Anti-CD16a antibody and use thereof

Abstract

The present invention belongs to the field of biological medicines, and relates to an anti-CD16A antibody and the use thereof. The present invention also relates to a nucleic acid molecule encoding the antibody, a vector and a host cell, and the use thereof in disease treatment.

Description

Anti-cd16a antibodies and uses thereof

[0001] The present application claims priority to Chinese patent application (application number: CN202410468117.6, invention title: Anti-cd16a antibodies and uses thereof) filed on April 16, 2024, and Chinese patent application (application number: CN202411057851.X, invention title: Anti-cd16a antibodies and uses thereof) filed on August 2, 2024, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and relates to anti-cd16a antibodies and uses thereof. The present application also relates to nucleic acid molecules, vectors and host cells encoding the antibodies and antigen-binding fragments thereof, and uses thereof for disease treatment. TECHNICAL BACKGROUND

[0003] NK cells (Natural Killer cells) are an important type of immune cells, which belong to a kind of lymphocytes, and their main function is to recognize and kill cells infected by pathogens and tumor cells. NK cells recognize abnormal cells through various receptors on their surface. Among them, CD16A (also known as FcγRIIIA) is an important receptor that can bind to the Fc region of antibodies. When antibodies bind to antigens on the surface of target cells, CD16A receptors recognize and bind to the Fc region of antibodies, thereby activating NK cells; activated NK cells release various cytotoxic substances, such as perforin and granzyme, to directly kill target cells, and this process is called antibody-dependent cellular cytotoxicity (ADCC). In addition, activated NK cells can also secrete various cytokines, such as interferon gamma (IFN-γ), to enhance immune response. Due to their ability to kill tumor cells, NK cells have important potential in anti-tumor immunotherapy. A common method of anti-tumor therapy is to use monoclonal antibodies, which can bind to antigens on the surface of tumor cells, activate CD16A receptors through the Fc region of antibodies, and thereby enhance the killing effect of NK cells.

[0004] Human CD16 has two subtypes: CD16A and CD16B (FcγRIIIB). CD16A is widely expressed on NK cells and some macrophages. While CD16B is mainly expressed on neutrophils. CD16A can mediate ADCC effect after activation. While the function of CD16B in neutrophils is not completely clear, but it may play a certain role in inflammation and immune regulation.

[0005] In addition, there is an important polymorphism of CD16A, i.e., CD16A-158V / F polymorphism. This is due to a single nucleotide polymorphism site on the CD16A gene, where V represents Valine and F represents Phenylalanine. This polymorphism site affects the affinity and function of the CD16A receptor. The CD16A-158V allele has higher binding affinity to antibodies and thus has stronger ADCC activity. In contrast, the CD16A-158F allele has lower binding affinity to antibodies and thus has weaker ADCC activity. The CD16A-158V / F polymorphism has important clinical significance. Some studies have shown that CD16A-158V allele carriers may have better therapeutic effect when receiving antibody therapy. SUMMARY

[0006] The inventors of the present application have obtained anti-CD16A antibodies. In some embodiments, the anti-CD16A antibodies can bind to human and cynomolgus monkey CD16 proteins with high affinity at the cellular and protein levels, and do not bind to human CD16B proteins. The inventors of the present application have further obtained bispecific antibodies that bind to both CD16A and tumor-associated antigens. In some embodiments, the anti-CD16A monoclonal antibodies and anti-CD16A bispecific antibodies of the present application can effectively promote the killing effect of NK cells on tumor cells and promote the killing ability of PBMCs on tumor cells. In addition, in some embodiments, the anti-CD16A antibodies of the present application also have species cross-binding activity and activation activity to human and monkey CD16A, thereby benefiting the preclinical evaluation research and the like in animal models.

[0007] Antibodies

[0008] In one aspect, the present application provides an anti-CD16A antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), and the anti-CD16A antibody comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 7; and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 8.

[0009] In certain embodiments, the anti-CD16A antibody comprises the HCDR1, HCDR2, and HCDR3 contained in any one of SEQ ID NOs: 9-13 and SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 contained in any one of SEQ ID NOs: 14-19 and SEQ ID NO: 8.

[0010] In certain embodiments, the anti-CD16A antibody of any of the preceding, comprises the HCDR1, HCDR2, and HCDR3 contained in SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 contained in SEQ ID NO: 8.

[0011] In certain embodiments, the HCDR1, HCDR2, and HCDR3 of the anti-CD16A antibody are identical to the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 9, respectively, and the LCDR1, LCDR2, and LCDR3 of the anti-CD16A antibody are identical to the LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 14, respectively.

[0012] In certain embodiments, the anti-CD16A antibody of any of the preceding, the 3 CDRs contained in the VH and / or the 3 CDRs contained in the VL are defined by the Kabat, IMGT, Abm, Contact, or Chothia numbering system. In certain embodiments, the CDRs are defined according to the Kabat numbering system.

[0013] In certain embodiments, the anti-CD16A antibody, wherein:

[0014] the HCDR1 comprises the sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the sequence set forth in SEQ ID NO: 2, the HCDR3 comprises the sequence set forth in SEQ ID NO: 3; and, the LCDR1 comprises the sequence set forth in SEQ ID NO: 4, the LCDR2 comprises the sequence set forth in SEQ ID NO: 5, the LCDR3 comprises the sequence set forth in SEQ ID NO: 6; the CDRs are defined by the Kabat numbering system.

[0015] In certain embodiments, the anti-CD16A antibody, the heavy chain variable region comprises the HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NOs: 1, 2, and 3, respectively, and, the light chain variable region comprises the LCDR1, LCDR2, and LCDR3 set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0016] In certain embodiments, the CDRs of the anti-CD16A antibody are defined by the Rabat numbering system.

[0017] In certain embodiments, the present application provides an anti-CD16A antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0018] (1) the VH of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 7 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 8 or a variant thereof; or

[0019] (2) the VH of the anti-CD16A antibody comprises a sequence set forth in any one of SEQ ID NOs: 9-13 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence set forth in any one of SEQ ID NOs: 14-19 or a variant thereof;

[0020] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it is derived; wherein “at least 80% sequence identity to the sequence from which it is derived” means at least 80% sequence identity to its parent sequence, e.g., “a variant of the sequence set forth in SEQ ID NO: 9” is a sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 9, and so on. In certain embodiments, the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived.

[0021] In certain embodiments, the anti-CD16A antibody comprises a framework region sequence derived from a murine immunoglobulin.

[0022] In certain embodiments, the anti-CD16A antibody of any of the preceding embodiments, the VH of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 7 or a variant thereof, the VL of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 8 or a variant thereof;

[0023] In certain embodiments, the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it was derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions, deletions or additions) compared to the sequence from which it was derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it was derived.

[0024] In certain embodiments, the anti-CD16A antibody of any of the preceding embodiments, the VH of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 7 or a variant thereof, the VL of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 8 or a variant thereof;

[0025] In certain embodiments, the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it was derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions, deletions or additions) compared to the sequence from which it was derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it was derived.

[0026] In certain embodiments, the anti-CD16A antibody of any of the preceding embodiments, the VH of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 7 or a variant thereof, the VL of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 8 or a variant thereof;

[0027] In certain embodiments, the anti-CD16A antibody is humanized, comprising framework region sequences derived from human immunoglobulins.

[0028] In certain embodiments, the anti-CD16A antibody comprises a framework region contained in an amino acid sequence encoded by a human antibody gene. In certain embodiments, the anti-CD16A antibody comprises a heavy chain framework region of a human antibody, and / or, a light chain framework region of a human antibody. In certain embodiments, the heavy chain framework region of a human antibody and / or the light chain framework region of a human antibody can optionally comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10) back mutations from a human-derived residue to a murine-derived residue. In certain embodiments, the heavy chain framework region of a human antibody and / or the light chain framework region of a human antibody comprises no more than 10 amino acid substitutions.

[0029] In certain embodiments, the anti-CD16A antibody of any one of the above, comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0030] (1) the VH of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 9 or a variant thereof, and the VL of the anti-CD16A antibody comprises any one of SEQ ID NOs: 14-19 or a variant thereof;

[0031] (2) the VH of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 10 or a variant thereof, and the VL of the anti-CD16A antibody comprises any one of SEQ ID NOs: 14-19 or a variant thereof;

[0032] (3) the VH of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 11 or a variant thereof, and the VL of the anti-CD16A antibody comprises any one of SEQ ID NOs: 14-19 or a variant thereof;

[0033] (4) the VH of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 12 or a variant thereof, and the VL of the anti-CD16A antibody comprises any one of SEQ ID NOs: 14-19 or a variant thereof; or

[0034] (5) the VH of the anti-CD16A antibody comprises a sequence set forth in SEQ ID NO: 13 or a variant thereof, and the VL of the anti-CD16A antibody comprises any one of SEQ ID NOs: 14-19 or a variant thereof;

[0035] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, deletions, or additions) compared with the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared with the sequence from which it is derived.

[0036] In certain embodiments, the anti-CD16A antibody of any of the preceding embodiments comprises:

[0037] (1) a VH as set forth in SEQ ID NO: 9, or a variant thereof, and a VL as set forth in any of SEQ ID NOs: 14-19, or a variant thereof;

[0038] (2) a VH as set forth in SEQ ID NO: 10, or a variant thereof, and a VL as set forth in any of SEQ ID NOs: 14-19, or a variant thereof;

[0039] (3) a VH as set forth in SEQ ID NO: 11, or a variant thereof, and a VL as set forth in any of SEQ ID NOs: 14-19, or a variant thereof;

[0040] (4) a VH as set forth in SEQ ID NO: 12, or a variant thereof, and a VL as set forth in any of SEQ ID NOs: 14-19, or a variant thereof; or

[0041] (5) a VH as set forth in SEQ ID NO: 13, or a variant thereof, and a VL as set forth in any of SEQ ID NOs: 14-19, or a variant thereof;

[0042] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared with the sequence from which it is derived.

[0043] In certain embodiments, the anti-CD16A antibody of any of the preceding embodiments comprises:

[0044] (1) a VH as set forth in SEQ ID NO: 9, and a VL as set forth in any one of SEQ ID NOs: 14-19;

[0045] (2) a VH as set forth in SEQ ID NO: 10, and a VL as set forth in any one of SEQ ID NOs: 14-19;

[0046] (3) a VH as set forth in SEQ ID NO: 11, and a VL as set forth in any one of SEQ ID NOs: 14-19;

[0047] (4) a VH as set forth in SEQ ID NO: 12, and a VL as set forth in any one of SEQ ID NOs: 14-19; or

[0048] (5) a VH as set forth in SEQ ID NO: 13, and a VL as set forth in any one of SEQ ID NOs: 14-19.

[0049] In certain embodiments, the anti-CD16A antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 14.

[0050] In certain embodiments, the anti-CD16A antibody comprises a constant region derived from a mammalian (e.g., human or murine) immunoglobulin.

[0051] In certain embodiments, the heavy chain of the anti-CD16A antibody comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG, such as IgGl, IgG2, IgG3, or IgG4), and / or the light chain of the anti-CD16A antibody comprises a light chain constant region derived from a human immunoglobulin (e.g., kappa or lambda).

[0052] In certain embodiments, the heavy chain constant region of the anti-CD16A antibody comprises L234F, L235E, and D265A mutations to remove ADCC, ADCP, and CDC effector functions.

[0053] In certain exemplary embodiments, the anti-CD16A antibody comprises a heavy chain constant region set forth in SEQ ID NO: 40, and / or a light chain constant region set forth in SEQ ID NO: 41.

[0054] In certain embodiments, the heavy chain of the anti-CD16A antibody comprises any one of SEQ ID NOs: 44-48 or a variant thereof, and the light chain of the anti-CD16A antibody comprises any one of SEQ ID NOs: 49-54 or a variant thereof.

[0055] In certain embodiments, the anti-CD16A antibody of any one of the preceding embodiments comprises a heavy chain and a light chain, wherein:

[0056] (1) the heavy chain comprises a sequence set forth in SEQ ID NO: 42 or a variant thereof, and the light chain comprises a sequence set forth in SEQ ID NO: 43 or a variant thereof;

[0057] (2) the heavy chain comprises a sequence set forth in SEQ ID NO: 44 or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54 or a variant thereof;

[0058] (3) the heavy chain comprises a sequence set forth in SEQ ID NO: 45 or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54 or a variant thereof;

[0059] (4) the heavy chain comprises a sequence set forth in SEQ ID NO: 46 or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54 or a variant thereof;

[0060] (5) the heavy chain comprises a sequence set forth in SEQ ID NO: 47 or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54 or a variant thereof; or

[0061] (6) the heavy chain comprises a sequence set forth in SEQ ID NO: 48, or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54, or a variant thereof;

[0062] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it is derived. In certain embodiments, the variant has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10) amino acid substitutions, deletions, or additions relative to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions relative to the sequence from which it is derived.

[0063] In certain embodiments, the anti-CD16A antibody of any of the preceding embodiments comprises:

[0064] (1) a heavy chain set forth in SEQ ID NO: 42, or a variant thereof, and a light chain set forth in SEQ ID NO: 43, or a variant thereof;

[0065] (2) a heavy chain set forth in SEQ ID NO: 44, or a variant thereof, and a light chain set forth in any one of SEQ ID NOs: 49-54, or a variant thereof;

[0066] (3) a heavy chain set forth in SEQ ID NO: 45, or a variant thereof, and a light chain set forth in any one of SEQ ID NOs: 49-54, or a variant thereof;

[0067] (4) a heavy chain set forth in SEQ ID NO: 46, or a variant thereof, and a light chain set forth in any one of SEQ ID NOs: 49-54, or a variant thereof;

[0068] (5) a heavy chain set forth in SEQ ID NO: 47, or a variant thereof, and a light chain set forth in any one of SEQ ID NOs: 49-54, or a variant thereof; or

[0069] (6) a heavy chain set forth in SEQ ID NO: 48, or a variant thereof, and a light chain set forth in any one of SEQ ID NOs: 49-54, or a variant thereof;

[0070] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared with the sequence from which it is derived.

[0071] In certain exemplary embodiments, the anti-CD16A antibody of any of the preceding items comprises:

[0072] (1) a heavy chain as set forth in SEQ ID NO: 42, and a light chain as set forth in SEQ ID NO: 43;

[0073] (2) a heavy chain as set forth in SEQ ID NO: 44, and a light chain as set forth in any one of SEQ ID NOs: 49-54;

[0074] (3) a heavy chain as set forth in SEQ ID NO: 45, and a light chain as set forth in any one of SEQ ID NOs: 49-54;

[0075] (4) a heavy chain as set forth in SEQ ID NO: 46, and a light chain as set forth in any one of SEQ ID NOs: 49-54;

[0076] (5) a heavy chain as set forth in SEQ ID NO: 47, and a light chain as set forth in any one of SEQ ID NOs: 49-54; or

[0077] (6) a heavy chain as set forth in SEQ ID NO: 48, and a light chain as set forth in any one of SEQ ID NOs: 49-54.

[0078] In certain exemplary embodiments, the anti-CD16A antibody of any of the preceding items comprises a heavy chain as set forth in SEQ ID NO: 44 and a light chain as set forth in SEQ ID NO: 49.

[0079] In certain exemplary embodiments, an anti-CD 16A antibody is provided that binds to the same epitope as or competes for binding to a human CD 16A antigen with any one of the preceding anti-CD 16A antibodies.

[0080] In certain exemplary embodiments, the anti-CD 16A antibody of any one of the preceding embodiments, wherein the anti-CD 16A antibody is an intact antibody or an antigen binding fragment.

[0081] In certain exemplary embodiments, the antigen binding fragment is selected from the group consisting of a Fab, a Fab', a (Fab')2, a Fv, a disulfide linked Fv, a scFv, a di-scFv, a (scFv)2, and a diabody.

[0082] In certain exemplary embodiments, the anti-CD 16A antibody is a murine, a humanized, or a chimeric antibody.

[0083] In certain exemplary embodiments, the anti-CD 16A antibody has at least one of the following functions:

[0084] (1) specifically binds to a human CD 16A antigen, and / or specifically binds to a cynomolgus monkey CD 16 antigen;

[0085] In certain exemplary embodiments, the anti-CD 16A antibody is capable of binding to CHO-K1 / hCD 16A-F cells with an EC 50 value of less than 20 nM (e.g., less than 20 nM, less than 15 nM, less than 11 nM, less than 10 nM, less than 9 nM, less than 8 nM, less than 7 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, or less), as measured by flow cytometric sorting techniques. 50 In certain exemplary embodiments, the EC 50 value is measured by the method of Example 7 of the present application.

[0086] In certain exemplary embodiments, the anti-CD 16A antibody specifically binds to hCD16A-158V, hCD16A-158F and / or cynoCD16 protein with a KD value of less than 3.00E-08 M (e.g., less than 2.00E-08 M, less than 1.00E-08 M, less than 9.00E-09 M, less than 8.00E-09 M, less than 7.00E-09 M, less than 6.00E-09 M, less than 5.00E-09 M, less than 4.00E-09 M, less than 3.00E-09 M, less than 2.00E-09 M, less than 1.00E-09 M or less), as detected by surface plasmon resonance technology method, in some embodiments, the KD value is detected by the method of Example 8 of the present application;

[0087] (2) does not bind to human CD 16B;

[0088] (3) promotes the killing ability of NK cells against tumor cells;

[0089] (4) promotes the killing ability of PBMCs against tumor cells.

[0090] In certain exemplary embodiments, an anti-CD 16A antibody is provided that binds to the same epitope as, or competes for binding to the human CD 16A antigen with, any of the foregoing anti-CD 16A antibodies.

[0091] Bispecific antibodies or multispecific molecules

[0092] In another aspect, the present application provides a bispecific antibody comprising a first antigen binding domain that specifically binds to a first antigen and a second antigen binding domain that specifically binds to a second antigen, wherein the first antigen is CD 16A and the first antigen binding domain is selected from the antigen binding domain of an anti-CD 16A antibody of the present application.

[0093] In certain embodiments, the second antigen is selected from a tumor associated antigen (TAA).

[0094] In certain embodiments, the TAA is selected from CD19, CD20, CD22, EGFR, BCMA, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CDH17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof.

[0095] In certain embodiments, the TAA is selected from EGFR or BCMA.

[0096] In certain embodiments, the second antigen is EGFR, the second antigen binding domain comprises a VH and a VL, wherein: the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 in the VH set forth in SEQ ID NO: 20, and the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 in the VL set forth in SEQ ID NO: 21; in certain embodiments, the 3 CDRs contained in the VH and / or the 3 CDRs contained in the VL are defined by the Kabat, IMGT, Abm, Contact, or Chothia numbering system. In certain embodiments, the CDRs are defined according to the Kabat numbering system.

[0097] In certain embodiments, the VH of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence set forth in SEQ ID NO: 22, HCDR2 comprises the sequence set forth in SEQ ID NO: 23, and HCDR3 comprises the sequence set forth in SEQ ID NO: 24; and

[0098] the VL of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence set forth in SEQ ID NO: 25, LCDR2 comprises the sequence set forth in SEQ ID NO: 26, and LCDR3 comprises the sequence set forth in SEQ ID NO: 27.

[0099] In certain embodiments, the heavy chain variable region of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 22, 23, and 24, respectively, and the light chain variable region of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 25, 26, and 27, respectively.

[0100] In certain embodiments, the CDRs are defined by the Kabat numbering system.

[0101] In certain embodiments, the VH of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 20, or a variant thereof, and the VL of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 21, or a variant thereof;

[0102] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it is derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived.

[0103] In certain embodiments, the second antigen binding domain comprises a VH set forth in SEQ ID NO: 20, or a variant thereof, and a VL set forth in SEQ ID NO: 21, or a variant thereof.

[0104] In certain embodiments, the second antigen binding domain comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3, and a VL comprising a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1 comprises the sequence set forth in SEQ ID NO: 30, the HCDR2 comprises the sequence set forth in SEQ ID NO: 31, the HCDR3 comprises the sequence set forth in SEQ ID NO: 32, the LCDR1 comprises the sequence set forth in SEQ ID NO: 33, the LCDR2 comprises the sequence set forth in SEQ ID NO: 34, and the LCDR3 comprises the sequence set forth in SEQ ID NO: 35.

[0105] In certain embodiments, the second antigen binding domain comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3, and a VL comprising a LCDR1, a LCDR2, and a LCDR3, wherein the HCDR1 comprises the sequence set forth in SEQ ID NO: 30, the HCDR2 comprises the sequence set forth in SEQ ID NO: 31, the HCDR3 comprises the sequence set forth in SEQ ID NO: 32, the LCDR1 comprises the sequence set forth in SEQ ID NO: 33, the LCDR2 comprises the sequence set forth in SEQ ID NO: 34, and the LCDR3 comprises the sequence set forth in SEQ ID NO: 35.

[0106] In certain embodiments, the second antigen is BCMA, the second antigen binding domain comprises a VH and a VL, wherein: the second antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 in the VH set forth in SEQ ID NO: 28, and the second antigen binding domain comprises a LCDR1, a LCDR2, and a LCDR3 in the VL set forth in SEQ ID NO: 29; in certain embodiments, the 3 CDRs contained in the VH and / or the 3 CDRs contained in the VL are defined by the Kabat, IMGT, Abm, Contact, or Chothia numbering system. In certain embodiments, the CDRs are defined according to the Kabat numbering system.

[0107] In certain embodiments, the VH of the second antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the sequence set forth in SEQ ID NO: 30, the HCDR2 comprises the sequence set forth in SEQ ID NO: 31, and the HCDR3 comprises the sequence set forth in SEQ ID NO: 32; and

[0108] the VL of the second antigen binding domain comprises a LCDR1, a LCDR2, and a LCDR3, wherein the LCDR1 comprises the sequence set forth in SEQ ID NO: 33, the LCDR2 comprises the sequence set forth in SEQ ID NO: 34, and the LCDR3 comprises the sequence set forth in SEQ ID NO: 35.

[0109] In certain embodiments, the heavy chain variable region of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 30, 31, and 32, respectively, and the light chain variable region of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 33, 34, and 35, respectively.

[0110] In certain embodiments, the CDRs are defined by the Kabat numbering system.

[0111] In certain embodiments, the VH of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 28, or a variant thereof, and the VL of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 29, or a variant thereof;

[0112] In certain embodiments, the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it is derived compared to the sequence from which it is derived. In certain embodiments, the variant has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10) amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived.

[0113] In certain embodiments, the second antigen binding domain comprises a VH set forth in SEQ ID NO: 28, or a variant thereof, and a VL set forth in SEQ ID NO: 29, or a variant thereof.

[0114] In certain embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO: 28, and a VL as set forth in SEQ ID NO: 29.

[0115] In certain embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO: 28, and a VL as set forth in SEQ ID NO: 29.

[0116] In certain embodiments, the first and second antigen binding domains are each independently selected from the group consisting of a Fab, a Fab', a (Fab')2, an Fv, a disulfide linked Fv, and an scFv.

[0117] In certain embodiments, the first or second antigen binding domain is murine, fully human, or chimeric.

[0118] In certain embodiments, the first antigen binding domain is a Fab.

[0119] In certain embodiments, the second antigen binding domain is an scFv, the VH of the second antigen binding domain is linked directly or via a peptide linker to the VL of the second antigen binding domain.

[0120] In certain embodiments, the peptide linker is (GmS)n, wherein m, n are each independently an integer from 1-10. In certain embodiments, wherein m, n are each independently 1, 2, 3, 4, 5, or 6.

[0121] In certain embodiments, the bispecific antibody comprises an immunoglobulin Fc.

[0122] In certain embodiments, the immunoglobulin Fc is linked directly or via a peptide linker to the first and second antigen binding domains.

[0123] In certain embodiments, the immunoglobulin Fc is an Fc fragment of human IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0124] In certain embodiments, the peptide linker is (GmS)n, where m, n are each independently an integer from 1-10. In certain embodiments, where m, n are each independently 1, 2, 3, 4, 5, or 6.

[0125] In certain embodiments, the immunoglobulin Fc fragment comprises L234F, L235E, and D265A mutations to remove ADCC, ADCP, and CDC effector functions.

[0126] In certain embodiments, the bispecific antibody is an IgG-scFv format bispecific antibody comprising peptide chains I-A, I-B, I-C, and I-D:

[0127] (1) a peptide chain I-A comprising, in order from N-terminus to C-terminus, a VL and a CL of the first antigen binding domain; optionally, the CL is a human immunoglobulin kappa or lambda light chain;

[0128] (2) a peptide chain I-B comprising, in order from N-terminus to C-terminus, a VH, a CH, a peptide linker 1, a VH of the second antigen binding domain, a peptide linker 2, a VL of the second antigen binding domain; optionally, the CH is a human IgGl, IgG2, IgG3, or IgG4 CH; optionally, the peptide linker 1 and the peptide linker 2 are each independently selected from (GmS)n, where m, n are each independently an integer from 1-10. In certain embodiments, m, n are each independently 1, 2, 3, 4, 5, or 6; in certain embodiments, where m is 4, and n is 2; in certain embodiments, where m is 2, and n is 6;

[0129] (3) a peptide chain I-C identical to the peptide chain I-B; and

[0130] (4) a peptide chain I-D identical to the peptide chain I-A.

[0131] In certain embodiments, a disulfide bond is formed between the peptide chain I-B and the peptide chain I-C; the disulfide bond can further enhance the stability of the bispecific antibody.

[0132] In certain embodiments, (1) the peptide chain I-A and the peptide chain I-D comprise a sequence set forth in SEQ ID NO: 37 or a variant thereof, and the peptide chain I-B and the peptide chain I-C comprise a sequence set forth in SEQ ID NO: 36 or a variant thereof; or

[0133] (2) the peptide chain I-A and the peptide chain I-D comprise a sequence as set forth in SEQ ID NO: 39, or a variant thereof, and the peptide chain I-B and the peptide chain I-C comprise a sequence as set forth in SEQ ID NO: 38, or a variant thereof;

[0134] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it is derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived.

[0135] In certain embodiments, the bispecific antibody comprises:

[0136] (1) a peptide chain I-A and a peptide chain I-D as set forth in SEQ ID NO: 37, or a variant thereof, and a peptide chain I-B and a peptide chain I-C as set forth in SEQ ID NO: 36, or a variant thereof; or

[0137] (2) a peptide chain I-A and a peptide chain I-D as set forth in SEQ ID NO: 39, or a variant thereof, and a peptide chain I-B and a peptide chain I-C as set forth in SEQ ID NO: 38, or a variant thereof;

[0138] wherein the variant has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence from which it is derived. In certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9 or up to 10 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; in certain embodiments, the substitutions are conservative substitutions. In certain embodiments, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived.

[0139] In certain embodiments, the bispecific antibody comprises:

[0140] (1) the peptide chain I-A and I-D as shown in SEQ ID NO: 37, and the peptide chain I-B and I-C as shown in SEQ ID NO: 36; or

[0141] (2) the peptide chain I-A and I-D as shown in SEQ ID NO: 39, and the peptide chain I-B and I-C as shown in SEQ ID NO: 38.

[0142] In certain embodiments, the bispecific antibody comprises 2 polypeptides as shown in SEQ ID NO: 37 and 2 polypeptides as shown in SEQ ID NO: 36; or the bispecific antibody comprises 2 polypeptides as shown in SEQ ID NO: 39 and 2 polypeptides as shown in SEQ ID NO: 38.

[0143] In certain embodiments, the bispecific antibody is a bispecific antibody in the format of scFv-scFv-Fc; in certain embodiments, the bispecific antibody comprises a peptide chain II-A;

[0144] In certain embodiments, the peptide chain II-A comprises, in order from N-terminus to C-terminus: the VH of the second antigen binding domain, a peptide linker 1, the VL of the second antigen binding domain, a peptide linker 2, the VH of the first antigen binding domain, a peptide linker 3, the VL of the first antigen binding domain, a peptide linker 4, and Fc;

[0145] In certain embodiments, the Fc is a human IgGl, IgG2, IgG3, or IgG4 Fc;

[0146] In certain embodiments, the peptide linker 1, the peptide linker 2, the peptide linker 3, and the peptide linker 4 are each independently selected from (GmS)n, wherein m, n are each independently an integer from 1 to 10; in certain embodiments, m, n are each independently 1, 2, 3, 4, 5, or 6; in certain embodiments, wherein m is 4 and n is 2; in certain embodiments, wherein m is 2 and n is 6;

[0147] In certain embodiments, the bispecific antibody comprises 2 peptide chains II-A; in certain embodiments, a disulfide bond is formed between the 2 peptide chains II-A;

[0148] In certain embodiments, the bispecific antibody, the peptide chain II-A comprises a sequence set forth in SEQ ID NO: 55, or a variant thereof having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 55; in certain embodiments, the variant has one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, deletions or additions) compared to the sequence of SEQ ID NO: 55; in certain embodiments, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 55; in certain embodiments, the variant has up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions compared to the sequence of SEQ ID NO: 55;

[0149] In certain embodiments, the bispecific antibody is a bispecific antibody in the format of scFv-VH-CH / VL-CL; in certain embodiments, the bispecific antibody comprises a peptide chain III-A and a peptide chain III-B, wherein: the peptide chain III-A comprises, in order from N-terminus to C-terminus: the VH of the second antigen binding domain, a peptide linker 1, the VL of the second antigen binding domain, a peptide linker 2, the VH of the first antigen binding domain, and CH; the peptide chain III-B comprises, in order from N-terminus to C-terminus: the VL of the first antigen binding domain, and CL; in certain embodiments, the CH is a human IgGl, IgG2, IgG3, or IgG4 CH; optionally, each of the peptide linker 1 and the peptide linker 2 is independently selected from (GmS)n, wherein each of m, n is independently an integer between 1 and 10; in certain embodiments, each of m, n is independently 1, 2, 3, 4, 5, or 6; in certain embodiments, wherein m is 4 and n is 2; in certain embodiments, wherein m is 2 and n is 6;

[0150] In certain embodiments, the bispecific antibody comprises 2 peptide chains III-A and 2 peptide chains III-B; in certain embodiments, a disulfide bond is formed between the 2 peptide chains III-A, and a disulfide bond is formed between III-A and the peptide chain III-B;

[0151] In certain embodiments, the bispecific antibody, the peptide chain III-A comprises a sequence as set forth in SEQ ID NO: 56 or a variant thereof having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 56, and the peptide chain III-B comprises a sequence as set forth in SEQ ID NO: 57 or a variant thereof having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 57; optionally, the variant has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10) amino acid substitutions, deletions, or additions compared with the sequence from which it was derived; in certain embodiments, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 55; optionally, the variant has up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions compared with the sequence from which it was derived; optionally, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 56 and 2 sequences as set forth in SEQ ID NO: 57.

[0152] In another aspect, the present application provides a multispecific molecule comprising an anti-CD16A antibody of the present application or a bispecific antibody of the present application.

[0153] In certain embodiments, the multispecific molecule specifically binds CD16A and additionally specifically binds one or more other targets.

[0154] In certain embodiments, the multispecific molecule further comprises at least one molecule (e.g., a third antibody) having a third binding specificity for a third target.

[0155] Preparation of antibodies

[0156] The antibodies of the present application can be prepared by various methods known in the art, for example, by genetic engineering recombinant technology. For example, a DNA molecule encoding the antibody of the present application is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and then a host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present application is expressed. The antigen-binding fragment of the present application can be obtained by hydrolyzing an intact antibody molecule.

[0157] In another aspect, the present application provides an isolated nucleic acid molecule encoding the anti-CD16A antibody, bispecific antibody, or multispecific molecule of the present application.

[0158] In another aspect, the present application provides a vector (e.g., a cloning vector or an expression vector) comprising the nucleic acid molecule of any one of the above. In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, and the like.

[0159] In another aspect, the present application provides a host cell comprising the nucleic acid molecule or the vector of any one of the above. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells, and the like).

[0160] In another aspect, there is provided a method of producing the anti-CD16A antibody, bispecific antibody, or multispecific molecule of the present application, comprising the steps of:

[0161] culturing the host cell of any one of the above under conditions permitting protein expression, and recovering the anti-CD16A antibody, bispecific antibody, or multispecific molecule from the culture of the cultured host cell.

[0162] Conjugates

[0163] In another aspect, the present application also provides a conjugate comprising the anti-CD16A antibody, bispecific antibody, or multispecific molecule of any one of the above, and a coupling moiety.

[0164] In certain embodiments, the antibody of the present application is conjugated to the coupling moiety, optionally, through a linker.

[0165] In certain embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, examples of which include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and how to select a suitable protein tag (e.g., a purification tag, a detection tag, or a tracking tag) depending on the desired purpose is known to one skilled in the art. In certain exemplary embodiments, the C-terminus of the antibody of the present application is linked to a purification tag.

[0166] In certain embodiments, the conjugated moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable label described herein can be any substance that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein (FITC), rhodamine, tetramethylrhodamine (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium esters), magnetic beads (e.g., Dynabeads®), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin reagents (e.g., streptavidin) modified to bind the above labels. In certain embodiments, such labels can be adapted for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, the detectable label described in any of the above can be linked to the antibody of the present application via linkers of varying lengths to reduce potential steric hindrance. )), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for use with avidin reagents (e.g., streptavidin) modified to bind the above labels. In certain embodiments, such labels can be adapted for use in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). In certain embodiments, the detectable label described in any of the above can be linked to the antibody of the present application via linkers of varying lengths to reduce potential steric hindrance.

[0167] In certain embodiments, the conjugated moiety is selected from a therapeutic agent, such as an antitumor drug or an immunosuppressant.

[0168] In certain embodiments, the conjugated moiety is selected from another biologically active polypeptide.

[0169] In certain embodiments, the conjugate is an antibody drug conjugate (ADC) comprising an anti-CD16A antibody, a bispecific antibody, a multispecific molecule as described in any of the above, and a conjugated moiety consisting of a cytotoxic drug linked to the anti-CD16A antibody, the bispecific antibody, or the multispecific molecule via a linker.

[0170] In certain embodiments, the linker is a non-cleavable linker (e.g., SMCC), a disulfide linker, a hydrazone linker, or a protease-cleavable linker. In certain embodiments, the protease-cleavable linker is selected from a cathepsin B substrate linker (e.g., a dipeptide linker Val-Cit, a dipeptide linker Val-Ala, or a tetrapeptide linker Gly-Gly-Phe-Gly), a pyrophosphorodiesters linker, a PEG linker, a beta-glucuronidase substrate linker, a beta-galactosidase substrate linker, or a sulfatase substrate linker.

[0171] The term "cytotoxic drug" refers to a substance that inhibits or stops cell function and / or causes cell destruction.

[0172] In certain embodiments, the cytotoxic drug is selected from paclitaxel, tubulysins, duostatins, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, tenoposide, camptothecin or analogs or derivatives thereof, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, maytansine or analogs or derivatives thereof, dactinomycin, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, a calicheamicin or analogs or derivatives thereof, an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), an alkylating agent (e.g., mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazin (DTIC), procarbazine, cisplatin and other platinum derivatives (e.g., carboplatin), duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin) or analogs or derivatives of CC-1065), dolastatin, auristatin, pyrrolo[2,1-c][1,4]benzodiazepine PDB, indolinobenzodiazepine (IGN) or an analog thereof, an antibiotic (e.g., dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), an anti-mitotic agent (e.g., microtubulin targeting agents), diphtheria toxin and related molecules (e.g., diphtheria A chain and active fragments and hybrid molecules thereof), ricin (e.g., ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, pseudomonas exotoxin, alorin, saporin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, kuwanon, ranunculin (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, jatropha toxic protein, phytolacca americana inhibitor, gossypol, mitogellin, restrictocin, phenomicin, antimicrobial / lytic peptides (e.g., CLIP, Magainin 2, Melittin, cecropin and P18), ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A and Pseudomonas endotoxin.

[0173] Pharmaceutical composition

[0174] In another aspect, the present application provides a pharmaceutical composition comprising the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, or conjugate according to any one of the above, and one or more pharmaceutically acceptable excipients.

[0175] In certain embodiments, the pharmaceutical composition can further comprise an additional anti-tumor agent and / or an immunosuppressive agent.

[0176] In certain embodiments, the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, or conjugate of the present application and the additional anti-tumor agent and / or immunosuppressive agent can be provided as separate components or as a mixed component in the pharmaceutical composition. Thus, the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, or conjugate of the present application and the additional anti-tumor agent and / or immunosuppressive agent can be administered simultaneously, separately or sequentially.

[0177] In certain embodiments, the one or more pharmaceutically acceptable excipients can comprise a sterile injectable liquid, such as an aqueous or non-aqueous suspension or solution. In certain exemplary embodiments, such sterile injectable liquid is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), stabilizers, surfactants, buffer solutions, and any combination thereof.

[0178] The pharmaceutical composition of the present application can comprise a "therapeutically effective amount" of the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, or conjugate of the present application. A "therapeutically effective amount" means an amount sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from the disease. The therapeutically effective amount can vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, body weight and gender, the mode of administration of the drug, and other therapies being administered concurrently, and the like.

[0179] Therapeutic applications

[0180] In another aspect, the present application provides a method of preventing and / or treating a tumor in a subject, comprising administering to a subject in need thereof an anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, conjugate of the present application, or a pharmaceutical composition of the present application. The present application also relates to the use of the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition for the manufacture of a medicament for preventing and / or treating a tumor in a subject. The present application also relates to an anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition as described above for use as a medicament for preventing and / or treating a tumor in a subject.

[0181] In certain embodiments, the tumor is selected from the group consisting of skin cancer, non-Hodgkin's lymphoma, renal cell carcinoma, lung cancer, brain glioma, gastric cancer, head and neck cancer, large intestine cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous carcinoma.

[0182] In certain embodiments, the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is used alone or in combination with another anti-tumor drug and / or immunosuppressant.

[0183] In certain embodiments, the subject is a mammal, such as a human.

[0184] The anti-CD16A antibodies, bispecific antibodies, multispecific molecules, nucleic acid molecules, vectors, host cells, conjugates, or pharmaceutical compositions of the present application can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0185] A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by incorporating the anti-CD16A antibodies of the present application in the required amount in an appropriate solvent with one or more of the other ingredients enumerated above, as desired. It is a further optional step to sterilize the solution. Moreover, sterile solutions can be prepared by incorporating the anti-CD16A antibodies of the present application in the required amount in the appropriate solvent with one or more of the other ingredients, as desired, followed by filtered sterilization. Alternatively, sterile solid compositions can be prepared by

[0186] The anti-CD16A antibodies, bispecific antibodies, multispecific molecules, nucleic acid molecules, vectors, host cells, conjugates, or pharmaceutical compositions of the present application can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracerebrospinal, inguinal, intravesical, local (e.g., powder, salve, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In certain embodiments, the anti-CD16A antibodies, bispecific antibodies, multispecific molecules, nucleic acid molecules, vectors, host cells, conjugates, or pharmaceutical compositions of the present application are administered by intravenous injection or bolus.

[0187] Definitions of Terms

[0188] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and other biological procedures described herein are in accordance with conventional techniques of the respective fields. In order to better understand the present application, the following definitions and explanations of terms are provided.

[0189] When the terms "for example," "e.g.," "for instance," "like," "such as," "include," "including," or "comprising" are used in this document, these terms are meant to be construed as "including but not limited to."

[0190] Unless otherwise indicated herein, or in the context of a contradiction with respect to the context, the terms "a" and "an" and the like are to be construed to cover both the singular and the plural. Thus, for example, reference to "a" or "an" entity is to be construed to cover instances in which the entity is alone or one of the plurality of entities.

[0191] The term "and / or" means both "and" and "or." For example, the phrase "A, B, and / or C" is intended to cover each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0192] As used herein, the term "CD16A" refers to the Fc gamma receptor IIIA on the surface of NK and the like cells. When an antibody binds to an antigen on the surface of a target cell, the CD16A receptor recognizes and binds to the Fc region of the antibody, thereby activating the NK cell, which in turn kills the target cell. The sequence of CD16A is well known to those skilled in the art (see, e.g., UniProt No. P08637).

[0193] As used herein, the term“tumor-associated antigen” refers to an antigen that is expressed at a higher level (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more higher) by a tumor cell than by a non-tumor cell. In certain embodiments, the tumor-associated antigen is presented on the surface of a tumor cell; in certain embodiments, the tumor-associated antigen is located on or within a tumor cell; in certain embodiments, the tumor-associated antigen is presented only by tumor cells, and not by normal cells (i.e., non-tumor cells); in certain embodiments, the tumor-associated antigen is expressed in both tumor and non-tumor cells, but is overexpressed on tumor cells compared to non-tumor cells. In certain embodiments, the tumor-associated antigen (TAA for short) is selected from CD19, CD20, CD22, EGFR, BCMA, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CDH17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, Lewis Y, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof; in certain embodiments, the tumor-associated antigen is selected from EGFR or BCMA.

[0194] As used herein, the term "antibody" refers to a proteinaceous molecule capable of specifically binding an antigen. An antibody can bind an antigen through at least one antigen- binding site located in its variable region. When the term "antibody" is used, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies; full-length antibodies and antigen binding fragments, so long as they exhibit the desired antigen-binding activity. A "full-length antibody" or "intact antibody" typically comprises two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). Antibody light chains can be assigned to a class, kappa (kappa) and lambda (lambda). Heavy chains can be assigned to a class, mu, delta, gamma, alpha, or epsilon, and define a specificity of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. A heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The constant domains of a typical IgG heavy chain include three domains (CH1, CH2, and CH3). A light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The constant domains do not directly participate in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions include domains of hypervariability (referred to as hypervariable regions or complementarity determining regions (CDRs)), which are interspersed with framework regions (FRs) that are relatively conserved. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable region of the heavy chain / light chain pair (VH and VL) forms an antigen binding site. Assignment of amino acids to each region or domain can follow the definition of Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0195] As used herein, the term "complementarity determining region" or "CDR" refers to amino acid residues in the variable region of an antibody that are responsible for binding to an antigen. There are three CDRs in each of the variable regions of the heavy and light chains (CDR1, CDR2, and CDR3), which are identified as HCDR1, HCDR2, and HCDR3 for the heavy chain and LCDR1, LCDR2, and LCDR3 for the light chain, respectively. The boundaries of the CDRs can be defined according to various numbering systems known in the art, for example, according to the definition in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the Abm numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), the Contact numbering system (MacCallum, R.M., Martin, A.C.R., & Thornton, J.M. (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody variable region, a skilled artisan can readily determine the CDRs according to the definition in the numbering system. And, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0196] As used herein, the term "framework region" or "FR" refers to the domains of an antibody variable region other than the CDR residues.

[0197] As used herein, the term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0198] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide that comprises a portion of an intact antibody that is capable of specifically binding to an antigen that the intact antibody binds to, and which differs from the intact antibody. Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide linked Fv, scFv, di-scFv, diabodies, and other polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding capacity of the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0199] As used herein, the term "Fd" means an antibody fragment consisting of a VH and CH1 domain; the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CH1 domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; the term "Fab' fragment" means a fragment obtained after reduction of the disulfide bond linking the two heavy chain fragments in a F(ab')2 fragment, consisting of one complete light chain and a Fd fragment of the heavy chain (consisting of a VH and CH1 domain).

[0200] As used herein, the term "Fv" means an antibody fragment consisting of a VL and VH domain of a single arm of an antibody.

[0201] As used herein, the term "Fc" means an antibody fragment formed by disulfide bond linkage of the second, third constant region of the first heavy chain with the second, third constant region of the second heavy chain. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding. "Effector functions" mediated by the Fc domain include Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation, etc. The Fc domain can include either a native Fc region or a variant Fc region. A native Fc region comprises an amino acid sequence identical to that of an Fc region found in nature, e.g., a native sequence human Fc region includes a native sequence human IgGl Fc region; a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification. In some embodiments, a variant Fc region can possess altered effector function (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) compared to a native Fc region.

[0202] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist, for example, of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used. In some cases, a disulfide bond can also exist between the VH and VL of the scFv. In certain embodiments of the application, the scFv can form a di-scFv, which refers to two or more individual scFv linked in series to form an antibody. In certain embodiments of the application, the scFv can form a (scFv)2, which refers to two or more individual scFv linked in parallel to form an antibody.

[0203] As used herein, the terms "diabodies", "diabody", and "di-chain antibodies" mean that the VHand VLdomains are expressed on a single polypeptide chain, but using a too-short linker that does not allow for pairing between the domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0204] As used herein, the term "multispecific molecule", also referred to as "multispecific antibody", refers to an antibody having binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen binding domains having binding specificity for different antigens (or epitopes), thereby being able to bind at least two different binding sites and / or target molecules. Each of the antigen binding domains comprised by a multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F(ab')2 fragment, or an scFv). In some cases, each of the antigen binding domains is connected by a peptide linker. A multispecific antibody comprises a bispecific antibody. The term "bispecific antibody" refers to an antibody having binding specificity for two different antigens (or epitopes).

[0205] In some embodiments, each of the above-described antibody fragments retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen.

[0206] Antigen binding fragments of antibodies (e.g., the above-described antibody fragments) can be obtained using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) from a given antibody (e.g., an antibody provided herein), and can be screened in the same manner as the intact antibody.

[0207] The terms "human antibody", "fully human antibody", or "human antibody" can be used interchangeably and mean an antibody that comprises only human immunoglobulin sequences. Nonetheless, a human antibody can contain mouse sugar chains if it is produced in a mouse, a mouse cell, or a hybridoma derived from a mouse cell, or it can contain rat sugar chains if it is produced in a rat, a rat cell, or a hybridoma derived from a rat cell. Human antibodies can be obtained by conventional phage and yeast display, transgenic animals, single B cells, and the like technologies.

[0208] As used herein, the term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) from a human-derived immunoglobulin (acceptor antibody). In certain embodiments, a humanized antibody has CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FR and / or constant region) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc. In some embodiments, the donor antibody can be a murine-derived antibody having the desired properties (e.g., antigen specificity, affinity, and / or reactivity, etc.). To prepare a humanized antibody, the CDR regions of the donor antibody can be inserted into a human-derived framework sequence using methods known in the art. In some cases, the human-derived framework sequence can contain amino acid mutations that are replaced by the corresponding non-human residues. In addition, a humanized antibody can also contain residues that are not found in either the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or the human-derived framework sequence, to further improve or optimize the performance of the humanized antibody.

[0209] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or / and heavy chain is derived from another antibody (which can be derived from the same or different species or belong to the same or different antibody class or subclass), but the antibody retains binding activity to the target antigen. In certain embodiments, the term "chimeric antibody" can include an antibody in which the heavy chain variable region and the light chain variable region of the antibody are from a first antibody, and the heavy chain constant region and the light chain constant region of the antibody are from a second antibody.

[0210] As used herein, the term "identity" is used in reference to the match of sequences between two polypeptides or between two nucleic acids. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions x 100%). In certain embodiments, the two sequences are the same length.

[0211] Determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0212] As used herein, the term "variant", in the context of a polypeptide (including a polypeptide), refers to a polypeptide or peptide that comprises an altered (by amino acid residue substitution, deletion, and or addition) amino acid sequence. In certain instances, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not by way of limitation, a polypeptide can be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivatized polypeptide or peptide can be produced by chemical modification using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Furthermore, a variant has similar, the same, or improved function as the polypeptide or peptide from which it is derived.

[0213] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. In the present application, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Typically, an antibody binds an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1000 nM or less, e.g., about 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, or less.

[0214] The specific binding properties between two molecules can be determined using methods known in the art. For example, the activity of an antibody to bind an antigen can be determined by measuring the rate of antigen binding site / antigen complex formation and dissociation. Both the "association rate constant" (kaor kon) and the "dissociation rate constant" (kdisor koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of kdis / konis equal to the dissociation constant KD. KD, kon, and kdisvalues can be measured using any effective method. In certain embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore.

[0215] The term "epitope" refers to a region (area or region) on an antigen that is capable of being specifically bound by an antibody. An epitope can be formed by contiguous amino acids (linear epitopes) or non-contiguous amino acids (conformational epitopes) that are brought into spatial orientation by the folding (i.e., tertiary folding of the antigen by the nature of the protein) of the antigen. For example, an epitope can comprise at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind a particular epitope (i.e., those that bind the same epitope) can be performed using methods known in the art, including but not limited to alanine scanning, peptide mapping (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0216] When "compete" is used in reference to antigen binding proteins (e.g., antibodies) that compete for the same epitope, it is meant that the antigen binding proteins compete with one another for binding to a common antigen, which can typically be determined by an assay in which the antigen binding protein (e.g., antibody) to be tested inhibits (e.g., reduces) the specific binding of a reference antigen binding protein (e.g., reference antibody) to the antigen. Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another antigen binding protein, including, but not limited to, solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeled RIA with 125I-labeled ligand (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15), and the like. In some embodiments, the binding of a reference antibody (e.g., an anti-CD16A antibody) to an antigen is inhibited by at least 40% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 90%, 95%, 97%, or 98% or more) by an antibody that competes for binding thereto.

[0217] As used herein, the detectable label of the present application can be any substance that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein (FITC), rhodamine, tetramethylrhodamine (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or a cyanine dye derivative (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent agents such as acridinium esters, luminol and its derivatives, ruthenium derivatives such as trispyridyl ruthenium), magnetic beads (e.g., Dynabeads®), and the like. ), a calorimetric label such as gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above labels. In certain embodiments, the detectable label is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin. In certain embodiments, the detectable label can be attached to the antibodies of the application via linkers of varying lengths to reduce potential steric hindrance.

[0218] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a polynucleotide inserted into it, the vector is referred to as an expression vector. A vector can be introduced into a host cell by transformation, transduction or transfection, so that the host cell takes up (and perhaps expresses) the genetic material carried by the vector. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 -derived artificial chromosomes (PACs) ; bacteriophages such as lambda phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), pox viruses, baculoviruses, papillomaviruses, papova viruses (e.g., SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional start sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can contain a replication origin.

[0219] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or mammalian cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0220] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the replacement of an amino acid residue by another residue having similar side chain properties, such as replacing an amino acid residue with a residue that is physically or functionally similar (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a preferred substitution is one in which the replaced amino acid residue is replaced with another amino acid residue from the same side chain family. Methods of identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0221] The nomenclature used herein to refer to the twenty conventional amino acids follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art (amino acid three-letter and one-letter codes are set forth in J. Biol. Chem, 243, p 3558 (1968)). For example, alanine can be represented by A or Ala.

[0222] As used herein, the term "pharmaceutically acceptable excipient" refers to an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, and preservatives.

[0223] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome for a subject who exhibits symptoms of the disease or is diagnosed with the disease. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (e.g., survival without treatment).

[0224] As used herein, the term "subject" refers to a mammal, such as a human or a non-human primate (eg, cynomolgus monkey). In certain embodiments, the subject has a solid tumor, a hematological tumor, or an autoimmune disease.

[0225] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0226] In the present invention, unless otherwise specified, the "first" (e.g., the first antigen or the first antigen-binding domain) and the "second" (e.g., the second antigen or the second antigen-binding domain) are mainly for reference distinction and do not have a typical order meaning.

[0227] Advantageous Effects of the Invention

[0228] In some embodiments, the present application provides anti-CD16A antibodies that can bind to human and cynomolgus monkey CD16 proteins with high affinity at both cellular and protein levels, and do not bind to human CD16B protein. The present application further provides bispecific antibodies that bind to both CD16A and tumor-associated antigens. The anti-CD16A antibodies and anti-CD16A bispecific antibodies of the present application can effectively promote the killing effect of NK cells on tumor cells, and promote the killing ability of PBMCs on tumor cells. In addition, the anti-CD16A antibodies of the present application also have species cross-binding activity and activation activity to human and monkey CD16A, thereby benefiting the preclinical evaluation studies and the like in animal models. In some embodiments, the anti-CD16A antibodies of the present application can effectively inhibit the growth of tumors in vivo.

[0229] Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the preferred embodiments and the accompanying drawings, various objects and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0230] Figure 1: Binding of anti-CD16A chimeric antibody M01 to human CD16A-158V protein.

[0231] Figure 2: Binding of anti-CD16A chimeric antibody M01 to human CD16A-158F protein.

[0232] Figure 3: Binding of anti-CD16A chimeric antibody M01 to monkey CD16 protein.

[0233] Figure 4: Binding of anti-CD16A chimeric antibody M01 to human CD16B protein.

[0234] Figure 5: Binding of anti-CD16A chimeric antibody M01 to primary NK cells.

[0235] Figure 6: Binding of anti-CD16A chimeric antibody M01 to primary neutrophils.

[0236] Figure 7: Schematic diagram of anti-CD16A / EGFR bispecific antibody structure.

[0237] Figure 8: Schematic diagram of anti-CD16A / BCMA bispecific antibody structure.

[0238] Figure 9: Binding of anti-CD16A / EGFR bispecific antibody C01 and anti-CD16A / BCMA bispecific antibody C02 to CHO-K1 / hCD16A-F cells.

[0239] Figure 10: Anti-CD16A / EGFR bispecific antibody C01 can activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner.

[0240] Figure 11: Anti-CD16A / BCMA bispecific antibody C02 can activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner.

[0241] Figure 12: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell killing of target cells A431 (EGFR+) experimental results graph.

[0242] Figure 13: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell killing of target cells HT-29 (EGFR+) experimental results graph.

[0243] Figure 14: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell activation experimental results graph (target cells A431 (EGFR+)).

[0244] Figure 15: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell activation experimental results graph (target cells HT-29 (EGFR+)).

[0245] Figure 16: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell IL-6 secretion experimental results graph (target cells A431 (EGFR+)).

[0246] Figure 17: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell TNF-a secretion experimental results graph (target cells A431 (EGFR+)).

[0247] Figure 18: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell IL-6 secretion experimental results graph (target cells HT-29 (EGFR+)).

[0248] Figure 19: Anti-CD16A / EGFR bispecific antibody C01 promotes NK cell TNF-a secretion experimental results graph (target cells HT-29 (EGFR+)).

[0249] Figure 20: Anti-CD16A / EGFR bispecific antibody C03 structure schematic (scFv-scFv-Fc).

[0250] Figure 21: Anti-CD16A / EGFR bispecific antibody C04 structure schematic (scFv-VH-CH / VL-CL).

[0251] Figure 22: Results of the binding experiment of anti-CD16A / EGFR bispecific antibodies C03 and C04 to CHO-K1 / hCD16A-F cells.

[0252] Figure 23: Anti-CD16A / BCMA bispecific antibodies C03 and C04 can activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner.

[0253] Figure 24: Results of the experiment that anti-CD16A / EGFR bispecific antibodies C03 and C04 promote NK cells to kill target cells HT-29 (EGFR+).

[0254] Figure 25: Results of the experiment that anti-CD16A / EGFR bispecific antibodies C03 and C04 promote NK cells to activate (target cells HT-29 (EGFR+)).

[0255] Figure 26: Results of the detection of the body weight of mice in each group in the in vivo efficacy experiment of anti-CD16A / EGFR bispecific antibodies.

[0256] Figure 27: Results of the detection of the tumor volume in each group in the in vivo efficacy experiment of anti-CD16A / EGFR bispecific antibodies.

[0257] Figure 28: Results of the detection of the proportion of different immune cells in each group in the in vivo efficacy experiment of anti-CD16A / EGFR bispecific antibodies.

[0258] Sequence information

[0259] Table 1: Information of the sequences involved in the present application is described in the following table:

[0260] Note: The CDRs are defined according to the Kabat numbering system DETAILED DESCRIPTION

[0261] The present application will now be described in the following non-limiting examples.

[0262] Those skilled in the art will appreciate that the examples describe the present application by way of example only, and are not intended to limit the scope of the application as claimed. The experimental methods in the examples are conventional methods unless otherwise specified. The specific conditions not indicated in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

[0263] Example 1: Construction of CD16 monoclonal stable cell strain

[0264] The cDNA encoding human CD16A-158V (short: CD16A-V, UniProt: P08637, F158V), CD16A-158F (short: CD16A-F, UniProt: P08637), CD16B (UniProt: O75015) and cynomolgus CD16 (cynoCD16, UniProt: A3RFZ7) were cloned into expression vectors (Invitrogen, Cat# V79020) to get the corresponding plasmid clones, which were then transfected into CHO-K1 cells by transient transfection and expressed on the membrane as human CD16A-158V, CD16A-158F, CD16B and cynomolgus CD16, respectively.

[0265] The positive identified stable cell pools were subcloned by limiting dilution and cultured at 37°C, 5% (v / v) CO2, and after about 2 weeks, some of the selected monoclonal wells were amplified to 6-well plates. The amplified clones were screened by flow cytometry using APC anti-human CD16 (Biolegend, 980104) antibody. The cell lines with good growth, high fluorescence intensity and monoclonality were selected for further expansion and cryopreservation in liquid nitrogen, i.e. CHO-K1 / hCD16A-F, CHO-K1 / hCD16A-V, CHO-K1 / cynoCD16 and CHO-K1 / hCD16B monoclonal stable cell strains were obtained, which can stably express.

[0266] Example 2: Preparation of monoclonal hybridoma clones of human CD16A antibody and sequencing of the clones

[0267] 2-1. Immunization and fusion

[0268] The immunization was performed using 6-8 week old Balb / c and SJL mice (purchased from Shanghai Sylab). After receiving the mice, they were raised under SPF conditions. The primary immunization: human CD16A-158F protein (Acro Biosystems, CDA-H5220, same below) was emulsified with Freund's complete adjuvant and injected intraperitoneally at 0.20 mL, 100 μg of protein per mouse. The booster immunization: CD16A-158F protein was emulsified with Freund's incomplete adjuvant and injected intraperitoneally at 0.20 mL, 50 μg of protein per mouse. The interval between the primary immunization and the first booster immunization was 2 weeks, and the interval between each subsequent booster immunization was 2 weeks. Blood was collected 7 days after each booster immunization, and the antibody titer and specificity in the serum were detected by ELISA. After two booster immunizations, the mouse with the highest titer was selected. The selected mouse was given a final immunization of 50 μg of purified CD16A-158F protein intraperitoneally, and the mouse was sacrificed 4 days later. The spleen cells were collected and fused with the mouse myeloma cell line SP2 / 0 cells (ATCC) by electrofusion and diluted for culture.

[0269] 2-2. Screening of hybridoma clones

[0270] Hybridoma clones were screened at large scale with human CD16A-158F protein and cynomolgus CD16 protein (Acro Biosystems, FC6-C52H9, same below) and CHO-K1 / hCD16A-F and CHO-K1 / cynoCD16 monoclonal stable cell lines, respectively, and positive clones with OD 450nm > 1.0 and MFI values in iQue3 (Sartorius) more than 10 times of negative control values were selected and expanded to 24-well plates. Hybridoma clones were subcloned by limiting dilution method to ensure monoclonality. Monoclonal hybridoma cells were screened with human CD16A-158V protein (Acro Biosystems, CD8-H52H4, same below), CD16A-158F protein, CD16B-NA1 (Acro Biosystems, CDB-H5227, same below), CD16B-NA2 (Acro Biosystems, CDB-H5222, same below) and cynomolgus CD16 protein and CHO-K1 / hCD16A-F, CHO-K1 / hCD16A-V, CHO-K1 / cynoCD16 and CHO-K1 / hCD16B monoclonal stable cell lines, respectively, and clones with OD 450nm > 1.0 and MFI values in iQue3 more than 10 times of negative control values were selected as monoclonal positive hybridomas. Monoclonal positive hybridoma clones were inoculated into SFM serum-free medium (Invitrogen, 12045076) containing 2% (w / w) FBS and expanded at 37°C, 5% (v / v) CO2, and the selected positive hybridoma cells were frozen in liquid nitrogen.

[0271] 2-3. Determination of hybridoma VH and VL sequences

[0272] Positive clones were cultured to appropriate concentration, centrifuged to collect 5 x 106hybridoma cells, and RNA of hybridoma clone cells was extracted using TRIzol Plus RNA Purification Kit (Invitrogen, 12183555). First-strand cDNA was synthesized using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, 6210A) and PCR reagent kit was Premix Taq (Takara, RR006A). 7 TM Plus RNA Purification Kit (Invitrogen, 12183555). First-strand cDNA was synthesized using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, 6210A) and PCR reagent kit was Premix Taq (Takara, RR006A). TM TM ​​(Takara, RR003A) was performed. 5 μL of the PCR product was taken for agarose gel electrophoresis detection, and the positive sample was purified using a column recovery kit, which was Gel & PCR Clean-up (MACHEREY-NAGEL, 740609). The ligation reaction was performed: sample 50 ng, T vector 50 ng, ligase 0.5 μL, buffer 1 μL, reaction system 10 μL, and the ligation product was obtained by reacting at 16°C for half an hour, wherein the T vector ligation kit was pMD TM 19-T Vector Cloning Kit (Takara, 6013); 5 μL of the ligation product was added to 100 μL of F-DH5α competent cells (Weidi Biology, DF1001) for ice bath for 5 minutes. It was coated on the LB solid medium containing antibiotics which had been preheated at 37°C and incubated at 37°C overnight. The next day, 30 μL of PCR system was prepared using primers M13F and M13R on the T vector, and colony PCR was performed. The colony was dipped in the PCR reaction system with a pipette gun head and blown and sucked, and 0.5 μL was spotted on another LB solid culture dish containing 100 μg / mL ampicillin to preserve the strain. After the PCR reaction, 5 μL was taken for agarose gel electrophoresis detection, and the positive sample was sequenced to obtain the sequences of the heavy chain variable region and the light chain variable region of the antibody A01. The variable region sequence composition is shown in Table 2 and Table 3.

[0273] Table 2: Sequence composition of the heavy chain variable region of the anti-CD16A antibody A01

[0274] Note: The CDRs are defined according to the Kabat numbering system

[0275] Table 3: Sequence composition of the light chain variable region of the anti-CD16A antibody A01

[0276] Note: The CDRs are defined according to the Kabat numbering system

[0277] Example 3: Obtaining and preparing of anti-human CD16A chimeric antibody

[0278] According to the sequences of the antibody heavy chain variable region and light chain variable region obtained in Example 2, the DNA encoding the heavy chain variable region was directionally cloned into an expression vector (the expression vector was purchased from Invitrogen, item number V79020) containing a signal peptide and a human antibody heavy chain IgG1_L234F_L235E_D265A constant region (SEQ ID NO: 40) to obtain an A01-VH recombinant vector; the DNA encoding the light chain variable region was directionally cloned into an expression vector (the expression vector was purchased from Invitrogen, item number V79020) containing a signal peptide and a human antibody light chain kappa constant region (SEQ ID NO: 41) to obtain an A01-VL recombinant vector. The recombinant vectors were transfected into ExpiCHO cells (Gibco, A29127) using ExpiFectamine TM CHO transfection kit (Invitrogen, A29130) was transiently transfected into ExpiCHO cells (Gibco, A29127) and ExpiCHO TM Expression Medium (Gibco, A2910002) was cultured in a CO2 incubator at 37°C with shaking. After 8-10 days, the cell culture solution was collected, centrifuged to remove the cell components, and filtered with a 0.45 μm filter to clarify the culture supernatant. The supernatant was subjected to MabSelect Prism A (Cytiva, 17-5498-02) affinity chromatography. The MabSelect Prism A chromatography column was first regenerated with 0.1 M NaOH, then washed with pure water and equilibrated with PBS. After the supernatant was bound, PBS was used for washing until the A280 reading decreased to the baseline. The target protein was eluted with 0.1 M acetic acid buffer at pH = 3.5, and neutralized with 1 M Tris HCl, pH 9.0. After appropriate concentration of the eluted sample, further purification was performed using a gel chromatography Superdex 200 (Cytiva, 28-9893-35) equilibrated with PBS, and the target protein was concentrated to an appropriate concentration by collecting the receiving tubes. The anti-human CD16A chimeric antibody M01 was obtained, the sequence composition of which is shown in Table 4 below, and was used in subsequent examples.

[0279] Table 4: Sequence composition of anti-CD16A chimeric antibody M01

[0280] Example 4: Detection of the binding ability of anti-human CD16A chimeric antibody

[0281] The desired CHO-K1 / hCD16A-F, CHO-K1 / hCD16A-V, CHO-K1 / cynoCD16 and CHO-K1 / hCD16B monoclonal stable cell lines were expanded in T-75 cell culture flasks to 90% confluency, the culture medium was discarded, washed twice with PBS buffer, then the cells were digested with Trypsin-EDTA (Gibco, 25200-072). The cells were collected and washed twice with PBS buffer. The collected cells were suspended in FACS buffer (PBS + 2% FBS, the percentage is volume percentage) to 2 x 10 6 Cells / mL, 50 μL per well was added to a 96-well V-bottom plate (NEST, 701201), and an equal volume of serially diluted test antibody was added. Incubate on ice for 1 hour, wash twice with FACS buffer, and add 100 μL per well of 1:1000 diluted goat anti-human IgG, Fc Fragment Specific secondary antibody (Jackson, 109-605-098) to the cells, and incubate on ice for 1 hour. Wash twice with FACS buffer. Resuspend the cells with 100 μL of FACS buffer, and detect and analyze the results with a flow cytometer iQue3. Fit the dose response data with the fluorescence signal with a four-parameter logistic model by GraphPad Prism.

[0282] The results are shown in Figures 1-4. At the cellular level, M01 can dose-dependently bind to cell surface human CD16A-158V, CD16A-158F and monkey CD16 proteins, and does not bind to human CD16B protein. The anti-GP120 antibody b12 in the literature Zwick MB, et al. J Virol. 2003 was used as a negative control antibody, which does not bind to cells.

[0283] Example 5: Binding of anti-human CD16A chimeric antibodies to primary NK cells and neutrophils

[0284] NK cells were isolated from PBMC cells (Rui Bio) using an NK Cell Isolation Kit (Miltenyi Biotec, 130-092-657) (for detailed steps, refer to the kit instructions), and then resuspended in FACS buffer to 2 x 10 6 Cells / mL. Neutrophils were resuspended in FACS buffer to 2 x 10 6Cells / mL were added to a 96-well V-bottom plate at 50 pL per well, and an equal volume of serially diluted test antibody was added. After 1 hour of incubation on ice, the cells were washed twice by centrifugation in FACS buffer, and 100 pL per well of a 1 : 1000 dilution of goat anti-human IgG, Fc Fragment Specific secondary antibody (Jackson, 109-605-098) was added. After 1 hour of incubation on ice, the cells were washed twice by centrifugation in FACS buffer. The cells were resuspended in 100 pL FACS buffer, and the results were detected and analyzed using a flow cytometer, iQue3. Dose response data were fit to a four-parameter logistic model with fluorescence signal using GraphPad Prism.

[0285] Results are shown in Figures 5 and 6, M01 strongly binds to CD16A on the surface of primary NK cells with a half maximal effective concentration (EC 50 ) of 14.16 nM, and M01 does not bind to CD16B on the surface of primary neutrophils.

[0286] Example 6: Humanization of Antibodies

[0287] For antibody humanization, the main method of CDR grafting with back mutation was used for design. Briefly, first, humanized sequence analysis and selection of high frequency of human IGHV and IGKV genes existing in human antibody library and highly homologous to mouse antibodies by sequence alignment (NCBI Igblast) were performed as humanization templates. After selecting the human antibody skeleton, by homology modeling, the key amino acids in the mouse variable region that may determine the structure were predicted, and back mutation design was performed on the grafted skeleton region. According to the above principles, for the humanization of M01, germline genes IGHV146*01 (77.35%) + IGHJ6*01 and IGKV113*02 (74.1%) + IGKJ4*01 were selected as variable region grafting skeletons for CDR grafting and important amino acid back mutation design: 5 heavy chain variable region sequences (VH1, VH2, VH3, VH4, VH5) and 6 light chain variable region sequences (VL1, VL2, VL3, VL4, VL5, VL6) were designed, respectively, followed by cross combination for expression, a total of 30 expression combinations (see Table 5 below), among which the 3 CDR sequences of the heavy chain variable region and the 3 CDR sequences of the light chain variable region of the humanized antibody were exactly the same as those of the chimeric antibody M01 from which they were derived.

[0288] Table 5: Humanized antibody expression combinations

[0289] The heavy chain gene of the humanized antibody sequence was cloned into an expression vector containing a signal peptide and a human antibody heavy chain IgG1_L234F_L235E_D265A constant region to obtain M01-Hu-VH recombinant vector; the light chain gene of the humanized antibody sequence was cloned into an expression vector containing a signal peptide and a human antibody light chain kappa constant region to obtain M01-Hu-VL recombinant vector. The humanized antibody B01-B30 was obtained by expression and purification using the same method as in Example 3, the sequence composition thereof is shown in Table 6 below, and is used in subsequent examples.

[0290] Table 6: Sequence composition of the heavy chain variable region of the humanized CD16A antibody

[0291] Example 7: Detection of the binding ability of the anti-human CD16A humanized antibody

[0292] The binding ability of the CD16A humanized antibody to the CHO-K1 / hCD16A-F monoclonal stable cell strain was detected according to the method of Example 4.

[0293] The results are shown in Table 7, and all the humanized antibodies can bind to the CHO-K1 / hCD16A-F cells, and have good binding ability.

[0294] Table 7: Binding ability of the anti-human CD16A humanized antibody to CHO-K1 / hCD16A-F

[0295] Example 8: Determination of the affinity constant of the anti-human CD16A humanized antibody

[0296] The affinities and kinetics of the antibodies and human CD16A-158V, CD16A-158F, CD16B-NA1, CD16B-NA2 and cynomolgus CD16 proteins were determined by using Biacore 8K Plus (GE healthcare) based on the principle of surface plasmon resonance (SPR). The Protein A chip (Cytiva, 29127555) was used to capture the candidate antibodies to be tested at a concentration of 1 pg / mL, and the different CD16 proteins were gradient diluted (25 nM as the starting concentration, 2-fold dilution, 5 concentration points) to flow through the sensor chip at a flow rate of 10 pL / min. The running buffer was 1x PBS-P+ (Cytiva, 50-105-5354), and the detection conditions of the CD16 proteins were as follows: capture time 60 s; antigen binding time 120 s; dissociation time 600 s; regeneration condition was glycine 1.5 (Cytiva, BR100354) for 30 s. After double subtraction (control channel and zero concentration), the experimental data were fitted by the "1:1 binding" model by Biacore 8K Plus insight evaluation soft to obtain the affinity and kinetic data.

[0297] The experimental results show that the anti-human CD16A humanized antibodies of the present application have good binding ability to human CD16A and monkey CD16 proteins. For example, the affinities of the antibodies to human CD16A-V, CD16A-F, CD16B-NA1, CD16B-NA2 and monkey CD16 proteins were determined by using Biacore 8K Plus as shown in Table 8. The results show that the affinity of M01 to human CD16A-V and CD16A-F is significantly higher than that of 4LS21 antibody (the sequence is from the anti-CD16A antibody of PCT Publication No. WO2006125668A2, the heavy chain is SEQ ID NO: 9, and the light chain is SEQ ID NO: 16), and neither binds to human CD16B-NA1 and CD16B-NA2. After humanization, the affinity of B01 to CD16A-V, CD16A-F and monkey CD16 proteins is equivalent to that of chimeric antibody M01, and still does not bind to CD16B-NA1 and CD16B-NA2.

[0298] Table 8: Affinity constants of M01 humanized antibodies to CD16 proteins

[0299] Note: N.B is the abbreviation of "No binding", indicating no binding.

[0300] Example 9: Preparation of anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies

[0301] In order to verify whether the anti-CD16A antibody obtained by the present application has the ability to mediate NK cell activation and killing, the present embodiment constructs a bispecific antibody by combining the anti-CD16A antibody with anti-EGFR and anti-BCMA antibodies, respectively. The structural form of the bispecific antibody adopts huIgG(H)-scFv, and the specific construction and expression method is as follows:

[0302] The DNA encoding the complete heavy chain of the anti-CD16A / EGFR bispecific antibody (SEQ ID NO: 36) is directionally cloned into an expression vector containing a signal peptide (the expression vector is purchased from Invitrogen, item number V79020), obtaining a CD16A / EGFR-BsAb heavy chain recombinant vector; the DNA encoding the complete light chain of the anti-CD16A / EGFR bispecific antibody (SEQ ID NO: 37) is directionally cloned into an expression vector containing a signal peptide (the expression vector is purchased from Invitrogen, item number V79020), obtaining a CD16A / EGFR-BsAb light chain recombinant vector. The recombinant vectors are transfected into ExpiCHO cells (Gibco, A29127) using ExpiFectamine TM CHO transfection kit (Invitrogen, A29130) is transiently transfected into ExpiCHO cells (Gibco, A29127) and ExpiCHO TM Expression Medium (Gibco, A2910002) is cultured in a CO2 incubator at 37°C with shaking. After 8-10 days, the cell culture solution is collected, centrifuged to remove the cell components, and filtered to clarify the culture supernatant using a 0.45 μM filter membrane. The supernatant is subjected to MabSelect PrismA (Cytiva, 17-5498-02) affinity chromatography. The MabSelect PrismA chromatography column is first regenerated with 0.1 M NaOH, then washed with pure water and equilibrated with PBS. After the supernatant is bound, PBS is used for washing until the A280 reading drops to the baseline. The target protein is eluted with 0.1 M acetic acid buffer at pH = 3.5, and neutralized with 1 M Tris-HCl, pH 9.0. After appropriate concentration of the eluted sample, further purification is performed using PBS equilibrated gel chromatography Superdex200 (Cytiva, 28-9893-35), and the target protein is concentrated to an appropriate concentration by collecting the receiving tubes. The anti-CD16A / EGFR bispecific antibody C01 is prepared, as shown in the structural schematic diagram of FIG. 7, and is used in the subsequent embodiments.

[0303] The DNA encoding the complete heavy chain of the anti-CD16A / EGFR bispecific antibody (SEQ ID NO: 38) was directionally cloned into an expression vector containing a signal peptide (the expression vector was purchased from Invitrogen, Cat# V79020) to obtain a CD16A / EGFR-BsAb heavy chain recombinant vector. The DNA encoding the complete light chain of the anti-CD16A / EGFR bispecific antibody (SEQ ID NO: 39) was directionally cloned into an expression vector containing a signal peptide (the expression vector was purchased from Invitrogen, Cat# V79020) to obtain a CD16A / EGFR-BsAb light chain recombinant vector. The expression and purification methods were the same as in Example 3. The anti-CD16A / EGFR bispecific antibody C01 was prepared, the structural schematic diagram of which is shown in Figure 8, and was used in the subsequent examples.

[0304] Other anti-CD16A antibodies of the present application were used to construct bispecific antibodies by the same method above.

[0305] Example 10: Binding of anti-CD16A / EGFR bispecific antibody and anti-CD16A / BCMA bispecific antibody to CD16A monoclonal stable cell line

[0306] The binding ability of the anti-CD16A / EGFR bispecific antibody C01 and the anti-CD16A / BCMA bispecific antibody C02 to the CHO-K1 / hCD16A-F monoclonal stable cell line was detected according to the method of Example 4.

[0307] The experimental results showed that the bispecific antibodies of the present application could dose-dependently bind to CHO-K1 / hCD16A-F. Exemplary experimental results are shown in Figure 9, and the anti-CD16A / EGFR bispecific antibody C01 and the anti-CD16A / BCMA bispecific antibody C02 could dose-dependently bind to CHO-K1 / hCD16A-F. Anti-GP120, as a negative control antibody, did not bind to CHO-K1 / hCD16A-F.

[0308] Example 11: Luciferase reporter assay for anti-CD16A / EGFR bispecific antibody C01 and anti-CD16A / BCMA bispecific antibody C02 function

[0309] The bispecific antibody activates CD16A signal. The Jurkat-NFAT-CD16A cell line expressing human CD16A-158V (Jurkat-NFAT-CD16A, Acro Biosystems, SCJUR-STF067) was used to detect the activation of CD16A signal by the bispecific antibody. The Jurkat-NFAT-CD16A cells were resuscitated and cultured in RPMI-1640 (Gibco, 22400105) complete medium containing 10% inactivated fetal bovine serum (Gibco, A5669701), 20 μg / ml hygromycin B (Gibco, A5669701) and 5 μg / ml puromycin (Gibco, A1113803). The Jurkat-NFAT-CD16A cells were washed three times with RPMI-1640 analysis medium containing 10% inactivated fetal bovine serum, and the density was adjusted to 2x10 6 / ml after resuspension. The A431 cells (KeHui Bio) were digested with 0.25% trypsin-EDTA, centrifuged at 400g for 5 minutes, and the density was adjusted to 2x10 6 / ml after resuspension. The NCI-H929 cells (KeHui Bio) were centrifuged and resuspended, and the density was adjusted to 2x10 6 / ml. The bispecific antibodies C01 and C02 were diluted to 100 nM with RPMI-1640 analysis medium, and then 8 gradients were diluted by 10 times. 25 μL of Jurkat-NFAT-CD16A cells, 25 μL of tumor cells and 50 μL of antibody dilution were added to each well, mixed and incubated in a 5% CO2 incubator at 37°C for 6 hours. At the time of detection, 100 μL of ONE-Glo TM Luminescent Reporter Gene Detection Reagent (Promega, E6120) was added to each well, and the luminescent signal of luciferase was read after 5 minutes of dark incubation. The positive control AFM24 (the sequence is from PCT Publication No. WO2019175368A1, the heavy chain is SEQ ID NO: 28, and the light chain is SEQ ID NO: 29) and the anti-BCMA monoclonal antibody (the sequence is from PCT Publication No. US10072088B2, the heavy chain is SEQ ID NO: 27, and the light chain is SEQ ID NO: 28).

[0310] The experimental results show that the bispecific antibody of the present application can activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner. The experimental results are shown in Figures 10 and 11, and the bispecific antibodies C01 and C02 can both activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner. Among them, the EC 50 of C01 to activate Jurkat-NFAT-CD16A cells is 0.044 nM, which is lower than that of AFM24.50 about 5 times, indicating that C01 has a better effect of activating CD16A. Anti-GP120 as a negative control antibody does not activate Jurkat-NFAT-CD16A cells. The EC 50 of anti-BCMA mAb is 0.173 nM, and the EC 50 about 4.6 times, indicating that C02 has a better effect of activating CD16A than the mAb.

[0311] Example 12: Evaluation of the activity of anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies in promoting NK cells to kill target cells

[0312] In this example, PBMC cells, tumor cells A431 (Key Biotech) and HT-29 (Key Biotech) were incubated with anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies, and the death of tumor cells was characterized by detecting the release of calcein from target cells. The specific experimental method is as follows: PBMC cells were resuscitated using RPMI-1640 analysis medium containing 10% inactivated fetal bovine serum, centrifuged at 400g for 10 minutes, and then resuspended to a density of 1x10 7 / ml, ready for use. A431 cells were digested using 0.25% trypsin-EDTA. A431 and HT-29 cells were washed three times with serum-free RPMI-1640 medium, and then resuspended to a density of 1x10 6 / ml, and then added with Calcein-AM reagent (Invitrogen, C3099) to a final concentration of 2 μM, and incubated in a 5% CO2 incubator at 37°C for 1 hour. Then the cells were washed three times with RPMI-1640 analysis medium, and then resuspended to a density of 2x10 5 / ml. Bispecific antibodies C01 and C02 were diluted to 100 nM using RPMI-1640 analysis medium, and then diluted by 10 times to 8 gradients. 50 μL of PBMC cells, 50 μL of tumor cells and 100 μL of antibody diluent were added to each well of the experimental group, mixed well, and then incubated in a 5% CO2 incubator at 37°C for 4 hours. When detecting, the 96-well plate was centrifuged at 400g for 5 minutes, and then 100 μL of supernatant was carefully aspirated and added to a 96-well plate, and the fluorescence signal at 514 nm was detected using an enzyme-labeled instrument.

[0313] The experimental results show that the bispecific antibodies of the present application can promote NK cells to kill tumor cells in a tumor antigen-specific manner.

[0314] Exemplary experimental results are shown in FIG. 12 and FIG. 13, and the results show that the bispecific antibody C01 of the present application can promote the killing of tumor cells A431 and HT-29 by NK cells in a tumor antigen specific manner, wherein the EC50 of AFM24 in HT-29 cells is 0.0398 nM, and the EC50 of C01 is 0.0069 nM, which is about 5.7 times smaller than that of AFM24. 50 Exemplary experimental results are shown in FIG. 12 and FIG. 13, and the results show that the bispecific antibody C01 of the present application can promote the killing of tumor cells A431 and HT-29 by NK cells in a tumor antigen specific manner, wherein the EC50 of AFM24 in HT-29 cells is 0.0398 nM, and the EC50 of C01 is 0.0069 nM, which is about 5.7 times smaller than that of AFM24. 50 Exemplary experimental results are shown in FIG. 12 and FIG. 13, and the results show that the bispecific antibody C01 of the present application can promote the killing of tumor cells A431 and HT-29 by NK cells in a tumor antigen specific manner, wherein the EC50 of AFM24 in HT-29 cells is 0.0398 nM, and the EC50 of C01 is 0.0069 nM, which is about 5.7 times smaller than that of AFM24. 50 Exemplary experimental results are shown in FIG. 12 and FIG. 13, and the results show that the bispecific antibody C01 of the present application can promote the killing of tumor cells A431 and HT-29 by NK cells in a tumor antigen specific manner, wherein the EC50 of AFM24 in HT-29 cells is 0.0398 nM, and the EC50 of C01 is 0.0069 nM, which is about 5.7 times smaller than that of AFM24.

[0315] Example 13: Evaluation of the activity of anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies in promoting the activation of NK cells

[0316] This example evaluates the ability of anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies to promote the activation of NK cells by detecting the expression level of CD107a on NK cells in PBMC by flow cytometry. Cells and antibodies are treated according to the method of Example 12, and the 96-well plate is incubated in a 37°C incubator with 5% CO2 for 4 hours. Then the 96-well plate is centrifuged at 400g for 5 minutes, washed three times with FACS buffer, and anti-CD3-APC / Cy7 (Biolegend, 300318), anti-CD56-PerCP / Cy5.5 (Biolegend, 362506) and anti-CD107a-APC (Biolegend, 328620) are added at a concentration of 1:1000, and cell death and viability dye (Invitrogen, C34554) is added at a concentration of 1:1000, and incubated on ice for 1 hour. Wash three times with FACS buffer. Resuspend the cells with 200 μL FACS buffer, and detect and analyze the percentage of CD107a expression on NK cells (CD3-, CD56+) by flow cytometry (BD, FACS CelestaTM).

[0317] The experimental results show that the bispecific antibodies of the present application can promote the increase of CD107a expression on the surface of NK cells in a tumor antigen specific manner, indicating that they can promote the activation of NK cells.

[0318] Exemplary experimental results are shown in FIG. 14 and FIG. 15, and the results show that the EC50 of AFM24 in HT-29 cells is 0.0698 nM, and the EC50 of C01 is 0.015 nM, which is about 4.6 times smaller than that of AFM24. 50 Exemplary experimental results are shown in FIG. 14 and FIG. 15, and the results show that the EC50 of AFM24 in HT-29 cells is 0.0698 nM, and the EC50 of C01 is 0.015 nM, which is about 4.6 times smaller than that of AFM24. 50 Exemplary experimental results are shown in FIG. 14 and FIG. 15, and the results show that the EC50 of AFM24 in HT-29 cells is 0.0698 nM, and the EC50 of C01 is 0.015 nM, which is about 4.6 times smaller than that of AFM24. 50 Exemplary experimental results are shown in FIG. 14 and FIG. 15, and the results show that the EC50 of AFM24 in HT-29 cells is 0.0698 nM, and the EC50 of C01 is 0.015 nM, which is about 4.6 times smaller than that of AFM24.

[0319] Example 14: Evaluation of the activity of anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies in promoting PBMCs to release cytokines

[0320] This example evaluates the activity of anti-CD16A / EGFR bispecific antibodies and anti-CD16A / BCMA bispecific antibodies in promoting PBMCs to release cytokines by detecting the cytokine levels in cell culture supernatants. Cells and antibodies are treated according to the method of Example 12, and the 96-well plates are incubated in a 37°C incubator with 5% CO2for 24 hours. Then the 96-well plates are centrifuged at 400g for 5 minutes, and 100 μL supernatant is carefully aspirated for cytokine detection. The specific experimental methods are described in the instructions of TNF-a ELISA Kit (Invitrogen, 88-7346-88) and IL-6 ELISA Kit (Invitrogen, 88-7066-88).

[0321] The experimental results show that the bispecific antibodies of the present application can promote PBMCs to release TNF-a and IL-6 in a tumor antigen specific manner. Exemplary experimental results are shown in Figures 16-19, which show that bispecific antibody C01 can promote PBMCs to release TNF-a and IL-6 in a tumor antigen specific manner.

[0322] Example 15: Preparation of anti-CD16A / EGFR bispecific antibodies (C03 and C04)

[0323] C03 and C04 bispecific antibodies are prepared according to the method of Example 9 of the present application, the structure of C03 is shown in Figure 20, and the structure of C04 is shown in Figure 21. The prepared C03 and C04 are used in subsequent examples.

[0324] Example 16: Binding of anti-CD16A / EGFR bispecific antibodies (C03 and C04) to CD16A monoclonal stable cell lines

[0325] The binding ability of anti-CD16A / EGFR bispecific antibodies C03 and C04 to CHO-K1 / hCD16A-F monoclonal stable cell lines is detected according to the same method of Example 10 of the present application.

[0326] The experimental results are shown in Figure 22, which show that anti-CD16A / EGFR bispecific antibodies C03 and C04 can dose-dependently bind to CHO-K1 / hCD16A-F.

[0327] Example 17: Detection of the function of anti-CD16A / EGFR bispecific antibodies (C03 and C04) by luciferase reporter gene

[0328] The function of anti-CD16A / EGFR bispecific antibodies C03 and C04 was detected by the same method as in Example 11.

[0329] The experimental results are shown in Figure 23, which shows that both bispecific antibodies C03 and C04 can activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner. The EC50 of AFM24 for activating Jurkat-NFAT-CD16A cells is 1.15 nM, and the EC50 of C03 and C04 for activating Jurkat-NFAT-CD16A cells are 0.015 nM and 0.011 nM, respectively, which is about 100 times better than that of AFM24. In addition, Figure 23 shows that the maximum effect of luciferase luminescence signal of AFM24 is 46917, and the maximum effect of luciferase luminescence signal of C03 and C04 are 401301 and 424109, respectively, which is about 9 times better than that of AFM24. 50 50 The experimental results are shown in Figure 23, which shows that both bispecific antibodies C03 and C04 can activate Jurkat-NFAT-CD16A cells in a tumor antigen specific manner. The EC50 of AFM24 for activating Jurkat-NFAT-CD16A cells is 1.15 nM, and the EC50 of C03 and C04 for activating Jurkat-NFAT-CD16A cells are 0.015 nM and 0.011 nM, respectively, which is about 100 times better than that of AFM24. In addition, Figure 23 shows that the maximum effect of luciferase luminescence signal of AFM24 is 46917, and the maximum effect of luciferase luminescence signal of C03 and C04 are 401301 and 424109, respectively, which is about 9 times better than that of AFM24.

[0330] The experimental results show that, compared with the control AFM24, C03 and C04 can better activate CD16A.

[0331] Example 18: Evaluation of the activity of anti-CD16A / EGFR bispecific antibodies (C03 and C04) in promoting NK cells to kill target cells

[0332] The activity of anti-CD16A / EGFR bispecific antibodies C03 and C04 in promoting NK cells to kill target cells was evaluated by the same method as in Example 12.

[0333] The experimental results are shown in Figure 24, which shows that the bispecific antibodies C03 and C04 of the application can promote NK cells to kill tumor cells HT-29 in a tumor antigen specific manner. The EC50 of AFM24 for promoting NK cells to kill tumor cells HT-29 is 0.020 nM, and the EC50 of C03 and C04 for promoting NK cells to kill tumor cells HT-29 are 0.011 nM and 0.011 nM, respectively. 50 50 In addition, Figure 24 shows that the maximum effect of specific cell lysis of AFM24 is 14.6%, and the maximum effect of specific cell lysis of C03 and C04 are 30.2% and 27.3%, respectively.

[0334] The experimental results show that, compared with the control AFM24, C03 and C04 can better promote NK cells to kill target cells.

[0335] Example 19: Evaluation of the activity of anti-CD16A / EGFR bispecific antibodies (C03 and C04) in promoting NK cells to activate

[0336] ​​The anti-CD16A / EGFR bispecific antibodies C03 and C04 were evaluated for their activity in promoting NK cell activation according to the same method as in Example 13.

[0337] The experimental results are shown in Figure 25. The results show that the bispecific antibodies C03 and C04 of the application can promote the increase of CD107a expression on the surface of NK cells in a tumor antigen specific manner, indicating that they can promote the activation of NK cells. The EC 50 of AFM24 was 0.011 nM, and the EC 50 of C03 and C04 were 0.005 nM and 0.005 nM, respectively; the maximum effect of AFM24 was 9.69%, and the maximum effects of C03 and C04 were 13.9% and 13.0%, respectively.

[0338] The experimental results show that C03 and C04 can better promote the activation of NK cells than the control AFM24.

[0339] Example 20: In vivo efficacy of anti-CD16A / EGFR bispecific antibodies

[0340] Purpose of the experiment: To evaluate the pharmacological efficacy of the test articles AFM24, C03 and C04 in a human epidermal carcinoma A-431 subcutaneous tumor model.

[0341] Experimental materials: Female B-human CD16A transgenic mice were used for the experiment, 6-8 weeks old or 18-20 g.

[0342] Experimental method: The experimental animals were all raised in a specific pathogen-free animal room barrier, and the experimental animals were adapted for 3 days before the experiment. Human epidermal carcinoma A-431 cells were cultured in vitro in monolayer in DMEM complete medium (added with a final concentration of 10% fetal bovine serum) at 37°C in an atmosphere containing 5% CO2. The tumor cells were routinely subcultured 2-3 times per week, and the cells in the exponential growth phase were collected and counted for tumor cell inoculation. 5x10 6 A-431 tumor cells were resuspended in 0.1 ml PBS and matrigel, and inoculated subcutaneously on the right side of each mouse to form tumors. When the average tumor volume of the tumor-bearing mice grew to 100 mm 3When the tumor volume reached 100-200mm3, the tumor-bearing mice were randomly divided into groups, with 5 mice in each group, and a total of 7 groups. The groups were G1 (PBS), G2 (AFM24, 15mg / kg), G3 (AFM24, 45mg / kg), G4 (C03, 15mg / kg), G5 (C03, 45mg / kg), G6 (C04, 15mg / kg), and G7 (C04, 45mg / kg). All the tested products were administered via the tail vein, twice a week for a total of 8 times. During the experiment, the tumor volume and body weight of each group were monitored twice a week. At the end of the experiment, the animals were euthanized, the tumor weight was measured, the tumor volume growth inhibition rate was calculated, and the tumors of the mice in groups G1 to G7 were collected for flow cytometry detection. The main detection indicators included immune cells, macrophages (Macrophage), NK cells, granulocyte-like myeloid-derived suppressor cells (G-MDSC), and monocyte-like myeloid-derived suppressor cells (M-MDSC).

[0343] Tumor volume inhibition rate (TGI TV ): TGI TV (%) = [1-(Ti-T0) / (Ci-C0)]x100%

[0344] (Ti: mean tumor volume of the treatment group on the i-th day of administration, T0: mean tumor volume of the treatment group on the 0-th day of administration; Ci: mean tumor volume of the control group on the i-th day of administration, C0: mean tumor volume of the control group on the 0-th day of administration)

[0345] Experimental results: This experiment tested the effects of AFM24, C03, and C04 on the growth of human epidermoid carcinoma A-431 subcutaneous transplanted tumors. All animals had good activity and eating status during the administration period, and the body weight increased to a certain extent, indicating that the animals were well tolerated to the tested products. On the 28th day after grouping and administration, compared with the G1 (PBS) group, the tumor volume growth inhibition rates TGI TV of groups G2 to G7 were 77.8%, 80.2%, 88.9%, 97.2%, 75.8%, and 92.1%, respectively, with p values all less than 0.0001. All the tested products could significantly inhibit the growth of human epidermoid carcinoma A-431 subcutaneously transplanted tumors, and had a dose-dependent relationship. In particular, at a high dose of 45mg / kg, the C03 and C04 groups of the present application had significant differences in tumor growth inhibition compared with the AFM24 group (p values were all less than 0.05).

[0346] The flow cytometry results at the end of the experiment (Figure 28) showed that the percentage of NK cells and Macrophages cells in mCD45 was significantly increased in the high-dose 45 mg / kg groups of C03, C04 and AFM24 compared with the G1 control; the percentage of G-MDSC positive cells in mCD45 tended to decrease with the increase of the dose of C03 and C04, and the percentage of G-MDSC positive cells in mCD45 was lower in the high-dose groups of C03 and C04 than in the high-dose group of AFM24; the percentage of M-MDSC positive cells in mCD45 was decreased in AFM24, C03 and C04, and the percentage of M-MDSC positive cells in mCD45 was significantly lower in the high-dose group of C03 than in the high-dose group of AFM24.

[0347] In summary, the experimental animals had good tolerance to all the tested products. AFM24, C03 and C04 had significant dose-dependent inhibitory effects on the growth of human epidermal carcinoma A-431 subcutaneous transplanted tumors, and the high-dose groups of C03 and C04 had better tumor inhibition effect than AFM24. Therefore, the therapeutic effects of C03 and C04 of the present application are better than AFM24.

[0348] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of the teachings disclosed herein without departing from the scope of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An anti-CD16A antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), and the anti-CD16A antibody comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH having at least 80% sequence identity to SEQ ID NO: 7; and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL having at least 80% sequence identity to SEQ ID NO:

8.

2. The anti-CD16A antibody of claim 1, wherein: the anti-CD16A antibody comprises HCDR1, HCDR2 and HCDR3 contained in any one of SEQ ID NOs: 9-13 and SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 contained in any one of SEQ ID NOs: 14-19 and SEQ ID NO: 8; optionally, the HCDR1 of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the sequence set forth in SEQ ID NO: 2, and the HCDR3 comprises the sequence set forth in SEQ ID NO: 3; and, the LCDR1 of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 4, the LCDR2 comprises the sequence set forth in SEQ ID NO: 5, and the LCDR3 comprises the sequence set forth in SEQ ID NO: 6; optionally, the heavy chain variable region of the anti-CD16A antibody comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NOs: 1, 2 and 3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NOs: 4, 5 and 6, respectively.

3. An anti-CD16A antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (1) the VH of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 7 or a variant thereof, and the VL of the anti-CD16A antibody comprises the sequence set forth in SEQ ID NO: 8 or a variant thereof; or (2) the VH of the anti-CD16A antibody comprises any one of SEQ ID NOs: 9-13 or a variant thereof, and the VL of the anti-CD16A antibody comprises any one of SEQ ID NOs: 14-19 or a variant thereof; wherein the variant has at least 80% sequence identity to the sequence from which it is derived; optionally, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived; optionally, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived.

4. The anti-CD16A antibody according to any one of claims 1 to 3, wherein the anti-CD16A antibody comprises a framework region sequence derived from a human immunoglobulin; optionally, the anti-CD16A antibody comprises a heavy chain framework region of a human-derived antibody, and / or a light chain framework region of a human-derived antibody; Optionally, the heavy chain framework region and / or the light chain framework region of the anti-CD16A antibody comprises one or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from human-derived residues to murine-derived residues; Optionally, the anti-CD16A antibody, wherein: (1) the VH of the anti-CD16A antibody comprises a sequence as set forth in SEQ ID NO: 9 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 14-19 or a variant thereof; (2) the VH of the anti-CD16A antibody comprises a sequence as set forth in SEQ ID NO: 10 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 14-19 or a variant thereof; (3) the VH of the anti-CD16A antibody comprises a sequence as set forth in SEQ ID NO: 11 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 14-19 or a variant thereof; (4) the VH of the anti-CD16A antibody comprises a sequence as set forth in SEQ ID NO: 12 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 14-19 or a variant thereof; or (5) the VH of the anti-CD16A antibody comprises a sequence as set forth in SEQ ID NO: 13 or a variant thereof, and the VL of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 14-19 or a variant thereof; wherein the variant has at least 80% sequence identity compared to the sequence from which it is derived; Optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; Optionally, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived; Optionally, the anti-CD16A antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO:

14.

5. The anti-CD16A antibody according to any one of claims 1 to 4, wherein The anti-CD16A antibody comprises a constant region; Optionally, the heavy chain of the anti-CD16A antibody comprises a heavy chain constant region (CH) derived from a human immunoglobulin, and / or the light chain of the anti-CD16A antibody comprises a light chain constant region (CL) derived from a human immunoglobulin; Optionally, the heavy chain of the anti-CD16A antibody comprises a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, and / or the light chain of the anti-CD16A antibody comprises a human kappa or lambda light chain constant region; Optionally, the heavy chain of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 44-48 or a variant thereof, and the light chain of the anti-CD16A antibody comprises a sequence as set forth in any one of SEQ ID NOs: 49-54 or a variant thereof; Optionally, the anti-CD16A antibody, wherein: (1) the heavy chain comprises a sequence as set forth in SEQ ID NO: 42 or a variant thereof, and the light chain comprises a sequence as set forth in SEQ ID NO: 43 or a variant thereof; (2) the heavy chain comprises a sequence set forth in SEQ ID NO: 44, or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54, or a variant thereof; (3) the heavy chain comprises a sequence set forth in SEQ ID NO: 45, or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54, or a variant thereof; (4) the heavy chain comprises a sequence set forth in SEQ ID NO: 46, or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54, or a variant thereof; (5) the heavy chain comprises a sequence set forth in SEQ ID NO: 47, or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54, or a variant thereof; or (6) the heavy chain comprises a sequence set forth in SEQ ID NO: 48, or a variant thereof, and the light chain comprises any one of SEQ ID NOs: 49-54, or a variant thereof; wherein the variant has at least 80% sequence identity to the sequence from which it is derived; optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; optionally, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived; optionally, the anti-CD16A antibody comprises a heavy chain as set forth in SEQ ID NO: 44 and a light chain as set forth in SEQ ID NO:

49.

6. The anti-CD16A antibody according to any one of claims 1 to 5, wherein the anti-CD16A antibody is an intact antibody or an antigen-binding fragment; optionally, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide linked Fv, scFv, di-scFv, (scFv)2, and diabody (diabody); optionally, the anti-CD16A antibody is a murine antibody, a humanized antibody, or a chimeric antibody; optionally, the anti-CD16A antibody has at least one of the following functions: (1) specifically binds to human CD16A antigen, and / or specifically binds to cynomolgus monkey CD16 antigen; (2) does not bind to human CD16B; (3) promotes the killing ability of NK cells against tumor cells; (4) promotes the killing ability of PBMCs against tumor cells.

7. An anti-CD16A antibody that binds to the same epitope as, or competes for binding to human CD16A antigen with, the anti-CD16A antibody of any one of claims 1-6.

8. A bispecific antibody comprising a first antigen binding domain that specifically binds to a first antigen and a second antigen binding domain that specifically binds to a second antigen, wherein, the first antigen is CD16A, and the first antigen binding domain is selected from the antigen binding domain of the anti-CD16A antibody of any one of claims 1-7; optionally, the second antigen is selected from a tumor associated antigen (TAA); optionally, the second antigen is selected from a tumor associated antigen (TAA); Optionally, the TAA is selected from CD19, CD20, CD22, EGFR, BCMA, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CDH17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof. Optionally, the TAA is selected from EGFR or BCMA.

9. The bispecific antibody of claim 8, the second antigen is EGFR, the second antigen binding domain comprises a VH and a VL, wherein, the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 in a VH set forth in SEQ ID NO: 20, and the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 in a VL set forth in SEQ ID NO: 21; Optionally, the bispecific antibody, wherein: the VH of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence set forth in SEQ ID NO: 22, HCDR2 comprises the sequence set forth in SEQ ID NO: 23, and HCDR3 comprises the sequence set forth in SEQ ID NO: 24; and the VL of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence set forth in SEQ ID NO: 25, LCDR2 comprises the sequence set forth in SEQ ID NO: 26, and LCDR3 comprises the sequence set forth in SEQ ID NO: 27; Optionally, the heavy chain variable region of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NOs: 22, 23, and 24, respectively, and the light chain variable region of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NOs: 25, 26, and 27, respectively; Optionally, the VH of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 20 or a variant thereof, and the VL of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 21 or a variant thereof; wherein the variant has at least 80% sequence identity compared to the sequence from which it is derived. Optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence from which it is derived. Optionally, the TAA is selected from CD19, CD20, CD22, EGFR, BCMA, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CDH17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof. Optionally, the TAA is selected from EGFR or BCMA. Optionally, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived; Optionally, the second antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO: 20 and a light chain variable region as set forth in SEQ ID NO:

21.

10. The bispecific antibody of claim 8, the second antigen is BCMA, the second antigen binding domain comprises a VH and a VL, wherein, the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 in a VH as set forth in SEQ ID NO: 28, and the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 in a VL as set forth in SEQ ID NO: 29; Optionally, the second antigen binding domain, wherein: the VH of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the sequence set forth in SEQ ID NO: 30, HCDR2 comprises the sequence set forth in SEQ ID NO: 31, and HCDR3 comprises the sequence set forth in SEQ ID NO: 32; and the VL of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the sequence set forth in SEQ ID NO: 33, LCDR2 comprises the sequence set forth in SEQ ID NO: 34, and LCDR3 comprises the sequence set forth in SEQ ID NO: 35; Optionally, the heavy chain variable region of the second antigen binding domain comprises HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 30, 31, and 32, respectively, and the light chain variable region of the second antigen binding domain comprises LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 33, 34, and 35, respectively; Optionally, the VH of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 28 or a variant thereof, and the VL of the second antigen binding domain comprises the sequence set forth in SEQ ID NO: 29 or a variant thereof; wherein the variant has at least 80% sequence identity compared to the sequence from which it is derived; Optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; Optionally, the variant has at most 10 amino acid substitutions compared to the sequence from which it is derived; Optionally, the second antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO: 20 and a light chain variable region as set forth in SEQ ID NO:

21.

11. The bispecific antibody of any one of claims 8 to 10, wherein, the first and second antigen binding domains are antigen binding fragments; optionally, the first and second antigen binding domains are each independently selected from the group consisting of Fab, Fab’, (Fab’)2, Fv, disulfide linked Fv, and scFv; Optionally, the first or second antigen binding domain is murine, fully human, or chimeric; Optionally, the first antigen binding domain is a Fab; Optionally, the second antigen binding domain is an scFv, the VH of the second antigen binding domain is directly or via a peptide linker connected to the VL of the second antigen binding domain; Optionally, the peptide linker is (GmS)n, wherein m, n are each independently an integer from 1 to 10; optionally, m, n are each independently 1, 2, 3, 4, 5, or 6.

12. The bispecific antibody of any one of claims 8 to 11, wherein, The bispecific antibody comprises an immunoglobulin Fc; Optionally, the immunoglobulin Fc is directly or via a peptide linker connected to the first and second antigen binding domains; Optionally, the immunoglobulin Fc is an Fc of human IgG1, IgG2, IgG3, or IgG4; Optionally, the peptide linker is (GmS)n, wherein m, n are each independently an integer from 1 to 10; optionally, m, n are each independently 1, 2, 3, 4, 5, or 6. Optionally, the immunoglobulin Fc comprises L234F, L235E, and D265A mutations.

13. The bispecific antibody of any one of claims 8 to 12, wherein, The bispecific antibody is selected from any one of A, B, or C: A) the bispecific antibody comprises peptide chains I-A, I-B, I-C, and I-D, wherein: 1) peptide chain I-A comprises, from N-terminus to C-terminus, the VL and CL of the first antigen binding domain; optionally, the CL is a human immunoglobulin kappa or lambda light chain; 2) peptide chain I-B comprises, from N-terminus to C-terminus, the VH, CH, peptide linker 1, VH of the second antigen binding domain, peptide linker 2, VL of the second antigen binding domain of the first antigen binding domain; optionally, the CH is a human IgG1, IgG2, IgG3, or IgG4 CH; optionally, the peptide linker 1 and peptide linker 2 are each independently selected from (GmS)n, wherein m, n are each independently an integer from 1 to 10; optionally, m, n are each independently 1, 2, 3, 4, 5, or 6; 3) peptide chain I-C is identical to peptide chain I-B; 4) peptide chain I-D is identical to peptide chain I-A; optionally, a disulfide bond is formed between the peptide chain I-B and the peptide chain I-C; B) the bispecific antibody comprises peptide chain II-A, which comprises, from N-terminus to C-terminus: the VH, peptide linker 1, VL of the second antigen binding domain, peptide linker 2, VH of the first antigen binding domain, peptide linker 3, VL of the first antigen binding domain, peptide linker 4, and Fc of the first antigen binding domain; optionally, the Fc is a human IgG1, IgG2, IgG3, or IgG4 Fc; optionally, the peptide linker 1, peptide linker 2, peptide linker 3, and peptide linker 4 are each independently selected from (GmS)n, wherein m, n are each independently an integer from 1 to 10; optionally, m, n are each independently 1, 2, 3, 4, 5, or 6; optionally, the bispecific antibody comprises 2 peptide chains II-A; optionally, a disulfide bond is formed between the 2 peptide chains II-A; C) the bispecific antibody comprises a peptide chain III-A and a peptide chain III-B, wherein: the peptide chain III-A comprises, in N-terminal to C-terminal order: the VH of the second antigen binding domain, a peptide linker 1, the VL of the second antigen binding domain, a peptide linker 2, the VH of the first antigen binding domain, and a CH; the peptide chain III-B comprises, in N-terminal to C-terminal order: the VL of the first antigen binding domain, and a CL; optionally, the CH is a human IgGl, IgG2, IgG3, or IgG4 CH; optionally, the peptide linker 1 and the peptide linker 2 are each independently selected from (GmS)n, wherein m, n are each independently an integer of 1-10; optionally, m, n are each independently 1, 2, 3, 4, 5, or 6; optionally, the bispecific antibody comprises 2 peptide chains III-A and 2 peptide chains III-B; optionally, a disulfide bond is formed between the 2 peptide chains III-A, and a disulfide bond is formed between III-A and the peptide chain III-B; optionally, the bispecific antibody, wherein, a) the peptide chain I-A comprises a sequence as set forth in SEQ ID NO: 37 or a variant thereof, and the peptide chain I-B comprises a sequence as set forth in SEQ ID NO: 36 or a variant thereof; or the peptide chain I-A comprises a sequence as set forth in SEQ ID NO: 39 or a variant thereof, and the peptide chain I-B comprises a sequence as set forth in SEQ ID NO: 38 or a variant thereof; wherein the variant has at least 80% sequence identity to the sequence from which it is derived; optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence from which it is derived; optionally, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived; optionally, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 37 and 2 sequences as set forth in SEQ ID NO: 36; or the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 39 and 2 sequences as set forth in SEQ ID NO: 38; b) the peptide chain II-A comprises a sequence as set forth in SEQ ID NO: 55 or a variant thereof, the variant having at least 80% sequence identity to the sequence of SEQ ID NO: 55; optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence of SEQ ID NO: 55; optionally, the variant has up to 10 amino acid substitutions compared to the sequence of SEQ ID NO: 55; optionally, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 55; and / or b) the peptide chain II-A comprises a sequence as set forth in SEQ ID NO: 55 or a variant thereof, the variant having at least 80% sequence identity to the sequence of SEQ ID NO: 55; optionally, the variant has one or several amino acid substitutions, deletions, or additions compared to the sequence of SEQ ID NO: 55; optionally, the variant has up to 10 amino acid substitutions compared to the sequence of SEQ ID NO: 55; optionally, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 55; and / or c) the peptide chain III-A comprises a sequence as set forth in SEQ ID NO: 56 or a variant thereof having at least 80% sequence identity to the sequence of SEQ ID NO: 56, and the peptide chain III-B comprises a sequence as set forth in SEQ ID NO: 57 or a variant thereof having at least 80% sequence identity to the sequence of SEQ ID NO: 57; optionally, the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived; optionally, the variant has up to 10 amino acid substitutions compared to the sequence from which it is derived; optionally, the bispecific antibody comprises 2 sequences as set forth in SEQ ID NO: 56 and 2 sequences as set forth in SEQ ID NO:

57.

14. A multispecific molecule comprising the anti-CD16A antibody of any one of claims 1 to 7 or the bispecific antibody of any one of claims 8 to 13. Optionally, the multispecific molecule specifically binds CD16A and specifically binds one or more other antigens.

15. An isolated nucleic acid molecule encoding the anti-CD16A antibody of any one of claims 1 to 7, the bispecific antibody of any one of claims 8 to 13, or the multispecific molecule of claim 14.

16. A vector comprising the nucleic acid molecule of claim 15; optionally, the vector is a cloning vector or an expression vector.

17. A host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16.

18. A method of producing the anti-CD16A antibody of any one of claims 1 to 7, the bispecific antibody of any one of claims 8 to 13, or the multispecific molecule of claim 14, comprising culturing the host cell of claim 17 under conditions that allow expression of the antibody, and recovering the antibody from the cultured host cell.

19. A conjugate comprising the anti-CD16A antibody of any one of claims 1 to 7, the bispecific antibody of any one of claims 8 to 13, or the multispecific molecule of claim 14, and a coupling moiety; Optionally, the coupling moiety is selected from a protein tag, e.g., a purification tag; a detectable label, e.g., an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin; a therapeutic agent, e.g., an antitumor drug or an immunosuppressant; or, a further biologically active polypeptide; Optionally, the conjugate is an antibody conjugate, the coupling moiety is a cytotoxic drug, and the coupling moiety is linked to the anti-CD16A antibody of any one of claims 1 to 7, the bispecific antibody of any one of claims 8 to 13, or the multispecific molecule of claim 14 via a linker.

20. A pharmaceutical composition comprising the anti-CD16A antibody of any one of claims 1 to 7, the bispecific antibody of any one of claims 8 to 13, the multispecific molecule of claim 14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17, or the conjugate of claim 19; and one or more pharmaceutically acceptable excipients; Optionally, the pharmaceutical composition further comprises an additional anti-tumor agent and / or an immunosuppressive agent.

21. Use of the anti-CD16A antibody of any one of claims 1 to 7, the bispecific antibody of any one of claims 8 to 13, the multispecific molecule of claim 14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17, the conjugate of claim 19, or the pharmaceutical composition of claim 20 for the manufacture of a medicament for the prevention and / or treatment of a tumor in a subject; Optionally, the tumor is selected from skin cancer, non-Hodgkin lymphoma, renal cell carcinoma, lung cancer, brain glioma, gastric cancer, head and neck cancer, large intestine cancer, ovarian cancer, esophageal cancer, pancreatic cancer, cervical cancer, mesothelioma, breast cancer, colorectal cancer, or oral squamous carcinoma; Optionally, the anti-CD16A antibody, bispecific antibody, multispecific molecule, nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition is used alone or in combination with an additional anti-tumor agent and / or an immunosuppressive agent; Optionally, the subject is a human.

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