos: 51, 71; SEQ ID Nos: 58, 72; SEQ ID Nos: 60, 72; SEQ ID Nos: 60, 73; SEQ ID Nos: 59, 72; SEQ ID Nos: 61, 73; SEQ ID Nos: 62, 73; SEQ ID Nos: 58, 72; SEQ ID Nos: 74, 75; SEQ ID Nos: 76, 77; SEQ ID Nos: 78, 79; SEQ ID Nos: 80, 81; SEQ ID Nos: 82, 83; SEQ ID Nos: 84, 85; SEQ ID Nos: 86, 87; SEQ ID Nos: 88, 89;

    • b) amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence in a);
    • c) amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence in a); and/or


VHm of the heavy chain and VLm of the light chain respectively comprise amino acid sequences selected from the group consisting of: d) SEQ ID Nos: 90, 91; SEQ ID Nos: 92, 93; SEQ ID Nos: 94, 95; SEQ ID Nos: 96, 97; SEQ ID Nos: 98, 99; SEQ ID Nos: 100, 101; SEQ ID Nos: 102, 103; SEQ ID Nos: 104, 105; SEQ ID Nos: 106, 107; SEQ ID Nos: 108, 109; SEQ ID Nos: 110, 111; SEQ ID Nos: 112, 113; SEQ ID Nos: 114, 115; SEQ ID Nos: 116, 117; SEQ ID Nos: 118, 119; SEQ ID Nos: 193, 194;

    • e) amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one amino acid sequence in d);
    • f) amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one amino acid sequence in d).


In some embodiments, in the antibody of the present invention:

    • a) CH3-b of the fusion peptide and CH3-a of the heavy chain have substitution pairs forming knob-into-hole structures, for example, T366 in one CH3 domain is substituted by a larger amino acid residue, such as Tyrosine (Y) or Tryptophan (W), and Y407 in the other CH3 domain is substituted by a smaller amino acid residue, such as Threonine (T), Alanine (A), or Valine (V), and for example, comprises one or more substitutions in Table 15;
    • b) CH3-b of the fusion peptide and CH3-a of the heavy chain have substitution pairs forming ionic bonds, for example, one of the CH3 domains comprises one or more substitutions by amino acid residues having a positive charge under physiological conditions, while the other CH3 domain comprises one or more substitutions by one or more amino acid residues having a negative charge under physiological conditions; for example, the amino acid residue having a positive charge is Arginine (R), Histidine (H) or Lysine (K); for example, the amino acid residue having a negative charge may be Aspartic acid (D) or Glutamic acid (E); for example, the substituted amino acid residues include one or more of D356, L368, K392, D399 and K409, such as one or more substitutions in Table 16;
    • c) CH3-b of the fusion peptide and CH3-a of the heavy chain have substitution pairs forming disulfide bonds, for example, the substitutions in Table 17; and/or
    • d) CH3-b of the fusion peptide and CH3-a of the heavy chain have substitutions leading to weakened binding capability with protein A, for example, H435 and Y436 in one of the CH3 domains are substituted by Arginine and Phenylalanine, respectively, as shown in Table 18.


In some embodiments, the Fc fragment in the antibody of the present invention comprises CH2 having a sequence selected from any one of SEQ ID Nos: 155-161 and 192 and/or CH3 having a sequence selected from any one of SEQ ID Nos:162-183.


In some embodiments, the heavy chain or the heavy chain of the fusion peptide of the antibody of the present invention comprises a human or humanized Fc fragment, such as a human IgG Fc fragment, for example, IgG1, IgG2, IgG3, IgG4, and IgG5 Fc fragments.


In some embodiments, compared with wild-type antibodies, the Fc fragment of the heavy chain of the antibody, the heavy chain of the fusion peptide, and/or the fusion peptide of the present invention comprises one or more substitutions that form knob-into-hole structural pairs between the heavy chain and the fusion peptide.


In some embodiments, the Fc fragment of the heavy chain and/or the fusion peptide of the antibody of the present invention comprises one or more substitutions that form salt bridge pairs between the heavy chain and the fusion peptide.


In some embodiments, the antibody of the present invention comprises Y101, Y102, Y103, Y104, Y105, Y150-8-3, Y150-F8-4, Y150-F8-5, Y150-F8-6, Y150-F8-7, Y150-F8-8, Y150-F8-9, Y150-F8-10, Y150-F8-11, Y150-F8-12, Y150-F8-13, Y150-F8-14, Y150-F8-15, Y150-F9-7, Y150-F9-11, Y150-F9-12, MS-hCD3-IC15, MS-hCD3-IC16, MS-hCD3-IC17 and MS-hCD3-IC18, and wherein according to the order of components in the fusion peptide VHs-linker1-VLs-hinge 1-CH2-CH3-b, the heavy chain VHm-CH1-hinge 2-CH2-CH3-a, and the light chain VLm-CL,


Y101 respectively comprises SEQ ID Nos: 45, 129, 63, 142, 159, 167, 106, 154, 139, 159, 166, 107, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y102 respectively comprises SEQ ID Nos: 48, 129, 63, 142, 159, 167, 106, 154, 139, 159, 166, 107, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y103 respectively comprises SEQ ID Nos: 48, 129, 71, 142, 159, 167, 106, 154, 139, 159, 166, 107, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y104 respectively comprises SEQ ID Nos: 49, 129, 63, 142, 139, 167, 106, 154, 139, 159, 166, 107, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y105 respectively comprises SEQ ID Nos: 49, 129, 71, 142, 139, 167, 106, 154, 139, 159, 166, 107, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-8-3 respectively comprises SEQ ID Nos: 45, 129, 63, 141, 157, 167, 90, 154, 139, 157, 166, 91, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-4 respectively comprises SEQ ID Nos: 48, 129, 63, 141, 157, 167, 90, 154, 139, 157, 166, 91, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-5 respectively comprises SEQ ID Nos: 49, 129, 71, 141, 139, 167, 90, 154, 139, 157, 166, 91, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-6 respectively comprises SEQ ID Nos: 51, 129, 71, 141, 139, 167, 90, 154, 139, 157, 166, 91, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-7 respectively comprises SEQ ID Nos: 49, 129, 71, 144, 158, 167, 90, 154, 139, 158, 166, 91, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-8 respectively comprises SEQ ID Nos: 49, 129, 71, 144, 161, 167, 90, 154, 139, 161, 166, 91, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-9 respectively comprises SEQ ID Nos: 49, 129, 71, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-10 respectively comprises SEQ ID Nos: 58, 129, 72, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-11 respectively comprises SEQ ID Nos: 60, 129, 72, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-12 respectively comprises SEQ ID Nos: 60, 129, 73, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-13 respectively comprises SEQ ID Nos: 59, 129, 72, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-14 respectively comprises SEQ ID Nos: 61, 129, 73, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F8-15 respectively comprises 62, 129, 73, 144, 161, 167, 96, 154, 139, 161, 166, 97, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F9-7 respectively comprises 49, 129, 71, 141, 139, 167, 92, 154, 139, 157, 166, 93, 150; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F9-11 respectively comprises 49, 129, 71, 144, 161, 167, 92, 154, 139, 161, 166, 93, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


Y150-F9-12 respectively comprises 49, 129, 71, 144, 192, 167, 92, 154, 139, 192, 166, 93, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


MS-hCD3-IC15 respectively comprises 49, 129, 71, 141, 159, 167, 118, 154, 139, 159, 166, 119, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


MS-hCD3-IC16 respectively comprises 49, 129, 71, 141, 157, 167, 118, 154, 139, 157, 166, 119, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


MS-hCD3-IC17 respectively comprises 49, 129, 71, 141, 161, 167, 118, 154, 139, 161, 166, 119, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences;


MS-hCD3-IC18 respectively comprises 58, 129, 72, 141, 161, 167, 118, 154, 139, 161, 166, 119, 148; or amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid identity with at least one of the above amino acid sequences; or amino acid sequences having one or more (preferably one or several, and more preferably 1, 2, or 3) different amino acids with at least one of the above amino acid sequences.


In some embodiments, the antibody or an antigen binding fragment thereof of the present invention can bind to a target with KD less than about 10−8 M, for example, less than about 10−8 M, 10−9 M, 10−10 M, or less, or binds to a target with EC50 less than about 100 nM, for example, less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or smaller, and preferably, the antigen binding fragment is selected from F(ab′)2, F(ab)2, Fab′, Fab, Fv, Fd, and scFv.


In some embodiments, the invention provides a polynucleotide, which codes the antibody or an antigen binding fragment thereof described herein.


In some embodiments, the invention provides an expression vector, comprising the polynucleotide described herein.


In some embodiments, the invention provides a host cell, comprising the polynucleotide or the expression vector described herein.


In some embodiments, the invention provides a method for preparing the antibody of the present invention, comprising introducing the polynucleotide or the expression vector described herein into a host cell, so as to prepare the antibody.


In some embodiments, the invention provides an antibody conjugate, comprising the antibody or an antigen binding fragment thereof described herein and a conjugating moiety conjugated thereto, preferably, the conjugating moiety is selected from purification tags (e.g., a His tag), cytotoxic agents, detectable marks, radioactive isotopes, luminescent substances, colored substances, enzymes, or polyethylene glycol.


In some embodiments, the invention provides an antibody conjugate, wherein the antibody may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.


In some embodiments, the antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound enhancing agent, a non-radioactive label, a combination thereof and other such agents known in the art.


In some embodiments, the antibodies can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.


In some embodiments, the antibodies can also be detectably labeled using fluorescence emitting metals such as 152Eu, or other labels of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various groups to an antibody are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies “84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. (52:119-58 (1982)).


In some embodiments, the invention provides a fusion protein, comprising the antibody or an antigen binding fragment thereof described herein.


In some embodiments, the invention provides a pharmaceutical composition, comprising the antibody or an antigen binding fragment thereof, the antibody conjugate, or the fusion protein described herein, and optionally, further comprising a pharmaceutically acceptable carrier and/or excipient.


In some embodiments, the pharmaceutical composition described herein is a formulation suitable for oral administration to gastrointestinal (GI) tract, preferably, the formulation is selected from tablet, capsule, pill, powder, granule, emulsion, micro-emulsion, solution, suspension, syrup, and elixir; or the drug is a formulation suitable for subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, and intralesional injection.


In some embodiments, the invention provides a kit, comprising the antibody or an antigen binding fragment thereof, the antibody conjugate, or the fusion protein described herein, and preferably, further comprising a secondary antibody that specifically recognizes the antibody or an antigen binding fragment thereof, the antibody conjugate, or the fusion protein described herein; wherein, optionally, the secondary antibody further comprises a detectable label, such as a radioactive isotope, a luminescent substance, a colored substance, or an enzyme.


In some embodiments, the invention provides the antibody or an antigen binding fragment thereof described herein used for treating a disease, or a use of the antibody or an antigen binding fragment thereof described herein in treating a disease, or a use of the antibody or an antigen binding fragment thereof described herein in preparing a medicament for treating a disease.


In some embodiments, the antibodies provided herein can be used in combination with another therapeutic agent (e.g., a therapeutic agent used to treat tumors or cancer).


In some embodiments, the invention provides a kit that includes the antibody or antigen-binding fragment thereof provided herein and a pharmaceutical acceptable carrier, instructions for use, and optional another therapeutic agent (e.g., a therapeutic agent used to treat tumors or cancer).


In some embodiments, in the compositions and/or kits provided herein, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic moiety, an enzyme, a radioactive compound, a cytokine, an interferon, a target, or a reporter moiety.


In some embodiments, the antibodies or antigen-binding fragments thereof provided herein can be used to treat a disease such as cancer or tumors.


In some embodiments, the invention provides the use of the antibody or antigen-binding fragment thereof for the treatment of a disease such as cancer or tumors.


In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are used in the preparation of drugs for the treatment of a disease such as cancer or tumors.


In some embodiments, the antibodies or antigen-binding fragments provided herein can be used to treat a disease such as cancer or tumors, including but not limited to multiple myeloma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell cancer), etc.


In some embodiments, the invention provides a method of humanizing CD3 antibodies and the obtained humanized sequences. Monoclonal antibodies and multifunctional antibodies prepared based on the humanized antibody sequence have suitable affinity, high stability and good cell killing ability.


In some embodiments, compared with control antibodies such as the original antibody SP34 and CD3 antibodies with high homology provided in other documents, the humanized CD3 antibody provided herein shows better biological activity and/or stability than other CD3 antibodies in terms of the biological activity and stability of the multifunctional antibody.


In some embodiments, the invention provides a multifunctional antibody and a preparation method, the antibody comprising: (a) a light chain-heavy chain pair having specificity to tumor cells or microorganisms; and (b) a fusion peptide comprising a single chain variable fragment (scFv) and an Fc fragment comprising a CH2 domain and/or a CH3 domain, wherein the fusion peptide has specificity to immune cells.


In some embodiments, the light chain-heavy chain pair or VLm-VHm pair of the antibody of the invention has specificity to a tumor antigen. In some embodiments, the tumor antigen is selected from: PD-L1, SLAMF7, CD38, BCMA and the like. In some embodiments, the light chain-heavy chain pair or VLm-VHm pair has specificity to a protein that is overexpressed on a tumor cell compared to a corresponding non-tumor cell.


In some aspects, the light chain-heavy chain pair or VLm-VHm pair has specificity to a virus or bacterium. In one aspect, the light chain-heavy chain pair or VLm-VHm pair has specificity to an endotoxin.


In some embodiments, the immune cell is selected from the group consisting of T cells, CIK cells, NKT cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, macrophages, natural killer cells, eosinocytes, basophils and mast cells.


In some embodiments, the ScFv or VLs-VHs pair has specificity to the antigens including, for example, CD3, CD4, CD8, CD40L, CD152, CD16, CD56, CD94, CD158, CD161, CD19, CD20, CD21, CD40. In some embodiments, the antigen is CD3.


In some embodiments, the light chain is bound to the heavy chain or fusion heavy chain through a disulfide bond. In some embodiments, the heavy chain is bound to the fusion peptide through one or more disulfide bonds. In some embodiments, the fusion heavy chain 1 is bound to the fusion heavy chain 2 through one or more disulfide bonds. In some embodiments, the heavy chain or fusion heavy chain comprises a human or humanized Fc fragment. In some embodiments, the Fc fragment of the heavy chain or fusion heavy chain comprises a human IgG Fc fragment. In some embodiments, the Fc fragment of the fusion peptide comprises a human or a humanized Fc fragment. In some embodiments, the Fc fragment of the fusion peptide comprises a human IgG Fc fragment.


In some aspects, the Fc fragment of the heavy chain, the fusion heavy chain and/or the fusion peptide comprises one or more substitutions that form knobs-into-holes structure pairing between the heavy chain and the fusion peptide, as compared to a wild-type antibody fragment. The pairing can significantly improve the heterodimer pairing efficiency of the heavy chain and the fusion peptide.


In some aspects, the Fc fragment of the heavy chain and/or the fusion peptide comprise one or more substitutions that form a salt-bridge pairing between the heavy chain and the fusion peptide. The pairing can significantly improve the heterodimer pairing efficiency of the heavy chain and the fusion peptide.


In some aspects, the CH2 domain of the fusion peptide is located between the scFv fragment and the CH3 domain. In one aspect, the fusion peptide does not contain a CH1 domain.


In one embodiment, the application also provides a composition comprising the antibody in any of the above embodiments. In one aspect, the carrier is a drug carrier.


Another embodiment provides a complex comprising the antibody of any of the above embodiments that binds to one or more antigens.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1A is a schematic structural diagram of an antibody. FIG. 1B is a schematic diagram of a primary structure of protein of each component of the antibody.



FIG. 2 is a schematic diagram of a primary structure of protein of each component of a monoclonal antibody.



FIG. 3 illustrates expression levels of humanized CD3 monoclonal antibodies.



FIG. 4 illustrates binding capabilities of the monoclonal antibodies with the CD3+ T cells.



FIG. 5 illustrates a transient transfection expression level of a multi-functional antibody assembled from the humanized CD3 antibody in CHO cells.



FIG. 6 illustrates the cell affinity of monoclonal antibodies of existing CD3 antibodies.



FIG. 7 illustrates in vitro T-cell activation capabilities of different multi-functional antibodies according to the present invention.



FIG. 8 illustrates in vitro killig capability of different multi-functional antibodies against multiple myeloma cells MC/CAR.



FIG. 9 illustrates in vitro killig capability of different multi-functional antibodies against lung cancer cells H358.



FIG. 10 illustrates accelerated thermal stability detection at 40° C. of a multi-functional antibody Y105 according to the present invention and reference antibodies Y106, CT-F4, CT-F5 and CT-F6 in a citric acid buffer system.



FIG. 11 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 according to the present invention and comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system.



FIG. 12 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 according to the present invention and comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system.



FIG. 13 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 according to the present invention and comparative antibodies IC-2 to IC-7 in a citric acid buffer system.



FIG. 14 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 according to the present invention and comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 in a histidine buffer system.



FIG. 15 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 according to the present invention and comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 in a histidine buffer system.



FIG. 16 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 according to the present invention and comparative antibodies IC-2 to IC-7 in a histidine buffer system.



FIG. 17 illustrates acid-resistant stability detection of a multi-functional antibody Y105 according to the present invention and comparative antibodies Y106, CT-F4, CT-F5 and CT-F6 in a citric acid buffer system with pH 3.5.



FIG. 18 illustrates acid-resistant stability detection of multi-functional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 according to the present invention and comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system with pH 3.5.



FIG. 19 illustrates acid-resistant stability detection of multi-functional antibodies Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 according to the present invention and comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system with pH 3.5.



FIG. 20 illustrates acid-resistant stability detection of multi-functional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 according to the present invention and comparative antibodies IC-2 to IC-7 in a citric acid buffer system with pH 3.5.



FIG. 21 illustrates in vivo efficacy and tumor volume monitoring of different multi-functional antibodies in a mouse tumor model, wherein a multi-functional antibody Y150-F8-8 according to the present invention is used in FIG. 21A; a multi-functional antibody F8-9 according to the present invention is used in FIG. 21B; a multi-functional antibody F9-11 according to the present invention is used in FIG. 21C; the anti-CD3 antibody sequences are all VH2a and VL5, and the anti-CD38 antibody sequences are all different.





DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Definitions

There are six “complementarily determining regions” or “CDRs” in naturally occurring antibodies which are specifically positioned to form the antigen-binding domain. The remainder of the amino acids in the antigen-binding domains, referred to as “framework” regions, show less inter-molecular variability. The framework regions largely adopt a β-sheet configuration and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids of the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987)).


The term “complementarity determining region” (“CDR”) is used herein to describe the noncontinous antigen binding sites within the variable regions of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. MoI. Biol. 196:901-917 (1987). The CDRs include overlapping amino acid residues or amino acid substructure when compared against each other according to Kabat and Chothia's definitions. Nevertheless, use of definition of CDRs of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.


Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, independent of any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described by Kabat et al., also in U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).


The Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues behind the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue behind the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue behind the end of CDR-H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., behind a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue behind the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue behind the end of CDR-L2 (i.e., behind a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.


Antibodies described herein may be from any animal source including birds and mammals, including primates. Preferably, the antibody is a human, baboon, rhesus monkey, cynomolgus monkey, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibody.


The humanized antibodies described herein are capable of specifically binding to CD3, such as primate CD3, including, for example, human and/or monkey CD3.


As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present application may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain. Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). It will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.


The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.


As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.


A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.


The subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. The term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.


As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.


As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).


As used herein the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to position 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).


As used herein, the term “chimeric antibody” will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant disclosure) is obtained from a second species. In certain embodiments the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is from huma.


As used herein, “percent humanization” is calculated by determining the number of framework amino acid differences (i.e., non-CDR difference) between the humanized domain and the germline domain, subtracting that number from the total number of amino acids, and then dividing that by the total number of amino acids and multiplying by 100.


By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.” In some embodiments, the antibody of the present invention bind to the target with an KD of less than about 10−8M, 10−9M, 10−19M or less. In some embodiments, the antibody of the present invention bind to the target with an EC50 of less than about 100 nM, such as less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less.


It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a multifunctional antibody,” is understood to represent one or more multifunctional antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.


As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by nonnaturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.


The term “isolated” as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides is meant to encompass both purified and recombinant polypeptides.


As used herein, the term “recombinant” as it pertains to polypeptides or polynucleotides intends a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.


“Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the sequences of the present disclosure.


A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. Biologically equivalent polynucleotides are those having the above-noted specified percent homology and encoding a polypeptide having the same or similar biological activity.


The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence which has a certain degree of homology with another nucleic acid or with the complement thereof. In one aspect, homologs of a nucleic acid are capable of hybridizing to the nucleic acid or complement thereof. Likewise, “an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent sequence retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence.


Hybridization reactions can be performed under conditions of different “stringency”. In general, a low stringency hybridization reaction is carried out at about 40° C. in about 10×SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50° C. in about 6×SSC, and a high stringency hybridization reaction is generally performed at about 60° C. in about 1×SSC. Hybridization reactions can also be performed under “physiological conditions” which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg2+ normally found in a cell.


A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The term “polymorphism” refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a “polymorphic region of a gene”. A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.


The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are nonlimiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.


The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.


As used herein, the term “detectable label” intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a “labeled” composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A reaction that is simply detected generally comprises a reaction whose existence merely is confirmed, whereas a reaction that is quantified generally comprises a reaction having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluorescence assays, the detectable reaction may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.


As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.


The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab′)2, F(ab)2, Fab′, Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen is recognized as an intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.


A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin. In some aspects, the regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the properties of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art, such as those described in U.S. Pat. No. 5,892,019.


Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA, IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly within the scope of the present disclosure, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.


Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein) Immunoglobulin or antibody molecules of the disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.


Light chains are classified as either kappa or lambda (K, 4 Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.


Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.


As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VK domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y configuration. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VK chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).


As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Conditions in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.


By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, primates (for example, humans, monkeys such as cynomolgus, macaques, baboons, and chimpanzees, etc.), and so on.


As used herein, phrases such as “a patient in need of treatment” or “a subject in need of treatment” includes mammalian subjects, such as a human that would benefit from administration of an antibody or composition used in the present application, e.g., for detection, for a diagnostic procedure and/or for treatment.


Multifunctional Antibody


One embodiment of the present disclosure provides a heterodimer antibody, which comprises two different antigen-binding polypeptide units. In some aspects, the heterodimer differs in size from its corresponding homodimer, and the size difference can be utilized to facilitate separation of hetero- and homo-dimers.


In some aspects, as shown in FIG. 1, one of the two antigen-binding polypeptide units comprises a light chain-heavy chain pair like a wild-type antibody. Throughout the disclosure, this unit is also referred to as a “monovalent unit.”


In some aspects, as shown in FIG. 1, the other antigen-binding polypeptide unit, comprises a single chain variable fragment (scFv). Such a scFv can be fused to the N-terminus of the constant fragment (Fc) of an antibody, which is called a fusion peptide. Throughout the disclosure, this fusion peptide is also referred to as “single-chain unit”.


The present application provides a multifunctional antibody and a preparation method, the antibody comprising: (a) a light chain-heavy chain pair having specificity to tumor cells; and (b) a fusion peptide comprising a single chain variable fragment (scFv) and an Fc fragment comprising a CH2 domain and/or a CH3 domain, wherein the fusion peptide has specificity to immune cells. This antibody is called a multifunctional antibody.


Any of the antibodies or polypeptides described above may further include additional polypeptides, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.


It will also be understood by one of ordinary skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.


Furthermore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at “non-essential” amino acid regions may be made. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.


In certain embodiments, an antigen-binding polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, a single chain Fv antibody fragment of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).


Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non-classical amino acids.


In other embodiments, the antigen-binding polypeptides of the present disclosure may contain conservative amino acid substitutions.


A “conservative amino acid substitution” is one in which the amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably substituted with another amino acid residue from the same side chain family In another embodiment, a string of amino acids can be substituted with a structurally similar string that differs in order and/or composition of side chain family members.


Methods of Making Antibodies


Methods of making antibodies are well known in the art and described herein. In certain embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present disclosure are fully human Fully human antibodies can be made using techniques described in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140 which are incorporated by reference in their entireties.


In certain embodiments, the prepared antibodies will not elicit a deleterious immune response in the animal to be treated, e.g., in a human. In one embodiment, antigen binding polypeptides, variants, or derivatives thereof of the disclosure are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by substitution of surface residues.


De-immunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term “de-immunization” includes alteration of an antibody to modify T-cell epitopes (see, e.g., International Application Publication Nos.: WO/9852976 A1 and WO/0034317 A2). For example, variable heavy chain and variable light chain sequences from the starting antibody are analyzed and a human T-cell epitope “map” from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence is created. Individual T-cell epitopes from the T-cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative variable heavy and variable light sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides. Typically, between 12 and 24 variant antibodies are generated and tested for binding and/or function. Complete heavy and light chain genes comprising modified variable and human constant regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.


The binding specificity of antigen-binding polypeptides of the present disclosure can be determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).


Techniques described for the production of single-chain units (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be adapted to produce single-chain units of the present disclosure. Single-chain units are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain fusion peptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242: 1038-1041 (1988)).


Examples of techniques which can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.


Humanized antibodies are antibody molecules derived from a non-human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen-binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated by reference in its entirety).


Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.


Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.


Completely human antibodies which recognize a selected epitope can also be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/Technology 72:899-903 (1988). See also, U.S. Pat. No. 5,565,332, which is incorporated by reference in its entirety.)


In some embodiments, DNA encoding desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney cell 293 or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture of antibodies as described in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.


Additionally, using routine recombinant DNA techniques, one or more of the CDRs of the antigen-binding polypeptides of the present disclosure, may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes a polypeptide that specifically binds to at least one epitope of a desired polypeptide, e.g., LIGHT. Preferably, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in formation of intra-chain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present disclosure and within the skill of the art.


In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA: 851-855 (1984); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule, of appropriate antigen specificity, together with genes from a human antibody molecule of appropriate biological activity can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.


Yet another highly efficient means for generating recombinant antibodies is disclosed by Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique results in the generation of primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. Moreover, this technique is also described in commonly assigned U.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which is incorporated herein by reference.


In some embodiments, antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the disclosure as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.


In some embodiments, standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody of the present disclosure, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid subsitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference variable heavy chain region, CDRH1, CDR-H2, CDR-H3, variable light chain region, CDR-L1, CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity.


Treatment and Diagnostic Methods


As described herein, the antigen-binding polypeptides, variants or derivatives of the present disclosure may be used in certain treatments and diagnostic methods associated with cancer or an infectious disease.


The present disclosure is further directed to antibody-based therapies which involve administering the bispecific antibodies of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to, antibodies of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein).


The antibodies of the disclosure can also be used to treat, inhibit or prevent diseases, disorders or conditions including malignant diseases, disorders, or conditions associated with such diseases or disorders, such as diseases associated with immune response. In some embodiments, the antibodies of the invention can be used as immunosuppressive agents. In some embodiments, the antibodies of the invention can be used to treat autoimmune diseases. The antigen-binding polypeptides, variants or derivatives thereof of the disclosure are used to inhibit the growth, development and/or metastasis of cancer, especially those listed above or in the following paragraphs.


Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and/or prognosed with the antibodies or variants, or derivatives thereof of the disclosure include, but are not limited to cancer or tumors, including the development and/or metastasis of malignant tumors, and related diseases, such as multiple myeloma, lung cancer (such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), etc.


A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antigen-binding polypeptide, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.


Methods of administration of the antigen-binding polypeptides, variants or derivatives thereof include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.


The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.


Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.


It may be desirable to administer the antigen-binding polypeptides or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, or fibers. Preferably, when administering a protein, including an antibody, of the disclosure, care must be taken to use materials to which the protein does not absorb.


The amount of the antibodies of the disclosure which will be effective in the treatment, inhibition and prevention of an inflammatory, immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.


The methods for treating an infectious or malignant disease, condition or disorder comprising administration of an antibody, variant, or derivative thereof of the disclosure are typically tested in vitro, and then in vivo in an acceptable animal model, for the desired therapeutic or prophylactic activity, prior to use in humans. Suitable animal models, including transgenic animals, are well known to those of ordinary skill in the art. For example, in vitro assays to demonstrate the therapeutic utility of antigen-binding polypeptide described herein include the effect of an antigen-binding polypeptide on a cell line or a patient tissue sample. The effect of the antigen-binding polypeptide on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art, such as the assays disclosed elsewhere herein. In accordance with the disclosure, in vitro assays which can be used to determine whether administration of a specific antigen-binding polypeptide is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.


Antibody Structure Information


The structure of monoclonal antibody is: symmetrical monospecific antibody, including two identical light chains and two identical heavy chains, with light-heavy chain pairings and a heavy chain-heavy chain pairing; the light-heavy chain pairings target the same kind target.


The structure of the multifunctional antibody is: asymmetric bispecific antibody, including a light chain, a heavy chain and a fusion peptide, with a light-heavy chain pairing and a heavy chain-fusion peptide pairing; the light-heavy chain pairing targets tumor antigen, and the fusion peptide ScFv targets the immune cell antigen CD3.


In some aspects, the heavy chain is bound to the fusion peptide through one or more disulfide bonds, or one or more disulfide bonds are formed between two different fusion heavy chains. In one aspect, the one or more disulfide bonds are formed between the amino acid residues at the hinge region between the CH1 (or VLs) and the CH2 domains.


In some aspects, the CH2 domain of the fusion peptide is located between the scFv fragment and the CH3 domain. In other words, the scFv fragment is connected at the CH2 end of the Fc fragment. In some aspects, the single chain unit does not contain a CH1 domain.


In one aspect, either or both of the monovalent unit and the single-chain unit comprise human antibody sequences or humanized sequences. For instance, in one aspect, the heavy chain of the monovalent unit comprises a human or humanized Fc fragment. In a particular aspect, the Fc fragment of the heavy chain comprises a human IgG Fc fragment.


In one aspect, the Fc fragment of the fusion peptide comprises a human or humanized Fc fragment. In a particular aspect, the Fc fragment of the fusion peptide comprises a human IgG Fc fragment.



FIG. 1A is a schematic structural diagram of a multi-functional antibody 1. FIG. 1B is a schematic diagram of a primary structure of protein of each component of the antibody.


Humanized CD3 Antibody Engineered According to the Present Invention


(1) CDR and FR Sequences of the Variable Region of the Humanized CD3 Antibody









TABLE 1







CDR and FR sequences of the variable


regions of the humanized CD3 antibody










Amino acid sequences (those




in bold and underlined being
Sequence


Domain
replaceable amino acids
No.












CDR-H1
TYAMN
1





CDR-H2
RIRSKYNNYATYYADSVKD
2





CDR-H3
HGNFGNSYVSWFAY
3





CDR-H3a
HGNFGNSYVTWFAY
4





CDR-H3b
HGNFGNSYVSYFAY
5





CDR-H3c
HGNFGNSYVSFFAY
6





CDR-H3d
HGNFGNSYVSWLAY
7





CDR-H3e
HGNFGNSYVSWVAY
8





CDR-H3f
HGNFGNSYVSWIAY
9





CDR-H3g
HGNFGNSYVSWAAY
10





CDR-H3h
HGNFGNSYVSWYAY
11





CDR-H3i
HGNFGNSYVSWFVY
12





CDR-H3j
HGNFGNSYVSWFLY
13





CDR-H3k
HGNFGNSYVSWFIY
14





CDR-H3l
HGNFGNSYVSWGAY
190





CDR-H3m
HGNFGNSYVSWFGY
191





FR-H1


E
VQLVESGGGLVQPGGSLRLSCAASGFTFS

15





FR-H1a


Q
VQLVESGGGVVQPGRSLRLSCAASGFTFS

16





FR-H2
WVRQAPGKGLEWVA
17





FR-H3
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
18





FR-H3a
RFTISRDDSKNSLYLQMNSLRAEDTAVYYCAR
19





FR-H3b
RFTISRDDSKNSLYLQMNSLRAEDTAVYYCVR
20





FR-H3c
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
21





FR-H3d
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR
22





FR-H3e
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCAR
23





FR-H3f
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR
24





FR-H4
WGQGTLVTVSS
25





CDR-L1
RSSTGAVTTSNYAN
26





CDR-L2
GTNKRAP
27





CDR-L3
ALWYSNLWV
28





FR-L1
EIVLTQSPATLSLSPGERATLSC
29





FR-L1a
EIVMTQSPATLSLSPGERATLSC
30





FR-L1b
QTVVTQEPSLTVSPGGTVTLTC
31





FR-L2
WFQQKPGQAPRALIY
32





FR-L2a
WFQQKPGQAPRGLIG
33





FR-L2b
WVQQKPGQAPRALIG
34





FR-L2c
WYQQKPGQAPRALIY
35





FR-L2d
WVQQKPGQAPRGLIG
36





FR-L2e
WVQQKPGQAPKGLIG
37





FR-L2f
WVQQKPGKAPKLLIG
38





FR-L3
GVPARFSGSLSGTDATLTISSLQPEDFAVYYC
39





FR-L3a


WT
PARFSGSLLGGKAALTLSGVQPEDEAEYYC

40





FR-L3b
GVPARFSGSLLGGKAALTLSGVQPEDEAEYYC
41





FR-L3c
GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC
42





FR-L4
FGGGTKVEIK
43





FR-L4a
FGGGTKLTVL
44









(2) New Humanized CD3 Antibody Sequences (Some Examples)









TABLE 2







Sequences of the variable regions of the new humanized CD3 antibody












Amino acid sequences of the variable regions of the humanized CD3 antibody
Sequence


Domian
Code
(those in bold and underlined being CDR regions)
No.





Heavy
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDNAK
45


chain

NSLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS



variable
VH1a
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
46


region

NSLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS



(VHs)
VH1b
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
47




NSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS




VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDNSK
48




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS




VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
49




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS




VH2b
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDNSK
50




NTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS




VH2c
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
51




NTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS




VH2d
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
52




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVTWFAYWGQGTLVTVSS




VH2e
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
53




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSYFAYWGQGTLVTVSS




VH2f
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
54




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSFFAYWGQGTLVTVSS




VH2g
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
55




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWLAYWGQGTLVTVSS




VH2h
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
56




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWVAYWGQGTLVTVSS




VH2i
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
57




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWIAYWGQGTLVTVSS




VH2j
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
58




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWAAYWGQGTLVTVSS




VH2k
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
59




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWYAYWGQGTLVTVSS




VH2l
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
60




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFVYWGQGTLVTVSS




VH2m
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
61




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFLYWGQGTLVTVSS




VH2n
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSK
62




NTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFIYWGQGTLVTVSS






Light
VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
63


chain 

QPEDFAVYYCALWYSNLWVFGGGTKVEIK



variable
VL3a
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPKGLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
64


region

QPEDFAVYYCALWYSNLWVFGGGTKVEIK



(VLs)
VL3b
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKLLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
65




QPEDFAVYYCALWYSNLWVFGGGTKVEIK




VL3c
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKSLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
66




QPEDFAVYYCALWYSNLWVFGGGTKVEIK




VL3d
EIVMTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPKGLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
67




QPEDFAVYYCALWYSNLWVFGGGTKVEIK




VL3e
EIVMTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKLLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
68




QPEDFAVYYCALWYSNLWVFGGGTKVEIK




VL3f
EIVMTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKSLIGGTNKRAPGVPARFSGSLSGTDATLTISSL
69




QPEDFAVYYCALWYSNLWVFGGGTKVEIK




VL4
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRALIYGTNKRAPWTPARFSGSLLGGKAALTLS
70




GVQPEDEAEYYCALWYSNLWVFGGGTKLTVL




VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLS
71




GVQPEDEAEYYCALWYSNLWVFGGGTKVEIK




VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGGKAALTLS
72




GVQPEDEAEYYCALWYSNLWVFGGGTKVEIK




VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRALIGGTNKRAPGVPARFSGSLLGGKAALTLS
73




GVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Note:


VHs and VLs may be paired up arbitrarily.






Antibody Sequence Information


(1) Antibodies of Targeted Immune Cell Antigens









TABLE 3







Sequences of the variable regions of existing anti-CD3 antibodies









Amino acid sequences of the variable regions of anti-CD3 antibodies (those in bold


Antibody code
and underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHs, VHs1 or VHs2
No.
VLs, VLs1 or VLs2
No.





SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMN
74
QAVVTQESALTTSPGETVTLTCRSSTGAVTT
75


(WO2007042261
WVRQAPGKGLEWVARIRSKYNNYATYYADSVKD



SNYAN
WVQEKPDHLFTGLIGGTNKRAPGVP




A2)
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGN

ARFSGSLIGDKAALTITGAQTEDEAIYFCAL






FGNSYVSWFAY
WGQGTLVTVSS




WYSNLWV
FGGGTKLTVL







L2K
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTM
76
DIQLTQSPAIMSASPGEKVTMTCRASSSVSY
77


(U.S. Pat. No.


H
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDK




MN
WYQQKSGTSPKRWIYDTSKVASGVPYR




7,112,324)
ATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD

FSGSGSGTSYSLTISSMEAEDAATYYCQQWS






HYCLDY
WGQGTTLTVSS




SNPLT
FGAGTKLELK







DiL2K
DVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTM
78
DIVLTQSPATLSLSPGERATLSCRASQSVSYM
79


(U.S. Pat. No.


H
WVRQAPGQGLEWIGYINPSRGYTNYADSVKGRF




N
WYQQKPGKAPKRWIYDTSKVASGVPARFS




8,076,459)
TITTDKSTSTAYMELSSLRSEDTATYYCARYYDDHY

GSGSGTDYSLTINSLEAEDAATYYCQQWSSN






CLDY
WGQGTTVTVSS




PLT
FGGGTKVEIK







OKT3
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTM
80
QIVLTQSPAIMSASPGEKVTMTCSASSSVSY
81





H
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDK




MN
WYQQKSGTSPKRWIYDTSKLASGVPAH





ATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDD

FRGSGSGTSYSLTISGMEAEDAATYYCQQW






HYCLDY
WGQGTTLTVSS




SSNPFT
FGSGTKLEIN







AbII
QVQLQQSGAELARPGASVKMSCKASGYTFTRSTM
82
QVVLTQSPAIMSAFPGEKVTMTCSASSSVSY
83


(U.S. Pat. No.


H
WVKQRPGQGLEWIGYINPSSAYTNYNQKFKDKA




MN
WYQQKSGTSPKRWIYDSSKLASGVPARF




8,236,308)
TLTADKSSSTAYMQLSSLTSEDSAVYYCASPQVHY

SGSGSGTSYSLTISSMETEDAATYYCQQWSR






DYNGFPY
WGQGTLVTVSA




NPPT
FGGGTKLQIT







UCTH1
EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMN
84
DIQMTQTTSSLSASLGDRVTISCRASQDIRN
85



WVKQSHGKNLEWMGLINPYKGVSTYNQKFKDK



YLN
WYQQKPDGTVKLLIYYTSRLHSGVPSK





ATLTVDKSSSTAYMELLSLTSEDSAVYYCARSGYYG

FSGSGSGTDYSLTISNLEQEDIATYFCQQGNT






DSDWYFDV
WGAGTTVTVSS




LPWT
FAGGTKLEIK







CD3 antibody 1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMN
86
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTT
87


(U.S. Pat. No.
WVRQAPGKGLEWVARIRSKYNNYATYYADSVKD



SNYAN
WVQQKPGQAPRGLIGGTNKRAPGTP




8,846,042B2)
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGN

ARFSGSLLGGKAALTLSGVQPEDEAEYYCAL






FGNSYVS
WFAYWGQGTMVTVSS




WYSNLWV
FGGGTKLTVL







CD3 antibody 2
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMN
88
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTT
89


(U.S. Pat. No.
WVRQAPGKGLEWVGRIRSKYNNYATYYADSVKG



SNYAN
WVQQKPGQAPRGLIGGTNKRAPGV




9,650,446B2)
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGN

PARFSGSLLGGKAALTLSGAQPEDEAEYYCA






FGNSYVSWFAY
WGQGTLVTVSS




LWYSNLWV
FGGGTKLEIK










(2) Antibodies Targeting Tumor Antigens or Other Antigens









TABLE 4







Sequences of the variable regions of anti-CD38 antibodies








Antibody
Amino acid sequences of the variable regions of anti-CD38 antibodies (those in bold and


code
underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHm
No.
VLm
No.





Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMS
90
EIVLTQSPATLSLSPGERATLSCRASQSVSSYL
91


(U.S. Pat. No.
WVRQAPGKGLEWVSAISGSGGGTYYADSVKGRF



AW
YQQKPGQAPRLLIYDASNRATGIPARFSG




9,040,050)
TISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILW

SGSGTDFTLTISSLEPEDFAVYYCQQRSNWP






FGEPVFDY
WGQGTLVTVSS




PT
FGQGTKVEIK







MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYM
92
DIELTQPPSVSVAPGQTARISCSGDNLRHYY
93


(U.S. Pat. No.


N
WVRQAPGKGLEWVSGISGDPSNTYYADSVKGR




VYW
YQQKPGQAPVLVIYGDSKRPSGIPERFS




8,088,896)
FTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPL

GSNSGNTATLTISGTQAEDEADYYCQTYTG






VYTGFAY
WGQGTLVTVSS




GASLV
FGGGTKLTVL







SAR
QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWM
94
DIVMTQSHLSMSTSLGDPVSITCKASQDVST
95


(U.S. Pat. No.


Q
WVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGK




VVA
WYQQKPGQSPRRLIYSASYRYIGVPDRF




8,153,765)
ATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYY

TGSGAGTDFTFTISSVQAEDLAVYYCQQHYS






GSNSLDY
WGQGTSVTVSS




PPYT
FGGGTKLEIK







2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFS
96
DIQMTQSPSSLSASVGDRVTITCRASQGISS
97


(U.S. Pat. No.
WVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRV



WLA
WYQQKPEKAPKSLIYAASSLQSGVPSR




9,040,050)
TITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAAL

FSGSGSGTDFTLTISSLQPEDFATYYCQQYNS






GPFDY
WGQGTLVTVSS




YPRT
FGQGTKVEIK

















TABLE 5







Sequences of the variable regions of anti-BCMA antibodies









Amino acid sequences of the variable regions of anti-BCMA antibodies (those in bold


Antibody code
and underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHm
No.
VLm
No.














B50
QVQLVQSGAEVKKPGASVKVSCKASGYSFPDYYIN
98
DIVMTQTPLSLSVTPGQPASISCKSSQSLVHS
99


(U.S. Pat. No.
WVRQAPGQGLEWMGWIYFASGNSEYNQKFTGRV



NGNTYLH
WYLQKPGQSPQLLIYKVSNRFSG




9,598,500)
TMTRDTSINTAYMELSSLTSEDTAVYFCASLYDYD

VPDRFSGSGSGTDFTLKISRVEAEDVGIYYCS






WYFDV
WGQGTMVTVSS




QSSIYPWT
FGQGTKLEIK







B140153
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS
100
LPVLTQPPSASGTPGQRVTISCSGRSSNIGSNS
101


(WO2016090320
WVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTI

VNWYRQLPGAAPKLLIYSNNQRPPGVPVRF



A1)
TADKSTSTAYMELSSLRSEDTAVYYCARGGYYSHD

SGSKSGTSASLAISGLQSEDEATYYCATWDD






MWSED
WGQGTLVTVSS




NLNVHYV
FGTGTKVTVLG







B140174
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIG
102
SYELTQPPSASGTPGQRVTMSCSGTSSNIGSH
103


(WO2016090320
WVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVT



S
VNWYQQLPGTAPKLLIYTNNQRPSGVPDR




A1)
ISADKSISTAYLQWSSLKASDTAMYYCARYSGSFD

FSGSKSGTSASLAISGLQSEDEADYYCAAWD






N
WGQGTLVTVSS




GSLNGLV
FGGGTKLTVLG







B69
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFW
104
SYVLTQPPSVSVAPGQTARITCGGNNIGSKS
104


(U.S. Pat. No.


G
WIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTIS




VH
WYQQPPGQAPVVVVYDDSDRPSGIPERF




2,017,051,068
VDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAG

SGNSNGNTATLTISRVEAGDEAVYYCQVWD



A1)


LFDY
WGQGTLVTVSSA




SSSDHVV
FGGGTKLTVL

















TABLE 6







Sequences of the variable regions of anti-PD-Ll antibodies









Amino acid sequences of the variable regions of anti-PD-Ll antibodies (those in bold


Antibody code
and underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHm
No.
VLm
No.





S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAIS
106
EIVLTQSPATLSLSPGERATLSCRASOSVSSYL
107


(U.S. Pat. No.
WVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRV



A
WYQQKPGQAPRLLIYDASNRATGIPARFSG




7,943,743B2)
TITADESTSTAYMELSSLRSEDTAVYFCARKFHFVS

SGSGTDFTLTISSLEPEDFAVYYCQQRSNWP






GSPFGMDV
WGQGTTVTVSS




T
FGQGTKVEIK







Durvalumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIH
108
DIQMTQSPSSLSASVGDRVTITCRASQDVSTA
109


(WO2010077634
WVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFT



VA
WYQQKPGKAPKLLIYSASFLYSGVPSRFS




A1)
ISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPG

GSGSGTDFTLTISSLQPEDFATYYCQQYLYHP






G
FDYWGQGTLVTVSS




AT
FGQGTKVEIK







Avelumab
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMS
110
EIVLTQSPGTLSLSPGERATLSCRASQRVSSS
111


(WO2011066389
WVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRF



YLA
WYQQKPGQAPRLLIYDASSRATGIPDRF




A1)
TISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGW

SGSGSGTDFTLTISRLEPEDFAVYYCQQYGSL






FGELAFDY
WGQGTLVTVSS




PWT
FGQGTKVEIK







BMS-936559
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMM
112
QSALTQPASVSGSPGQSITISCTGTSSDVGGY
113


(WO2013079174
WVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTI



NYVS
WYQQHPGKAPKLMIYDVSNRPSGVS




A1)
SRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTV

NRFSGSKSGNTASLTISGLQAEDEADYYCSS






TTVDY
WGQGTLVTVSS




YTSSSTRV
FGTGTKVTVL

















TABLE 7







Sequences of the variable regions of anti-SLAMF7 antibodies









Amino acid sequences of the variable regions of anti-SLAMF7 antibodies (those in bold


Antibody code
and underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHm
No.
VLm
No.





Elotuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMS
114
DIQMTQSPSSLSASVGDRVTITCKASQDVGI
115



WVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISR



A
VAWYQQKPGKVPKLLIYWASTRHTGVPDR




(WO2004100898
DNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWY

FSGSGSGTDFTLTISSLQPEDVATYYCQQYSS



A2)


FDV
WGQGTLVTVSS




YPYT
FGQGTKVEIK

















TABLE 8







Sequences of the variable regions of anti-CEA antibodies









Amino acid sequences of the variable regions of anti-CEA antibodies (those in bold


Antibody code
and underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHm
No.
VLm
No.





hPR1A3
QVQLVQSGSELKKPGASVKVSCKASGYTFTVFGM
116
DIQMTQSPSSLSASVGDRVTITCKASQNVGT
117


(Cancer


N
WVRQAPGQGLEWMGWINTKTGEATYVEEFKG




NVA
WYQQKPGKAPKLLIYSASYRYSGVPSR




Immunol
RFVFSLDTSVSTAYLQISSLKADDTAVYYCARWDF

FSGSGSGTDFTFTISSLQPEDIATYYCHQYYT



Immunother


YDYVEAMDY
WGQGTTVTVSS




YPLFT
FGQGTKVEIKR




(1999) 47:






299-306)
















TABLE 9







Sequences of the variable regions of anti-luciferase antibodies








Antibody
Amino acid sequences of the variable regions of anti-luciferase antibodies (those in bold


code
and underlined being CDR regions)











(sequence

Sequence

Sequence


source)
VHm
No.
VLm
No.





4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWM
118
DVVMTQTPLSLPVSLGDQASISCRSSQSLVH
119





N
WVRQSPEKGLEWVAQIRNKPYNYETYYSDSVK




SNGNTYLR
WYLQKPGQSPKVLIYKVSNRFS







G
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSY


GVPDRFSGSGSGTDFTLKISRVEAEDLGVYF






YGMDY
WGQGTSVTVSS


CSQSTHVPWTFGGGTKLEIK
















TABLE 35







Sequences of the variable regions of anti-Claudin 18.2 antibodies









Amino acid sequences of the variable regions of anti-luciferase antibodies (those in


Antibody code
bold and underlined being CDR regions)











(sequence

Sequence

Sequence


 source)
VHm
No.
VLm
No.





IMAB362
QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWIN
193
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLN
194


(U.S. Pat. No.
WVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKAT



SGNQKNYLT
WYQQKPGQPPKWYWASTR




20,090,169,547
LTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNS



ES
GVPDRFTGSGSGTDFTLTISSVQAEDLAVY




A1)


FDY
WGQGTTLTVSS


YCQNDYSYPFTFGSGTKLEIK









Sequences of Other Domains


(1) Amino Acid Sequences of Linker Domains









TABLE 10







Amino acid sequences of linkers













Sequence


Domain
Code
Amino acid sequence
No.





Linker
Lin1
GGGGS
120






Lin2
GGGSAAA
121






Lin3
GGGGSAS
122






Lin4
GRPGSGRPGS
123






Lin5
GGGGSGGGGSAS
124






Lin6
GKSSGSGSESKS
125






Lin7
GSTSGSGKSSEGKG
126






Lin8
EPKSSDKTHTSPPS
127






Lin9
GGGGSDKTHTSPPS
128






Lin10
GGGGSGGGGSGGGGS
129






Lin11
GGGGSGGGGSGGGGSAS
130






Lin12
GSTSGSGKSSEGSGSTKG
131






Lin13
GSTSGSGKPGSGEGSTKG
132






Lin14
GGGGSGGGGSGGGGSGGGGS
133






Lin15
GGGGSGGGGSGGGGSGGGGSGGGGSAS
134






Lin16
GGGGSGGGGSGGGGSGGGGSGGGGSGG
135




GGSAS







Lin17
AGGGSGGGGSGGGGSGGGGSGGGGSGG
136




GGSGGGGSAS







Lin18
AGGGSGGGGSGGGGSGGGGSGGGGSGG
137




GGSGGGGSGGGGSAS







Lin19
AGGGSGGGGSGGGGSGGGGSGGGGSGG
138




GGSGGGGSGGGGSGGGGSAS









(2) Amino Acid Sequences of Hinge Domains









TABLE 11







Amino acid sequences of hinges













Sequence


Domain
Code
Amino acid sequence
No.





Hinge 2/Hinge 4
Hin1
EPKSCDKTHTCP
139





Hinge 1/Hinge 3/
Hin2
EPKSSDKTHTCP
140


Hinge5/Hinge 6
Hin3
GGGGSDKTHTCP
141



Hin4
RGRGSDKTHTCP
142



Hin5
DGDGSDKTHTCP
143



Hin6
GRGRGSDKTHTCP
144



Hin7
GDGDGSDKTHTCP
145



Hin8
RGRGSSDKTHTCP
146



Hin9
DGDGSSDKTHTCP
147









(3) Amino Acid Sequences of CL Domains of Light Chain Constant Regions









TABLE 12







Amino acid sequences of CL













Se-


Do-


quence


main
Code
Amino acid sequence
No.





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP
148




REAKVQWKVDNALQSGNSQESVTEQDSKDSTYS





LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK





SFNRGEC







Lc2
GQPKANPTVTLFPPSSEELQANKATLVCLISDFY
149




PGAVTVAWKADGSPVKAGVETTKPSKQSNNKYA





ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV





APTECS







Lc3
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFY
150




PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA





ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV





APTECS







Lc4
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFY
151




PGAVTVAWKADSSPAKAGVETTTPSKQSNNKYAA





SSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVA





PTECS







Lc5
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFY
152




PGAVKVAWKADGSPVNTGVETTTPSKQSNNKYA





ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV





APAECS







Lc6
GQPKAAPTVTLFPPSSEELQANKATLVCLISDFY
153




PGAVKVAWKADSSPAKAGVETTTPSKQSNNKYA





ASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTV





APTECS









(4) Amino Acid Sequences of CH1 Domains of Heavy Chain Constant Regions









TABLE 13







Amino acid sequence of CH1













Sequence


Domain
Code
Amino acid sequence
No.





CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLV
154




KDYFPEPVTVSWNSGALTSGVHTFPAVLQ





SSGLYSLSSVVTVPSSSLGTQTYICNVNHK





PSNTKVDKKV









The Fc amino acid numbering follows the Kabat numbering. The “Kabat numbering” refers to a numbering system described by Kabat et al., which is set forth in the United States Department of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). See the table below for specific numbering:









TABLE 14





Fc amino acid numbering based on the Kabat numbering scheme








































216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241





E
P
K
S
C
D
K
T
H
T
C
P
P
C
P
A
P
E
L
L
G
G
P
S
V
F





242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267





L
F
P
P
K
P
K
D
T
L
M
I
S
R
T
P
E
V
T
C
V
V
V
D
V
S





268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293





H
E
D
P
E
V
K
F
N
W
Y
V
D
G
V
E
V
H
N
A
K
T
K
P
R
E





294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319





E
Q
Y
N
S
T
Y
R
V
V
S
V
L
T
V
L
H
Q
D
W
L
N
G
K
E
Y





320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345





K
C
K
V
S
N
K
A
L
P
A
P
I
E
K
T
I
S
K
A
K
G
Q
P
R
E





346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371





P
Q
V
Y
T
L
P
P
S
R
D
E
L
T
K
N
Q
V
S
L
T
C
L
V
K
G





372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397





F
Y
P
S
D
I
A
V
E
W
E
S
N
G
Q
P
E
N
N
Y
K
T
T
P
P
V





398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423





L
D
S
D
G
S
F
F
L
Y
S
K
L
T
V
D
K
S
R
W
Q
Q
G
N
V
F





424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448





S
C
S
V
M
H
E
A
L
H
N
H
Y
T
Q
K
S
L
S
L
S
P
G
K










wherein,


amino acids at position 221-227 are the hinge domain,


amino acids at position 228-340 are the second constant region CH2 domain of heavy chains, and


amino acids at position 341-447 are the third constant region CH3 domain of heavy chains.


An antibody may be modified to improve the heterodimer pairing efficiency. For example, in some aspects, compared with wild-type antibody fragments, the Fc fragment of the monovalent unit heavy chain and/or the Fc fragment of the fusion peptide may comprise one or more substitutions, and knob-into-hole structural pairs are formed between these substitutions. The knob-into-hole configuration is known in the art. See, for example, Ridgway, et al., “‘Knob-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization,” Protein Engineering 9(7):617-21 (1996).


In one aspect, T366 on one CH3 domain is substituted by a larger amino acid residue, such as Tyrosine (Y) or Tryptophan (W). Then, Y407 on the other CH3 domain may be substituted by a smaller amino acid residue, such as Threonine (T), Alanine (A), or Valine (V).









TABLE 15







Fc amino acid substitution combinations form knob-into-hole structural


pairs between monovalent units and single-chain units to improve the


heterodimer pairing efficiency









Combination
Substitution on one
Substitution on the other


No.
CH3
CH3





1
T366W
Y407A


2
T366W
Y407V


3
T366Y
Y407A


4
T366Y
Y407V


5
T366W
T366S, L368A, Y407V









In one aspect, one of the CH3 domains comprises one or more substitutions by amino acid residues having a positive charge under physiological conditions, while the other CH3 domain comprises one or more substitutions by one or more amino acid residues having a negative charge under physiological conditions. In one aspect, the amino acid residue having a positive charge may be Arginine (R), Histidine (H) or Lysine (K). In another aspect, the amino acid residue having a negative charge may be Aspartic acid (D) or Glutamic acid (E). Amino acid residues that may be substituted include, but are not limited to, D356, L368, K392, D399 and K409.









TABLE 16







CH3 amino acid substitution combinations form ionic bonds


between monovalent units and single-chain units to improve the


heterodimer pairing efficiency











Combination
substitution(s) on one
substitution(s) on the other



No.
CH3
CH3







1
D356K D399K
K392D K409D



2
L368R D399K
K392D K409D



3
L368K D399K
K392D K409D



4
L368R D399K
K409D



5
L368K D399K
K409D



6
L368R
K409D



7
L368K
K409D










In one aspect, S354 on one of the CH3 domains is substituted by Cysteine, and Y349 on the other CH3 domain is also substituted by Cysteine. The residues on the two substitution positions form a disulfide bond.









TABLE 17







CH3 amino acid substitution combinations form disulfide bonds


between monovalent units and single-chain units to improve the


heterodimer pairing efficiency









Combination




No.
Substitution on one CH3
Substitution on the other CH3





1
S354C
Y349C









In one aspect, H435 and Y436 on one of the CH3 domains are substituted by Arginine and Phenylalanine, respectively. This substitution leads to significantly weakened binding capability between Fc and protein A, such that the heterodimer and homodimer have different protein A binding activities, and it is easy to separate the two during affinity chromatography.









TABLE 18







One CH3 amino acid substitution leads to weakened


binding capability with protein A










Combination




No.
Substitution on CH3







1
H435R, Y436F

















TABLE 19







CH2 amino acid sequences of different Fc









Combination

Sequence


Code
CH2 amino acid sequences
No.





WT
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
155



EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL




HQDWLNGKEYKCKVSNKALPAPIEKTISKAK






AAG
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
156



EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL




HQDWLNGKEYKCKVSNKALGAPIEKTISKAK






FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
157



EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL




HQDWLNGKEYKCKVSNKALPASIEKTISKAK






N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
158



EDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVL




HQDWLNGKEYKCKVSNKALPAPIEKTISKAK






N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
159



EDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVL




HQDWLNGKEYKCKVSNKALPAPIEKTISKAK






LALA
PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
160



EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL




HQDWLNGKEYKCKVSNKALPAPIEKTISKAK






SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
161



DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH




QDWLNGKEYKCKVSNKGLPAPIEKTISKTK






G2D
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
192



APEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH




QDWLNGKEYKCKVSNKGLPAPIEKTISKTK
















TABLE 20







CH3 amino acid sequences of Fc that form heterodimer











Combination

Sequence

Sequence


Code
CH3-a amino acid sequences
No.
CH3-b amino acid sequences
No.





WT
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
162
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
163



DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS




SPGK

LSLSPGK






W: SAV
GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS
164
GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYP
165



DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS




SPGK

LSLSPGK






CW: CSAV
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
166
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYP
167



SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS




LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS




LSPGK

LSLSPGK






CW: CSAVRF
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYP
168
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYP
169



SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS




LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

KLTVDKSRWQQGNVFSCSVMHEALHNRFTQKS




LSPGK

LSLSPGK






WDD: RKA
GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS
170
GQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYP
171



DIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDL

SDIAVEWESNGQPENNYKTTPPVLKSDGSFFLAS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS




SPGK

LSLSPGK






DD: KK
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
172
GQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYP
173



DIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDL

SDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSL-SL

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS




SPGK

LSLSPGK






SAV: W
GQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPS
174
GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYP
175



DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS




SPGK

LSLSPGK






CSAV: CW
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
176
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY
177



DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK




SPGK

SLSLSPGK






CSAVRF: CW
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPS
178
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFY
179



DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKL

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY




TVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSL

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK




SPGK

SLSLSPGK






RKA: WDD
GQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPS
180
GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYP
181



DIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKL

SDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL


DLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS





SPGK

LSLSPGK






KK: DD
GQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPS
182
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP
183



DIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKL

SDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYS




TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL


DLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS





SPGK

LSLSPGK









Specific sequences of antigens









TABLE 21







Amino acid sequences of tumor antigens









Name (source) of tumor

Sequence


antigen
Amino acid sequence
No.





Human CD38
VPRWRQQWSGPGTTKRFPETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEE
184


(Source: UniProtKB -
DYQPLMKLGTQTVPCNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTS



P28907)
KINYQSCPDWRKDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVH




NLQPEKVQTLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPED




SSCTSEI






Human BCMA
MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNA
185


(Source: UniProtKB -




Q02223)







Human PD-L1
FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSY
186


(Source: UniProtKB -
RQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILV



Q9NZQ7)
VDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIF




YCTFRRLDPEENHTAELVIPELPLAHPPNER






Human SLAMF7
SGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNRERVDFPDG
187


(Source: UniProtKB -
GYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVTMGLQSNKNGTCVTNL



Q9NQ25)
TCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARK




LCEGAADDPDSSM






Human CEA
KLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGR
188


(Source: UniProtKB -
EIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAF



P06731)
TCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDS




VILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNN




SGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWV




NNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPS




YTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHS




RTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSN




GNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCH




SASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPG




LSA






Human Claudin18.2
MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTEC
195


(Source: UniProtKB -
RGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVS



P56856)
GLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIG




GVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDE




VQSYPSKHDYV
















TABLE 22







Amino acid sequences of immune cell antigens









Name




(source)




of immune

Se-


cell

quence


antigen
Amino acid sequence
No.





Human CD3ϵ
DGNEEMGGITQTPYKVSISGTTVILTCPQYP
189


(Source:
GSEILWQHNDKNIGGDEDDKNIGSDEDHLSL



UniProtKB -
KEFSELEQSGYYVCYPRGSKPEDANFYLYLR



P07766)
ARVCENCMEMD









Example 1: Humanized Modified SP34

(1) SP34 Sequence Analysis


The amino acid sequence of the variable region of SP34 heavy chain (SP34VH) is as follows, wherein those in bold and underlined are CDR regions and the others are FR regions:










SP34VH



1                            30                             60                           90


EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR


|←----------FR-H1----------→|CDR-H1|←--FR-H2-→|←-----CDR-H2-----→|←-----------FR-H3-----------→|





                    120  125




HGNFGNSYVSWFAY
WGQGTLVTVSS



←---CDR-H3--→|←--FR-H4--→|






The amino acid sequence of the variable region of SP34 light chain (SP34VL) is as follows, wherein those in bold and underlined are CDR regions and the others are FR regions:










SP34VL



1                            30                            60                             90


QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWV


|←------FR-L1------→|←--CDR-L1--→|←---FR-L2---→|CDR-L2|←------------FR-L3------------→|-CDR-L3-|





         109


FGGGTKLTVL


←-FR-L4-→|






(2) Humanized Modification


(2.1) Modification of the Heavy Chain Variable Region:


All full-human or humanized antibody sequences that have been on the market are analyzed and FR sequences of human heavy chain variable regions are selected as follows:


(i) The first group of FR (VHFR-1), . . . is expressed as CDR:









VHFR-1 (1)


EVQLVESGGGLVQPGGSLRLSCAASGFTFS  . . WVRQAPGKGLEWV





S  . . RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR . . . WGQ





GTLVTVSS






SP34VH and VHFR-1 are compared, the blocks are CDR regions, - indicates regions where the amino acids are identical, and * indicates that amino acids are different at the positions:











SP34VH VHFR-1 Homology analysis


embedded image









According to the homology analysis and conservative substitution of amino acids, the first group of humanized antibody VHs sequences is obtained as follows:















Amino acid sequence of the heavy chain variable region of the humanized
Sequence


Code
CD3 antibody (those in bold and underlined being CDR regions)
No.







VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
45



RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






VH1a
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
46



RFTISRDDSKNSLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






VH1b
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
47



RFTISRDDSKNSLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS









(ii) The second group of FR (VHFR-2), . . . is expressed as CDR:









VHFR-2 (1)


QVQLVESGGGVVQPGRSLRLSCAASGFTFS  . . WVRQAPGKGLEWV





A . . . RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR . . . WGQ





GTLVTVSS






SP34VH and VHFR-2 are aligned, the blocks are CDR regions, - indicates regions where the amino acids are identical, and * indicates that amino acids are different at the positions:











SP34VH VHFR-2 Homology analysis


embedded image









According to the homology analysis and conservative substitution of amino acids, the second group of humanized antibody VHs sequences is obtained as follows:















Amino acid sequence of the heavy chain variable region of the humanized
Sequence


Code
CD3 antibody (those in bold and underlined being CDR regions)
No.







VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
48



RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
49



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






VH2b
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
50



RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






VH2c
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
51



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






VH2d
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
52



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVTWFAYWGQGTLVTVSS






VH2e
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
53



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSYFAYWGQGTLVTVSS






VH2f
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
54



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSFFAYWGQGTLVTVSS






VH2g
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
55



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWLAYWGQGTLVTVSS






VH2h
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
56



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWVAYWGQGTLVTVSS






VH2i
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
57



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWIAYWGQGTLVTVSS






VH2j
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
58



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWAAYWGQGTLVTVSS






VH2k
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
59



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWYAYWGQGTLVTVSS






VH2l
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
60



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFVYWGQGTLVTVSS






VH2m
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
61



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFLYWGQGTLVTVSS






VH2n
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD
62



RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFIYWGQGTLVTVSS









(2.2) Modification of the Light Chain Variable Region:


The selected human FR sequences are as follows:


(i) Light chain variable region sequences of all full-human or humanized antibody sequences that have been on the market are analyzed and the first group of FR (VLFR-1), . . . is expressed as CDR:









VLFR-1 (1)


EIVLTQSPGTLSLSPGERATLSC . . . WYQQKPGQAPRLLIY . .





. GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ . . . FGQGTKV





EIK






SP34VL and VLFR-1 are aligned, the blocks are CDR regions, - indicates regions where the amino acids are identical, and * indicates that amino acids are different at the positions:











SP34VL VLFR-1 Homology analysis


embedded image









According to the homology analysis and conservative substitution of amino acids, the first group of humanized antibody VLs sequences is obtained as follows:















Amino acid sequence of the light chain variable region of the humanized
Sequence


Code
CD3 antibody (those in bold and underlined being CDR regions)
No.







VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP
63



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK






VL3a
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPKGLIGGTNKRAP
64



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK






VL3b
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKLLIGGTNKRAP
65



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK






VL3c
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKSLIGGTNKRAP
66



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK






VL3d
EIVMTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPKGLIGGTNKRAP
67



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK






VL3e
EIVMTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKLLIGGTNKRAP
68



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK






VL3f
EIVMTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGKAPKSLIGGTNKRAP
69



GVPARFSGSLSGTDATLTISSLQPEDFAVYYCALWYSNWVFGGGTKVEIK









(ii) NCBI-IgBlast is used to search for antibody light chain variable regions that are highly homologous with FR of SP34VL, and the second group of FR (VHFR-2), . . . is expressed as CDR:









VLFR-2 (1)


QTVVTQEPSLTVSPGGTVTLTC . . . WFQQKPGQAPRALIY . . .





WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC . . . FGGGTKVEIK






SP34VL and VLFR-2 are aligned, the blocks are CDR regions, - indicates regions where the amino acids are identical, and * indicates that amino acids are different at the positions:











SP34VL VLFR-2 Homology analysis


embedded image









According to the homology analysis and conservative substitution of amino acids, the second group of humanized antibody VLs sequences is obtained as follows:















Amino acid sequence of the heavy chain variable region of the humanized
Sequence


Code
CD3 antibody (those in bold and underlined being CDR regions)
No.







VL4
QAVVTQEPSLTVFVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRALIYGTNKRAP
70



WTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL






VL5
QTVVTQEPSLTVFVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP
71



GVPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK






VL5a
QTVVTQEPSLTVFVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAP
72



GVPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK






VL5b
QTVVTQEPSLTVFVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRALIGGTNKRAP
73



GVPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK









Example 2: Humanized Antibody Preparation and Antibody Activity Detection





    • 1. Method for construction of antibody expression plasmids. pcDNA3.1 is used as the vector.

    • (1) Amplify a target fragment DNA by Polymerase Chain Reaction (PCR), the polymerase being DNA polymerase (2× PrimeSTAR Max Premix, TaKaRa, Article No. R405A). Obtain the DNA sequence by performing reverse translation of the amino acid sequence No. 45-73. The obtained PCR product is the target fragment DNA.

    • (2) cleave the vector plasmid with a restriction endonuclease. The restriction endonuclease is, for example, Notl, Nrul, BamHI-HF, or the like.
      • The obtained cleavage product is the cleaved vector DNA.
        • There are two types of vectors: a heavy chain expression vector and a light chain expression vector, wherein the heavy chain expression vector comprises signal peptide and human IGG1 heavy chain constant region DNA sequences, including CH1, hinge, CH2 and CH3, and heavy chain variable region, and the cleavage site is between the 3′ end of the signal peptide and the 5′ end of CH1; and wherein the light chain expression vector comprises signal peptide and human kappa light chain constant region DNA sequences, as well as the light chain variable region, and the cleavage site is between the 3′ end of the signal peptide and the 5′ end of the light chain constant region.

    • (3) Purify the PCR product or digestion product with a DNA purification kit from Tiangen, and see the instructions included inside the kit from Tiangen for specific operation steps. The obtained purification product is the purified target fragment DNA and purified cleaved vector DNA.

    • (4) Recombination of the target fragment with a recombinase (Exnase II, Vazyme, Article No. C112-01).
      • Heavy chain fragments are recombined onto digested heavy chain expression vector DNA, and light chain fragments are recombined onto digested light chain expression vector DNA.

    • (5) Heat shock transformation
      • Take 10 μl recombination product and add the same into 100 μl Trans10 competent cells, gently mix well, place the same in an ice bath for 30 min, place the tube in 42° C. water bath for 60 s without shaking, quickly transfer to the ice bath for 2 min, add 600 μl LB liquid culture medium (containing antibiotics), culture on a shaker at 37° C. for 1 h, take a proper amount of the bacterial solution and coat the same on LB plates containing corresponding antibiotics, place the plates upside down in a 37° C. constant-temperature incubator for overnight culture. Pick single colonies, and send the samples to Wuhan Genecreate for sequencing. Select single colonies that are sequenced to be correct for expanded culture, and perform plasmid maxiprep for transfection experiments on mammal cells. Plasmids obtained from recombination of heavy chain fragments on digested heavy chain expression vector DNA are referred to as heavy chain expression plasmids, and plasmids obtained from recombination of light chain fragments on digested light chain expression vector DNA are referred to as light chain expression plasmids.





1. Antibody Expression Methods


There are two transient transfection expression systems, CHO—S and 293E, which are described in detail below:


(1) CHO-S transient transfection steps (taking a total transfection volume of 100 ml as an example)

    • a) Subculture on the day before cell transfection. For example, CD-CHO may be used for cell subculture, the suspension cell density is adjusted to 1×106 cells/ml, the volume is 90 ml, it is ensured that the cells are in the logarithmic growth phase, and the cell density can reach 2×106 cells/ml for transfection on the second day;
    • b) Overnight culture on a shaker at 37° C., 125 rpm, and 5% CO2;
    • c) On the day of transfection, pre-heat FectoPRO transfection reagent to room temperature and gently mix well;
    • d) Take 10 ml serum-free culture medium, such as opti PRO-SFM, to dilute 50 μg DNA, gently mix well, add the mixture into 100 μl transfection reagent, mix well, and incubate at room temperature for 10 min to form a transfection complex;
    • e) Add the transfection complex into 90 ml of the prepared cells in the logarithmic growth phase, mix well immediately after the addition, and place on a shaker for culture at 37° C., 125 rpm, and 5% CO2;
    • f) At 2 to 4 h after the transfection, add 75 μl transfection booster Fecto PRO® Booster;
    • g) At 18 to 24 h after the transfection, cool down to 32° C. for culture;
    • h) feed at 3, 5, and 7 days after the transfection, the volume of fed culture medium being 3.5% of the total cell volume;
    • i) Harvest the cells when the cell viability is lower than 70%, the expression time being 9 to 13 days.


(2) 293E transient transfection steps (taking a total transfection volume of 20 ml as an example)

    • a) Subculture on the day before cell transfection. For example, FreeStyle™ 293 may be used for cell subculture, the suspension cell density is adjusted to 0.6-0.8×106 cells/ml, the volume is 20 ml, it is ensured that the cells are in the logarithmic growth phase, and the cell density can reach 1.2-1.6×106 cells/ml for transfection on the second day
    • b) Overnight culture on a shaker at 37° C., 125 rpm, and 5% CO2;
    • c) On the day of transfection, pre-heat LPEI to room temperature before use and gently mix well;
    • d) Use 0.67 ml serum-free culture medium, such as FreeStyle™ 293, to dilute 20 μg DNA, and mix well;
    • e) Use 0.67 ml serum-free culture medium, such as FreeStyle™ 293, to dilute 40 μg LPEI, and mix well;
    • f) Add the LPEI diluted in step 5 into DNA diluted in step 4, quickly mix well, and incubate at room temperature for 15 min to form a transfection complex;
    • g) Add the transfection complex in step 6 into 20 ml of the prepared cells in the logarithmic growth phase, mix well immediately after the addition, and place on a shaker for culture at 37° C., 125 rpm, and 5% CO2;
    • h) feed at 1 and 3 days after the transfection, the volume of fed culture medium being 5% of the total cell volume;
    • i) Harvest on the 6th day of expression.


(3) The transfection is co-transfection, which transfects any one kind of light chain expression plasmid and any one kind of heavy chain expression plasmid in equal ratio into the above-mentioned mammal cells, the antibody expressed is a monoclonal antibody having a bivalent symmetric Y-type structure that is consistent with that of natural antibodies. FIG. 2 is a schematic diagram of a primary structure of this structure.


(4) Codes and expression levels (in 293E cells) of monoclonal antibodies are as follows:









TABLE 23







Codes of humanized CD3 monoclonal antibodies














Corre-
Amino acid sequences of heavy

Corre-
Amino acid sequences of light



Anti-
sponding
chain variable regions (those
Se-
sponding
chain variable regions (those
Se-


body
VHs
in bold and underlined being
quence
VLs
in bold and underlined being
quence


code
code
CDR regions)
No.
code
CDR regions)
No.





B8
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63




AMNWVRQAPGKGLEWVARIRSKYNNYATY




AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B9
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63




AMNWVRQAPGKGLEWVARIRSKYNNYATY




AVTTSNYAN
WVQQKPGQAPRGLIGG






YADSVKDRFTISRDNSKNTLYLQMNSLRAEDT




TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B 10
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3a
EIVLTQSPATLSLSPGERATLSCRSSTG
64




AMNWVRQAPGKGLEWVARIRSKYNNYATY




AVTTSNYAN
WVQQKPGQAPKGLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B11
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3b
EIVLTQSPATLSLSPGERATLSCRSSTG
65




AMNWVRQAPGKGLEWVARIRSKYNNYATY




AVTTSNYAN
WVQQKPGKAPKLLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B12
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
66




AMNWVRQAPGKGLEWVARIRSKYNNYATY




AVTTSNYAN
WVQQKPGKAPKSLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B13
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3d
EIVMTQSPATLSLSPGERATLSCRSSTG
67






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPKGLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B14
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3e
EIVMTQSPATLSLSPGERATLSCRSSTG
68






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGKAPKLLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B15
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL3f
EIVMTQSPATLSLSPGERATLSCRSSTG
69






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGKAPKSLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLSGTDATLTISS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


LQPEDFAVYYCALWYSNLWVFGGGT





VSS


KVEIK






B16
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3a
EIVLTQSPATLSLSPGERATLSCRSSTG
64






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPKGLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B17
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3b
EIVLTQSPATLSLSPGERATLSCRSSTG
65






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGKAPKLLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B18
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3c
EIVLTQSPATLSLSPGERATLSCRSSTG
66






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGKAPKSLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B19
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3d
EIVMTQSPATLSLSPGERATLSCRSSTG
67






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPKGLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B20
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3e
EIVMTQSPATLSLSPGERATLSCRSSTG
68






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGKAPKLLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B21
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL3f
EIVMTQSPATLSLSPGERATLSCRSSTG
69






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGKAPKSLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B22
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL4
QAVVTQEPSLTVSPGGTVTLTCRSSTG
70






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRALIYG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
WTPARFSGSLLGGKAALTLS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKLTVL






B23
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL4
QAVVTQEPSLTVSPGGTVTLTCRSSTG
70






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRALIYG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
WTPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKLTVL






B24
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
45
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDNAKNSLYLQMNSLRAED





TNKRAP
GVPARFSGSLLGGKAALTLS






TAVYYCARHGNFGNSYVSWFAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B25
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
48
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B26
VH1a
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
46
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNSLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B27
VH1b
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
47
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNSLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCVRHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B28
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
49
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B29
VH2b
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
50
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCVRHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B30
VH2c
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
51
VL3
EIVLTQSPATLSLSPGERATLSCRSSTG
63






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLSGTDATLTISS






AVYYCVRHGNFGNSYVSWFAYWGQGTLVTV


LQPEDFAVYYCALWYSNLWVFGGGT





SS


KVEIK






B31
VH1a
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
46
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNSLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B32
VH1b
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTY
47
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNSLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCVRHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B33
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
49
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B34
VH2b
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
50
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDNSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCVRHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B35
VH2c
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
51
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTG
71






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCVRHGNFGNSYVSWFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B36
VH2d
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
52
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVTWFAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B37
VH2e
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
53
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSYFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B38
VH2f
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
54
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSFFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B39
VH2g
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
55
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWLAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B40
VH2h
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
56
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWVAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK
72





B41
VH2i
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
57
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG







AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWIAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B42
VH2j
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
58
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWAAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B43
VH2k
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
59
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWYAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B44
VH2l
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
60
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFVYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B45
VH2m
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
61
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFLYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B46
VH2n
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
62
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTG
72






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WFQQKPGQAPRGLIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFIYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B47
VH2d
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
52
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVTWFAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B48
VH2e
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
53
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSYFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B49
VH2f
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
54
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSFFAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B50
VH2g
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
55
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWLAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B51
VH2h
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
56
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWVAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B52
VH2i
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
57
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWIAYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK






B53
VH2j
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
58
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWAAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B54
VH2k
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
59
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWYAYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B55
VH2l
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
60
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFVYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B56
VH2m
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
61
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFLYWGQGTLVT


GVQPEDEAEYYCALWYSNLWVFGG





VSS


GTKVEIK






B57
VH2n
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTY
62
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTG
73






AMN
WVRQAPGKGLEWVARIRSKYNNYATY





AVTTSNYAN
WVQQKPGQAPRALIGG








YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDT





TNKRAP
GVPARFSGSLLGGKAALTLS






AVYYCARHGNFGNSYVSWFIYWGQGTLVTV


GVQPEDEAEYYCALWYSNLWVFGG





SS


GTKVEIK









Monoclonal antibodies of the above B8-B57 have the same heavy chain constant regions and light chain constant regions, and the specific sequences are as follows:









TABLE 24







Constant region sequences of monoclonal antibodies










Constant


Sequence


region
Domain
Amino acid sequence
No.





Heavy
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
154


chain

LGTQTYICNVNHKPSNTKVDKKV



constant
Hinge
EPKSCDKTHTCP
139


regions
CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
155




RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK




CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS
162




RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK






Light
CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
148


chain

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC



constant





regions









The specific sequences of SP34 monoclonal antibody are as follows:















SP34





monoclonal


Sequence


antibody
Domain
Specific sequences (those in bold and underlined being CDR regions)
No.


















SP34
VH
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSV
74


monoclonal



KD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS




antibody
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
154


heavy

VPSSSLGTQTYICNVNHKPSNTKVDKKV



chain
Hinge
EPKSCDKTHTCP
139



CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
155




NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK




CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL





TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
162





SP34
VL
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGSLI
75


monoclonal

GDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL



antibody
CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
148


light

STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC



chain










FIG. 3 illustrates expression levels of humanized CD3 monoclonal antibodies. From the antibody expression levels, it can be seen that monoclonal antibodies, such as B25-B33, B35-B42, B46-B53, B56-B57, and the like, have a transient transfection expression level of more than 15 mg/L.


2. Antibody Purification


Antibody purification is performed mainly through affinity chromatography, specifically:

    • (1) Harvest: centrifuge a cell culture broth for antibody expression at 3000×g for 10 mM, take the supernatant, filter it with a 0.22 μm filter, and store at 4° C. for later use;
    • (2) Affinity chromatography (MabSelect SuRe GE 17-5438-01, taking 18 ml column volume as an example)
      • a) Equilibrate: use the binding buffer (25 mM Tris, pH 7.0-7.4) to equilibrate the column until the UV detector and conductance value become stable or reach baseline, and equilibrate at least 5 column volumes;
      • b) Load: load the filtered supernatant at a flow rate of 5 ml/min;
      • c) wash to equilibrate: use the binding buffer to wash for 5 column volumes;
      • d) Elute: use an elution buffer (50 mM citrate-citric acid, pH 3.4±0.1) to elute samples at a flow rate of 5 ml/min, elute for 5 column volumes, and collect eluting peaks;
      • e) Neutralize: neutralize the eluate with 1 M Tris pH 8.0, and adjust pH of the sample to 6.0±0.1.


The antibody obtained through purification is a monoclonal antibody and has a bivalent symmetric Y-type structure that is consistent with that of natural antibodies.


3. Antibody Activity Detection


In this example, antibody activity detection mainly refers to detection of the binding activity between an antibody and CD3 positive cells.

    • 1) Cell preparation: CD3 positive CIK cells from induced culture/T cells separated from human whole blood are used for CD3 end affinity detection. Take a sufficient amount of cells, centrifuge at 300 g for 5 mM, discard the supernatant, use 1% FBS-PBS to re-suspend the cells, adjust the density to 4×106/ml, take 50 μl for each well, and plate the cells at 2×105 per well. The centrifuge is at 4 degrees and 300×g, centrifuge for 5 min, discard the supernatant, and plating the cells on ice;
    • 2) Antibody addition: according to the experiment design, subject the antibodies to gradient dilution, and perform the antibody dilution on ice. If the initial concentration of antibody dilution is 3000 nM, dilute 3×, and dilute by 11 concentration grades. Add the diluted antibody into the cell wells at 50 μl per well, gently pipette well, and incubate for 2 h at 4 degrees with shaking at 1100 rpm/min;
    • 3) Wash: use 150 μl 1% FBS-PBS to resuspend the cells, centrifuge at 4 degrees and 300×g for 5 min, and discard the supernatant. Repeat the washing once;
    • 4) Secondary antibody incubation: add diluted secondary antibody PE anti-human IgG FC (Biolegend, 409304), the final concentration of the secondary antibody is 8 μg/ml, and the volume is 50 μl/well. At the same time, provide wells only added with the cells and the secondary antibody as control, gently pipette well, and incubate in dark for 1 h at 4 degrees with shaking at 1100 rpm/min;
    • 5) Wash: use 150 μl 1% FBS-PBS to resuspend the cells, centrifuge at 4 degrees and 300×g for 5 min, and discard the supernatant. Repeat the washing once;
    • 6) Fixation: add 200 μl 2% paraformaldehyde into each well to resuspend the cells and fix cells at room temperature for 20 min. Centrifuge at 300×g for 5 min, and discard the supernatant;
    • 7) Cell re-suspension: use 200 μl 1% FBS-PBS to resuspend the cells, centrifuge at 300 g for 5 min, and discard the supernatant;
    • 8) loading for flow cytometry: use 150 μl 1% FBS-PBS to resuspend the cells, and detect on a flow cytometer;
    • 9) Data analysis: use software FlowJo 7.6 of the flow cytometer to analyze data, and use Graphpad Prism 5 to plot graphs to calculate EC50 values.


Detection results of binding activities between the antibodies and human and monkey T cells are shown in FIG. 4. FIG. 4 illustrates binding capabilities of the monoclonal antibodies with the CD3+T cells.


From the detection results of cell binding activities, it can be seen that, compared with sp34 monoclonal antibody, these antibodies all have higher affinity (EC50<100 nM) and can bind with both human and monkey CD3.


Example 3: Multi-Functional Antibody Preparation According to the Present Invention

I. Plasmid Construction Method


Operation steps are the same as those in “1. Method for construction of antibody expression plasmids” in Example 2 of the present application. Specifically, the construction of three plasmids is involved: light chain expression plasmid (pL), heavy chain expression plasmid (pH), and fusion peptide expression plasmid (pF1).


The multi-functional antibody expression method is the same as that in “2. Antibody expression methods” in Example 2 of the present application. During transfection, it is a co-transfection of three plasmids: to express the multi-functional antibody shown in FIG. 1, plasmids pL, pH and pF1 are needed for co-transfection to CHO-S or 293E cells for expression.


The multi-functional antibody according to the present invention consists of three polypeptides:

    • (1) Fusion peptide consisting of heavy chain variable region (VHs), linker1, light chain variable region (VLs), hinge 1, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3-b). See Table 10 for the linker1 sequence, the Hin2-9 sequence in Table 11 is the hinge 1, the CH2 sequence is in Table 19, and the CH3 sequence is the CH3-b sequence in Table 20. Sequences of VHs and VLs are all from the sequences of new humanized SP34 in the present application, and see Table 2 for details.
    • (2) Heavy chain consisting of heavy chain variable region (VHm), heavy chain constant region 2 (CH1), hinge, heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3-a). See Table 13 for the CH1 sequence, the Hin1 sequence in Table 11 is the hinge sequence, the CH2 sequence is consistent with CH2 of the fusion peptide (see Table 19), and the CH3 sequence is the CH3-a sequence in Table 20; CH3-a corresponds, one to one, to CH3-b in Table 20.
    • (3) Light chain consisting of light chain variable region (VLm) and light chain constant region (CL). See Table 12 for the CL sequence;


See FIG. 1B for a schematic diagram of the composition of the multi-functional antibody.


II. Purification Method for the Multi-Functional Antibody:


Antibody purification is performed mainly through affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and molecular sieve, specifically:

    • (1) Harvest: centrifuge a cell culture broth containing expressed antibody at 3000×g for 10 min, take the supernatant, filter with a 0.22 μm filter, and store at 4° C. for later use;
    • (2) Affinity chromatography (MabSelect SuRe GE 17-5438-01, taking 18 ml column volume as an example)
      • a) Equilibrate: use the binding buffer (25 mM Tris, pH 7.0-7.4) to equilibrate the column until the UV detector and conductance value become stable or reach baseline, and equilibrate at least 5 column volumes;
      • b) Load: load the filtered supernatant at a flow rate of 5 ml/min;
      • c) wash to equilibrate: use the binding buffer to wash for 5 column volumes;
      • d) Elute: use an elution buffer (50 mM citric acid, pH 3.4±0.1) to elute the samples at a flow rate of 5 ml/min for 5 column volumes, and collect fractions of elution peaks;
      • e) Neutralize: neutralize the eluate with 1 M Tris pH 8.0, and adjust pH of the sample to 6.0±0.1.
    • (3) Ion exchange chromatography (taking cation exchange chromatography as an example, HiTrap SP-HP GE 17-1151-01, 5 ml column volume)
      • a) Sample preparation: subject the sample for affinity chromatography to microfiltration, dilute the sample with ultra-pure water such that the conductance is lower than 5 mS/cm, and then adjust pH to 6.0±0.1;
      • b) Equilibrate and load: use 5 column volumes of the buffer B (25 mM citric acid+1 M sodium chloride, the conductance should be 80 to 90 mS/cm, and pH is 6.0±0.1) to equilibrate, then further use at least 5 column volumes of the buffer A (25 mM citric acid, the conductance should be lower than 5 mS/cm, and pH is 6.0±0.1) to equilibrate the column until the baselines of conductance, pH, and UV become stable, and then load the sample at a flow rate of 3 ml/min;
      • c) wash to equilibrate: use 5 column volumes of the buffer A to wash at a flow rate of 5 ml/min;
      • d) Elute: 20 column volumes of 0-30% buffer B; 10 column volumes of 100% buffer B, at a flow rate of 3 ml/min throughout the process, collect the eluate in different tubes, and detect the collected eluate.
    • (4) Hydrophobic chromatography (Capto phenyl ImpRes filler GE XK16/20 11.5 cm/23 ml)
      • a) Sample processing: use 5 M sodium chloride to adjust the sample to 1 M sodium chloride, and adjust pH to 6.0;
      • b) Equilibrate and load: first, use 5 column volumes of the buffer A (25 mM Citrate+1 M sodium chloride, and pH is 6.0±0.1) to equilibrate at a flow rate of 5 ml/min; and then load the sample at a flow rate of 3.3 ml/min;
      • c) Wash to equilibrate: use 5 column volumes of the buffer A to wash at a flow rate of 5 ml/min; use 5 column volumes of 10% buffer B (25 mM Citrate, and pH is 6.0±0.1) to wash at a flow rate of 5 ml/min;
      • d) Elute: elute with 90% buffer B at a flow rate of 5 ml/min, collect the eluting peaks in different tubes, and detect the collected eluting peaks;
    • (5) Molecular sieve (HiLoad Superdex 200 pg GE 28989336 26/600)
      • a) Equilibrate and load: use a buffer (20 mM histidine+0.15 M sodium chloride, and pH is 6.0±0.1) to equilibrate for 2 column volumes, and then load the sample at a flow rate of 3 ml/min;
        • Elute: elute with a buffer B for 2 column volumes, collect the eluting peaks in different tubes, and detect the collected eluting peaks.


See the table below for codes of some antibodies that are specifically expressed and amino acid sequences of corresponding antibody variable regions:









TABLE 25







Codes of some multi-functional antibodies and amino acid sequences of variable regions according


to the present invention












Anti-




Se-


body
Poly-



quence


code
peptide
Domain
Code
Amino acid sequences (those in bold and underlined being CDR)
No.















Y101
Fusion
VHs
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS
45



Peptide




VKD
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
63






SLSGTDATLTISSLQPEDFAVYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin4
RGRGSDKTHTCP
142




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y102
Fusion
VHs
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
48



Peptide




SVKD
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




Vls
VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
63






SLSGTDATLTISSLQPEDFAVYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin4
RGRGSDKTHTCP
142




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VL
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y103
Fusion
VHs
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
48



Peptide




SVKD
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin4
RGRGSDKTHTCP
142




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y104
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49



Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
63






SLSGTDATLTISSLQPEDFAVYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin4
RGRGSDKTHTCP
142




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





HingE 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y105
Fusion
VH2
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
49



peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin4
RGRGSDKTHTCP
142




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VL
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADS
45


F8-3
Peptide




VKD
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
63






SLSGTDATLTISSLQPEDFAVYYCALWYSNLWVFGGGTKVEIK





Hinge1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
90



chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
48


F8-4
Peptide




SVKD
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL3
EIVLTQSPATLSLSPGERATLSCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
63






SLSGTDATLTISSLQPEDFAVYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
90



chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
91



chain


DFTLTISSLEPEDFAVYYCQQRRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


F8-5
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FEX
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
90



chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2c
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
51


F8-6
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
90



chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VH2
Vh2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


F8-7
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
158






QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
90



chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297A
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
158






QYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


F8-8
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
90



chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
49


F8-9
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2j
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
58


F8-10
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWAAYWGQGTLVTVSS






Linker1
Lin10 
GGGGSGGGGSGGGGS
129




VLs
Vl5a
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPGVPARFSG
72






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge1
Hin6
GRGRGSDKTHTCP
144




CH2
G2D
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQ
192






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
G2D
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQ
192






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VL
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH21
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
60


F8-11
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFVYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPGVPARFSG
72






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VH2
VH21
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
60


F8-12
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFVYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRALIGGTNKRAPGVPARFSG
73






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



Chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2k
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
59


F8-13
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWYAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPGVPARFSG
72






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2m
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
61


F8-14
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFLYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRALIGGTNKRAPGVPARFSG
73






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2n
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
62


F8-15
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFIYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5b
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRALIGGTNKRAPGVPARFSG
73






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
2F5
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQ
96



chain




G
RVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
2F5
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGS
97



chain


GTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
49


F9-7
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSV
92



chain




KG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSG
93



chain


NTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVL





CL
Lc3
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA
150






ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS






Y150-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


F9-11
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSV
92



chain




KG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSG
93



chain


NTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVL





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


F9-12
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin6
GRGRGSDKTHTCP
144




CH2
G2D
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQ
192






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
SAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSV
92



chain




KG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
G2D
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEAPEVQFNWYVDGVEVHNAKTKPREEQ
192






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VL
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSG
93



chain


NTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVL





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






MS-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
49


hCD3-
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS




IC15

Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Ms-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


hCD3-
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS




IC16

Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






MS-
Fusion
VHs
VH2a
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
49


hCD3-
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS




IC17

Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
71






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Ms-
Fusion
VHs
VH2j
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVAD
58


hCD3-
Peptide




SVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWAAYWGQGTLVTVSS




IC18

Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
VL5a
QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPGVPARFSG
72






SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKVEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
SG2
PSPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
161






FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK







Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC









See FIG. 5 for an expression level of the multi-functional antibody according to the present invention. FIG. 5 illustrates a transient transfection expression level of the multi-functional antibody assembled from the humanized CD3 antibody in CHO cells.


It can be seen from FIG. 5 that multi-functional antibodies assembled from different CD3 antibodies have different transient transfection expression levels in CHO cells. Y102, Y150-F8-3/F8-4/F8-6/F8-7/F8-8/F8-9/F8-10/F9-11/F9-12, and MS-hCD3-IC15/IC16/IC17 have significantly high expression levels, and the antibody expression level is not less than 40 mg/L.


Example 4: Biological Activity Detection of the Multi-Functional Antibody

1. Cell Affinity

    • 1) Cell preparation: CD3 positive T cells separated from human whole blood are used for CD3 terminal affinity detection of multi-functional antibody molecules, and the affinity detection of tumor antigens is conducted on positive tumor cells of the corresponding antigens: for example, CD38 positive MM.1S cells (purchased from the Cell Resources Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences) or RPMI 8226 cells (purchased from the Cell Resources Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences) are used for CD38 antigen detection, H358 cells (purchased from the Cell Resources Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences) are used for PD-L1 antigen detection, and the like. Take a sufficient amount of cells, centrifuge at 300×g for 5 min, discard the supernatant, use 1% FBS-PBS to re-suspend the cells, adjust the density to 4×106/ml, take 50 μl for each well, and plate the cells at 2×105 per well. The centrifuge at 4 degrees and 300×g for 5 min, discard the supernatant, and plate the cells on ice;
    • 2) Antibody addition: according to the experiment design, subject the antibodies to gradient dilution, and perform the antibody dilution on ice. If the initial concentration of antibody dilution is 3000 nM, dilute 3×, and dilute by 11 concentration grades. Add the diluted antibody into the cell wells at 50 μl per well, gently pipette well, and incubate for 2 h at 4 degrees with shaking at 1100 rpm/min;
    • 3) Wash: use 150 μl 1% FBS-PBS to resuspend the cells, centrifuge at 4 degrees and 300×g for 5 min, and discard the supernatant. Repeat the washing once;
    • 4) Secondary antibody incubation: add diluted secondary antibody PE anti-human IgG FC (Biolegend, 409304), the final concentration of the secondary antibody is 8 ug/ml, and the volume is 50 μl/well. At the same time, provide wells only added with the cells and the secondary antibody as control, gently pipette well, and incubate in dark for 1 h at 4 degrees with shaking at 1100 rpm/min;
    • 5) Wash: use 150 μl 1% FBS-PBS to resuspend the cells, centrifuge at 4 degrees and 300 g for 5 min, and discard the supernatant. Repeat the washing once;
    • 6) Fixation: add 200 μl 2% paraformaldehyde into each well to resuspend the cells and fix cells at room temperature for 20 min. Centrifuge at 300×g for 5 min, and discard the supernatant;
    • 7) Cell re-suspension: use 200 μl 1% FBS-PBS to resuspend the cells, centrifuge at 300×g for 5 min, and discard the supernatant;
    • 8) loading for flow cytometry: use 150 μl 1% FBS-PBS to resuspend the cells, and detect on a flow cytometer;
    • 9) Data analysis: use analysis software FlowJo 7.6 of the flow cytometer to analyze data, and use Graphpad Prism 5 to plot graphs and calculate Kd values.


2. T Cell Activation

    • 1) Take tumor cells in good culture states (non-small cell lung cancer cell H358 purchased from the Cell Resources Center of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences; myeloma cells MC/CAR purchased from ATCC), prepare single cell suspension, and plate the single cell suspension according to 2E4/well cell number into a 96-well plate
    • 2) Isolate PBMC from whole blood of healthy volunteers by density gradient centrifugation, and add PBMC into the tumor cell plate according to an effect/target ratio (E:T) designed in the experiment
    • 3) Perform a series of concentration gradient dilution on the antibodies according to the experimental design, add antibodies of various concentrations
    • 4) Place the 96-well plate in a 37° C. and 5% CO2 incubator for incubation until the detection time. Collect suspended PBMC cells, add corresponding CD3, CD69 detecting antibodies, after 1 h of incubation, wash off excess antibodies, resuspend the cells, determine CD3 and CD69 dual positive cell percent by flow cytometry, i.e., a percent of activated T cells in PBMC induced by the antibodies. Specifically, the calculation is as follows:








CD

3






and






CD

69






dual





positive





cell





percent






(
%
)


=




CD

3






and






CD

69






dual





positive





cell





number


total





number





of






CD

3






positive





cells


×
100

%









      • Use the GraphPad Prism 5 software to perform nonlinear fitting with the double antibody concentration as the X axis, the CD3&CD69)% value as the Y axis (log(agonist) vs. response—Variable slope), and calculate to obtain the T cell activation curve and EC50 value.







3. In Vitro Killing

    • 1) Take a sufficient amount of tumor cells (e.g., H358 cells and MC/CAR cells), and prepare single cell suspensions;
    • 2) CFSE stained tumor cells: centrifuge a certain amount of cell suspension (300×g, 5 min), and discard the supernatant; add 2 ml CFSE solution prepared with PBS, which has a final CFSE concentration of 5 μM; place the cells in a 5% CO2, 37° C. incubator, and incubate for 15 min; take out the cells, wash with PBS, centrifuge (300×g, 5 min), discard the supernatant, repeat washing for three times, use a complete medium to resuspend the cells, and take the suspension for cell count;
    • 3) plating the tumor cells: use the complete medium to resuspend the cell to a density of 2×105/ml, and add the same into a 96-well plate according to 2×104 cells/well, i.e., 100 μl/well;
    • 4) Add effector cell PBMC (isolated from human whole blood): resuspend the effector cells using the complete medium used by tumor cells in the experiment system, add a corresponding number of the effector cells that is converted according to the effect/target ratio in the experiment design, and add according to 50 μl volume/well;
    • 5) Add the diluted antibody: according to the experiment design, the highest antibody concentration is 10 μg/ml. Since 50 μl of the antibody is added, which is ¼ of the total volume of 200 μl, all the antibodies must be prepared at 4× concentration for addition. Therefore, the antibodies are first diluted to 40 μg/ml, 10× dilution is performed starting from 40 μg/ml with 9 grades, and the volume is 50 μl/well;
    • 6) Observe the 96-well plate under a microscope, ensuring that the cells are evenly distributed in the culture wells, place the plate in a 5% CO2, 37° C. cell incubator for incubation, and leave it for detection;
    • 7) When the detection time arrives, adherent cells are processed: pipette out the cell supernatant, wash with 30 μl/well PBS, pipette out the washing solution and combine the washing solution with the supernatant that was pipetted out previously; add 30 μl Trypsin/well into the cell wells, and place the plate in the 5% CO2, 37° C. cell incubator for 3-5 min of digestion, add the previously collected supernatant, and pipette cells in each well to form a single cell suspension; suspended cells are processed: pipette for multiple times to mix well;
    • 8) Add PI into each sample at 10-15 min before detection by the flow cytometer (the final concentration is 10 μg/ml), 10 ul/well;
    • 9) Perform detection on the flow cytometer; use the FlowJo software to analyze detection results from the flow cytometer, output the data analysis to Microsoft Excel, use GraphPad for table preparation and analysis, and the cell killing calculation formula is: the percent of CFSE, PI dual positive cells in CFSE positive cells is the mortality rate of target cells.
      • The calculation formula is as follows:







mortality





rate





of





target





cells






(
%
)


=



pI





and





CFSE





dual





positive





cell





numb





er


CFES





positive





cell





number


×
100







    • 10) Calculation of killing of an antibody to tumor cells: according to the calculation formula for the mortality rate of target cells, calculate the mortality rate of target cells under each antibody concentration, plot with the antibody concentration as the X axis and the mortality rate of target cells as the Y axis, and obtain EC50 values using Graphpad Prism 5 as the data analysis software, which is the killing capacity of this antibody.





See Table 28 for specific binding activities of some multi-functional antibodies.


See FIG. 7 and Table 29 for specific T-cell activation capabilities of some multi-functional antibodies.


See FIGS. 8 and 9 and Table 30 and Table 31 for cytotoxicity of some multi-functional antibodies.


Comparative Example 1

I. Alignment of Sequences of Humanized Antibodies and Existing CD3 Antibodies:


The variable region sequence of CD3 antibody 1 is from U.S. Pat. No. 8,846,042B2, wherein the sequence number of the heavy chain variable region in this patent is 44, and the sequence number of the light chain variable region in this patent is 56;


The variable region sequence of CD3 antibody 2 is from U.S. Pat. No. 9,650,446B2, wherein the sequence number of the heavy chain variable region in this patent is 85, and the sequence number of the light chain variable region in this patent is 194.


(1) Alignment of Heavy Chain Variable Regions:













  VH2a CD3 antibody 1 VH CD3 antibody 2 VH


embedded image

        





Homology analysis
(1) *---------*----*-------------*TYAMN-------------*RIRSKYNNYATYYADSVK*---




   |←-----------FR-H1----------→|CDR-H1|←-FR-H2-→|←----CDR-H2---H→|←--










embedded image






                     -----------------------------HGNFGNSYVSYNAY-----*-----    




                     ------------FR-H3----------→|←--CDR-H3--→|←-FR-H4-→|






Between VH2a and CD3 antibody 1 VH, the amino acid sequence similarity is 96.8%, and the difference appears at FR-H1 and FR-H4;


Between VH2a and CD3 antibody 2 VH, the amino acid sequence similarity is 95.2%, and the difference appears at FR-H1, FR-H2 and CDR-H2;


(2) Alignment of Light Chain Variable Regions:











  VL5 CD3 antibody 1 VL CD3 antibody 2 VL


embedded image





Homology analysis
(1) -*--------------------*SSTGAVTTSNYAN---------------GTNKRAP-*----------



   |←------FR-L1-------→|←--CDR-L1--→|←---FR-L2---→|CDR-L2|←---------








embedded image





                                   ---------*----------ALWYSNLWV------****



                                   ----FR-L3--------→|-CDR-L3--|←-FR-H4→|






Between VL5 and CD3 antibody 1 VL, the amino acid sequence similarity is 94.5%, and the difference appears at FR-L1, FR-L3 and FR-L4;


Between VL5 and CD3 antibody 2 VL, the amino acid sequence similarity is 96.3%, and the difference appears at FR-L1, CDR-L1, FR-L3 and FR-L4;


II. Comparison of Biological Activity Between Humanized Antibodies and Monoclonal Antibodies of Existing CD3 Antibodies:


(1) Affinity Detection of Existing CD3 Antibodies









TABLE 26







Codes and amino acid sequences of existing CD3 antibodies











Code of






comparative
Poly-


Sequence


antibody
peptide
Domain
Amino acid sequences (those in bold and underlined being CDR)
No.





SP34
Heavy
VH
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATY
 74


monoclonal
chain



YADSVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSVVFAYWGQGTLVT




antibody


VSS





CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
154





LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge
EPKSCDKTHTCP
139




CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
155





PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
162





FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VL
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPA
 75



chain

RFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL





CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
148





STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CD3Ab1
Heavy
VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATY
 86



chain



YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVS









WFAY
WGQGTMVTVSS






CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
154





LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge
EPKSCDKTHTCP
139




CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
155





PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
162





FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VL
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPA
 87



chain

RFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL





CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
148





STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CD3Ab2
Heavy
VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATY
 88



chain



YADSVKG
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVT







VSS





CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
154





LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge
EPKSCDKTHTCP
139




CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
155





PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
162





FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VL
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPA
 89



chain

RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
148





STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CD3Ab3
Heavy
VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATY
 86



chain



YADSVKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVS









WFA
YWGQGTMVTVSS






CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
154





LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge
EPKSCDKTHTCP
139




CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
155





PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
162





FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VL
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPA
 89



chain

RFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
148





STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC









The preparation method for existing monoclonal antibodies is the same as the antibody preparation method in Examples 2 and 3. See FIG. 6 for detection data regarding cell binding activities. FIG. 6 illustrates the cell affinity of monoclonal antibodies of existing CD3 antibodies.


Comparative Example 2

(4) Codes and Variable Region Amino Acid Sequences of Some Comparative Multi-Functional Antibodies See the Table Below for Details:









TABLE 27







Codes and variable region amino acid sequences of comparative multi-functional antibodies












Code of







comparative
Poly-



Sequence


antibody
peptide
Domain
Code
Amino acid sequences (those in bold and underlined being CDR)
No.





Y106
Fusion
VHs
SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
 91



Peptide




SVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
SP34
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGS
75






LIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL
142




Hinge 1
Hin4
RGRGSDKTHTCP





CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



Chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-F
Fusion
VHs
SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
 74


8-1
Peptide




SVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
SP34
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGS
 75






LIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin7
GDGDGSDKTHTCP
145




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
 90



Chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWEGEPVEDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
 91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






Y150-F
Fusion
VHs
SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
 74


8-2
Peptide




SVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNEGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
SP34
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGS
 75






LIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin7
GDGDGSDKTHTCP
145




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
 90



Chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWEGEPVEDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
 91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC







Fusion
VHs
SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
 74


Y150-F
Peptide




SVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS




9-6

Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
SP34
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGS
 75






LIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin7
GDGDGSDKTHTCP
145




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
MOR
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSV
 92



Chain




KG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
MOR
DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSG
 93



chain


NTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVL





CL
Lc1
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYA
150






ASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS






CT-F1
Fusion
VHs
CD3
EQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG
 87





antibody 1
SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
 90



Chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWEGEPVEDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
 91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CT-F2
Fusion
VHs
CD3
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYAD
 88



Peptide

antibody 2


SVKG
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNEGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
 90



Chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWEGEPVEDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
 91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CT-F3
Fusion
VHs
CD3
EQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
Dara
EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVK
 90



Chain




G
RFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWEGEPVEDYWGQGTLVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
Dara
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
 91



chain


DFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CT-F4
Fusion
VHs
CD3
EQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG
 87





antibody 1
SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



Chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CT-F5
Fusion
VHs
CD3
EQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYAD
 88



Peptide

antibody 2


SVKG
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



Chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






CT-F6
Fusion
VHs
CD3
EQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
S70
QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQ
106



Chain




G
RVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
S70
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT
107



chain


DFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






IC-2
Fusion
VHs
SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
 74



Peptide




SVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
SP34
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGS
 75






LIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin7
GDGDGSDKTHTCP
145




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



Chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






IC-3
Fusion
VHs
SP34
EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD
 74



Peptide




SVKD
RFTISRDDSQSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
SP34
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNKRAPGVPARFSGS
 75






LIGDKAALTITGAQTEDEAIYFCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin7
GDGDGSDKTHTCP
145




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



Chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






IC-4
Fusion
VHs
CD3
EQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYVADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG
 87





antibody 1
SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



Chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






IC-5
Fusion
VHs
CD3
EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYAD
 88



Peptide

antibody 2


SVKG
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



Chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






IC-6
Fusion
VHs
CD3
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
157






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



Chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
167






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
118




CH2
FES
PCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
154






QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
139




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
157



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
166






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC






IC-7
Fusion
VHs
CD3
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS
 86



Peptide

antibody 1


VKD
RFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS






Linker1
Lin10
GGGGSGGGGSGGGGS
129




VLs
CD3
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSG
 89





antibody 2
SLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLEIK





Hinge 1
Hin3
GGGGSDKTHTCP
141




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVS
167




b
CSAV
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Heavy
VHm
4420
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD
118



Chain




SVKG
RFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSS






CH1
CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
154






VTVPSSSLGTQTYICNVNHKPSNTKVDKKV





Hinge 2
Hin1
EPKSCDKTHTCP
139




CH2
N297Q
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
159






QYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3-
CW:
GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
166




a
CSAV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK




Light
VLm
4420
DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDR
119



chain


FSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK





CL
Lc1
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
148






LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC









(2) Cell Affinity Detection for the Multi-Functional Antibodies According to the Present Invention and Comparative Multi-Functional Antibodies


See the table below for affinity detection results for multi-functional antibodies:









TABLE 28







Cell affinity of different multi-functional antibodies














Human
Monkey




Tumor
CD3
CD3




cell
positive
positive




binding
T cell
T cell




EC50
binding
binding




value
EC50
EC50


Notes
Antibody code
(nM)
value (nM)
value (nM)














Multi-
Y102
1.43
453.60
723.37


functional
Y103
1.33
1135.00
NA


antibodies
Y104
1.20
107.30
203.99


according to
Y105
0.95
144.60
168.39


the present
Y150-F8-5
1.53
192.32
290.55


invention
Y150-F8-6
1.24
24.04
29.19



Y150-F8-7
2.69
91.90
142.06



Y150-F8-8
1.76
107.14
158.92



Y150-F8-9
51.86
88.10
94.13



Y150-F8-10
42.71
416.24
439.95



Y150-F8-12
47.88
561.44
606.87



Y150-F8-14
49.85
564.74
597.69



Y150-F9-7
103.16
426.46
422.11



Y150-F9-11
105.60
437.18
461.05



MS-hCD3-IC15
NA
193.76
377.81



MS-hCD3-IC16
NA
170.02
294.63



MS-hCD3-IC17
NA
121.89
221.79



MS-hCD3-IC18
NA
581.70
680.27


Comparative
Y106
1.48
3.60
6.89


multi-
Y150-F8-1
2.91
101.40
151.79


functional
Y150-F8-2
2.39
85.50
101.88


antibodies
Y150-F9-6
27.1
111.33
130.5



CT-F1
3.47
176.64
346.56



CT-F2
3.21
125.54
173.53



CT-F3
2.17
170.88
229.03



CT-F4
1.48
187.18
326.21



CT-F5
1.38
148.45
189.52



CT-F6
1.33
156.42
240.32



IC-2
NA
171.91
311.81



IC-3
NA
155.22
285.07



IC-4
NA
197.27
296.13



IC-5
NA
122.13
160.60



IC-6
NA
118.00
153.82









Table 28 shows that, after the CD3 humanized antibodies according to the present invention are combined into multi-functional antibodies with various CD38 monoclonal antibodies, the affinity at two ends is affected to various degrees.


(3) Detection of T-Cell Activation Level of the Multi-Functional Antibodies According to the Present Invention

    • See FIG. 7 for detection of human T-cell activation level of the multi-functional antibodies according to the present invention, Y150-F8-9, Y150-F8-10, Y150-F8-12, Y150-F9-11, and MS-hCD3-IC-17 (wherein the amount ratio of effector cells, human PBMC, to the target cells MC/CAR is 5:1, and the processing time is 48 h). FIG. 7 illustrates in vitro T-cell activation capabilities of different multi-functional antibodies according to the present invention.









TABLE 29







EC50 values of in vitro T-cell activation by different


multi-functional antibodies












Antibody
EC50 values of T-cell




code
activation (pM)







Multi-functional
Y150-F8-9
  0.96



antibodies
Y150-F8-10
 14.11



according to the
Y150-F8-12
6531*



present
Y150-F9-11
 83.55



invention







*indicates that plateau has not been reached and the curve fitting is not accurate. The actual EC50 value may be higher than the number in the table.






In FIG. 7, Y150-F8-9 has the strongest T-cell activation capability, and the binding capability of this antibody with two antigens (CD38 and CD3) are both the strongest in the antibodies in the figure; Y150-F8-8 has the T-cell activation capability at a similar level as that of Y150-F8-9 (data not shown); for the antibodies of Y150-F8-9, Y150-F8-10 and Y150-F8-12, the anti-CD38 antibody sequences are completely the same, and the affinities with CD38 are also the same, but the anti-CD3 antibody sequences are not completely consistent, there is a difference of 1 to 3 amino acid point mutations in the affinity with CD3, and there is also significant difference in T-cell activation capability: Y150-F8-9 has the strongest T-cell activation capability, followed by Y150-F8-10, while Y150-F8-12 has a weak T-cell activation capability. Y150-F9-11 has significant T-cell activation capability.


(4) Cytotoxicity Detection for the Multi-Functional Antibodies According to the Present Invention and Comparative Multi-Functional Antibodies


See FIG. 8 for detection results of cytotoxicity of the multi-functional antibodies according to the present invention, Y150-F8-5 and Y150-F8-6, and the comparative multi-functional antibody Y150-F8-1 against multiple myeloma cells MC/CAR (wherein the amount ratio of effector cells, human PBMC, to the target cells MC/CAR is 5:1, and the processing time is 72 h):



FIG. 8 illustrates in vitro cytotoxicity of different multi-functional antibodies against multiple myeloma cells MC/CAR.









TABLE 30







EC50 values of cytotoxicity against tumor cells MC/CAR by different


multi-functional antibodies











Cytotoxicity EC50



Antibody code
(ng/ml)





Multi-functional
Y150-F8-5
0.1


antibodies
Y150-F8-6
0.1


according to the




present




invention




Comparative
Y150-F8-1
3.8


multi-functional




antibody




Control
CD38
Too weak to be calculated


antibodies
monoclonal




antibody




MS-hCD3-IC17
No cytotoxicity









In FIG. 8, the anti-CD3 scFv sequences of Y150-8-5 and Y150-8-6 are both new humanized CD3 antibody sequences. In addition, sequences of other parts of the antibodies (including Fab and Fc) are completely the same as those of Y150-F8-1. The anti-CD3 scFv sequence of Y150-F8-1 is the sequence of a known antibody SP34. It can be seen from the above data that the multi-functional antibodies according to the present invention have similar to or even stronger cytotoxicity against tumor cells. The control antibodies do not have cytotoxicity, indicating that the cytotoxicity of the multi-functional antibodies is generated from bispecific and targeted binding to tumor and immune cells, thereby inducing the immune cells to attack the tumor cells. The CD38 monoclonal antibody is the CD38 antibody drug DARZALEX® that has been marketed.


See FIG. 9 for detection results of cytotoxicity of the multi-functional antibody Y105 according to the present invention against lung cancer cells H358 (wherein the amount ratio of effector cells, human PBMC, to the target cells MC/CAR is 10:1, and the processing time is 48 h).



FIG. 9 illustrates in vitro cytotoxicity of different multi-functional antibodies against lung cancer cells H358.









TABLE 31







EC50 values of cytotoxicity against tumor cells H358 by


different multi-functional antibodies













Cytotoxicity EC50




Antibody code
(ng/ml )















Multi-functional
Y105
1.8



antibodies





according to the





present





invention





Comparative
Y106
1.1



multi-functional





antibody





Control
S70
No cytotoxicity



antibodies
monoclonal





antibody





MS-hCD3-IC15
171.8










In FIG. 9, the anti-CD3 scFv sequence of Y105 is a new humanized CD3 antibody sequence. In addition, sequences of other parts of the antibody (including Fab and Fc) are completely the same as those of Y106. The sequence of anti-CD3 scFv sequence of Y106 is the sequence of a known antibody SP34. It can be seen from the above data that the multi-functional antibodies according to the present invention have similar to or even stronger cytotoxicity against tumor cells. The control antibodies have very weak cytotoxicity, indicating that the cytotoxicity of the multi-functional antibodies is generated from bispecific and targeted binding to tumor and immune cells, thereby inducing the immune cells to attack the tumor cells. S70 mAb is the PD-L1 antibody drug Tecentriq® that has been marketed.


(5) Stability Detection for the Multi-Functional Antibodies According to the Present Invention and Comparative Multi-Functional Antibodies


Experiment I: Detection of Accelerated Thermal Stability at 40° C. with the Following Specific Operation Steps





    • 1. Place a sample in a specific buffer, and the buffer has the following composition: (a) citric acid buffer: 20 mM citric acid, pH 5.5, or (b) histidine buffer: 50 mM histidine, pH 5.5, and adjust the sample concentration to 1 mg/mL;

    • 2. Add the sample to tubes at 500 μL per tube, seal and place the tubes in a 40° C. water bath, and take samples at every 24 h for HPLC-SEC detection. The water batch time is 14 days in total.





See FIGS. 10-16 for detection results.



FIG. 10 illustrates accelerated thermal stability detection at 40° C. of a multi-functional antibody Y105 according to the present invention and comparative antibodies Y106, CT-F4, CT-F5 and CT-F6 in a citric acid buffer system. It can be seen from FIG. 10 that the multi-functional antibody according to the present invention has excellent thermal stability, the antibody does not experience significant changes after 14 days of treatment at 40° C., which is similar to the comparative antibody Y100, but is significantly superior to CT-F4, CT-F5 and CT-F6, wherein Y105 has the same Fab and Fc sequences as all the comparative antibodies, but a different ScFv. Y105 is the CD3 antibody VH2a and VL5 sequences according to the present invention, Y106 is the SP34 antibody sequence, CT-F4 is the CD3 antibody 1 sequence, CT-F5 is the CD3 antibody 2 sequence, and CT-F6 is the CD3 antibody 1VH and CD3 antibody 2VL sequences.



FIG. 11 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 according to the present invention and comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system. In FIG. 11, all the multi-functional antibodies have the same Fab sequence, and Y150-F8-5, Y150-F8-6, Y150-F8-1, CT-F1, CT-F2 and CT-F3 have the same Fc sequence; the CD3 antibody sequences of Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 are VH2a and VL5; the CD3 antibody sequences of Y150-F8-1 and Y150-F8-2 are SP34, the CD3 antibody sequence of CT-F1 is the CD3 antibody 1, the CD3 antibody sequence of CT-F2 is the CD3 antibody 2, and the CD3 antibody sequences of CT-F3 are the CD3 antibody 1VH and CD3 antibody 2VL. It can be seen from the data in the figure that the multi-functional antibodies according to the present invention have thermal stability significantly superior to the thermal stability of the comparative antibodies.



FIG. 12 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 according to the present invention and comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system. In FIG. 12, Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 are multi-functional antibodies according to the present invention, and the CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (F8-10), or VH21 and VL5b (F8-12); Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 are comparative multi-functional antibodies, and the CD3 antibody sequences are SP34 (Y150-F8-1, Y150-F9-6), CD3 antibody 1 (CT-F1), CD3 antibody 2 (CT-F2), and CD3 antibody 1VH and CD3 antibody 2VL (CT-F3), respectively. It can be seen from the data in FIG. 12 that the multi-functional antibodies according to the present invention have thermal stability significantly superior to the thermal stability of the comparative antibodies.



FIG. 13 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 according to the present invention and comparative antibodies IC-2 to IC-7 in a citric acid buffer system. In FIG. 13, all antibodies have exactly the same Fab sequence, wherein MS-hCD3-IC15, IC16, IC17 and IC18 are multi-functional antibodies according to the present invention, and the CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (IC18). IC-2 to IC-7 are comparative multi-functional antibodies, and the CD3 antibody sequences are SP34 (IC-2 and IC-3), CD3 antibody 1 (IC-4), CD3 antibody 2 (IC-5), and CD3 antibody 1VH and CD3 antibody 2VL (IC-6 and IC-7), respectively. It can be seen from the data in the figure that the multi-functional antibodies according to the present invention have thermal stability significantly superior to the thermal stability of the comparative antibodies.



FIG. 14 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 according to the present invention and comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 in a histidine buffer system. In FIG. 14, all multi-functional antibodies have the same Fab sequence, and Y150-F8-5, Y150-F8-6, Y150-F8-1, CT-F1, CT-F2 and CT-F3 have the same Fc sequence; the CD3 antibody sequences of Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 are VH2a and VL5; the CD3 antibody sequences of Y150-F8-1 and Y150-F8-2 are SP34, the CD3 antibody sequence of CT-F1 is the CD3 antibody 1, the CD3 antibody sequence of CT-F2 is the CD3 antibody 2, and the CD3 antibody sequences of CT-F3 are the CD3 antibody 1VH and CD3 antibody 2VL. It can be seen from the data in the figure that the multi-functional antibodies according to the present invention all have excellent thermal stability, while the thermal stability of the comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 is significantly weaker than those of the multi-functional antibodies according to the present invention.



FIG. 15 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 according to the present invention and comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 in a histidine buffer system. In FIG. 15, Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 are multi-functional antibodies according to the present invention, and the CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (F8-10), or VH21 and VL5b (F8-12); Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 are comparative multi-functional antibodies, and the CD3 antibody sequences are SP34 (Y150-F8-1 and Y150-F9-6), CD3 antibody 1 (CT-F1), CD3 antibody 2 (CT-F2), and CD3 antibody 1VH and CD3 antibody 2VL (CT-F3), respectively. It can be seen from the data in FIG. 15 that the multi-functional antibodies according to the present invention have thermal stability significantly superior to the thermal stability of the comparative antibodies.



FIG. 16 illustrates accelerated thermal stability detection at 40° C. of multi-functional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 according to the present invention and comparative antibodies IC-2 to IC-7 in a histidine buffer system. In FIG. 16, all antibodies have exactly the same Fab sequence, wherein MS-hCD3-IC15, IC16, IC17 and IC18 are multi-functional antibodies according to the present invention, and the CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (IC18). IC-2 to IC-7 are comparative multi-functional antibodies, and the CD3 antibody sequences are SP34 (IC-2 and IC-3), CD3 antibody 1 (IC-4), CD3 antibody 2 (IC-5), and CD3 antibody 1VH and CD3 antibody 2VL (IC-6 and IC-7), respectively. It can be seen from the data in the figure that the multi-functional antibodies according to the present invention have thermal stability significantly superior to the thermal stability of the comparative antibodies.


Experiment II: Low-pH Stability Detection

Low-pH stability is also referred to as acid resistance, which investigates whether an antibody molecule can maintain its original state after being treated in an acidic environment for a period of time and then neutralized to physiological conditions. The specific method is as follows: when protein A affinity chromatography is performed on an antibody molecule, the antibody solution eluted from the acid eluting step (using pH 3.5 citric acid buffer) is not neutralized; after staying in the buffer for a period of time, samples are taken at 30 min and 60 min, added 1/10 volume of 1M Tris-HCl (pH8.0) for neutralization, and HPLC-SEC detection is performed on the samples.



FIG. 17 illustrates acid-resistant stability detection of a multi-functional antibody Y105 according to the present invention and comparative antibodies Y106, CT-F4, CT-F5 and CT-F6 in a citric acid buffer system with pH 3.5. It can be seen from FIG. 17 that the multi-functional antibody Y105 according to the present invention has excellent acid resistance, the antibody does not experience significant changes after 60 min treatment at low pH, and its acid resistance is significantly superior to that of Y106, CT-F4, CT-F5 and CT-F6.



FIG. 18 illustrates acid-resistant stability detection of multi-functional antibodies Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 according to the present invention and comparative antibodies Y150-F8-1, Y150-F8-2, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system with pH 3.5. In FIG. 18, all multi-functional antibodies have the same Fab sequence, and Y150-F8-5, Y150-F8-6, Y150-F8-1, CT-F1, CT-F2 and CT-F3 have the same Fc sequence; the CD3 antibody sequences of Y150-F8-5, Y150-F8-6, Y150-F8-7 and Y150-F8-8 are VH2a and VL5; the CD3 antibody sequences of Y150-F8-1 and Y150-F8-2 are SP34, the CD3 antibody sequence of CT-PI is the CD3 antibody 1, the CD3 antibody sequence of CT-F2 is the CD3 antibody 2, and the CD3 antibody sequences of CT-F3 are the CD3 antibody 1VH and CD3 antibody 2VL. It can be seen from the data in the figure that the multi-functional antibodies according to the present invention have acid resistance significantly superior to that of the comparative antibodies.



FIG. 19 illustrates acid-resistant stability detection of multi-functional antibodies Y150-F8-9, F8-10, F8-12, F9-7 and F9-11 according to the present invention and comparative antibodies Y150-F8-1, Y150-F9-6, CT-F1, CT-F2 and CT-F3 in a citric acid buffer system with pH 3.5. In FIG. 19, Y150-F8-9, Y150-F9-7 and Y150-F9-11 are multi-functional antibodies according to the present invention, and the CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (F8-10), or VH21 and VL5b (F8-12); Y150-F8-1, CT-F1, CT-F2 and CT-F3 are comparative multi-functional antibodies, and the CD3 antibody sequences are SP34 (Y150-F8-1 and Y150-F9-6), CD3 antibody 1 (CT-F1), CD3 antibody 2 (CT-F2), and CD3 antibody 1VH and CD3 antibody 2VL (CT-F3), respectively. It can be seen from the data in FIG. 19 that the multi-functional antibodies according to the present invention have acid resistance significantly superior to that of the comparative antibodies.



FIG. 20 illustrates acid-resistant stability detection of multi-functional antibodies MS-hCD3-IC15, IC16, IC17 and IC18 according to the present invention and comparative antibodies IC-2 to IC-7 in a citric acid buffer system with pH 3.5. In FIG. 20, all antibodies have exactly the same Fab sequence, wherein MS-hCD3-IC15, IC16, IC17 and IC18 are multi-functional antibodies according to the present invention, and the CD3 antibody sequences are VH2a and VL5, or VH2j and VL5a (IC18). IC-2 to IC-7 are comparative multi-functional antibodies, and the CD3 antibody sequences are SP34 (IC-2 and IC-3), CD3 antibody 1 (IC-4), CD3 antibody 2 (IC-5), and CD3 antibody 1VH and CD3 antibody 2VL (IC-6 and IC-7), respectively. It can be seen from the data in the figure that the multi-functional antibodies according to the present invention have acid resistance significantly superior to that of the comparative antibodies.


(6) In Vivo Efficacy Experiment Using Multi-Functional Antibodies According to the Present Invention and Comparative Multi-Functional Antibodies


I. Experimental Materials

Cells: Daudi (human multiple myeloma cell line purchased from ATCC), human PBMC;


Mice: NOD/SCID 5-week old, female, Beijing Vital River Laboratory Animal Technology Co., Ltd.


Inoculation method: Daudi cells—Subcutaneously on right back; human PBMC—tail vein, inoculated with Daudi and PBMC cells on DO;


drugs to be tested: (B) Y150-F8-8, (C) Y150-F8-9, (D) Y150-F9-11; Negative control: (A) blank control; (H) MS-hCD3-IC-17; Positive control: (G) CD38mAb (CD38 monoclonal antibody, Darzalex®);


Administration mode: (1) Y150-F8-8, Y150-F8-9, Y150-F9-11 and MS-hCD3-17, administered via tail vein at different doses, respectively, start administration on DO, TIWx2; (2) CD38mAb, administered on D0 and D7, the dose on D0 is 5 mg/kg, the dose on D7 is 15 mg/kg; 6 animals per group;


Weight: measure weight 3 times per week during drug administration and 2 times per week thereafter


Tumor volume: for 9-20 days of the tumor latency period and when the average tumor volume reaches 30 mm3, measure the length and width of the tumor 2 times per week with a monitoring period of about 30 days, or when the average tumor volume of the negative control group reaches 2000 mm3, take photos of all remaining tumor-bearing mice. When the tumor volume of a group gets close to 2000 mm3 or the tumor volume of an individual mice reaches 3000 mm3, end this group.


II. Experimental Results

Experimental results of Y150-F8-8 are shown in FIG. 21A, experimental results of Y150-F8-9 are shown in FIG. 21B, and experimental results of Y150-F9-11 are shown in FIG. 21C.



FIG. 21 illustrates in vivo efficacy and tumor volume monitoring of different multi-functional antibodies in a mouse tumor model, wherein Y150-F8-8 (A), F8-9 (B) and F9-11 (C) are all multi-functional antibodies according to the present invention, the anti-CD3 antibody sequences are all VH2a and VL5, and the anti-CD38 antibody sequences are all different.









TABLE 32







In vivo efficacy of different multi-functional antibodies (29 days after drug


administration)













Dose
TV
T/C


Antibody
Group
(mg/kg)
(mm3)
(%)














Blank control
A
0
2382
100.00


Y150-F8-8
B1
0.05
0
0.00



B2
0.005
344.8
14.48


Y150-F8-9
C1
0.05
0
0.00



C2
0.01
0
0.00



C3
0.002
229.9
9.65


Y150-F9-11
D1
10
0
0.00



D2
3
0
0.00



D3
1
0
0.00



D4
0.3
567.5
23.82


CD38mAb
G
5(D0), 15(D7)
0
0.00


MS-hCD3-IC17
H
0.05
2167
90.99









From FIG. 21, it can be seen that the multi-functional antibodies Y150-F8-8, F8-9 and F9-11 according to the present invention have significant tumor-inhibiting effect, and there is no significant difference when compared with the control monoclonal antibody, all of which can completely inhibit tumors at an effective dose, and the animals do not show significant toxic side effect.


(7) Monkey Toxicity Experiment by Using Multi-Functional Antibodies According to the Present Invention


2F5mAb is an anti-CD38 monoclonal antibody, which can cross bind to human and monkey CD38s, and the specific sequences are:









TABLE 33







2F5 monoclonal antibody sequences


Code of











comp-






arative
Poly-

Amino acid sequences
Sequence


antibody
peptide
Domain
(those in bold and underlied being CDR)
No.





2F5mAb
Heavy
VH
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQA
 96



chain

PGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYM






DLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSS





CH1
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW






NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC
154





NVNHKPSNTKVDKKV





Hinge
EPKSCDKTHTCP
139




CH2
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE






DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
155





LHQDWLNGKEYKCKVSNKALPAPIEKTISKAK





CH3
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW






ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
162





VFSCSVMHEALHNHYTQKSLSLSPGK




Light
VL
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPE
 97



chain

KAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA






TYYCQQYNSYPRTFGQGTKVEIK





CL
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW






KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
148





HKVYACEVTHQGLSSPVTKSFNRGEC









Monkey toxicity experiments are conducted on multi-functional antibodies Y150-F8-10, F9-11, and F9-12, as well as comparative antibody Y150-F9-6 and monoclonal antibody 2F5mAb, respectively, the drugs are administered once via intravenous infusion, and the doses are listed in the table below:









TABLE 34







Drug doses administered in the monkey toxicity experiments










Antibody
Amount
Dose (mg/kg)
Toxic response





Y150-F9-6
2
0.5
Mortality rate 100%


Y150-F8-10
2
1.0
No death


Y150-F9-11
2
1.0
No death


Y150-F9-12
2
1.0
No death









For the Y150-F8-10, F9-11, and F9-12 groups, cell numbers in the lymphocyte subpopulation CD38+CD20+ in monkeys all decrease significantly within 24 h after the drug administration, while for the 2F5mAb group (the dose is 20 mg/kg), the number of cells in the subpopulation decrease to around 30% of the number prior to the drug administration, and the cells are not completely eliminated. These data show that Y150-F8-10, F9-11, and F9-12 molecules have the effect of significantly eliminating CD38+ cells. From Table 34, it can be seen that Y150-F8-10, Y150-F9-11, and Y150-F9-12 have weaker toxicity than that of Y150-F9-6.

Claims
  • 1. An antibody or an antigen binding fragment thereof specifically binding to human CD3, wherein the antibody or the antigen binding fragment comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-62;
  • 2. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region and the light chain variable region respectively comprise the amino acid sequences of: SEQ ID NOs: 46 and 63;SEQ ID NOs: 47 and 63;SEQ ID NOs: 49 and 63;SEQ ID NOs: 50 and 63;SEQ ID NOs: 51 and 63;SEQ ID NOs: 46 and 71;SEQ ID NOs: 47 and 71;SEQ ID NOs: 49 and 71;SEQ ID NOs: 51 and 71;SEQ ID NOs: 52 and 72;SEQ ID NOs: 53 and 72;SEQ ID NOs: 54 and 72;SEQ ID NOs: 55 and 72;SEQ ID NOs: 56 and 72;SEQ ID NOs: 57 and 72;SEQ ID NOs: 58 and 72;SEQ ID NOs: 62 and 72;SEQ ID NOs: 52 and 73;SEQ ID NOs: 53 and 73;SEQ ID NOs: 54 and 73;SEQ ID NOs: 55 and 73;SEQ ID NOs: 56 and 73;SEQ ID NOs: 57 and 73;SEQ ID NOs: 58 and 73;SEQ ID NOs: 61 and 73;SEQ ID NOs: 62 and 73;SEQ ID NOs: 45 and 63;SEQ ID NOs: 48 and 63;SEQ ID NOs: 45 and 64;SEQ ID NOs: 45 and 67;SEQ ID NOs: 48 and 64;SEQ ID NOs: 48 and 67;SEQ ID NOs: 45 and 71;SEQ ID NOs: 48 and 71;SEQ ID NOs: 50 and 71;SEQ ID NOs: 61 and 72;SEQ ID NOs: 60 and 73;SEQ ID NOs: 60 and 72; orSEQ ID NOs: 59 and 72.
  • 3. A polynucleotide, which encodes the antibody or antigen binding fragment thereof according to claim 1.
  • 4. A pharmaceutical composition, comprising the antibody or antigen binding fragment thereof according to claim 1 and a pharmaceutically acceptable carrier or excipient.
  • 5. The antibody or antigen binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 49 and the light chain variable region comprises amino acid sequences of SEQ ID NO: 71.
PCT Information
Filing Document Filing Date Country Kind
PCT/CN2019/075901 2/22/2019 WO
Publishing Document Publishing Date Country Kind
WO2020/168555 8/27/2020 WO A
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Related Publications (1)
Number Date Country
20220041721 A1 Feb 2022 US