Antibodies and their uses

Abstract

The present invention provides an antibody or antigen-binding protein thereof, which is capable of specifically binding to human CD3 or human GPRC5D protein or MUC16, or to human CD3 and a second antigen. The present invention further provides nucleic acid molecules encoding the antibody, expression vectors for expressing the antibody, host cells, and methods of preparation thereof. The present invention also provides diagnostic and therapeutic methods using the antibody of the present invention.

Description

[Technical field]

[0001] The present invention provides antibodies or antigen-binding proteins thereof, and asymmetric bispecific antibodies constructed thereon. The present invention further provides nucleic acid molecules encoding said antibodies, expression vectors for expressing said antibodies, host cells, and methods for their preparation. The present invention also provides diagnostic and therapeutic methods using the antibodies of the present invention. [Background technology]

[0002] CD3 is a homodimeric or heterodimeric antigen expressed on T cells that binds to the T cell receptor complex (TCR) and is considered to be necessary for T cell activation. Functional CD3 is formed by dimeric association of two of four different chains: ε, ζ, δ, and γ. CD3 dimeric sequences include γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to aggregate CD3 on T cells, thereby triggering T cell activation in a manner similar to TCR engagement via peptide-loaded MHC molecules. Therefore, it has been proposed that anti-CD3 antibodies can be used for therapeutic purposes involving T cell activation. It has also been proposed that bispecific antibodies that can bind to CD3 and target tumor surface antigens can link tumor cells and T cells, thereby directly activating T cells and releasing granzymes, perforins, and cytokines to kill tumors, thereby achieving the therapeutic purpose of suppressing tumors.

[0003] G protein-coupled receptor family C group 5 member D (GPRC5D) as an orphan receptor has been found to be highly expressed in plasma cells of multiple myeloma patients and correlates with the survival rate of the patients (Atamaniuk, J., et al. (2012). "Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma." Eur J Clin Invest 42(9):953-960.). Further studies have shown that GPRC5D is expressed on the plasma cell surface in normal human blood cells, while GPRC5D expression on the surface of all other blood cells is negative, and GPRC5D is highly expressed on the surface of CD38+CD138+ plasma cells isolated from the bodies of multiple myeloma patients (Kodama, T., et al. (2019). "Anti-GPRC5D / CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma." Mol Cancer Ther 18(9):1555~1564.), and analysis of multiple myeloma cells from patients found no correlation between GPRC5D and BCMA expression (Smith, EL, et al. (2019). "GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells." Sci Transl Med 11(485).) (Pillarisetti, K., et al.(2020). "A T-cell-redirecting bispecific G-protein-coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma." Blood 135(15):1232~1243.), thus indicating that GPRC5D may also function as a tumor-specific target in multiple myeloma.GPRC5D may also function as a tumor-specific target in other tumors.

[0004] Since its discovery, MUC16 protein has been used as a specific marker for ovarian cancer, and is also thought to be associated with poor prognosis of ovarian cancer. MUC16 is a type I transmembrane glycoprotein with a large molecular weight, which consists of an extracellular domain, a transmembrane segment, and an intracellular portion. The extracellular domain is divided into two parts, the first half of which is highly glycosylated, and the second half of which consists of about 60 tandemly linked repeat domains, each of which has 156 amino acids. The N-terminus of MUC16 protein contains 56 SEA domains, and MUC16 protein is cleaved at the first or second SEA domain at the membrane-proximal end of the extracellular domain and releases free CA125 (Das, S. and SK Batra (2015). "Understanding the Unique Attributes of MUC16 (CA125): Potential Implications in Targeted Therapy." Cancer Res 75(22):4669-4674, CA125 enters the circulation by being shed from the full-length MUC16 at the cell surface, Das, S., et al. (2015). "Membrane proximal ectodomain cleavage of MUC16 occurs in the acidifying Golgi / post-Golgi compartments" Sci Rep 5:9759). In addition to ovarian cancer, CA125 index has also been found to be elevated in other tumors, such as breast cancer, pancreatic cancer, colorectal cancer, and non-small cell lung cancer. Studies have shown that MUC16 promotes tumor invasion, metastasis, and suppression of immune responses, etc., and based on this, MUC16 may be a potential target for treating cancer.Although clinical research has been conducted on therapies targeting MUC16 for a long time, the clinical results have not met expectations (Aithal, A., et al. (2018), "MUC16 as a novel target for cancer therapy", Expert Opin Ther Targets 22(8):675-686). The main reason is that most of the antibodies already developed can bind to free CA125 in the circulation, thereby reducing the amount of binding between the antibody and membrane MUC16, and reducing the antibody's ability to penetrate the tumor.

[0005] In recent years, immune checkpoint inhibitors (ICIs) have opened a new era of tumor immunotherapy, relieving immunosuppression by blocking the binding of immune checkpoint molecules (e.g., PD-1 or CTLA4) to their ligands, thereby restoring T cell function. To date, drugs of the immune checkpoint inhibitor class have been approved for sale in several indications, including melanoma, non-small cell lung cancer, and lymphoma (Pico de Coana, Y., et al. (2015). "Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system." Trends Mol Med 21(8): 482-491) (Bu, X., et al. (2017). "Immune Checkpoint Blockade in Breast Cancer Therapy." Adv Exp Med Biol 1026: 383-402) (Hargadon, KM, et al. (2018). "Immune checkpoint blockade therapy for cancer: An overview of FDA-approved immune checkpoint inhibitors." Int Immunopharmacol 62: 29-39). Nevertheless, only a small proportion of ovarian cancer patients benefit from treatment with immune checkpoint inhibitors (Borella, F., et al. (2020). "Immune Checkpoint Inhibitors in Epithelial Ovarian Cancer: An Overview on Efficacy and Future Perspectives." Diagnostics (Basel) 10 (3)) (Lorusso, D., et al. (2020). "Emerging role of immune checkpoint inhibitors in the treatment of ovarian cancer." Expert Opin Emerg Drugs 25 (4): 445-453).The immune system mainly uses T cells to eliminate tumor cells in the body, but tumor cells use various methods to avoid being killed by the immune system. T cell engagers are a type of bispecific antibody that can simultaneously bind to CD3 on T cells and tumor-associated antigens (TAA) on tumor cells (Labrijn, AF, et al. (2019). "Bispecific antibodies: a mechanistic review of the pipeline." Nat Rev Drug Discov 18(8):585-608.) (Ellerman, D. (2019). "Bispecific T-cell engagers: Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety" Methods 154:102-117). This bispecific antibody binds to both T cells and tumor cells, promotes the formation of an immune synapse between the cells, and kills tumor cells by activating T cells. The feasibility of this theory has been demonstrated by blinatumomab as the first commercially available T cell engager, with many more T cell engagers in early development and widely used to treat hematological and solid tumors (Goebeler, ME and RC Bargou (2020). "T cell-engaging therapies-BiTEs and beyond." Nat Rev Clin Oncol 17(7):418-434.).

[0006] Therefore, there is a need in the art for the development of antibodies that specifically bind to CD3, antibodies that specifically bind to CD3 and other antigens, and bispecific antibodies constructed based on them, for the treatment of cancer. Summary of the Invention

[0007] The present invention provides an anti-CD3 antibody or antigen-binding fragment thereof that targets human CD3, which has the following advantages: 1) highly specific binding to human CD3 and target cells expressing human CD3; 2) suitability as a genetic engineering element; 3) Low immunogenicity.

[0008] The present invention further provides anti-MUC16 antibodies that target human MUC16 and have one or more of the following advantages: (1) binds with high affinity to MUC16 (e.g., human or monkey MUC16) and to target cells expressing human MUC16; (2) it does not bind to free circulating CA125; (3) Suitable as a genetic engineering element; (4) low immunogenicity; (5) High expression purity; (6) Maintain high affinity even after humanization.

[0009] The present invention provides novel anti-GPRC5D antibodies and bispecific antibodies with CD3 antibodies constructed using the same. In some embodiments, the anti-GPRC5D antibodies of the present invention have high binding affinity to human GPRC5D and can recognize human and monkey (e.g., cynomolgus monkey) GPRC5D.

[0010] In some embodiments, the anti-GPRC5D antibody can specifically bind to GPRC5D (eg, human GPRC5D and / or monkey (eg, cynomolgus) GPRC5D) with high affinity and has binding activity significantly superior to that of the control antibody GC5B596.

[0011] The present invention further provides CD3-based bispecific antibodies, eg, bispecific antibodies that specifically bind to CD3 and MUC16.

[0012] The anti-CD3xMUC16 bispecific antibodies of the invention have one or more of the following properties: (1) highly specific binding to human CD3 and target cells expressing human CD3, and at the same time, highly affinity binding to human MUC16 and target cells expressing human MUC16; (2) to target more T cells to cancer cells that do not bind to free circulating CA125 and that simultaneously express MUC16; (3) Activating T cells and inducing cytotoxic activity against cancer cells expressing MUC16, thereby effectively killing the cancer cells; (4) low immunogenicity; (5) Excellent tumor suppression effect.

[0013] The anti-CD3 / GPRC5D bispecific antibody of the present invention has one or more of the following properties and is therefore expected to have excellent therapeutic effects in GPRC5D-related indications: (1) in a cell (e.g., a T cell, e.g., a Jurkat cell), by binding to CD3 expressed on the cell; (2) binding to human and / or monkey (e.g., cynomolgus monkey) GPRC5D, e.g., binding to GPRC5D expressed on a cell, e.g., binding to GPRC5D with high affinity; (3) the CD3 signaling pathway is activated only in the presence of cells expressing GPRC5D, e.g., only in the presence of cells expressing GPRC5D; (4) In the absence of cells expressing GPRC5D, the CD3 signaling pathway is activated only at high concentrations. (5) There is little non-specific activation of the CD3 signaling pathway. For example, in cells that do not express GPRC5D (e.g., HEK293T cells), the signaling pathway is activated only at high concentrations and in a concentration-dependent manner. (6) To induce more specific T cells that kill cells expressing GPRC5D without non-specific activation or visible non-specific activation, for example, to kill only target cells expressing GPRC5D and not cells that do not express GPRC5D; (7) Having better cytokine induction specificity, for example, only in cells expressing GPRC5D (e.g., HEK293T-GPRC5D cells), the cells are induced to release cytokines such as interferon (e.g., IFNγ), tumor necrosis factor (e.g., TNFα), and / or interleukin (e.g., IL-6); (8) It is capable of specifically binding to human and monkey (e.g., cynomolgus monkey) CD3 proteins in an in vitro binding assay such as Fortebio, i.e., it has species cross-reactivity; (9) Optimal CD3 affinity, e.g., affinity K in in vitro binding assays such as those by Fortebio D is between 1 and 1000 nM, e.g., the binding affinity K D is between 1 and 100 nM, for example between 10 and 100 nM, for example between 50 and 100 nM, e.g., the binding affinity K D is between 1 and 1000 nM, for example, between 1 and 100 nM, for example, between 10 and 100 nM, for example, between 50 and 100 nM, for example, between 100 and 1000 nM, for example, between 200 and 1000 nM; (10) It has better tumor inhibition effect, for example, the tumor inhibition rate is 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, and even it can completely regress the tumor. (11) Capable of binding to human and monkey (e.g., cynomolgus monkey) GPRC5D expressed on cells and / or capable of binding to human and monkey (e.g., cynomolgus monkey) CD3 expressed on cells, and having cross-species activity against GPRC5D and / or CD3. [Brief description of the drawings]

[0014] [Figure 1] Humanized anti-CD3 antibodies Roche-CD3 / 017-Roche-CD3, DA023AH23L2 (Figure 1A) and 017-2 (Figure 1B) activate the Jurkat / NFAT-luc reporter gene system. [Diagram 2] Mouse anti-GPRC5D antibody binds to cells expressing human GPRC5D protein (top) and cynomolgus monkey GPRC5D protein (bottom). [Diagram 3] Humanized anti-GPRC5D antibodies bind to engineered cells that express the human GPRC5D protein. [Figure 4] Schematic diagram of bispecific molecule structure. [Diagram 5] The anti-CD3 / anti-GPRC5D bispecific antibody binds to Jurkat cells stably expressing the human CD3 protein. [Figure 6] A and B: Anti-CD3 / anti-GPRC5D bispecific antibody binds to HEK293T cells stably expressing human GPRC5D protein; C: Anti-CD3 / anti-GPRC5D bispecific antibody Bi-29H6-6 binds to HEK293T cells stably expressing cynomolgus monkey GPRC5D protein; D-F: Anti-CD3 / GPRC5D bispecific antibody Bi-29H6-6 does not bind to cells expressing GPRC5A, GPRC5B or GPRC5C proteins. [Figure 7] The anti-CD3 / GPRC5D bispecific antibody is capable of activating the CD3 signaling pathway in the presence of cells expressing human GPRC5D. [Figure 8] The anti-CD3 / GPRC5D bispecific antibody is unable to activate the CD3 signaling pathway in the presence of cells that do not express human GPRC5D. [Figure 9] Anti-CD3 / GPRC5D bispecific antibodies induce PBMCs to specifically kill tumor cells expressing GPRC5D. [Figure 10] Anti-CD3 / GPRC5D bispecific antibody-mediated killing of irrelevant target cells, Raji, by PBMCs. [Figure 11] Detection of T cell released secretory factors IFNγ (A), IL-6 (B), and TNFα (C) of anti-CD3 / GPRC5D bispecific antibodies in the NCI-H929 system. [Figure 12]Detection of T cell-released secretory factors IFNγ (A), IL-6 (B), and TNFα (C) by anti-CD3 / GPRC5D bispecific antibodies in the Raji line, which does not express GPRC5D. [Figure 13] A: Antitumor effect of anti-CD3 / anti-GPRC5D bispecific antibody in human CD3E transgenic mouse MC38-huGPRC5D model. B: Antitumor effect of anti-CD3 / anti-GPRC5D bispecific antibody in NCI-H929 tumor model inoculated with human PBMC transplanted in NCG mouse. [Figure 14] SDS-PAGE electrophoresis results of mouse anti-MUC16 antibody 776.1 and its mutants (left panel: non-reduced SDS-PAGE electrophoresis results, right panel: reduced SDS-PAGE electrophoresis results). [Figure 15] Flow cytometry detection of binding of mouse anti-human MUC16 antibody 776.1 and its variants to 293T cells expressing human Muc16 protein. [Figure 16] Flow cytometry detection of binding of mouse anti-human MUC16 antibody 776.1 and its variants to 293T cells expressing rhesus MUC16 protein. [Figure 17] ELISA results showing binding of humanized anti-MUC-16 antibody Hu-L4H7 to 293T cells expressing human MUC16 protein. [Figure 18] ELISA results showing that the humanized anti-MUC-16 antibody Hu-L4H7 does not bind to human CA125. [Figure 19] Results of immunohistochemical detection of MUC16 and CD8 in tumor tissues. [Figure 20] Schematic diagram of the bispecific antibody molecule structure [Figure 21] Anti-CD3 / anti-MUC16 bispecific antibody binds to OVCAR3 cells. [Figure 22] The anti-CD3 / anti-MUC16 bispecific antibody binds to 293T / rhesus MUC16 cells. [Diagram 23] The anti-CD3 / anti-MUC16 bispecific antibody binds to Jurkat cells. [Figure 24] Anti-CD3 / anti-MUC16 bispecific antibody activates Jurkat / NFAT-luc cells in the presence of OVCAR3 tumor cells. [Diagram 25] Anti-CD3 / anti-MUC16 bispecific antibodies do not activate Jurkat / NFAT-luc cells in the presence of 293T cells. [Figure 26] A: Anti-CD3 / anti-MUC16 bispecific antibody specifically mediates T cell killing of tumor cells. B: Anti-CD3 / anti-MUC16 bispecific antibody activity in specifically mediating T cell killing of OVCAR3 tumor cells. Incubated for 72 hours. [Figure 27] As a result of inducing cytokine release after T cell activation with anti-CD3 / anti-MUC16 bispecific antibody, the release of cytokines IFNγ, TNFα, and IL-6 factors was compared when co-cultured with OVCAR3 or 293T cells, respectively. [Figure 28A] Results of tumor volume increase in each group after administration. [Figure 28B] Results of relative tumor volume increase in each group after administration. [Figure 29] Detection of T cell activation, exhaustion, and apoptosis in TDCC experiments. [Diagram 30] Anti-CD3 / anti-MUC16 bispecific antibodies specifically mediate repeated killing of tumor cells by T cells. [Diagram 31] Detection of T cell activation, exhaustion, and apoptosis in TDCC repeated killing experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention It is to be understood that the invention is not limited to the particular methodology, configurations, and reagents described herein, as these may vary, and that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the scope of the claims.

[0016] I. Definition The following definitions are used to interpret this specification, and where appropriate, terms used in the singular may include the plural and vice versa. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0017] The term "about," when used in conjunction with numerical values, is meant to encompass numerical values ​​within a range having a lower limit of 10% less than the specified numerical value and an upper limit of 10% greater than the specified numerical value.

[0018] As used herein, the term "and / or" means any one of available options, or two or more or all of available options.

[0019] As used herein, the term "comprising" or "including" means including the recited elements, integers, or steps, but not excluding any other elements, integers, or steps. When the term "comprising" or "including" is used herein, unless otherwise specified, it also includes the case where it consists of the recited elements, integers, or steps. For example, when referring to an antibody variable region that "comprises" a particular specific sequence, it is also intended to encompass an antibody variable region that consists of that specific sequence.

[0020] References in this specification to "first" and "second" are merely intended to distinguish between two domains or two chains and do not in any way indicate the location of the two domains.

[0021] The term "CD3" as used herein refers to an antigen expressed on T cells as part of the multi-molecular T cell receptor (TCR), i.e., the T cell binding antigen T cell surface glycoprotein CD3, which consists of a homodimer or heterodimer formed by two of the four receptor chains CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-ε (hCD3ε) comprises the amino acid sequence described in UniProtKB / Swiss-Prot:P07766. Human CD3-δ (hCD3δ) comprises the amino acid sequence described in UniProtKB / Swiss-Prot:P04234. In some embodiments, CD3 as described herein refers to CD3 from a human or monkey (e.g., a cynomolgus monkey).

[0022] The term "antibody that binds CD3" or "anti-CD3 antibody" as used herein includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ, or ζ), as well as antibodies and antigen-binding fragments thereof that specifically recognize and bind to a dimeric complex of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). The antibodies and antigen-binding fragments of the invention can bind to soluble CD3, bound CD3, and / or CD3 expressed on the cell surface. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants, such as monomeric and dimeric CD3 structures that lack a transmembrane domain or are otherwise not bound to a cell membrane. In one embodiment, the antigen-binding region that binds to CD3 in the anti-CD3 antibody or antigen-binding fragment thereof (e.g., ScFv) or bispecific antibody of the invention may have relatively low binding activity for CD3 or cells expressing CD3 (e.g., T cells). The binding affinity of the antibody to CD3 can be detected by flow cytometry detection or biofilm layer optical interference technology detection, for example, the measurement test described in Example 6.5 or Example 10.2. Preferably, in the bispecific antibody molecule of the present invention, the binding affinity of the antigen-binding region that binds to CD3 to human or monkey (cynomolgus monkey) CD3 is 1 to 1000 nM. In some embodiments, the anti-CD3 antibody or antigen-binding fragment thereof, or the antigen-binding region that binds to CD3 in the bispecific antibody of the present invention binds to human and / or monkey (e.g., cynomolgus monkey) CD3 with a relatively low binding affinity, and thus can activate human and / or monkey (e.g., cynomolgus monkey) T cells.

[0023] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2, and regulatory T cells (Tregs). Effector cells may further include natural killer cells, macrophages, granulocytes, plasma cells, or B cells (lymphocytes).

[0024] The term "GPRC5D" refers to tumor associated antigen G protein-coupled receptor family C group 5 member D (e.g., human GPRC5D protein of accession number NP_061124.1 or cynomolgus GPRC5D protein of XP_005570249.2). In one embodiment, the human GPRC5D protein of the invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 57, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, the cynomolgus GPRC5D protein of the invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, the antigen-binding region of the anti-GPRC5D antibody or its antigen-binding fragment (e.g., Fab) or bispecific antibody of the present invention that binds to GPRC5D has high affinity binding activity to cells expressing human GPRC5D, for example, has higher binding affinity than a control antibody (e.g., GC5B596). In one embodiment, the measurement is performed by flow cytometry measurement, for example, by the measurement experiment described in Example 6.2. In one embodiment, the antigen-binding region of the anti-GPRC5D antibody or its antigen-binding fragment (e.g., Fab) or bispecific antibody of the present invention that binds to GPRC5D has cross-reactivity with human and monkey (e.g., cynomolgus monkey) GPRC5D, i.e., can bind to human and monkey (e.g., cynomolgus monkey) GPRC5D.

[0025] The terms "whole antibody", "full length antibody", "complete antibody" and "intact antibody" may interchangeably refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) connected to each other via disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs)) with conserved regions (framework regions (FRs)) intervening therebetween. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in binding of the antibody to the antigen, but exhibits several effector functions. In some embodiments, the antibody heavy chain constant region HC of the invention is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises a LALA mutation. In some embodiments, the heavy chain constant region comprises D265A and P329A mutations. In some embodiments, the heavy chain constant region comprises a LALA mutation, a D265A and a P329A mutation. In some embodiments, the heavy chain constant region of the bispecific antibody molecule of the invention comprises a "knobs-in-holes mutation". In some preferred embodiments, the antibody heavy chain constant region HC of the invention is (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 45 or 48; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 45 or 48; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 45 or 48.

[0026] In some embodiments, the antibody light chain constant region LC of the invention is a Lambda or Kappa light chain constant region. (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 47 or 50; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 47 or 50; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 47 or 50.

[0027] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0028] The term "antigen-binding fragment" refers to a portion or fragment of an intact or complete antibody that has fewer amino acid residues than the intact or complete antibody and can bind to an antigen or can bind to an antigen competitively with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be prepared by recombinant DNA technology or by cleaving an intact antibody by enzymatic or chemical means. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, and single domain antibody (sdAb). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; for example, a Fab fragment can be obtained by digesting a complete antibody with papain. Also, F(ab')2 produced by digesting a complete antibody below the disulfide bond in the hinge region with pepsin is a dimer of Fab' and is a bivalent antibody fragment. F(ab')2 can be reduced by breaking the disulfide bonds in the hinge region under neutral conditions, which converts the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (see Fundamental Immunology, edited by WE Paul, Raven Press, NY (1993) for a detailed description of additional antibody fragments). The Fv fragment consists of the single arm VL and VH domains of an antibody. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be recombinantly linked by a synthetic linking peptide that allows the two domains to be produced as a single protein chain, in which the VL and VH regions pair to form a single chain Fv (scFv). The antibody fragment can be obtained by chemical methods, DNA recombinant methods, or protease digestion methods.

[0029] The terms "Fab fragment" or "Fab" can be used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains, comprising an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL and a light chain constant domain CL, wherein one polypeptide chain comprises, from N-terminus to C-terminus, VH and one constant domain selected from CH1 and CL, and the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant domain selected from CL and CH1, wherein the VH domain and the VL domain pair to form an antigen-binding site. In the present specification, a Fab chain comprising the heavy chain constant domain CH1 is also referred to as a "Fab heavy chain", and a Fab chain comprising the light chain constant domain CL is also referred to as a "Fab light chain".

[0030] The term "target" refers to an entity to which a binding molecule binds. A target may be an antigen, or a ligand or receptor.

[0031] The term "antigen" refers to a molecule that elicits an immune response. Such immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that almost any macromolecule, including any protein or peptide, may serve as an antigen. Antigens may also be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to that portion of an antigen that specifically interacts with an antibody molecule.

[0032] The term "target binding region" as used herein refers to the portion of a multispecific binding molecule, e.g., a bispecific binding molecule, that binds to a specific target or antigen. The target binding region may, for example, be an antibody or immunoglobulin itself, or an antibody fragment. Such a target binding region may or may not have a tertiary structure independent of the rest of the bispecific antibody molecule, and may or may not bind to its target as a separate entity. The target binding region may also be a receptor or a ligand, or a domain of a receptor that can bind to a ligand. In the case of a multispecific or bispecific antibody, the "target binding region" is also referred to as the "antigen binding region". In one embodiment, the antigen binding region used in the bispecific antibody molecule of the invention comprises a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), which may be comprised in a single polypeptide chain (e.g., scFv) or in two isolated polypeptide chains (e.g., Fab heavy and Fab light chains, respectively).

[0033] The term "monospecific" antibody as used herein refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The term "multispecific" antibody as used herein refers to an antibody having at least two antigen binding sites, of which at least one antigen binding site binds to a different antigen epitope, e.g., a different epitope on the same antigen or a different epitope on a different antigen, relative to the remaining antigen binding sites. For example, the present invention provides an antibody against GPRC5D, an antibody against CD3, and a bispecific antibody against GPRC5D and CD3. The present invention further provides a bispecific antibody against CD3 and MUC16.

[0034] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an IgG class immunoglobulin is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has one heavy chain variable region (VH), also called the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has one light chain variable region (VL), also called the light chain variable domain, followed by one light chain constant domain (CL). The heavy chains of immunoglobulins can be classified into one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subclasses, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of immunoglobulins can be classified into one of two types called κ and λ, depending on the amino acid sequence of their constant domain. IgG immunoglobulins essentially consist of two Fab molecules connected via an immunoglobulin hinge region and two dimerized Fc regions (Fc dimers).

[0035] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains of natural antibodies usually have a similar structure, in which each domain contains four conserved framework regions (FR) and three complementarity determining regions.

[0036] "Complementarity determining regions", "CDR regions" or "CDRs" are regions in an antibody variable domain that are hypervariable in sequence and form structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of the heavy and light chains are usually called CDR1, CDR2 and CDR3 and are numbered sequentially from the N-terminus. The CDRs in the heavy chain variable domain of an antibody are called HCDR1, HCDR2 and HCDR3, and the CDRs in the light chain variable domain of an antibody are called LCDR1, LCDR2 and LCDR3. For a given light or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined by any one or a combination of a number of known antibody CDR assignment methods, such as those proposed by Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), which are based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDepartment of Health and Human Services, National Institutes of Health (1987)), which are based on the variability of the antibody sequence; AbM (University of Bath), Contact (University College London), International ImMunoGeneTics (University College London), and others. The database (IMGT) (available on the World Wide Web at www.imgt.cines.fr / ) and a North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures.

[0037] Below are the CDR region ranges defined in the Kabat, AbM, Chothia, Contact and IMGT methods. [Table 1]

[0038] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" includes a CDR sequence determined by any one of the above methods. A CDR may be determined by having the same Kabat numbering position as the sequence of a reference CDR (e.g., any one of the exemplary CDRs of the present invention). Unless otherwise specified, in the present invention, when referring to a residue position in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position based on the Kabat numbering system of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0039] In one embodiment, the HCDRs and LCDRs in the antibodies of the invention are determined according to the Kabat scheme.

[0040] It should be appreciated that there may be differences in the boundaries of the CDRs of the variable regions of the same antibody obtained by different allocation schemes, i.e., there will be differences in the CDR sequences of the variable regions of the same antibody defined by different allocation schemes. Thus, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of said antibody also includes antibodies whose variable region sequences include said specific CDR sequences, but where a different scheme (e.g., different rules or combinations of allocation schemes) are used and such CDR boundaries differ from the specific CDR boundaries defined in the present invention.

[0041] The term "Fc domain" or "Fc region" or "Fc fragment" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of antibodies of the IgG, IgA and IgD classes, or the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) derived from the two heavy chains of antibodies of the IgM and IgE classes. Unless otherwise specified herein, amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also called EU index) described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) (https: / / pubmed.ncbi.nlm.nih.gov / 5257969 / ), see also http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html. As used herein, the term "Fc domain" or "Fc region" or "Fc fragment" does not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, and the heavy chain constant region CH1 and light chain constant region CL, but optionally includes the hinge region at the N-terminus of the heavy chain constant region, e.g., EPKSS or EPKSC. In some embodiments, the heavy chain constant region Fc suitable for the present invention is derived from an antibody heavy chain constant region, such as the constant region of human IgG1, IgG2, IgG3 or IgG4, preferably the constant region of IgG1. In some embodiments, the Fc region comprises a mutation that reduces binding to an Fcγ receptor, such as the LALA mutation, the D265A mutation and / or the P329A mutation, preferably the LALA mutation and the D265A and P329A mutations.In some embodiments, the Fc comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 49, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments of the invention, the Fc fragment dimerizes to form an Fc dimer. In some embodiments, the Fc fragment heterodimerizes to form an Fc heterodimer. When the Fc fragment heterodimerizes to form a heterodimer, the Fc fragment may comprise a mutation for heterodimerization, such as a knob-in-hole mutation.

[0042] Examples of immunoglobulin "effector functions" include C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated uptake of antigens by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0043] The term "chimeric antibody" refers to an antibody molecule in which (a) the constant region or a portion thereof has been altered, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered type, effector function and / or species or an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug) that confers new properties to the chimeric antibody, or (b) the variable region or a portion thereof has been altered, replaced or exchanged by a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by changing its constant region to a constant region derived from a human immunoglobulin. Due to the alteration to a human constant region, the chimeric antibody retains its specificity for antigen recognition and has reduced immunogenicity in humans compared to the original mouse antibody.

[0044] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) and is less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining parts of the antibody with their corresponding human-derived parts (i.e., replacing the parts of the constant and variable regions that are not involved in binding with the corresponding parts of a human antibody).

[0045] As used herein, the terms "anti", "binding" or "specifically binding" mean that the binding action is selective for a target or antigen and can be distinguished from unwanted or non-specific interactions. The binding ability of a binding moiety to a specific target or antigen can be measured by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or other conventional binding assays known in the art, such as radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0046] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its target Y is generally determined by the dissociation constant (K D ), and the dissociation constants are the dissociation rate constant and the association rate constant (k dis and k on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0047] "Percentage identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are the same as those in the specific amino acid sequences set forth herein, after aligning the candidate sequence with the specific amino acid sequences set forth herein and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and where any conservative substitutions are not considered as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention, which have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity, to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may include conservative changes or are conservatively modified variants.

[0048] With respect to a polypeptide sequence, a "conservative change" includes substitutions, deletions, or additions to the polypeptide sequence, but does not substantially alter the desired functional activity of the polypeptide sequence. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following eight groups of amino acids are conservative substitutes for each other: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative sequence changes" is intended to refer to amino acid modifications that do not significantly affect or alter the target antigen binding properties of the antibody or binding protein molecule of the invention that contains the amino acid sequence. For example, conservatively modified variants retain at least 80%, 85%, 90%, 95%, 98%, 99% or more, e.g., 100%-110% or more, of the binding affinity to the target antigen relative to the parent antibody or binding protein.

[0049] "Knob-in-hole" mutation or "knob-in-hole" mutation or "knob-in-hole" as used herein refers to the introduction of a mutation into a first Fc polypeptide and a second Fc polypeptide, respectively, by the "knob-in-hole" technique, thereby forming a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and the interface of the second Fc polypeptide. As is known in the art, the "knob-in-hole" technique can facilitate precise binding of the different chains of an antibody molecule by modifying the interface between the different chains of the antibody molecule. In general, the technique involves introducing a "protrusion / knob" at the interface of one chain and a corresponding "cavity / hole" at the interface of the other chain with which it is to be paired, such that the protrusion can be positioned in the cavity. One preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain with which it is to be paired. A protuberance can be constructed by replacing small amino acid side chains at the interface of the CH3 domain of the heavy chain constant domain from one chain with larger side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protuberance is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired by replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine). Another possible interface involves the CL domain of the light chain and the CH1 domain of the heavy chain, as in the Fab fragment described above, which promotes correct heterodimerization between the two chains of the Fab fragment by establishing a protuberance-cavity interaction.

[0050] The term "single-chain variable fragment" or "scFv" is used herein to refer to a single-chain antibody fragment comprising a heavy chain variable domain, VH, and a light chain variable domain, VL, linked by a linker, of which the VH and VL pair to form an antigen-binding site.

[0051] As used herein, the antibody constant region or antibody constant domain, including the CH1, CL and Fc domains, as well as the CH2, CH3 and optionally CH4 domains constituting the Fc domain, may be selected depending on the desired function of the antibody molecule. For example, the constant region may be an IgA, IgD, IgE, IgG or IgM region, in particular an immunoglobulin constant domain of human IgG, such as a constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably a constant domain of human IgG1. As an example, a Fab fragment of an antibody may include a CH and CL constant region derived from IgG1. As a further example, an Fc region of an antibody may include a CH2 and CH3 domain derived from IgG1. The immunoglobulin constant region may have a native sequence or a variant sequence.

[0052] The term "linker" as used herein refers to any molecule that can be directly linked to different portions of a bispecific binding molecule. Examples of linkers that establish a covalent bond between different molecular portions include peptide linkers and non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the linker is a peptide linker (also called a "linking peptide"), which refers to a short amino acid sequence consisting of amino acids such as glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, to link the amino acid sequence of a first portion of a binding molecule to a second portion of the binding molecule, or a hinge region from an immunoglobulin. For example, a peptide linker can link a first target binding region of a binding molecule to a second target binding region. For example, a peptide linker can also link one portion of an antibody to another portion of an antibody, such as a light chain variable region to a heavy chain variable region. Preferably, the peptide linker is long enough to link two entities such that they maintain a conformation relative to each other without interfering with the desired activity. In one embodiment, the linking peptide is 5-50 amino acids long, e.g., 10, 15, 20, 25, 30 amino acids long. In one embodiment, the linking peptide comprises the amino acid sequence (GS), (GGS), (GSGGS), (GGGGS), (GGGS), and (GGGGS)G, where n is an integer equal to or greater than 1, e.g., n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10. Useful linkers further include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the peptide linker is (GGGGS), where n=1, 2, 3, or 4, e.g., the sequence shown in SEQ ID NO:12.

[0053] In a further embodiment, the connecting peptide is a hinge region or a portion of a hinge region derived from an immunoglobulin, including a native hinge region or a portion thereof, or a mutated hinge region or a portion thereof. In one embodiment, the connecting peptide is, for example, an immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3 or IgG4) hinge region or a portion thereof (e.g., EPKSC), or a mutated hinge region or a portion thereof, e.g., EPKSS. Alternatively, a computer program may be used to simulate the three-dimensional structure of the protein and peptide, or a suitable flexible connecting peptide may be rationally designed by phage display methods.

[0054] Median Effective Concentration (EC 50 The term "maximum 50% response" refers to the concentration of a drug, antibody or toxic agent that induces a 50% response between baseline and maximum after a specific exposure time.

[0055] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", including the originally transformed cell and their derived progeny. Host cells may be any type of cell line that can be used to produce the antibody molecules of the invention, and include eukaryotic cells, such as mammalian cells, insect cells, yeast cells, and prokaryotic cells, such as E. coli cells. Host cells include cultured cells, including cells within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0056] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid linked to it. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. The term "expression vector" refers to a vector that contains a recombinant polynucleotide and includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) into which a recombinant polynucleotide has been incorporated.

[0057] The terms "individual" or "subject" can be used interchangeably and refer to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual refers to a human.

[0058] The term "treat" refers to reducing, interrupting, slowing, ameliorating, arresting, reducing, or reversing the onset of a symptom, complication, or biochemical manifestation of a disease, ameliorating a symptom, or preventing or inhibiting further progression of a disease, condition, or disease.

[0059] The term "prevention" includes the inhibition of the onset or progression of a disease, condition, or symptom of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. In the context of cancer generally, the term "prevention" refers to the administration of an agent before symptoms or symptoms of cancer arise, particularly before cancer occurs in a subject at risk of developing cancer.

[0060] The term "therapeutic agent" as used herein includes any substance effective in the treatment or prevention of a tumor, such as a cancer, including a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (e.g., an immunosuppressant).

[0061] The term "cytotoxic agent" is used herein to refer to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0062] A "chemotherapeutic agent" includes a chemical compound useful in the treatment of cancer or immune system disorders.

[0063] The term "small molecule drug" refers to a low molecular weight organic compound that can regulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, generally less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules are more cell permeable, less susceptible to degradation, and less likely to cause an immune response than large molecules.

[0064] The term "immunomodulatory agent" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates an immune response. The immune response may be a humoral or cellular response. Immunomodulatory agents include immunosuppressants. In some embodiments, immunomodulatory agents of the invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0065] The term "effective amount" refers to an amount or dosage that provides the desired effect in a patient in need of treatment or prevention after administration of one or more doses of an antibody or fragment, or composition or combination of the invention to the patient.

[0066] A "therapeutically effective amount" refers to an amount to effectively achieve a desired therapeutic result at the necessary dosage for the necessary period of time. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment or composition or combination are not outweighed by the beneficial effects of the treatment. A "therapeutically effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%, compared to an untreated subject.

[0067] A "prophylactically effective amount" refers to an amount to effectively achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is applied prior to or at an earlier stage of disease in a subject.

[0068] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells or tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein. The term "tumor" includes solid tumors and liquid tumors.

[0069] The terms "anti-tumor effect" or "tumor inhibitory effect" or "tumor suppressor effect" refer to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a decrease in tumor cell proliferation, or a reduction in tumor cell viability.

[0070] The term "auxiliary pharmaceutical material" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient, vector, stabilizer, etc., administered with an active substance.

[0071] The term "pharmaceutical composition" refers to a composition that is present in a form that effectively activates the biological activity of the active ingredient contained therein, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0072] The term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including, but not limited to, a pharmaceutical kit and a pharmaceutical composition. The term "non-fixed combination" refers to active ingredients (e.g., (i) an immunoconjugate of the present invention, and (ii) an additional therapeutic agent) being administered to a patient simultaneously in separate entities, without specific time restrictions, or sequentially at the same or different time intervals, where such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient's body. The term "fixed combination" refers to two or more active agents being administered to a patient simultaneously in the form of a single entity. Preferably, by selecting the doses and / or time intervals of the two or more active agents, the combination of each component can achieve a better effect in treating a disease or condition than any one component used alone. Each component may be in a single formulation, which may be the same or different.

[0073] The term "combination therapy" refers to the administration of two or more therapeutic agents or forms of treatment (e.g., radiation therapy and surgical therapy) to treat a disease as described herein. Such administration includes co-administration of these therapeutic agents at about the same time, e.g., in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders and liquids). The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. It should be noted that such administration includes the sequential use of each type of therapeutic agent at about the same time or at different times. In either case, the treatment plan provides the beneficial effect of the pharmaceutical combination in treating the disease or condition as described herein.

[0074] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample may be solid tissue, such as fresh, frozen and / or preserved organ or tissue samples, biopsy or aspirate samples, blood or any blood component, bodily fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites) or interstitial fluid, or cells from any stage of pregnancy or development in a subject. In some embodiments, the tissue sample is tumor tissue. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0075] II. Humanized CD3 Antibody One aspect of the present invention provides humanized CD3 antibodies that have better binding affinity to CD3 and are therefore more suitable for constructing antigen-binding regions in bispecific antibody molecules.

[0076] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the invention bind to CD3 (e.g., human CD3 or monkey CD3, such as cynomolgus monkey CD3) with a desired affinity. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the invention can bind to both human CD3 and monkey CD3, such as cynomolgus monkey CD3. In some embodiments, the affinity of the antibodies is measured by biolayer interferometry techniques or surface plasmon resonance.

[0077] In some embodiments, the anti-CD3 antibodies of the invention have an equilibrium dissociation constant (K D In some embodiments, the anti-CD3 antibodies of the invention bind to human CD3 or monkey CD3, such as cynomolgus monkey CD3, with a K D In some embodiments, the anti-CD3 antibodies of the invention have a K of between about 100 nM and 1000 nM (e.g., between about 200, 300, 400, or 500 and 1000 nM). DIt binds to human CD3.

[0078] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention bind to CD3 on the surface of effector cells. In some embodiments, the antibodies or antigen-binding fragments thereof can activate effector cells. In some embodiments, the effector cells are T cells. In some embodiments, the binding is detected using flow cytometry. In some embodiments, the activating effect of CD3 antibodies is detected using a reporter gene detection system (e.g., Jurkat / NFAT-luc reporter gene system).

[0079] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can activate effector cells to induce killing of tumor cells.

[0080] In some embodiments, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3.

[0081] In some embodiments, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention comprise three complementarity determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2 and LCDR3.

[0082] In some embodiments, an anti-CD3 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0083] In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH). In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the invention comprise a light chain variable region (VH). In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0084] In some embodiments, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention further comprise an antibody heavy chain constant region, HC. In some embodiments, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention further comprise an antibody light chain constant region, LC. In some embodiments, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention further comprise a heavy chain constant region, HC, and a light chain constant region, LC.

[0085] In some embodiments, the anti-CD3 antibody heavy chain variable region VH of the present invention is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:3; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:3; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3, and preferably, said amino acid changes do not occur in the CDR regions.

[0086] In some embodiments, the light chain variable region VL of an anti-CD3 antibody of the invention is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:4; or (ii) comprises or consists of the amino acid sequence of SEQ ID NO:4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, and preferably, said amino acid changes do not occur in the CDR regions.

[0087] In some embodiments, the three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region of the anti-CD3 antibody of the invention are (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in any one of SEQ ID NOs: 3; or (ii) a sequence containing at least one amino acid change in total in the three HCDR regions relative to any one of the sequences in (i), and containing no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

[0088] In some embodiments, the three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from the light chain variable region of the anti-CD3 antibody of the invention are (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in any one of SEQ ID NOs: 4; or (ii) a sequence containing at least one amino acid change in total in the three LCDR regions relative to any one of the sequences in (i), and containing no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

[0089] In some embodiments, the anti-CD3 antibodies of the invention include HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:5, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:6, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:7, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9, and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:10.

[0090] In some specific embodiments of the invention, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention comprise a VH and a VL, The VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0091] In some specific embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 3, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 4.

[0092] In some specific embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof of the present invention comprises an HCDR1 set forth in SEQ ID NO:5, an HCDR2 set forth in SEQ ID NO:6, an HCDR3 set forth in SEQ ID NO:7, an LCDR1 set forth in SEQ ID NO:8, an LCDR2 set forth in SEQ ID NO:9, and an LCDR3 set forth in SEQ ID NO:10.

[0093] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0094] In some embodiments, the anti-CD3 antibodies, or antigen-binding fragments thereof, of the invention have one or more of the following properties: (i) exhibiting the same or similar binding affinity and / or specificity for CD3 as an antibody of the invention; (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to CD3; (iii) binding to a similar or overlapping epitope to an antibody of the present invention; (iv) binding to CD3 competitively with the antibody of the present invention; (v) having one or more biological properties of the antibody of the invention.

[0095] In some embodiments, the anti-CD3 antibody of the invention is an IgG1 format antibody or an IgG2 format antibody or an IgG3 format antibody or an IgG4 format antibody, e.g., an IgG1 format antibody. In some embodiments, the light chain constant region of the anti-CD3 antibody of the invention is a kappa or lambda light chain constant region, e.g., a lambda light chain constant region.

[0096] In some embodiments, the anti-CD3 antibody is a monoclonal antibody.

[0097] In some embodiments, the anti-CD3 antibody is humanized.

[0098] In one embodiment, the anti-CD3 antibody of the present invention further comprises an antibody fragment thereof (e.g., an antigen-binding fragment), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab')2, single domain antibody (e.g., VHH), dAb (domain antibody) or linear antibody.

[0099] In one embodiment, the anti-CD3 antibody fragment of the invention has a VH and VL as described herein, and e.g., n=1, 2, 3, 4 or 5, e.g., n=3 or 4. (GGGGS) n For example, the scFv includes a linker peptide that is set forth in SEQ ID NO: 12. In some embodiments, the scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0100] In one embodiment, the anti-CD3 antibody of the invention is a single-chain antibody comprising an anti-CD3 antibody fragment, scFv, and a heavy chain constant region, Fc, optionally comprising a hinge region, or consisting of an scFv and a heavy chain constant region, Fc, optionally comprising a hinge region.

[0101] In some embodiments, the anti-CD3 single chain antibody of the invention comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0102] In one aspect, the present invention provides an anti-CD3 antibody or antigen-binding fragment thereof that targets human CD3, which has the following advantages: 1) highly specific binding to human CD3 and target cells expressing human CD3; 2) suitability as a genetic engineering element; 3) Low immunogenicity.

[0103] In one embodiment, the anti-CD3 antibody or antigen-binding fragment thereof is humanized.

[0104] In one specific embodiment, the anti-CD3 antibody or antigen-binding fragment thereof comprises: 1) A heavy chain variable region (VH) having the sequence set forth in SEQ ID NO:3 CD3 ), or a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 and the same CDRs; and 2) the light chain variable region (VL) whose sequence is set forth in SEQ ID NO:4 CD3 ) or a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and the same CDRs.

[0105] In another specific embodiment, the anti-CD3 antibody or antigen-binding fragment thereof comprises: 1) a heavy chain (H) whose sequence is set forth in SEQ ID NO:1 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1 and the same CDRs; and 2) A light chain (L) whose sequence is set forth in SEQ ID NO:2 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 and having the same CDRs.

[0106] In one embodiment, the antigen-binding fragment of the anti-CD3 antibody is an Fv, Fab, Fab', Fab'-SH, F(ab'), linear antibody, single chain antibody (e.g., scFv). In one specific embodiment, the antigen-binding fragment of the anti-CD3 antibody is an scFv, which comprises the sequence set forth in SEQ ID NO: 11, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11 and the same CDRs. In another embodiment, the anti-CD3 antibody comprises an scFv and an Fc region, which comprises the sequence set forth in SEQ ID NO: 13, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13 and the same CDRs.

[0107] III. Anti-GPRC5D antibody One aspect of the invention provides GPRC5D antibodies of the invention which have a higher binding affinity to GPRC5D and are therefore more suitable for constructing antigen-binding regions in, for example, bispecific antibody molecules.

[0108] In some embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof of the present invention bind to GPRC5D (e.g., human GPRC5D or monkey GPRC5D such as cynomolgus monkey GPRC5D) with higher affinity compared to a control antibody such as GC5B596. In some embodiments, the anti-GPRC5D antibodies or antigen-binding fragments thereof of the present invention can bind to both human GPRC5D and monkey GPRC5D such as cynomolgus monkey GPRC5D.

[0109] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention binds to GPRC5D expressed in a cell. In some embodiments, the affinity of the anti-GPRC5D antibody to GPRC5D expressed in a cell is measured by flow cytometry.

[0110] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2 and HCDR3.

[0111] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2 and LCDR3.

[0112] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0113] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH). In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VH). In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0114] In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention further comprises an antibody heavy chain constant region HC. In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention further comprises an antibody light chain constant region LC. In some embodiments, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region HC and a light chain constant region LC.

[0115] In some embodiments, the heavy chain variable region of the anti-GPRC5D antibody of the invention comprises: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38, or 40; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40, and preferably, said amino acid changes do not occur in the CDR regions.

[0116] In some embodiments, the light chain variable region of the anti-GPRC5D antibody of the invention comprises: (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19, or 21; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21, and preferably, said amino acid changes do not occur in the CDR regions.

[0117] In some embodiments, the three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region of the anti-GPRC5D antibody of the invention are (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in any one of SEQ ID NOs: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; or (ii) a sequence containing at least one amino acid change in total in the three HCDR regions and no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) relative to any one of the sequences of (i); For example, the CDRs are determined according to the Kabat method.

[0118] In some embodiments, the three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from the light chain variable region of the anti-GPRC5D antibody of the invention are (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in any one of SEQ ID NOs: 63, 73, 83, 17, 19 or 21; or (ii) a sequence containing at least one amino acid change in total in the three LCDR regions and no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) relative to any one of the sequences of (i); For example, the CDRs are determined according to the Kabat method.

[0119] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 64, 74 or 84, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 64, 74 or 84.

[0120] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 65, 75, 85, 39 or 41, or HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 65, 75, 85, 39 or 41.

[0121] In some embodiments, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 66, 76 or 86, or the HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 66, 76 or 86.

[0122] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 67, 77 or 87, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 67, 77 or 87.

[0123] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 68 or 78, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 68, 78.

[0124] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 69, 79 or 88, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 69, 79 or 88.

[0125] In some specific embodiments of the invention, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a VH and a VL, (i) the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (ii) the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 72 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iii) the VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 82 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 83 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iv) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, 19 or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0126] In some specific embodiments of the invention, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises: (i) HCDR1 as set forth in SEQ ID NO: 64, HCDR2 as set forth in SEQ ID NO: 65, 39 or 41, HCDR3 as set forth in SEQ ID NO: 66, LCDR1 as set forth in SEQ ID NO: 67, LCDR2 as set forth in SEQ ID NO: 68 and LCDR3 as set forth in SEQ ID NO: 69; (ii) HCDR1 as set forth in SEQ ID NO: 74, HCDR2 as set forth in SEQ ID NO: 75, HCDR3 as set forth in SEQ ID NO: 76, LCDR1 as set forth in SEQ ID NO: 77, LCDR2 as set forth in SEQ ID NO: 78, and LCDR3 as set forth in SEQ ID NO: 79, or (iii) HCDR1 shown in SEQ ID NO: 84, HCDR2 shown in SEQ ID NO: 85, HCDR3 shown in SEQ ID NO: 86, LCDR1 shown in SEQ ID NO: 87, LCDR2 shown in SEQ ID NO: 78, and LCDR3 shown in SEQ ID NO: 88.

[0127] In some specific embodiments of the present invention, the anti-GPRC5D antibody or antigen-binding fragment thereof of the present invention comprises a VH and a VL, wherein the VH and VL comprise or consist of the amino acid sequences shown below, respectively: (i) SEQ ID NO: 62 and SEQ ID NO: 63; (ii) SEQ ID NO: 72 and SEQ ID NO: 73; (iii) SEQ ID NO: 82 and SEQ ID NO: 83; (iv) SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 and SEQ ID NO: 21; (v) SEQ ID NO: 16 or 25 and SEQ ID NO: 17, or (vi) SEQ ID NO: 16 or 27 and SEQ ID NO: 19.

[0128] In some embodiments of the invention, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a heavy chain. In some embodiments of the invention, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a light chain. In some embodiments of the invention, the anti-GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain.

[0129] In some specific embodiments, the heavy chain of the anti-GPRC5D antibody comprises or consists of the amino acid sequence of SEQ ID NO: 60, 70, 80, 14, 22, 24, 26 or 28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequence.

[0130] In some specific embodiments, the light chain of the anti-GPRC5D antibody comprises or consists of the amino acid sequence of SEQ ID NO: 61, 71, 81, 15, 18 or 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the above amino acid sequence.

[0131] In some specific embodiments, the anti-GPRC5D antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, 22, 24, 26 or 28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15, 18 or 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0132] In some specific embodiments, the anti-GPRC5D antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain each comprise or consist of an amino acid sequence set forth in the following SEQ ID NO: or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto: (i) SEQ ID NO: 60 and SEQ ID NO: 61; (ii) SEQ ID NO: 70 and SEQ ID NO: 71; (iii) SEQ ID NO: 80 and SEQ ID NO: 81; (iv) SEQ ID NO: 14 or 24 and SEQ ID NO: 15; (v) SEQ ID NO: 14 or 26 and SEQ ID NO: 18; (vi) SEQ ID NO: 14, 22, 24, 26 or 28 and SEQ ID NO: 20.

[0133] In one embodiment of the present invention, the amino acid changes in the anti-GPRC5D antibodies described herein comprise amino acid substitutions, insertions or deletions. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0134] In one embodiment of the invention, the amino acid changes in the anti-GPRC5D antibodies described in the invention are changes that remove post-translational modifications, for example, in the CDRs of the anti-GPRC5D antibodies, for example, the HCDR2 of the anti-GPRC5D antibodies that contain N55S and / or G56E substitutions.

[0135] In some embodiments, the anti-GPRC5D antibodies, or antigen-binding fragments thereof, of the invention have one or more of the following properties: (i) exhibiting the same or similar binding affinity and / or specificity for GPRC5D as the antibodies of the present invention; (ii) inhibiting (e.g., competitively inhibiting) the binding of the antibody of the present invention to GPRC5D; (iii) binding to a similar or overlapping epitope to an antibody of the present invention; (iv) binding to GPRC5D competitively with the antibody of the present invention; (v) having one or more biological properties of the antibody of the invention.

[0136] In some embodiments, the anti-GPRC5D antibody of the invention is an IgG1 format antibody or an IgG2 format antibody or an IgG3 format antibody or an IgG4 format antibody, for example, an IgG1 format antibody. In some embodiments, the light chain constant region of the anti-GPRC5D antibody of the invention is a lambda or kappa light chain constant region, for example a kappa light chain constant region.

[0137] In some embodiments, the anti-GPRC5D antibody is a monoclonal antibody.

[0138] In some embodiments, the anti-GPRC5D antibody is humanized.

[0139] In some embodiments, the anti-GPRC5D antibody is a chimeric antibody.

[0140] In one embodiment, the anti-GPRC5D antibody of the present invention further comprises an antibody fragment thereof (e.g., an antigen-binding fragment), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab')2, single domain antibody (e.g., VHH), dAb (domain antibody) or linear antibody.

[0141] IV.MUC16 antibody The present invention provides anti-MUC16 antibodies that target human MUC16 and have one or more of the following advantages: (1) binds with high affinity to MUC16 (e.g., human or monkey MUC16) and to target cells expressing human MUC16; (2) it does not bind to free circulating CA125; (3) Suitable as a genetic engineering element; (4) low immunogenicity; (5) High expression purity; (6) Maintain high affinity even after humanization.

[0142] In one embodiment, the anti-MUC16 antibody, or antigen-binding fragment thereof, is humanized.

[0143] In some embodiments, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention comprise three complementarity determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3.

[0144] In some embodiments, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention comprise three complementarity determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2 and LCDR3.

[0145] In some embodiments, an anti-MUC16 antibody, or antigen-binding fragment thereof, of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0146] In some embodiments, the anti-MUC16 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH). In some embodiments, the anti-MUC16 antibodies or antigen-binding fragments thereof of the invention comprise a light chain variable region (VH). In some embodiments, the anti-MUC16 antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0147] In some embodiments, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention further comprise an antibody heavy chain constant region, HC. In some embodiments, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention further comprise an antibody light chain constant region, LC. In some embodiments, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention further comprise a heavy chain constant region, HC, and a light chain constant region, LC.

[0148] In some embodiments, the heavy chain variable region VH of an anti-MUC16 antibody of the invention is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 92 or 113; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 92 or 113; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 92 or 113, and preferably, said amino acid changes do not occur in the CDR regions.

[0149] In some embodiments, the light chain variable region V of an anti-MUC16 antibody of the invention is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114, and preferably, said amino acid changes do not occur in the CDR regions.

[0150] In some embodiments, the three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region of the anti-MUC16 antibodies of the invention are (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in any one of SEQ ID NOs: 92 or 113; or (ii) a sequence containing at least one amino acid change in total in the three HCDR regions relative to any one of the sequences in (i), and containing no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

[0151] In some embodiments, the three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from the light chain variable region of the anti-MUC16 of the invention are (i) the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in any one of SEQ ID NOs: 96, 100, 102 or 114; or (ii) a sequence containing at least one amino acid change in total in the three LCDR regions relative to any one of the sequences in (i), and containing no more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution).

[0152] In some embodiments, the anti-MUC16 antibodies of the invention include HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 93, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 94, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 95, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 97, 101 or 103, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 98, and / or LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 99.

[0153] In some specific embodiments of the invention, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention comprise a VH and a VL, The VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 92 or 113 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the VL comprises or consists of an amino acid sequence set forth in SEQ ID NO: 96, 100, 102 or 114 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0154] In some specific embodiments of the invention, the anti-MUC16 antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as set forth in SEQ ID NO: 92 or 113, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as set forth in SEQ ID NO: 96, 100, 102 or 114.

[0155] In some specific embodiments of the invention, the anti-MUC16 antibodies or antigen-binding fragments thereof of the invention comprise an HCDR1 set forth in SEQ ID NO:93, an HCDR2 set forth in SEQ ID NO:94, an HCDR3 set forth in SEQ ID NO:95, an LCDR1 set forth in SEQ ID NO:97, 101 or 103, an LCDR2 set forth in SEQ ID NO:98 and an LCDR3 set forth in SEQ ID NO:99.

[0156] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0157] In some embodiments, the anti-MUC16 antibodies, or antigen-binding fragments thereof, of the invention have one or more of the following properties: (i) exhibit the same or similar binding affinity and / or specificity for MUC16 as an antibody of the invention; (ii) inhibiting (e.g., competitively inhibiting) binding of an antibody of the invention to MUC16; (iii) binding to a similar or overlapping epitope to an antibody of the present invention; (iv) competitively binds to MUC16 with an antibody of the invention; (v) having one or more biological properties of the antibody of the invention.

[0158] In some embodiments, the anti-MUC16 antibody of the invention is an IgG1 format antibody or an IgG2 format antibody or an IgG3 format antibody or an IgG4 format antibody, e.g., an IgG1 format antibody. In some embodiments, the light chain constant region of the anti-MUC16 antibody of the invention is a kappa or lambda light chain constant region, e.g., a lambda light chain constant region.

[0159] In some embodiments, the anti-MUC16 antibody is a monoclonal antibody.

[0160] In some embodiments, the anti-MUC16 antibody is humanized.

[0161] In one embodiment, the anti-MUC16 antibodies of the invention further comprise an antibody fragment thereof (e.g., an antigen-binding fragment), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab')2, single domain antibody, e.g., VHH, dAb (domain antibody), or linear antibody.

[0162] In one specific embodiment, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises a VH and a VL, 1) the VH comprises an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, and an HCDR3 as set forth in SEQ ID NO: 95, and the VL comprises an LCDR1 as set forth in SEQ ID NO: 97, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, or 2) the VH comprises an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, and an HCDR3 as set forth in SEQ ID NO: 95, and the VL comprises an LCDR1 as set forth in SEQ ID NO: 101, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, or 3) the VH comprises an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, and an HCDR3 as set forth in SEQ ID NO: 95, and the VL comprises an LCDR1 as set forth in SEQ ID NO: 103, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, or In a specific embodiment, the anti-MUC16 antibody or antigen-binding fragment thereof comprises: 1) a VH whose sequence is set forth in SEQ ID NO: 92 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 92 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 96 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 96 and having the same CDRs, or 2) a VH whose sequence is set forth in SEQ ID NO: 92 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 92 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 100 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 100 and having the same CDRs, or 3) a VH whose sequence is set forth in SEQ ID NO: 92 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 92 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 102 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 102 and having the same CDRs, or 4) a VH whose sequence is set forth in SEQ ID NO: 113 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 113 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 114 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and having the same CDRs.

[0163] In another specific embodiment, the anti-MUC16 antibody, or antigen-binding fragment thereof, comprises: 1) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 106 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 106 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 107 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 107 and the same CDRs, or 2) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 106 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 106 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 108 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 108 and the same CDRs, or 3) a heavy chain (H) having a sequence as set forth in SEQ ID NO: 106 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 106 and having the same CDRs, and a light chain (L) having a sequence as set forth in SEQ ID NO: 109 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 109 and having the same CDRs, or 4) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 115 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 115 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 116 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116 and the same CDRs.

[0164] In some embodiments, the antibody comprises an amino acid change. In one embodiment of the present invention, the amino acid change described herein comprises an amino acid substitution, insertion or deletion. In a preferred embodiment, the amino acid change described herein occurs in a region outside the CDR (e.g., FR). More preferably, the amino acid change described herein occurs in a region outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid change described herein is an amino acid substitution, preferably a conservative substitution.

[0165] In some embodiments, the antibodies of the invention may be cysteine ​​engineered antibodies, such as "thiomAbs," in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0166] V. Bispecific antibodies In some embodiments, the anti-CD3 antibodies of the invention are bispecific or multispecific antibodies that comprise a first binding specificity that includes, e.g., CD3, and another binding specificity for one or more molecules (e.g., a tumor-associated antigen, e.g., GPRC5D or MUC16).

[0167] In some embodiments, the anti-GPRC5D antibodies of the invention are bispecific or multispecific antibodies that comprise, for example, a first binding specificity for GPRC5D and another binding specificity for one or more molecules (e.g., CD3).

[0168] In some embodiments, the anti-MUC16 antibodies of the invention are bispecific or multispecific antibodies that comprise, for example, a first binding specificity for MUC16 and another binding specificity for one or more molecules (e.g., CD3).

[0169] Thus, one aspect of the invention is a bispecific antibody comprising: The present invention relates to a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to a tumor-associated antigen and / or the second antigen-binding region specifically binds to CD3.

[0170] As used herein, a "tumor-associated antigen" refers to an antigen that is associated with a tumor, for example, a protein that is highly expressed in a tumor, such as GPRC5D or MUC16.

[0171] Thus, one aspect of the invention is a bispecific antibody comprising: The present invention relates to a bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to GPRC5D or MUC16 and / or the second antigen-binding region specifically binds to CD3.

[0172] In some embodiments, the first antigen-binding region is derived from an anti-GPRC5D antibody described herein, e.g., a Fab fragment of an anti-GPRC5D antibody. In some embodiments, the first antigen-binding region is derived from an anti-MUC16 antibody described herein, e.g., a Fab fragment of an anti-MUC16 antibody.

[0173] In some embodiments, the second antigen-binding region is derived from an anti-CD3 antibody, such as the SP34 antibody or a humanized antibody thereof, such as an anti-CD3 humanized antibody described herein, such as an scFv fragment of the anti-CD3 antibody.

[0174] A first antigen-binding region suitable for a bispecific antibody of the present invention may comprise or consist of an anti-GPRC5D or MUC16 full-length antibody or an antigen-binding fragment thereof of the present invention, so long as it is capable of specifically binding to GPRC5D or MUC16, and includes, but is not limited to, for example, a full-length antibody, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibody, VHH, or heavy chain antibody that specifically binds to GPRC5D.

[0175] The second antigen-binding region suitable for the bispecific antibody of the present invention may comprise or consist of a full-length anti-CD3 antibody or an antigen-binding fragment thereof, as long as it is capable of specifically binding to CD3, and includes, but is not limited to, for example, a full-length antibody, a single-chain Fv, Fab, Fab', (Fab)2, a single-domain antibody, a VHH, or a heavy-chain antibody that specifically binds to CD3.

[0176] In some embodiments, the bispecific antibody of the present invention is an IgG-like bispecific antibody. As used herein, "IgG-like bispecific antibody" refers to a bispecific antibody that comprises an Fc dimer. Thus, in some embodiments, the bispecific antibody of the present invention comprises an Fc dimer.

[0177] In some embodiments, the bispecific antibody of the invention is an IgG-like bispecific antibody comprising a Fab fragment as one antigen-binding region and an scFv fragment as another antigen-binding region, hi some embodiments, the IgG-like bispecific antibody comprises a Fab fragment that specifically binds to GPRC5D or MUC16 as a first antigen-binding region and an scFv that specifically binds to CD3 as a second antigen-binding region.

[0178] The bispecific antibody structure of the present invention In some embodiments, the present invention provides a bispecific antibody comprising one or two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, of which: (1) A Fab fragment that specifically binds to a first antigen is fused at the C-terminus of the CH1 of the Fab heavy chain to the N-terminus of an scFv fragment that specifically binds to a second antigen (e.g., the N-terminus of VH or VL), and the C-terminus of the scFv fragment (e.g., the C-terminus of VL or VH) is fused to the CH2 or hinge region of one of the Fc regions of an Fc heterodimer (e.g., an Fc region containing a knob or an Fc region containing a hole), e.g., a structure as shown in FIG. 4A ; (2) A first Fab fragment that specifically binds to a first antigen is fused at the C-terminus of the CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of an Fc heterodimer (e.g., an Fc region with a hole or an Fc region with a knob), and a second Fab fragment that specifically binds to the first antigen is fused at the C-terminus of the CH1 of the Fab heavy chain to the N-terminus of an scFv fragment that specifically binds to a second antigen (e.g., the N-terminus of VH or VL), and the C-terminus of the scFv fragment (e.g., the C-terminus of VL or VH) is fused to the CH2 or hinge region of another Fc region of the Fc heterodimer (e.g., an Fc region with a knob or an Fc region with a knob), for example, as shown in FIG. 4B or FIG. 20A ; (3) A Fab fragment that specifically binds to a first antigen is fused at the C-terminus of the CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of an Fc heterodimer (e.g., an Fc region containing a hole or an Fc region containing a knob), and the C-terminus of an scFv fragment that specifically binds to a second antigen (e.g., the C-terminus of the VL or VH) is fused to the CH2 or hinge region of another Fc region of the Fc heterodimer (e.g., an Fc region containing a knob or an Fc region containing a hole), e.g., a structure as shown in FIG. 4C or FIG. 20B ; or (4) A first Fab fragment that specifically binds to a first antigen is fused at the C-terminus of the CH1 of the Fab heavy chain to the N-terminus of the VH of a second Fab fragment that specifically binds to the first antigen, and the second Fab fragment is fused at the C-terminus of the CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of an Fc heterodimer (e.g., an Fc region containing a knob or an Fc region containing a hole), and the C-terminus (e.g., the C-terminus of the VL or VH) of an scFv fragment that specifically binds to a second antigen is fused to the CH2 or hinge region of another Fc region of the Fc heterodimer (e.g., an Fc region containing a hole or an Fc region containing a knob), e.g., a structure as shown in FIG. 20C.

[0179] In some embodiments, the fusion comprises a direct fusion or a fusion via a linker.

[0180] Thus, in some embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising one Fab fragment that specifically binds to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, of which the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the first Fc region constitutes the first heavy chain; the C-terminus of the CH1 of the Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to the CH2 or hinge region of a second Fc domain, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0181] In some further embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, wherein the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of CH1 of the first Fab fragment is fused to a CH2 or hinge region containing a first Fc region, thereby forming a first heavy chain; the C-terminus of the CH1 of the second Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the scFv fragment is fused to the CH2 or hinge region of a second Fc domain, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; Among them, the first Fab fragment and the second Fab fragment are homologous or different.

[0182] In some further embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising one Fab fragment that specifically binds to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, wherein the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of CH1 of the Fab fragment is fused to CH2 or a hinge region of a first Fc domain, thereby forming a first heavy chain; the C-terminus of the scFv fragment is fused to a CH2 or hinge region containing a second Fc region, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0183] In some further embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, wherein the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of the CH1 of the first Fab fragment is fused to the N-terminus of the VH of the second Fab fragment, and the C-terminus of the CH1 of the second Fab fragment is fused to the CH2 or hinge region of a first Fc domain, thereby forming a first heavy chain; the C-terminus of the scFv fragment is fused to the CH2 or hinge region of a second Fc domain, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; Among them, the first Fab fragment and the second Fab fragment are homologous or different.

[0184] In some embodiments, the first antigen is selected from GPRC5D or MUC16 (e.g., human GPRC5D or human MUC16). In some embodiments, the second antigen is CD3.

[0185] In some embodiments, the first Fc region comprises a knob mutation and the second Fc region comprises a hole mutation, In some embodiments, the first Fc region comprises a hole mutation and the second Fc region comprises a knob mutation.

[0186] In some embodiments, the C-terminus of the scFv fragment is the C-terminus of the VL of the scFv fragment. In some embodiments, the N-terminus of the scFv fragment is the N-terminus of the VH of the scFv fragment.

[0187] Fab fragments suitable for bispecific antibodies of the invention In some embodiments, a Fab fragment, which is one of the antigen-binding regions of a bispecific antibody, is composed of two polypeptide chains comprising antibody VH, CH1, VL and CL domains, in which the VH pairs with the VL and the CH1 pairs with the CL to form an antigen-binding region. In some embodiments, in the Fab, one chain comprises, from the N-terminus to the C-terminus, VH and CH1 (i.e., VH-CH1), and the other chain comprises, from the N-terminus to the C-terminus, VL and CL (i.e., VL-CL).

[0188] In some embodiments, in the bispecific antibody, the Fab may be fused to the N-terminus of an antibody Fc domain via the C-terminus comprising the VH chain, or to the N-terminus of an antibody Fc domain via another antigen-binding region, such as an scFv fragment, in which the Fc domain may or may not include a hinge region (e.g., EPKSS or EPKSC). Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and is fused to an antibody Fc domain via the C-terminus of the CH1 of the VH-CH1 chain, in which the Fc domain may or may not include a hinge region (e.g., EPKSS or EPKSC). Herein, the Fab chain connected to the Fc dimer is also referred to as the Fab heavy chain, and the Fab chain not connected to the Fc dimer is also referred to as the Fab light chain. In some embodiments, the fusion is a direct fusion or a fusion via a linker.

[0189] In some embodiments, the Fab fragment contained in the bispecific antibody of the present invention specifically binds to GPRC5D. In some embodiments, the Fab fragment contained in the bispecific antibody of the present invention is derived from the anti-GPRC5D antibody of the present invention comprising the heavy chain variable region VH and the light chain variable region VL of the anti-GPRC5D antibody of the present invention.

[0190] In some embodiments, the Fab heavy chain comprises a VH and a CH1, wherein the VH is the VH of an anti-GPRC5D antibody of the invention. In some embodiments, the CH1 is a CH1 derived from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 derived from IgG1. In some embodiments, the CH1 is (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 44; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 44; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 44.

[0191] In some embodiments, the Fab light chain comprises a VL-CL, wherein VL is the VL of an anti-GPRC5D antibody of the invention. In some embodiments, the CL is a light chain constant region from an antibody kappa or lambda light chain, preferably a light chain constant region from a kappa light chain. In some embodiments, the CL is a light chain constant region of an anti-GPRC5D antibody of the invention. In some embodiments, the Fab light chain is a light chain of an anti-GPRC5D antibody of the invention.

[0192] In some embodiments, a Fab fragment included in a bispecific antibody of the invention specifically binds to MUC16. In some embodiments, a Fab fragment included in a bispecific antibody of the invention is derived from an anti-MUC16 antibody of the invention comprising the heavy chain variable region, VH, and light chain variable region, VL, of the anti-MUC16 antibody of the invention.

[0193] In some embodiments, the Fab heavy chain comprises a VH and a CH1, wherein the VH is the VH of an anti-MUC16 antibody of the invention. In some embodiments, the CH1 is a CH1 derived from IgG1, IgG2, IgG3 or IgG4, preferably a CH1 derived from IgG1. In some embodiments, the CH1 is (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 44; (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 44; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 44.

[0194] In some embodiments, the Fab light chain comprises a VL-CL, where VL is the VL of an anti-MUC16 antibody of the invention. In some embodiments, the CL is a light chain constant region from an antibody kappa or lambda light chain, preferably a kappa light chain. In some embodiments, the CL is a light chain constant region of an anti-MUC16 antibody of the invention. In some embodiments, the Fab light chain is a light chain of an anti-MUC16 antibody of the invention.

[0195] scFv suitable for bispecific antibodies of the present invention In some embodiments, the scFv fragment, which is one of the bispecific antibody binding regions, consists of a single polypeptide chain containing antibody VH and VL domains, in which the VH and VL are connected (e.g., via a linker) to pair and form an antigen-binding site. In some embodiments, the scFv has a trans structure and includes, from the N-terminus to the C-terminus, a VH, a linker, and a VL (VH-linker-VL). In other embodiments, the scFv has a cis structure and includes, from the N-terminus to the C-terminus, a VL, a linker, and a VH (VH-linker-VL). In some embodiments, the linker is a peptide linker composed of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5 to 50 amino acids long, for example 5 to 30 amino acids long, for example 15 amino acids or 20 amino acids long. In one embodiment, the linker comprises the amino acid sequence (G4S)n, where n=1, 2, 3, 4 or 5, preferably n=3 or 4, more preferably n=3.

[0196] In some preferred embodiments, the scFv antigen-binding site comprised in the antibody molecules of the present invention is a disulfide bond stabilized scFv.

[0197] In some embodiments, in the bispecific antibody, the scFv can be fused via the C-terminus of the chain to the N-terminus of the antibody Fc domain. In some embodiments, in the bispecific antibody, the scFv is fused at the N-terminus of the chain to the C-terminus of another antigen-binding region (e.g., a Fab heavy chain) and at the C-terminus of the chain to the N-terminus of the antibody Fc domain.

[0198] In some embodiments, in the bispecific antibody, the scFv can be fused via the C-terminus of the chain comprising the VL to the N-terminus of the antibody Fc domain. In some embodiments, in the bispecific antibody, the scFv is fused at the N-terminus of the VH chain to the C-terminus of another antigen-binding region (e.g., a Fab heavy chain) and at the C-terminus of the VL chain to the N-terminus of the antibody Fc domain.

[0199] In some embodiments, in the bispecific antibody, the scFv can be fused via the C-terminus of the VH-containing chain to the N-terminus of the antibody Fc domain. In some embodiments, in the bispecific antibody, the scFv is fused at the N-terminus of the VL chain to the C-terminus of another antigen-binding region (e.g., a Fab heavy chain) and at the C-terminus of the VH chain to the N-terminus of the antibody Fc domain.

[0200] In some embodiments, the scFv contained in the bispecific antibody of the present invention specifically binds to CD3. In some embodiments, the scFv contained in the bispecific antibody of the present invention is derived from an anti-CD3 antibody of the present invention comprising the heavy chain variable region VH and the light chain variable region VL of the anti-CD3 antibody of the present invention.

[0201] Fc dimers suitable for bispecific antibodies of the present invention In one embodiment, the two Fc regions in the bispecific antibody of the invention dimerize to form a dimeric Fc. Preferably, the two Fc regions heterodimerize to form a heterodimeric Fc.

[0202] In some embodiments, the first and second Fc regions are the same, while in other embodiments, the first and second Fc regions are different and pair to form a heterodimer.

[0203] The Fc region fragment suitable for the antibody molecule of the present invention may be any antibody Fc region. The Fc region may include native sequence Fc regions and variant Fc regions. Native sequence Fc domains encompass various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). For example, the Fc region of the antibody of the present invention may comprise two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at the N-terminus, such as an IgG1 hinge region or a portion of an IgG1 hinge region. The hinge region may include mutations. In some embodiments, the hinge region may be EPKSS or EPKSC.

[0204] Preferably, the Fc region of an antibody of the invention comprises, from N- to C-terminus, CH2-CH3, or, from N- to C-terminus, hinge region-CH2-CH3. In some embodiments, a suitable Fc region for an antibody or bispecific antibody of the invention is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of an amino acid sequence of SEQ ID NO: 49, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto.

[0205] The Fc region in an antibody or bispecific antibody of the invention can be mutated to obtain desired properties. Mutations to the Fc region are known in the art.

[0206] In one embodiment, the Fc region is modified in terms of its effector function (e.g., complement activation function of the Fc region). In one embodiment, the effector function is reduced or eliminated as compared to a wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from using an Fc isotype that naturally has the reduced or eliminated effector function and modifying the Fc region.

[0207] In one preferred embodiment, the Fc region has a reduced Fc region-mediated effector function, e.g., has a reduced or eliminated ADCC or ADCP or CDC effector function, e.g., comprises mutations to achieve such function.

[0208] As will be appreciated by those skilled in the art, depending on the anticipated use of the antibody molecule of the present invention, the antibody molecule of the present invention may contain modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, in particular a human Fcγ receptor. In some embodiments, the Fc region contains a mutation that reduces binding to an Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has one or more of the L234A / L235A mutation, the D265A mutation, or the P329A mutation that reduces binding to an Fcγ receptor. In some embodiments, the Fc region used in the present invention has the L234A / L235A mutation, the D265A mutation, and the P329A mutation that reduces binding to an Fcγ receptor. In a further preferred embodiment, the Fc fragment may have a mutation that increases serum half-life, for example a mutation that improves binding of the Fc fragment to FcRn. In some embodiments, the Fc region comprising a mutation that reduces binding to an Fcγ receptor comprises or consists of an amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto, and comprises the L234A / L235A mutation, the D265A mutation, and the P329A mutation.

[0209] As will be appreciated by those skilled in the art, to facilitate the formation of the bispecific antibodies of the invention as heterodimers, the Fc regions contained in the bispecific antibodies of the invention may contain mutations that favor heterodimerization. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions.

[0210] Methods for promoting heterodimerization of Fc regions are known in the art, for example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are complementarily engineered such that each CH3 region (or the heavy chain containing it) cannot homodimerize with itself, but is forced to heterodimerize with the other complementarily engineered CH3 region (such that the first and second CH3 regions heterodimerize and no homodimers are formed between the two first CH3 regions or the two second CH3 regions).

[0211] Preferably, corresponding knob and hole mutations are introduced into the first and second monomeric Fc regions, respectively, based on the knob-in-hole technique, see, for example, Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.

[0212] In one particular embodiment, in the CH3 region of the Fc region, the threonine residue at position 366 is substituted with a tryptophan residue (T366W) (knob mutation), while in the CH3 region of another Fc region, the tyrosine residue at position 407 is substituted with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A) (EU index numbering).

[0213] In yet another embodiment, in the CH3 region of one Fc region, the knob mutations comprise or consist of substitution of the threonine residue at position 366 by a tryptophan residue (T366W) and substitution of the serine residue at position 354 by a cysteine ​​residue (S354C) or substitution of the glutamic acid residue at position 356 by a cysteine ​​residue (E356C), in particular substitution of the serine residue at position 354 by a cysteine ​​residue, and in the CH3 region of the other Fc region, the hole mutations comprise or consist of substitution of the tyrosine residue at position 407 by a valine residue (Y407V), optionally substitution of the threonine residue at position 366 by a serine residue (T366S) and substitution of the leucine residue at position 368 by an alanine residue (L368A) (numbering according to the EU index), optionally substitution of the tyrosine residue at position 349 by a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0214] In one specific embodiment, one Fc region comprises the amino acid substitutions S354C and T366W (knob mutation) and the other Fc region comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (hole mutation) (numbering according to the EU index).

[0215] Thus, in one specific embodiment, the two Fc regions comprised in the bispecific antibody of the present invention are heterodimerized, of which a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V; or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C; Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, e.g., one or more of an L234A / L235A mutation, a D265A mutation, or a P329A mutation, e.g., an L234A / L235A mutation, a D265A mutation, and a P329A mutation.

[0216] In some embodiments, the Fc region further comprises other mutations that are advantageous for purification of the heterodimer. For example, to facilitate purification of the heterodimer using Protein A, a H435R mutation (Eric J. Smith, Scientific Reports|5:17943|DOI:10.1038 / srep17943) can be introduced into one of the Fc regions of the heterodimer (e.g., the Fc region with the Hole mutation). In other embodiments, for heterodimeric monomers that include a hinge region, a mutation such as C220S can also be introduced into the hinge region to facilitate heterodimer formation.

[0217] In one specific embodiment, the two Fc regions of the bispecific antibody of the invention are heterodimerized, The first Fc region comprises a knob mutation comprising or consisting of an amino acid sequence of SEQ ID NO: 43 or 89, or at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto, in some embodiments the Fc region comprises an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto, and comprises the L234A / L235A mutation, the D265A mutation and the P329A mutation, and the knob mutation (e.g., S354C and T366W), in some embodiments the Fc region comprises or does not comprise the hinge region EPKSS or EPKSC, The second Fc region comprises or consists of an amino acid sequence of SEQ ID NO: 30, 42 or 59, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto. In some embodiments, the Fc region comprises an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto, and comprises the L234A / L235A, D265A and P329A mutations and the knob mutation, in some embodiments, the Fc region includes or does not include the hinge region EPKSS or EPKSC.

[0218] Exemplary Bispecific Antibody Molecules (I) Bispecific antibody that specifically binds to GPRC5D and CD3 In some preferred embodiments, the present invention provides bispecific antibodies comprising one or two Fab fragments of an anti-GPRC5D antibody, one scFv of an anti-CD3 antibody and an Fc heterodimer, of which (1) The Fab fragment of anti-GPRC5D is fused at the C-terminus of the CH1 of the Fab heavy chain to the N-terminus of the VH of an anti-CD3 antibody scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of one of the Fc regions of an Fc heterodimer (e.g., an Fc region containing a knob), e.g., a structure as shown in FIG. 4A , (2) The first GPRC5D Fab fragment is fused at the C-terminus of the CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of the Fc heterodimer (e.g., an Fc region containing a hole), and the second anti-GPRC5D Fab fragment is fused at the C-terminus of the CH1 of the Fab heavy chain to the N-terminus of the VH of an anti-CD3 antibody scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of another Fc region of the Fc heterodimer (e.g., an Fc region containing a knob), for example, as shown in FIG. 4B. (3) The Fab fragment of GPRC5D is fused at the C-terminus of the CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions of the Fc heterodimer (e.g., an Fc region containing a hole), and the C-terminus of the VL of the scFv fragment of the anti-CD3 antibody is fused to the CH2 or hinge region of another Fc region of the Fc heterodimer (e.g., an Fc region containing a knob), e.g., a structure as shown in Figure 4C.

[0219] In some embodiments, the fusion comprises a direct fusion or a fusion via a linker.

[0220] Thus, in some embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising a Fab fragment of one anti-GPRC5D antibody, an scFv of one anti-CD3 antibody and an Fc heterodimer, of which The Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL, The Fc region containing the hole mutation constitutes the first heavy chain, the C-terminus of the CH1 of the Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of an Fc domain that includes a knob mutation, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0221] In some embodiments, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, 34 or 35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0222] In some further embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising two Fab fragments of an anti-GPRC5D antibody, one scFv of an anti-CD3 antibody and an Fc heterodimer, wherein The Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL, the C-terminus of CH1 of the first Fab fragment is fused to CH2 or a hinge region of an Fc domain containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the CH1 of the second Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of an Fc domain containing a knob mutation, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; Among them, the first Fab fragment and the second Fab fragment are homologous or different.

[0223] In some embodiments, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 32, 33 or 36, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, 34 or 35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0224] In some embodiments, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0225] In some embodiments, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0226] In some embodiments, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0227] In some further embodiments, the present invention relates to a bispecific antibody that is an IgG-like bispecific antibody comprising a Fab fragment of one anti-GPRC5D antibody, an scFv of one anti-CD3 antibody and an Fc heterodimer, wherein The Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL, the C-terminus of CH1 of the Fab fragment is fused to CH2 or a hinge region of an Fc domain containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of an Fc domain containing a knob mutation, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0228] In some embodiments, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 32, 33 or 36, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0229] (II) A bispecific antibody that specifically binds to CD3 and MUC16 The present invention provides a bispecific antibody that simultaneously targets human CD3 and human MUC16 (anti-CD3xMUC16 bispecific antibody), which has the following advantages: (1) highly specific binding to human CD3 and target cells expressing human CD3, and at the same time, highly affinity binding to human MUC16 and target cells expressing human MUC16; (2) to target more T cells to cancer cells that do not bind to free circulating CA125 and that simultaneously express MUC16; (3) Activating T cells and inducing cytotoxic activity against cancer cells expressing MUC16, thereby effectively killing the cancer cells; (4) low immunogenicity; (5) Excellent tumor suppression effect.

[0230] In one embodiment, the invention provides an anti-CD3xMUC16 bispecific antibody comprising: (i) one heavy chain 1 having the structure of VH-CH1-Fc, one heavy chain 2 having the structure of VH-CH1-scFv-Fc, and two light chains having the structure of VL-CL, in which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize MUC16 molecules, and the scFv forms an antigen recognition site that recognizes CD3 molecules; or (ii) one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule; or (iii) one heavy chain 1 having the structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having the structure of scFv-Fc, and two light chains having the structure of VL-CL, wherein the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule.

[0231] In one embodiment, the invention provides an anti-CD3xMUC16 bispecific antibody comprising: (i) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 97, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH that recognizes CD3 (referred to herein as VH in order to distinguish it from the VH that recognizes MUC16). CD3 ) and VL that recognizes CD3 (referred to herein as VL to distinguish it from VL that recognizes MUC16). CD3 Among them, the above VH CD3 comprises HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:7, andCD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 101, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 103, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VLCD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises heavy chain 1, heavy chain 2, and light chain comprising LCDR1 as set forth in SEQ ID NO:8, LCDR2 as set forth in SEQ ID NO:9, and LCDR3 as set forth in SEQ ID NO:10; or (ii) one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 97, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 101, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 103, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3comprises heavy chain 1, heavy chain 2, and light chain comprising LCDR1 as set forth in SEQ ID NO:8, LCDR2 as set forth in SEQ ID NO:9, and LCDR3 as set forth in SEQ ID NO:10; or (iii) one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 97, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or a heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, a heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 101, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VLCD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 103, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10.

[0232] In one specific embodiment, the invention provides an anti-CD3xMUC16 bispecific antibody comprising: (i) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 92 or 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102, or 114, and the scFv that recognizes the CD3 molecule comprises a VH that recognizes CD3 (VH CD3 ) and VL that recognizes CD3 (referred to herein as VL to distinguish it from VL that recognizes MUC16). CD3 Among them, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4. (ii) one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 92 or 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102, or 114, and the scFv that recognizes the CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4; or (iii) one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 92 or 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102, or 114, and the scFv that recognizes the CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4.

[0233] In one embodiment, the two antigen recognition sites that recognize the MUC16 molecule contained in the bispecific antibody may have the same VH / VL sequence or different VH / VL sequences.

[0234] In another embodiment, the invention provides an anti-CD3xMUC16 bispecific antibody comprising: (i) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize the MUC16 molecule, and the scFv forms an antigen recognition site that recognizes the CD3 molecule, among which the VH that recognizes the MUC16 molecule includes the sequence of SEQ ID NO: 113 or has at least the sequence of SEQ ID NO: 113; and a VL that recognizes a MUC16 molecule comprises a sequence selected from SEQ ID NO: 114 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and the same CDRs, and an scFv that recognizes a CD3 molecule comprises a VH selected from SEQ ID NO: 114 or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and the same CDRs, CD3 and VL CD3 Among these, the above VH CD3 comprises a sequence as set forth in SEQ ID NO:3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 and having the same CDRs, and CD3 a heavy chain 1, a heavy chain 2, and a light chain comprising a sequence as set forth in SEQ ID NO:4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and having the same CDRs; or (ii) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and one light chain having a structure of VL-CL, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 113 or is at least 90% identical to SEQ ID NO: 113. and a VL that recognizes a MUC16 molecule comprises a sequence selected from SEQ ID NO: 114, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and the same CDRs, and an scFv that recognizes a CD3 molecule comprises a VH selected from SEQ ID NO: 114, or a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and the same CDRs, and CD3 and VL CD3 Among these, the above VH CD3 comprises a sequence as set forth in SEQ ID NO:3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 and having the same CDRs, and CD3 a heavy chain 1, a heavy chain 2, and a light chain comprising the sequence set forth in SEQ ID NO:4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and having the same CDRs; or (iii) one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, wherein the two light chains pair with two VH-CH1 structures in the heavy chain 1 to form two antigen recognition sites that recognize the MUC16 molecule, respectively, and the scFv forms an antigen recognition site that recognizes the CD3 molecule, and among them, the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 113 or a sequence selected from SEQ ID NO: 11 and a VL that recognizes a MUC16 molecule comprises a sequence selected from SEQ ID NO: 114, or a scFv that recognizes a CD3 molecule comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and the same CDRs, and a VL that recognizes a MUC16 molecule comprises a sequence selected from SEQ ID NO: 114, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and the same CDRs, and a VL that recognizes a CD3 molecule comprises a sequence selected from SEQ ID NO: 114, CD3 and VL CD3 Among these, the above VH CD3 comprises a sequence as set forth in SEQ ID NO:3 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 and having the same CDRs, and CD3 a heavy chain 1, a heavy chain 2, and a light chain comprising the sequence set forth in SEQ ID NO:4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and having the same CDRs.

[0235] In one embodiment, the present invention provides an anti-CD3xMUC16 bispecific antibody as provided above, wherein the Fc region may be selected from the native Fc region of any IgG, IgA, IgM class antibody known in the art, such as the consensus or germline IgG Fc region. In a specific embodiment, the IgG Fc region may be derived from human IgG1, IgG2, IgG3 or IgG4 isotypes. In a specific embodiment, the IgG Fc region is the Fc sequence of human IgG1. In a further embodiment, the Fc region comprises mutations such as mutations that increase the stability of the bispecific antibody dimer (e.g. knobs-into-holes), mutations that decrease or increase effector function.

[0236] In any of the above embodiments, the scFv of the anti-CD3xMUC16 bispecific antibody of the invention is connected to the Fc or CH1 by a linker, which may be any convenient flexible sequence known in the art or available in the future, typically a short flexible amino acid sequence, such as GGGSG, GGSGG, GSGGG, SGGGG, GGTGS, GTSPGG, GNGGGS, G4S-GGSGG-G4S-SGGGG, GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LQRDGERP, LRQKDGGGSERP, GSTSGSGKPGSGEGSTKG, etc. In a specific embodiment, the linker is (G4S)n, where n is an integer equal to or greater than 1, e.g., n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9. In a specific embodiment, the linker is selected from (G4S)3.

[0237] In a further embodiment, the invention provides an anti-CD3xMUC16 bispecific antibody comprising: (i) one heavy chain 1 having the structure of VH-CH1-Fc, one heavy chain 2 having the structure of VH-CH1-scFv-Fc, and two light chains having the structure of VL-CL, wherein the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, wherein heavy chain 1 comprises the sequence shown in SEQ ID NO: 118, heavy chain 2 comprises the sequence shown in SEQ ID NO: 121, and light chain comprises the sequence shown in SEQ ID NO: 116. (ii) one heavy chain 1 having the structure of VH-CH1-Fc, one heavy chain 2 having the structure of scFv-Fc, and one light chain having the structure of VL-CL, in which the light chain forms an antigen recognition site that recognizes a MUC16 molecule by pairing with the VH-CH1 structure of heavy chain 1, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which heavy chain 1 comprises the sequence shown in SEQ ID NO: 118, heavy chain 2 comprises the sequence shown in SEQ ID NO: 117, and light chain comprises the sequence shown in SEQ ID NO: 116; or (iii) one heavy chain 1 having the structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having the structure of scFv-Fc, and two light chains having the structure of VL-CL, wherein the two light chains pair with the two VH-CH1 structures in heavy chain 1 to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, wherein heavy chain 1 comprises the sequence shown in SEQ ID NO: 119, heavy chain 2 comprises the sequence shown in SEQ ID NO: 117, and light chain comprises the sequence shown in SEQ ID NO: 116.

[0238] VI. Nucleic Acids Encoding Antibodies and Host Cells Containing Them In one aspect, the invention provides a nucleic acid encoding any of the above anti-CD3 antibodies or anti-GPRC5D antibodies or MUC16 or bispecific antibodies or any chain thereof.

[0239] For example, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence selected from the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 11, 13, 14 to 29, 31 to 38, 40, 60 to 63, 70 to 73, 80 to 83, 92, 96, 100, 102, 106 to 109, 113 to 119, or 121, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 11, 13, 14 to 29, 31 to 38, 40, 60 to 63, 70 to 73, 80 to 83, 92, 96, 100, 102, 106 to 109, 113 to 119, or 121.

[0240] As will be appreciated by those of skill in the art, due to the degeneracy of the genetic code, each antibody or polypeptide amino acid sequence can be encoded by more than one nucleic acid sequence. Nucleic acid sequences encoding the molecules of the invention can be produced using methods well known in the art, for example, by de novo solid phase DNA synthesis or by PCR amplification.

[0241] In one aspect, the invention provides a nucleic acid encoding any of the above antibodies or any of the antibody chains, the polypeptide encoded by said nucleic acid being capable of exhibiting human CD3 and / or GPRC5D and / or MUC16 antigen (and / or monkey (e.g. rhesus or cynomolgus)) binding ability when expressed from a suitable expression vector.

[0242] In a further aspect, the invention provides nucleic acids encoding any of the bispecific antibodies described above. The polypeptides encoded by the nucleic acids, when expressed from a suitable expression vector, can exhibit binding capability for human or monkey (e.g. rhesus or cynomolgus) CD3 and another antigen (e.g. human or monkey (e.g. rhesus or cynomolgus) GPRC5D or MUC16 antigens). In one embodiment, the nucleic acids encoding each chain of the bispecific antibody may be in the same vector or different vectors. In further embodiments, the nucleic acids encoding each chain of the bispecific antibody may be introduced into homologous or distinct host cells for expression. Thus, in some embodiments, a method of producing a bispecific antibody of the invention comprises culturing a host cell comprising nucleic acid encoding each chain under conditions suitable for expression of each chain of the molecule, to produce a bispecific antibody of the invention.

[0243] In one embodiment, a vector is provided that comprises the nucleic acid. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. The vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC). In one embodiment, the vector is a pcDNA vector, for example, pcDNA3.1.

[0244] In one embodiment, a host cell is provided that comprises the nucleic acid or vector described above, for example a vector for cloning or expression encoding an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from a yeast cell, a mammalian cell, for example a CHO cell (for example a CHO-S, for example an ExpiCHO-S) or a 293 cell (for example a 293F or HEK293 cell) or other cell suitable for the preparation of an antibody or a fragment thereof. In one embodiment, the host cell is prokaryotic, for example a bacterial cell, for example an E. coli cell.

[0245] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for the preparation of antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains in which the glycosylation pathway has already been "humanized" produce antibodies with partial or complete human glycosylation patterns. Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells may also be used as hosts. For example, mammalian cell lines modified for suspension growth can be used. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7), human embryonic kidney cell lines (HEK293, 293F or 293T), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc., as well as myeloma cell lines such as Y0, NS0 and Sp2 / 0. Suitable mammalian host cell lines for the production of antibodies are known in the art.

[0246] VII. Production and purification of anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies of the present invention In one embodiment, there is provided a method of preparing an anti-CD3 or anti-GPRC5D or anti-MUC16 antibody or bispecific antibody of the invention, said method comprising culturing a host cell comprising nucleic acid encoding the anti-CD3 or anti-GPRC5D or anti-MUC16 antibody or bispecific antibody (e.g. any one and / or more polypeptide chains) or comprising an expression vector for said nucleic acid under conditions suitable for expression of the anti-CD3 or anti-GPRC5D or anti-MUC16 antibody or bispecific antibody or chains thereof as provided above, and optionally recovering said anti-CD3 or anti-GPRC5D or anti-MUC16 antibody or bispecific antibody from the host cell (or host cell medium).

[0247] The polynucleotides encoding the polypeptide chains of the anti-CD3 or anti-GPRC5D or anti-MUC16 or bispecific antibodies of the invention may be inserted into one or more vectors for further cloning and / or expression in a host cell. Expression vectors can be constructed by methods well known to those skilled in the art. Once an expression vector for expression, containing one or more nucleic acid molecules of the invention, has been prepared, the expression vector can be transfected or introduced into a suitable host cell. To this end, various techniques can be utilized, such as, for example, protoplast fusion, calcium phosphate co-precipitation, electroporation, retroviral transduction, viral transfection, particle gun, liposome-based transfection or other common techniques.

[0248] The anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies prepared as described herein can be purified by known conventional techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art.

[0249] The purity of the antibody molecules of the present invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0250] VIII. Assays for anti-CD3 antibodies or anti-GPRC5D antibodies or bispecific antibodies. A variety of assays known in the art can be used to identify, screen, or characterize the physical / chemical properties and / or biological activity of the anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies provided herein.

[0251] In one aspect, the anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies of the invention are tested for their target (e.g., antigen, e.g., free antigen or antigen expressed on a cell) binding activity by known methods, such as, for example, biolayer interferometry, ELISA, flow cytometry, etc. Binding to CD3 and / or GPRC5D and / or anti-MUC16 antibodies (or CD3 and / or GPRC5D and / or MUC16 expressed on a cell) may be measured by methods known in the art, exemplary methods being disclosed herein. In some embodiments, it is measured by radioimmunoassay (RIA) or biolayer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or flow cytometry (FACS).

[0252] The invention also provides an assay for identifying a biological activity of an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody, the biological activity being selected from a property of the anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention.

[0253] For example, the binding activity of the antibody molecules of the present invention to cells expressing CD3 and / or GPRC5D and / or MUC16 may be measured by methods known in the art, such as fluorescent reporter molecules and flow cytometry, or by exemplary methods disclosed in the Examples herein, for example by the methods shown in Examples 5, 6, 7, 8 or 10, measuring the binding of the antibody molecules of the present invention to CD3 and / or GPRC5D and / or MUC16 expressed on cells.

[0254] For example, the activation activity of the antibody molecules of the invention on T cells can be measured by methods known in the art, such as T cell activation test systems, e.g. NFAT-luc reporter gene system, such as the Jurkat / NFAT-luc reporter gene system, by detecting the CD3 signaling pathway in T cells, or by detecting the release of cytokines (e.g. interferons such as IFNγ, tumor necrosis factors, e.g. TNFα and / or interleukins such as IL-6) following activation of T cells, e.g. by the methods shown in Examples 1, 6, 10, 12, 13 or 14.

[0255] For example, the inhibitory activity or structural safety of the antibody molecule of the present invention against tumors can be measured by methods known in the art, such as tumor suppression experiments on mouse tumor models, for example the methods shown in Example 7 or 11.

[0256] Cells for use in any of the above in vitro assays can be primary cells or cell lines, including cells that naturally express or overexpress CD3 (e.g., human or monkey (e.g., cynomolgus monkey)) or GPRC5D (e.g., human or monkey (e.g., cynomolgus monkey) GPRC5D) or MUC16 (e.g., human or monkey (e.g., cynomolgus monkey) MUC16), e.g., cells that overexpress CD3 or GPRC5D or MUC16, e.g., T cells or 293 cells or CHO cells or Jurkat cells or myeloma cells, e.g., HEK293 or CHO-K1 or Jurkat / NFAT-Luc cells or NCI-H929.

[0257] As can be appreciated, any of the above assays can be accomplished using combinations of the antibodies of the present invention and other active agents.

[0258] IX. Immunoconjugates, pharmaceutical compositions, drug combination products and reagent kits of the present invention, anti-CD3 antibodies, anti-GPRC5D antibodies, anti-MUC16 antibodies or bispecific antibodies In some embodiments, the invention provides an immunoconjugate comprising any of the anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies described herein. Preferably, the immunoconjugate comprises one or more additional therapeutic agents (e.g., a cytotoxin or a small molecule compound) or markers.

[0259] In some embodiments, the invention provides a composition or medicament or formulation comprising any of the anti-CD3 antibody or anti-GPRC5D antibody or MUC16 antibody or bispecific antibody described herein, preferably the composition is a pharmaceutical composition.

[0260] In one embodiment, the composition further comprises a pharmaceutical auxiliary material. In one embodiment, the composition, e.g., the pharmaceutical composition, comprises a combination of an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention and one or more additional therapeutic agents.

[0261] The above compositions or medicaments or formulations of the present invention may further comprise suitable pharmaceutical auxiliary materials, such as pharmaceutical vectors known in the art, pharmaceutical excipients including buffering agents.

[0262] As used herein, "medicinal vectors" include any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible.

[0263] For a discussion of the use of pharmaceutical adjuvants and their applications, see also "Handbook of Pharmaceutical Excipients", 8th Edition, RC Rowe, PJ Eskey and SCOwen, Pharmaceutical Press, London, Chicago.

[0264] The composition or medicament or formulation of the present invention can be in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injections or eye drops), dispersions or suspensions, liposomes and suppositories. The preferred form is determined by the intended mode of administration and therapeutic application.

[0265] Drugs or formulations comprising the anti-CD3 or anti-GPRC5D antibodies or bispecific antibodies described herein can be prepared by mixing the anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies of the present invention having the desired purity with one or more optional pharmaceutical auxiliary materials, for example in the form of a lyophilized formulation or an aqueous solution.

[0266] The compositions or medicaments or formulations of the invention may further comprise one or more active ingredients, which are necessary for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other, e.g., where it is desirable to provide another therapeutic agent.

[0267] The invention further provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention and one or more additional therapeutic agents.

[0268] The present invention further provides a pharmaceutical kit set comprising the pharmaceutical combination, for example, the pharmaceutical kit set comprises, in the same package: a first container containing a pharmaceutical composition comprising an anti-CD3 antibody or an anti-GPRC5D antibody or an anti-MUC16 antibody or a bispecific antibody of the invention; a second container containing a pharmaceutical composition comprising another therapeutic agent.

[0269] In some embodiments, the other therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0270] X. Uses of anti-CD3 antibodies or anti-GPRC5D antibodies or anti-MUC16 antibodies or bispecific antibodies, and methods of using them. One aspect of the invention provides a method of preventing or treating a disease in a subject, the method comprising administering to the subject an anti-CD3 antibody, anti-GPRC5D antibody, anti-MUC16 antibody, or bispecific antibody of the invention, or an immunoconjugate, composition, or medicament or formulation comprising same. In some embodiments, the invention provides a method of specifically activating T cells in a subject (e.g., further enhancing, stimulating, or increasing an immune response in a subject), the method comprising administering to the subject a bispecific antibody of the invention, or an immunoconjugate, composition, or medicament or formulation comprising same.

[0271] In some embodiments, the disease is, for example, a tumor, such as cancer. The cancer may be an early, intermediate or late stage cancer or a metastatic cancer. In some embodiments, the cancer may be a solid tumor or a hematological tumor. In some embodiments, the cancer is ovarian cancer, myeloma, colon cancer, rectal cancer or colorectal cancer.

[0272] In some embodiments, the ovarian cancer is a cancer associated with abnormal ovarian cell proliferation, which includes a group of heterogeneous lesions, most commonly derived from epithelial cell type.In one embodiment, the ovarian cancer is, for example, ovarian cancer, particularly serous ovarian cancer, such as ovarian serous cystadenocarcinoma, ovarian cancer serous adenocarcinoma, ovarian cancer mucinous adenocarcinoma, endometrioid adenocarcinoma, ovarian cancer clear cell adenocarcinoma.

[0273] In some embodiments, the treatment of the above diseases benefits from activation of the CD3 signaling pathway and / or activation of T cells.

[0274] In some embodiments, the cancer is a cancer characterized by having elevated protein and / or nucleic acid levels (e.g., increased expression) of GPRC5D, e.g., tumor cells of the cancer have elevated protein and / or nucleic acid levels (e.g., increased expression) of GPRC5D, e.g., compared to the protein and / or nucleic acid levels of GPRC5D in the same tissue of a healthy subject, or compared to the protein and / or nucleic acid levels of GPRC5D in healthy tissue adjacent to the patient's tumor tissue.

[0275] In some embodiments, the cancer is a cancer characterized by having elevated MUC16 protein and / or nucleic acid levels (e.g., increased expression), e.g., tumor cells of the cancer have elevated MUC16 protein and / or nucleic acid levels (e.g., increased expression), e.g., compared to the MUC16 protein and / or nucleic acid levels in the same tissue of a healthy subject, or compared to the MUC16 protein and / or nucleic acid levels in healthy tissue adjacent to the patient's tumor tissue.

[0276] The patient or subject may be a mammal, such as a primate, and is preferably a higher primate, such as a human (e.g., a patient suffering from or at risk of suffering from a cancer described herein). In some embodiments, the subject will undergo or has undergone other treatments, such as chemotherapy and / or radiation treatments.

[0277] In one embodiment, the anti-MUC16 antibodies disclosed herein do not bind to free CA125 molecules in the circulation, but only to MUC16 molecules bound to cell membranes expressed on the cell surface. The MUC16 molecule is known to be a molecule specifically expressed by ovarian cancer, and therefore, a therapeutically effective amount of an anti-MUC16 antibody or an anti-CD3xMUC16 bispecific antibody can specifically bind to ovarian cancer cells expressing the MUC16 molecule, enhancing the immune system's killing of cancer cells, thereby helping to achieve therapeutic goals.

[0278] Thus, the invention provides the use of an anti-MUC16 antibody or an anti-CD3xMUC16 bispecific antibody in the preparation of a medicament for the treatment of a tumor (eg, ovarian cancer).

[0279] In another aspect, the invention relates to a method of preventing or treating a tumor (e.g., ovarian cancer) in a subject, comprising administering to the subject an effective amount of an anti-MUC16 antibody or anti-CD3xMUC16 bispecific antibody disclosed herein, or a pharmaceutical composition comprising same.

[0280] In one embodiment, the anti-GPRC5D antibody or anti-GPRC5DxCD3 bispecific antibody disclosed herein can be used in the treatment of cancer, such as myeloma, colon cancer, rectal cancer or colorectal cancer.Accordingly, the invention provides the use of an anti-MUC16 antibody or anti-GPRC5DxCD3 bispecific antibody in the preparation of a medicament for the treatment of cancer, such as myeloma, colon cancer, rectal cancer or colorectal cancer.

[0281] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer, e.g., myeloma, colon cancer, rectal cancer, or colorectal cancer) in a subject, comprising administering to the subject an effective amount of an anti-GPRC5D antibody or anti-CD3xGPRC5D bispecific antibody disclosed herein, or a pharmaceutical composition comprising same.

[0282] The anti-CD3 or anti-GPRC5D or anti-MUC16 antibodies or bispecific antibodies of the invention (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising them) may be administered by any suitable method, including parenteral, pulmonary and intranasal administration, and, where localized treatment is required, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Depending in part on whether administration is brief or chronic, it may be administered by any suitable route, for example, injection, such as intravenous or subcutaneous injection. A variety of administration schedules are included herein, including, but not limited to, a single dose or multiple doses at multiple time points, bolus administration, and pulse infusion.

[0283] The appropriate dose of the anti-CD3 or anti-GPRC5D or anti-MUC16 or bispecific antibody (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising them) of the invention for preventing or treating a disease (when used alone or in combination with one or more other therapeutic agents) is determined by the type of disease being treated, the type of antibody, the severity and progression of the disease, whether prophylactic or therapeutic administration is required, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient in a single treatment or over a series of treatments. In another aspect, the invention provides the use of the anti-CD3 or anti-GPRC5D or anti-MUC16 antibody or bispecific antibody of the invention, or an immunoconjugate or composition or combination product comprising them, in the production or preparation of a medicament, the medicament being used for the uses described herein, e.g., for the prevention or treatment of a related disease or condition as described herein.

[0284] In some embodiments, the anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody (and immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising same) may be administered in combination with one or more additional therapies, e.g., forms of treatment and / or other therapeutic agents, and used for the uses described herein, e.g., for the prevention or treatment of the associated diseases or conditions described herein.

[0285] In some embodiments, the form of treatment is, for example, surgery or radiation therapy.

[0286] In some embodiments, the other therapeutic agent is, for example, a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent (eg, an immunosuppressant).

[0287] XI. Diagnosis and detection In one aspect, the invention further relates to methods for the diagnosis and detection (e.g., for diagnostic or non-diagnostic purposes) of anti-CD3 antibodies or anti-GPRC5D antibodies or MUC16 or bispecific antibodies, and diagnostic and detection compositions comprising same.

[0288] In some embodiments, the anti-GPRC5D antibodies provided herein can be used to detect the presence of GPRC5D in a biological sample. In some embodiments, the bispecific antibodies provided herein can be used to detect the presence of CD3 and / or GPRC5D in a biological sample.

[0289] In some embodiments, the anti-MUC16 antibodies provided herein can be used to detect the presence of MUC16 in a biological sample. In some embodiments, the bispecific antibodies provided herein can be used to detect the presence of CD3 and / or MUC16 in a biological sample.

[0290] In some embodiments, the bispecific antibodies provided herein can be used to detect the presence of a first antigen and / or a second antigen in a biological sample. In some embodiments, the bispecific antibodies provided herein can be used to detect the presence of a first antigen and / or a second antigen in a biological sample.

[0291] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads conjugated with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In some embodiments, the biological sample is a body fluid, such as blood, serum, or plasma.

[0292] In some embodiments, the method comprises contacting a biological sample with an anti-GPRC5D antibody or bispecific antibody described herein under conditions that allow its binding to GPRC5D, and detecting whether a complex is formed between the anti-GPRC5D antibody or bispecific antibody and GPRC5D. The formation of a complex indicates the presence of GPRC5D. The method may be an in vitro or in vivo method.

[0293] In some embodiments, the method comprises contacting a biological sample with an anti-MUC16 antibody or bispecific antibody described herein under conditions that allow its binding to MUC16, and detecting whether a complex is formed between the anti-MUC16 antibody or bispecific antibody and MUC16. Formation of a complex indicates the presence of MUC16. The method may be an in vitro or in vivo method.

[0294] In some embodiments, a labeled anti-GPRC5D or anti-MUC16 antibody or bispecific antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels and radioactive labels) and indirectly detected moieties, such as enzymes or ligands, such as by enzymatic reactions or molecular interactions. In some embodiments, the label is a marker, such as, for example, biotin or hFc.

[0295] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-CD3 antibody or anti-GPRC5D antibody or anti-MUC16 antibody or bispecific antibody of the invention. In some embodiments, the sample is obtained prior to use of the other therapy. In some embodiments, the sample is obtained during or after treatment with the other therapy.

[0296] In some embodiments, GPRC5D or MUC16 is detected prior to treatment, for example, prior to initiation of treatment or prior to some treatment after a treatment interval.

[0297] In some embodiments, the first antigen and / or the second antigen are detected prior to treatment, for example, prior to initiation of treatment or prior to some treatment after a treatment interval.

[0298] XII. MODE FOR CARRYING OUT THE DISCLOSURE In some aspects, the present invention relates to the following specific embodiments: 1. A bispecific antibody, a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to a tumor-associated antigen and / or the second antigen-binding region specifically binds to CD3; wherein the second antigen-binding region is an scFv of an anti-CD3 antibody comprising a VH and a VL, and optionally, the VH and VL are connected via a linker comprising the amino acid sequence (G4S)n, e.g., n=1, 2, 3, 4, or 5, preferably n=3 or 4, more preferably n=3.

[0299] 2. The bispecific antibody of embodiment 1, wherein the VH contained in the scFv comprises three complementarity determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, from the heavy chain variable region, and the VL contained in the scFv comprises three complementarity determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, from the light chain variable region, among which: (i) HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH shown in SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 are selected from the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 4, respectively; or (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:5, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:6, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:7, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:8, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:9, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:10; Bispecific antibodies.

[0300] 3. The bispecific antibody according to embodiment 2, The VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO:3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; Bispecific antibodies.

[0301] 4. The bispecific antibody of any one of embodiments 1 to 3, wherein the first antigen-binding region is a Fab that specifically binds to the first antigen.

[0302] 5. The bispecific antibody of embodiment 4, wherein said Fab comprises a CH1, wherein said CH1 is derived from IgG1, IgG2, IgG3 or IgG4, preferably from IgG1.

[0303] 6. The CH1 comprises or consists of (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 44; or (ii) The bispecific antibody according to embodiment 5, comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 44.

[0304] 7. The bispecific antibody according to any one of embodiments 1 to 6, which is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are homologous or different.

[0305] 8. A bispecific antibody according to embodiment 7, wherein one or both of the Fc regions comprise a mutation that reduces binding to an Fcγ receptor, such as one or more of the L234A / L235A mutation, the D265A mutation and the P329A mutation, such as the L234A / L235A mutation, the D265A mutation and the P329A mutation.

[0306] 9. The bispecific antibody of embodiment 8, wherein one or both of the Fc regions comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46 or 49, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity thereto.

[0307] 10. The bispecific antibody according to any one of embodiments 7 to 9, wherein the two Fc regions are different, and preferably, a knob mutation and a hole mutation are introduced into the first monomer Fc region and the second monomer Fc region, respectively.

[0308] 11. The bispecific antibody according to embodiment 10, a) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V; or b) one Fc-region polypeptide comprises the knob mutations T366W and Y349C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and S354C; or c) one Fc-region polypeptide comprises the knob mutations T366W and S354C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and Y349C; Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, e.g., one or more of an L234A / L235A mutation, a D265A mutation, or a P329A mutation, e.g., an L234A / L235A mutation, a D265A mutation, and a P329A mutation. Bispecific antibodies.

[0309] 12. The bispecific antibody according to any one of embodiments 7 to 11, wherein one or both of the Fc regions comprises a hinge region, such as EPKSS or EPKSC.

[0310] 13. The bispecific antibody according to embodiment 12, (i) the first Fc region comprises a knob mutation, a) comprising or consisting of the amino acid sequence of SEQ ID NO: 43 or 89, or b) comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 43 or 89 and comprising the L234A / L235A mutations, the D265A mutation and the P329A mutation, and the knob mutations (e.g. S354C and T366W); and / or (ii) the second Fc region comprises a hole mutation, a) comprising or consisting of the amino acid sequence of SEQ ID NO: 30, 42 or 59; or b) comprising or consisting of an amino acid sequence having at least 90% identity, e.g. 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 30, 42 or 59 and including the L234A / L235A mutation, the D265A mutation and the P329A mutation, as well as the hole mutation (e.g. Y349C, T366S, L368A and Y407V); Bispecific antibodies.

[0311] 14. The bispecific antibody according to any one of embodiments 1 to 13, which is an IgG-like bispecific antibody comprising one Fab fragment that specifically binds to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; The Fc region containing the hole mutation constitutes the first heavy chain, the C-terminus of the CH1 of the Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc domain that contains a knob mutation, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0312] 15. The bispecific antibody according to any one of embodiments 1 to 13, which is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of CH1 of the first Fab fragment is fused to CH2 or a hinge region of an Fc domain containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the CH1 of the second Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc domain that includes a knob mutation, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; Among them, the first Fab fragment and the second Fab fragment are homologous or different.

[0313] 16. The bispecific antibody according to any one of embodiments 1 to 13, which is an IgG-like bispecific antibody comprising one Fab fragment that specifically binds to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of CH1 of the Fab fragment is fused to CH2 or a hinge region of an Fc domain containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc region containing a knob mutation, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0314] 17. The bispecific antibody according to any one of embodiments 1 to 13, which is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of the CH1 of the first Fab fragment is fused to the N-terminus of the VH of the second Fab fragment, and the C-terminus of the CH1 of the second Fab fragment is fused to the CH2 or hinge region of the Fc domain containing a hole mutation, thereby forming a first heavy chain; The C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc region that contains a knob mutation, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; Among them, the first Fab fragment and the second Fab fragment are homologous or different.

[0315] 18. The bispecific antibody according to any one of embodiments 1 to 18, wherein the first antigen is a tumor-associated antigen selected from, for example, GPRC5D or MUC16 (for example, human GPRC5D or human MUC16).

[0316] 19. The bispecific antibody according to embodiment 18, wherein the first antigen is human GPRC5D and the first antigen-binding region comprises three CDRs of the heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2 and LCDR3, (i) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH shown in any one of SEQ ID NOs: 62, 38 and 40, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO: 63, respectively; (ii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 72, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 73, respectively; or (iii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 82, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 83, respectively; Bispecific antibodies.

[0317] 20. The bispecific antibody according to embodiment 19, wherein the first antigen-binding region is (i) HCDR1 as set forth in SEQ ID NO: 64, HCDR2 as set forth in SEQ ID NO: 65, 39 or 41, HCDR3 as set forth in SEQ ID NO: 66, LCDR1 as set forth in SEQ ID NO: 67, LCDR2 as set forth in SEQ ID NO: 68 and LCDR3 as set forth in SEQ ID NO: 69; (ii) HCDR1 as set forth in SEQ ID NO: 74, HCDR2 as set forth in SEQ ID NO: 75, HCDR3 as set forth in SEQ ID NO: 76, LCDR1 as set forth in SEQ ID NO: 77, LCDR2 as set forth in SEQ ID NO: 78, and LCDR3 as set forth in SEQ ID NO: 79, or (iii) HCDR1 shown in SEQ ID NO: 84, HCDR2 shown in SEQ ID NO: 85, HCDR3 shown in SEQ ID NO: 86, LCDR1 shown in SEQ ID NO: 87, LCDR2 shown in SEQ ID NO: 78, and LCDR3 shown in SEQ ID NO: 88.

[0318] 21. The bispecific antibody according to embodiment 19 or 20, wherein the first antigen-binding region comprises a heavy chain variable region VH, wherein the heavy chain variable region is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; Bispecific antibodies.

[0319] 22. The bispecific antibody according to any one of embodiments 19 to 21, wherein the first antigen-binding domain comprises a light chain variable domain VL, wherein the light chain variable domain is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; Bispecific antibodies.

[0320] 23. The bispecific antibody according to any one of embodiments 19 to 22, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, (i) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (ii) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iii) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or (iv) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17, 19 or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; Bispecific antibodies.

[0321] 24. The bispecific antibody according to any one of embodiments 19 to 23, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL each comprise or consist of the amino acid sequences shown below: (i) SEQ ID NO: 62 and SEQ ID NO: 63; (ii) SEQ ID NO: 72 and SEQ ID NO: 73; (iii) SEQ ID NO: 82 and SEQ ID NO: 83; (iv) SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 and SEQ ID NO: 21; (v) SEQ ID NO: 16 or 25 and SEQ ID NO: 17, or (vi) SEQ ID NO: 16 or 27 and SEQ ID NO: 19.

[0322] 25. The bispecific antibody according to embodiment 18, wherein the first antigen is human MUC16 and the first antigen-binding region comprises three CDRs of the heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and three CDRs of the light chain variable region VL, LCDR1, LCDR2 and LCDR3, The HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 92 or 113, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 96, 100, 102 or 114, respectively; Bispecific antibodies.

[0323] 26. The bispecific antibody of embodiment 25, wherein the first antigen-binding region is comprising an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, an HCDR3 as set forth in SEQ ID NO: 95, an LCDR1 as set forth in SEQ ID NO: 97, 101 or 103, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, Bispecific antibodies.

[0324] 27. The bispecific antibody according to embodiment 25 or 26, wherein the first antigen-binding region comprises a heavy chain variable region VH, wherein the heavy chain variable region is comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 92 or 113, or comprising or consisting of the amino acid sequence of SEQ ID NO: 92 or 113; Bispecific antibodies.

[0325] 28. The bispecific antibody according to any one of embodiments 25 to 27, wherein the first antigen-binding region comprises a light chain variable region VL, wherein said light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114, or comprises or consists of the amino acid sequence of SEQ ID NO: 96, 100, 102 or 114.

[0326] 29. The bispecific antibody according to any one of embodiments 25 to 28, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 92 or 113, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 96, 100, 102 or 114, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; Bispecific antibodies.

[0327] 30, the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, in which VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 92, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 96, 100 or 102; or The bispecific antibody according to any one of embodiments 25 to 29, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 113, and the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 114.

[0328] 31. A nucleic acid molecule encoding any chain of the bispecific antibody according to any one of embodiments 1 to 30, or consisting of the above nucleic acid sequence.

[0329] 32. An expression vector comprising the nucleic acid molecule according to embodiment 31, preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0330] 33. A host cell comprising the nucleic acid molecule according to embodiment 31 or the expression vector according to embodiment 32, preferably the host cell is prokaryotic or eukaryotic, such as a 293 cell or a CHO cell, such as a 293F cell or a 293T cell or a CHO-S cell.

[0331] 34. A method for preparing a bispecific antibody according to any one of embodiments 1 to 30, comprising culturing a host cell of the nucleic acid molecule according to embodiment 31 or the expression vector according to embodiment 32 under conditions suitable for expression of said antibody chains, and optionally recovering said antibody from said host cell (or host cell medium).

[0332] 35. An immune complex comprising the bispecific antibody according to any one of embodiments 1 to 30.

[0333] 36. A pharmaceutical composition or a medicament or a preparation comprising a bispecific antibody according to any one of embodiments 1 to 30, or an immunoconjugate according to embodiment 35, and optionally a pharmaceutical auxiliary material.

[0334] 37. A pharmaceutical combination product comprising a bispecific antibody according to any one of embodiments 1 to 30, or an immunoconjugate according to embodiment 35, and one or more additional therapeutic agents (e.g., a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug or an immunomodulatory agent).

[0335] 38. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a bispecific antibody according to any one of embodiments 1 to 30, or an immunoconjugate according to embodiment 35, or a pharmaceutical composition or formulation according to embodiment 36, or a pharmaceutical combination product according to embodiment 37.

[0336] 39. The method of embodiment 38, wherein the tumor cells of the cancer have elevated protein and / or nucleic acid levels (e.g., elevated expression) of the first antigen.

[0337] 40. The method of embodiment 38, wherein the first antigen is selected from GPRC5D or MUC16.

[0338] 41. The method according to any one of embodiments 38 to 40, wherein the cancer is a solid tumor or a blood tumor, such as ovarian cancer (e.g., serous ovarian cancer, e.g., ovarian serous cystadenocarcinoma, ovarian serous adenocarcinoma, ovarian mucinous adenocarcinoma, endometrial adenocarcinoma, ovarian clear cell adenocarcinoma), myeloma, colon cancer, rectal cancer or colorectal cancer.

[0339] 42. The method of any one of embodiments 38 to 41, further comprising administering in combination with an additional therapy, such as a form of treatment (e.g., surgical therapy or radiation therapy) and / or an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent).

[0340] 43. A method for detecting the presence of a first antigen in a biological sample, comprising: (i) contacting a biological sample with the bispecific antibody according to any one of embodiments 1 to 30 under conditions allowing its binding to the first antigen, and (ii) detecting whether a complex is formed between the antibody or bispecific antibody and a first antigen; the formation of a complex therein indicates the presence of a first antigen, method.

[0341] In some aspects, the present invention relates to the following specific embodiments: 1. An anti-CD3 antibody or antigen-binding fragment thereof that specifically binds to human CD3, comprising: 1) A heavy chain variable region (VH) having a sequence shown in SEQ ID NO:3 CD3 ), or a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 and the same CDRs; and 2) A light chain variable region (VL) having a sequence shown in SEQ ID NO:4 CD3 ) or a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and the same CDRs.

[0342] 2. The anti-CD3 antibody or antigen-binding fragment thereof of embodiment 1 comprises: 1) a heavy chain (H) whose sequence is set forth in SEQ ID NO:1 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1 and the same CDRs; and 2) A light chain (L) whose sequence is set forth in SEQ ID NO:2 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 and having the same CDRs.

[0343] 3. An anti-MUC16 antibody or antigen-binding fragment thereof that specifically binds to human MUC16, comprising a VH and a VL, wherein: 1) the VH comprises an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, and an HCDR3 as set forth in SEQ ID NO: 95, and the VL comprises an LCDR1 as set forth in SEQ ID NO: 97, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, or 2) the VH comprises an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, and an HCDR3 as set forth in SEQ ID NO: 95, and the VL comprises an LCDR1 as set forth in SEQ ID NO: 101, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, or 3) the VH comprises an HCDR1 as set forth in SEQ ID NO: 93, an HCDR2 as set forth in SEQ ID NO: 94, and an HCDR3 as set forth in SEQ ID NO: 95, and the VL comprises an LCDR1 as set forth in SEQ ID NO: 103, an LCDR2 as set forth in SEQ ID NO: 98, and an LCDR3 as set forth in SEQ ID NO: 99, or

[0344] 4. An anti-MUC16 antibody or antigen-binding fragment thereof that specifically binds to human MUC16 according to embodiment 3, comprising: 1) a VH whose sequence is set forth in SEQ ID NO: 92 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 92 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 96 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 96 and having the same CDRs, or 2) a VH whose sequence is set forth in SEQ ID NO: 92 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 92 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 100 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 100 and having the same CDRs, or 3) a VH whose sequence is set forth in SEQ ID NO: 92 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 92 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 102 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 102 and having the same CDRs, or 4) a VH whose sequence is set forth in SEQ ID NO: 113 or a VH having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 113 and having the same CDRs, and a VL whose sequence is set forth in SEQ ID NO: 114 or a VL having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 114 and having the same CDRs.

[0345] 5. The anti-MUC16 antibody or antigen-binding fragment thereof of embodiment 3 or 4 comprises: 1) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 106 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 106 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 107 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 107 and the same CDRs, or 2) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 106 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 106 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 108 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 108 and the same CDRs, or 3) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 106 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 106 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 109 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 109 and the same CDRs, or 4) a heavy chain (H) whose sequence is set forth in SEQ ID NO: 115 or a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 115 and the same CDRs, and a light chain (L) whose sequence is set forth in SEQ ID NO: 116 or a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116 and the same CDRs.

[0346] 6. The anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 2, or the anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3 to 5, wherein the antigen-binding fragment is an Fv, Fab, Fab', Fab'-SH, F(ab')2, a linear antibody, or an scFv.

[0347] 7. The anti-CD3 antibody or antigen-binding fragment thereof of embodiment 6, wherein the scFv comprises a sequence as set forth in SEQ ID NO:11 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:11 and the same CDRs.

[0348] 8. Anti-CD3xMUC16 bispecific antibodies include: (i) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 96, an LCDR2 shown in SEQ ID NO: 97, and an LCDR3 shown in SEQ ID NO: 98, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 101, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, andCD3 a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 103, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 heavy chain 1, heavy chain 2, and light chain comprising LCDR1 as set forth in SEQ ID NO:8, LCDR2 as set forth in SEQ ID NO:9, and LCDR3 as set forth in SEQ ID NO:10; or (ii) one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 97, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 101, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 103, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises heavy chain 1, heavy chain 2, and light chain comprising LCDR1 as set forth in SEQ ID NO:8, LCDR2 as set forth in SEQ ID NO:9, and LCDR3 as set forth in SEQ ID NO:10; or (iii) one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 97, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, andCD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or a heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, a heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 101, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10; or one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises an HCDR1 shown in SEQ ID NO: 93, an HCDR2 shown in SEQ ID NO: 94, and an HCDR3 shown in SEQ ID NO: 95, the VL that recognizes the MUC16 molecule comprises an LCDR1 shown in SEQ ID NO: 103, an LCDR2 shown in SEQ ID NO: 98, and an LCDR3 shown in SEQ ID NO: 99, and the scFv that recognizes a CD3 molecule comprises a VH CD3 and VLCD3 Among these, the above VH CD3 comprises HCDR1 as set forth in SEQ ID NO:5, HCDR2 as set forth in SEQ ID NO:6, and HCDR3 as set forth in SEQ ID NO:7, and CD3 comprises a heavy chain 1, a heavy chain 2, and a light chain comprising an LCDR1 as set forth in SEQ ID NO:8, an LCDR2 as set forth in SEQ ID NO:9, and an LCDR3 as set forth in SEQ ID NO:10.

[0349] 9. The anti-CD3xMUC16 bispecific antibody according to embodiment 8 comprises: (i) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 92 or 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102, or 114, and the scFv that recognizes the CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4. (ii) one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 92 or 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102, or 114, and the scFv that recognizes the CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VHCD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4; or (iii) one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 92 or 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 96, 100, 102, or 114, and the scFv that recognizes the CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4.

[0350] 10. The anti-CD3xMUC16 bispecific antibody of embodiment 8 or 9 comprises: (i) one heavy chain 1 having a structure of VH-CH1-Fc, one heavy chain 2 having a structure of VH-CH1-scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises the sequence of SEQ ID NO: 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 114, and the scFv that recognizes the CD3 molecule comprises the VH CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4. (ii) one heavy chain 1 having a VH-CH1-Fc structure, one heavy chain 2 having an scFv-Fc structure, and one light chain having a VL-CL structure, in which the light chain pairs with the VH-CH1 structure of the heavy chain 1 to form an antigen recognition site that recognizes a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 114, and the scFv that recognizes the CD3 molecule comprises a VH CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence set forth in SEQ ID NO:4; or (iii) one heavy chain 1 having a structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having a structure of scFv-Fc, and two light chains having a structure of VL-CL, among which the two light chains pair with two VH-CH1 structures in the heavy chain 1, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, among which the VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 113, the VL that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 114, and the scFv that recognizes the CD3 molecule comprises a VH that recognizes the MUC16 molecule comprises a sequence selected from SEQ ID NO: 115, CD3 and VL CD3 Among these, the above VH CD3 comprises the sequence shown in SEQ ID NO: 3, and VL CD3 heavy chain 1, heavy chain 2, and light chain comprising the sequence shown in SEQ ID NO:4.

[0351] 11. The anti-CD3 x MUC16 bispecific antibody according to any one of embodiments 8 to 10, wherein the two antigen recognition sites that recognize the MUC16 molecule contained therein may have the same VH / VL sequence or different VH / VL sequences.

[0352] 12. The anti-CD3 x MUC16 bispecific antibody according to any one of embodiments 8 to 11, wherein the Fc region is selected from an Fc region of an antibody of the IgG class, such as an Fc region from the isotype IgG1, IgG2, IgG3 or IgG4, and preferably wherein the Fc region comprises mutations, such as mutations that increase the stability of the bispecific antibody dimer (e.g. knobs-into-holes), mutations that reduce or increase effector function.

[0353] 13. The anti-CD3 x MUC16 bispecific antibody of any one of embodiments 8 to 12, wherein said scFv is connected to Fc or CH1 by a linker, e.g. a flexible peptide linker.

[0354] 14. The anti-CD3xMUC16 bispecific antibody according to any one of embodiments 8 to 13, comprising: (i) one heavy chain 1 having the structure of VH-CH1-Fc, one heavy chain 2 having the structure of VH-CH1-scFv-Fc, and two light chains having the structure of VL-CL, wherein the two light chains pair with the VH-CH1 structures of heavy chains 1 and 2, respectively, to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, wherein heavy chain 1 comprises the sequence shown in SEQ ID NO: 118, heavy chain 2 comprises the sequence shown in SEQ ID NO: 121, and light chain comprises the sequence shown in SEQ ID NO: 116. (ii) one heavy chain 1 having the structure of VH-CH1-Fc, one heavy chain 2 having the structure of scFv-Fc, and one light chain having the structure of VL-CL, in which the light chain forms an antigen recognition site that recognizes a MUC16 molecule by pairing with the VH-CH1 structure of heavy chain 1, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, in which heavy chain 1 comprises the sequence shown in SEQ ID NO: 118, heavy chain 2 comprises the sequence shown in SEQ ID NO: 117, and light chain comprises the sequence shown in SEQ ID NO: 116; or (iii) one heavy chain 1 having the structure of VH-CH1-VH-CH1-Fc, one heavy chain 2 having the structure of scFv-Fc, and two light chains having the structure of VL-CL, wherein the two light chains pair with the two VH-CH1 structures in heavy chain 1 to form two antigen recognition sites that recognize a MUC16 molecule, and the scFv forms an antigen recognition site that recognizes a CD3 molecule, wherein heavy chain 1 comprises the sequence shown in SEQ ID NO: 119, heavy chain 2 comprises the sequence shown in SEQ ID NO: 117, and light chain comprises the sequence shown in SEQ ID NO: 116.

[0355] 15. A nucleic acid encoding an anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1-2 or 6-7, or an anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, or an anti-CD3xMUC16 bispecific antibody according to any one of embodiments 8-13.

[0356] 16. A vector comprising the nucleic acid according to embodiment 15, preferably said vector being an expression vector.

[0357] 17. A host cell comprising a nucleic acid according to embodiment 15 or a vector according to embodiment 16.

[0358] 18. A method for producing an anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1-2 or 6-7, an anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, or an anti-CD3xMUC16 bispecific antibody according to any one of embodiments 8-13, comprising the steps of: (i) culturing a host cell of the invention under conditions suitable for expression of an anti-CD3 antibody, anti-MUC16 or anti-CD3xMUC16 bispecific antibody of the invention, and, optionally, (ii) recovering the anti-CD3 antibody, anti-MUC16 or anti-CD3xMUC16 bispecific antibody of the invention.

[0359] 19. A pharmaceutical composition comprising an anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1-2 or 6-7, an anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, or an anti-CD3xMUC16 bispecific antibody according to any one of embodiments 8-13, and a pharma- ceutical acceptable vector.

[0360] 20. The pharmaceutical composition according to embodiment 19, further comprising another therapeutic agent, preferably said other therapeutic agent being selected from chemotherapeutic agents, cytotoxic agents.

[0361] 21. Use of an anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1-2 or 6-7, an anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, or an anti-CD3xMUC16 bispecific antibody according to any one of embodiments 8-13, or a pharmaceutical composition according to any one of embodiments 19-20, in the preparation of a medicament for the treatment, prevention and / or diagnosis of ovarian cancer.

[0362] 22. A method for treating, preventing and / or diagnosing ovarian cancer, comprising administering to a subject in need thereof a therapeutically or diagnostically effective amount of an anti-CD3 antibody or antigen-binding fragment thereof according to any one of embodiments 1-2 or 6-7, an anti-MUC16 antibody or antigen-binding fragment thereof according to any one of embodiments 3-6, or an anti-CD3xMUC16 bispecific antibody according to any one of embodiments 8-13, or a pharmaceutical composition according to any one of embodiments 19-20.

[0363] 23. The use according to embodiment 21 or the method according to embodiment 22, wherein the ovarian cancer is an ovarian cancer, such as a serous ovarian cancer, such as an ovarian serous cystadenocarcinoma, an ovarian cancer serous adenocarcinoma, an ovarian cancer mucinous adenocarcinoma, an endometrioid adenocarcinoma, an ovarian cancer clear cell adenocarcinoma.

[0364] In some aspects, the present invention relates to the following specific embodiments: 1. An anti-GPRC5D antibody or an antigen-binding fragment thereof, comprising three CDRs of a heavy chain variable region VH, HCDR1, HCDR2 and HCDR3, and three CDRs of a light chain variable region VL, LCDR1, LCDR2 and LCDR3, (i) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH shown in any one of SEQ ID NOs: 62, 38 and 40, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO: 63, respectively; (ii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 72, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 73, respectively; or (iii) the HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 82, respectively, and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 83, respectively; An anti-GPRC5D antibody or an antigen-binding fragment thereof.

[0365] 2. An anti-GPRC5D antibody or an antigen-binding fragment thereof, (i) HCDR1 as set forth in SEQ ID NO: 64, HCDR2 as set forth in SEQ ID NO: 65, 39 or 41, HCDR3 as set forth in SEQ ID NO: 66, LCDR1 as set forth in SEQ ID NO: 67, LCDR2 as set forth in SEQ ID NO: 68 and LCDR3 as set forth in SEQ ID NO: 69; (ii) HCDR1 as set forth in SEQ ID NO: 74, HCDR2 as set forth in SEQ ID NO: 75, HCDR3 as set forth in SEQ ID NO: 76, LCDR1 as set forth in SEQ ID NO: 77, LCDR2 as set forth in SEQ ID NO: 78, and LCDR3 as set forth in SEQ ID NO: 79, or (iii) HCDR1 shown in SEQ ID NO: 84, HCDR2 shown in SEQ ID NO: 85, HCDR3 shown in SEQ ID NO: 86, LCDR1 shown in SEQ ID NO: 87, LCDR2 shown in SEQ ID NO: 78, and LCDR3 shown in SEQ ID NO: 88.

[0366] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; An antibody or an antigen-binding fragment thereof.

[0367] 4. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising a light chain variable region VL, wherein the light chain variable region is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; An antibody or an antigen-binding fragment thereof.

[0368] 5. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, (i) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (ii) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iii) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or (iv) the heavy chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17, 19 or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; An antibody or an antigen-binding fragment thereof.

[0369] 6. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL each comprise or consist of the amino acid sequences shown below: (i) SEQ ID NO: 62 and SEQ ID NO: 63; (ii) SEQ ID NO: 72 and SEQ ID NO: 73; (iii) SEQ ID NO: 82 and SEQ ID NO: 83; (iv) SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 and SEQ ID NO: 21; (v) SEQ ID NO: 16 or 25 and SEQ ID NO: 17, or (vi) SEQ ID NO: 16 or 27 and SEQ ID NO: 19.

[0370] 7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, further comprising an antibody heavy chain constant region HC, for example, the antibody heavy chain constant region HC being an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region.

[0371] 8. The antibody or antigen-binding fragment thereof of embodiment 7 comprises: (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 45 or 48; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 45 or 48.

[0372] 9. The antibody or antigen-binding fragment thereof of embodiment 7 or 8, wherein the heavy chain constant region comprises a mutation that reduces binding to an Fcγ receptor, such as a LALA mutation, a D265A mutation and / or a P329A mutation, preferably a LALA mutation as well as D265A and P329A mutations.

[0373] 10. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 9, comprising a light chain constant region, for example, the light chain constant region being a lambda or kappa light chain constant region.

[0374] 11. The antibody or antigen-binding fragment thereof of embodiment 10, wherein the light chain constant region is (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 47 or 50; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 47 or 50; An antibody or an antigen-binding fragment thereof.

[0375] 12. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 11, comprising a heavy chain, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 60, 70, 80, 14, 22, 24, 26 or 28, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 60, 70, 80, 14, 22, 24, 26 or 28, or consists of such an amino acid sequence.

[0376] 13. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 12, comprising a light chain, wherein the light chain comprises an amino acid sequence of SEQ ID NO: 61, 71, 81, 15, 18 or 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 61, 71, 81, 15, 18 or 20, or consisting of such an amino acid sequence.

[0377] 14. The antibody or antigen-binding fragment thereof according to embodiment 12 or 13, comprising a heavy chain and a light chain, (i) the heavy chain comprises or consists of an amino acid sequence of SEQ ID NO: 60 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of an amino acid sequence of SEQ ID NO: 61 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (ii) the heavy chain comprises or consists of an amino acid sequence of SEQ ID NO: 70 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of an amino acid sequence of SEQ ID NO: 71 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iii) the heavy chain comprises or consists of an amino acid sequence of SEQ ID NO: 80 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of an amino acid sequence of SEQ ID NO: 81 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (iv) the heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14, 22, 24, 26 or 28, or an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15, 18 or 20, or an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (v) the heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 14 or 24, or an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (vi) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 14 or 26, or an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and said light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (vii) comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 14, 22, 24, 26 or 28, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and said light chain comprising or consisting of an amino acid sequence as set forth in SEQ ID NO: 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, An antibody or an antigen-binding fragment thereof.

[0378] 15. The antibody or antigen-binding fragment thereof according to embodiment 14, comprising a heavy chain and a light chain, wherein the heavy chain and the light chain each comprise the amino acid sequence shown in the following SEQ ID NO: or consist of the amino acid sequence shown in the following SEQ ID NO: (i) SEQ ID NO: 60 and SEQ ID NO: 61; (ii) SEQ ID NO: 70 and SEQ ID NO: 71; (iii) SEQ ID NO: 80 and SEQ ID NO: 81; (iv) SEQ ID NO: 14 or 24 and SEQ ID NO: 15; (v) SEQ ID NO: 14 or 26 and SEQ ID NO: 18; (vi) SEQ ID NO: 14, 22, 24, 26 or 28 and SEQ ID NO: 20.

[0379] 16. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 15, wherein the antibody is a humanized antibody or a chimeric antibody.

[0380] 17. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, wherein the antibody is a monoclonal antibody.

[0381] 18. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17, wherein the antigen-binding fragment is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single chain antibody (e.g., scFv), (Fab')2, single domain antibody (e.g., VHH), dAb (domain antibody), or linear antibody.

[0382] 19. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the antibody is a bispecific or multispecific antibody having a first binding specificity for GPRC5D and another binding specificity for one or more molecules.

[0383] 20. The antibody or antigen-binding fragment thereof of embodiment 19, which is a bispecific antibody having a first binding specificity for GPRC5D and a second binding specificity for CD3.

[0384] 21. The bispecific antibody of embodiment 20, comprising a first antigen-binding region and a second antigen-binding region, The first antigen-binding region specifically binds to GPRC5D and comprises a VH and a VL, said VH comprises HCDR1, HCDR2, HCDR3 as defined in embodiment 1 or 2, and said VL comprises LCDR1, LCDR2 and LCDR3 as defined in embodiment 1 or 2; or The VH and VL are as defined in any one of embodiments 3 to 6; The second antigen-binding region specifically binds to CD3. Bispecific antibodies.

[0385] 22. The bispecific antibody according to embodiment 21, wherein the second antigen-binding region comprises a VH comprising three complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region, and a VL comprising three complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 from the light chain variable region, (i) HCDR1, HCDR2 and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH shown in any one of SEQ ID NOs: 3, respectively, and LCDR1, LCDR2 and LCDR3 are selected from the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL shown in any one of SEQ ID NOs: 4; (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:5, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:6, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:7, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:8, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:9, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:10; Bispecific antibodies.

[0386] 23. The bispecific antibody of embodiment 22, wherein the second antigen-binding region comprises a VH and a VL, The VH comprises or consists of an amino acid sequence as set forth in SEQ ID NO:3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; Bispecific antibodies.

[0387] 24. The bispecific antibody of any one of embodiments 21 to 23, wherein the first antigen-binding domain is a Fab of an anti-GPRC5D antibody as defined in any one of embodiments 1 to 18.

[0388] 25. The bispecific antibody of embodiment 24, wherein the Fab comprises a CH1, wherein the CH1 is derived from IgG1, IgG2, IgG3, or IgG4, preferably from IgG1.

[0389] 26. The CH1 comprises or consists of (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 44; or (ii) The bispecific antibody according to embodiment 25, comprising or consisting of an amino acid sequence selected from SEQ ID NO: 44.

[0390] 27. The bispecific antibody of any one of embodiments 21 to 26, wherein the second antigen-binding region is an scFv.

[0391] 28. The bispecific antibody of embodiment 27, wherein the scFv comprises a linker comprising the amino acid sequence (G4S)n, e.g., n=1, 2, 3, 4 or 5, preferably n=3 or 4, more preferably n=3.

[0392] 29. The bispecific antibody according to any one of embodiments 21 to 28, which is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are homologous or different.

[0393] 30. A bispecific antibody according to embodiment 29, wherein one or both of the Fc regions comprise a mutation that reduces binding to an Fcγ receptor, such as one or more of the L234A / L235A mutation, the D265A mutation and the P329A mutation, such as the L234A / L235A mutation, the D265A mutation and the P329A mutation.

[0394] 31. The bispecific antibody of embodiment 30, wherein one or both of the Fc regions comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46 or 49, or an amino acid sequence having at least 90% identity thereto, such as 95%, 96%, 97%, 99% or more identity thereto.

[0395] 32. The bispecific antibody according to any one of embodiments 29 to 31, wherein the two Fc regions are different, and preferably, a knob mutation and a hole mutation are introduced into the first monomer Fc region and the second monomer Fc region, respectively.

[0396] 33. The bispecific antibody according to embodiment 32, a) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V; or b) one Fc-region polypeptide comprises the knob mutations T366W and Y349C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and S354C; or c) one Fc-region polypeptide comprises the knob mutations T366W and S354C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and Y349C; Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, e.g., one or more of an L234A / L235A mutation, a D265A mutation, or a P329A mutation, e.g., an L234A / L235A mutation, a D265A mutation, and a P329A mutation. Bispecific antibodies.

[0397] 34. The bispecific antibody of any one of embodiments 29 to 33, wherein one or both of the Fc regions comprises a hinge region, e.g., EPKSS or EPKSC.

[0398] 35. The bispecific antibody according to embodiment 33, (i) the first Fc region comprises a knob mutation, a) comprising or consisting of the amino acid sequence of SEQ ID NO: 43 or 89, or b) comprising or consisting of an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 43 or 89 and comprising the L234A / L235A mutations, the D265A mutation and the P329A mutation, and the knob mutations (e.g. S354C and T366W); and / or (ii) the second Fc region comprises a hole mutation, a) comprising or consisting of the amino acid sequence of SEQ ID NO: 30, 42 or 59; or b) comprising or consisting of an amino acid sequence having at least 90% identity, e.g. 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 30, 42 or 59 and including the L234A / L235A mutation, the D265A mutation and the P329A mutation, as well as the hole mutation (e.g. Y349C, T366S, L368A and Y407V); Bispecific antibodies.

[0399] 36. The bispecific antibody of any one of embodiments 21 to 35, comprising a first antigen-binding region that is a Fab fragment that specifically binds to GPRC5D, a second antigen-binding region that is an scFv that specifically binds to CD3, and an Fc dimer.

[0400] 37. A bispecific antibody according to embodiment 36, comprising one first antigen-binding region Fab, one second antigen-binding region scFv and an Fc dimer, wherein the Fab comprises VH-CH1 and VL-CL, the scFv comprises VH-linker-VL, and one of the Fc regions in the Fc dimer comprises a hole mutation and the other comprises a knob mutation, The Fc region containing the hole mutation constitutes the first heavy chain, the C-terminus of the CH1 of the Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of an Fc domain that contains a knob mutation, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0401] 38. The bispecific antibody according to embodiment 37, wherein the first heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 30, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, 34 or 35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; Bispecific antibodies.

[0402] 39. A bispecific antibody according to embodiment 36, comprising two first antigen-binding regions Fab, one second antigen-binding region scFv and an Fc dimer, wherein the Fab comprises VH-CH1 and VL-CL, the scFv comprises VH-linker-VL, and one of the Fc regions in the Fc dimer comprises a hole mutation and the other comprises a knob mutation; the C-terminus of CH1 of the first Fab fragment is fused to CH2 or a hinge region of an Fc domain containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the CH1 of the second Fab fragment is fused to the N-terminus of the VH of the scFv fragment, and the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of an Fc domain containing a knob mutation, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; Among them, the first Fab fragment and the second Fab fragment are homologous or different.

[0403] 40. The bispecific antibody according to embodiment 39, (i) the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or the light chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; (ii) the first heavy chain comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 32, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or (iii) the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 36, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0404] 41. A bispecific antibody according to embodiment 36, comprising one first antigen-binding region Fab, one second antigen-binding region scFv and an Fc dimer, wherein said Fab comprises VH-CH1 and VL-CL, said scFv comprises VH-linker-VL, and one of the Fc regions in the Fc dimer comprises a hole mutation and the other comprises a knob mutation, the C-terminus of CH1 of the Fab fragment is fused to CH2 or a hinge region of an Fc domain containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the VL of the scFv fragment is fused to the CH2 or hinge region of an Fc domain containing a knob mutation, thereby forming a second heavy chain; and The VL-CL of the above Fab fragment constitutes the light chain.

[0405] 42. The bispecific antibody according to embodiment 41, the first heavy chain comprises or consists of an amino acid sequence set forth in SEQ ID NO: 32, 33 or 36, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the second heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or The light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO:20, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0406] 43. A nucleic acid molecule encoding any chain of the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 20, or any chain of the bispecific antibody according to any one of embodiments 21 to 42, or consisting of said nucleic acid sequence.

[0407] 44. An expression vector comprising the nucleic acid molecule of embodiment 43, preferably, the expression vector is pCDNA, such as pCDNA3.1.

[0408] 45. A host cell comprising a nucleic acid molecule according to embodiment 43 or an expression vector according to embodiment 44, preferably the host cell is prokaryotic or eukaryotic, such as a 293 cell or a CHO cell, such as a 293F cell or a 293T cell or a CHO-S cell.

[0409] 46. ​​A method for preparing an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 20, or a bispecific antibody according to any one of embodiments 21 to 42, comprising culturing a host cell of the nucleic acid molecule according to embodiment 43 or the expression vector according to embodiment 44 under conditions suitable for expression of the chains of said antibody, and optionally recovering said antibody from said host cell (or host cell medium).

[0410] 47. An immune complex comprising the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 20, or the bispecific antibody according to any one of embodiments 21 to 42.

[0411] 48. A pharmaceutical composition or a medicament or a formulation comprising an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 20, or a bispecific antibody according to any one of embodiments 21 to 42, or an immunoconjugate according to embodiment 47, and optionally a pharmaceutical auxiliary material.

[0412] 49. A pharmaceutical combination product comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 20, or a bispecific antibody according to any one of embodiments 21 to 42, or an immunoconjugate according to embodiment 47, and one or more additional therapeutic agents (e.g., a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug or an immunomodulatory agent).

[0413] 50. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 20, or a bispecific antibody according to any one of embodiments 21 to 42, or an immunoconjugate according to embodiment 47, or a pharmaceutical composition or formulation according to embodiment 48, or a pharmaceutical combination product according to embodiment 49.

[0414] 51. The method of embodiment 50, wherein the tumor cells of said cancer have elevated protein and / or nucleic acid levels (e.g., elevated expression) of GPRC5D.

[0415] 52. The method of embodiment 50 or 51, wherein the cancer is a solid tumor or a hematological tumor, such as myeloma, colon cancer, rectal cancer or colorectal cancer.

[0416] 53. The method of any one of embodiments 50 to 52, further comprising administering in combination with an additional therapy, such as a form of treatment (e.g., surgical therapy or radiation therapy) and / or an additional therapeutic agent (e.g., a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug or an immunomodulatory agent).

[0417] 54. A method for detecting the presence of GPRC5D in a biological sample, comprising: (i) contacting a biological sample with an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 20, or a bispecific antibody according to any one of embodiments 21 to 42, under conditions allowing its binding to GPRC5D; and (ii) detecting whether a complex is formed between the antibody or bispecific antibody and GPRC5D; The formation of the complex indicates the presence of GPRC5D. method. EXAMPLES

[0418] Example 1: Preparation and characterization of humanized CD3 antibodies 1.1 Preparation of CD3 antibody humanization The SP34 antibody is a mouse antibody that specifically binds to the CD3 epsilon subunit (CD3ε) and has cross-species activity with humans and monkeys (Pessano, S., et al. (1985). "The T3 / T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits." EMBO J 4(2):337-344.). In this example, humanization was carried out using the antibody SP34 as a candidate molecule.

[0419] Mouse anti-SP34 was humanized by CDR grafting technology. Briefly, the above SP34 VH and SP34 VL sequences were searched in the IMGT database and aligned to obtain human germline gene sequences IGHV3-73*01 and IGLV7-46*01, which have relatively high homology with the SP34 heavy chain variable region and light chain variable region, respectively, and used as the framework for the humanized antibody variable region. The CDRs of mouse anti-SP34 were grafted into the corresponding humanized antibody heavy chain and light chain variable region frameworks, respectively, to form a humanized anti-CD3 antibody variable region. In order to maintain the affinity of mouse antibody SP34, it is necessary to perform back mutation on the obtained humanized antibody variable region.

[0420] As a result, the humanized heavy chain variable region sequence (hu34H1) and light chain variable region sequence (hu34L2) of the SP34 antibody were obtained, and further a humanized SP34 antibody was obtained (see Table 1 for specific sequences).

[0421] [Table 2] The humanized control antibody (Roche-CD3 / 017-Roche-CD3) sequence is derived from patent US 2016 / 0075785 A1, the heavy chain amino acid sequence is SEQ ID NO: 51 and the light chain is SEQ ID NO: 52, see in particular the sequence listing.

[0422] The amino acid sequences of the variable regions of Table 1 and the control antibody Roche-CD3 were sent to General Biology System (Anhui) Co., Ltd. for codon optimization and gene synthesis. Genes encoding the VH and VL regions of the Roche-CD3 antibody were sequentially inserted into the expression vector pcDNA3.1(+) containing a gene encoding the human IgG1 heavy chain constant region (amino acid sequence of SEQ ID NO: 45) and a gene encoding the lambda light chain constant region (amino acid sequence of SEQ ID NO: 47), to obtain a plasmid expressing the full-length heavy chain hu34H1 (SEQ ID NO: 1) of the anti-CD3 humanized antibody 017-2 and a plasmid expressing the full-length light chain hu34L2 (SEQ ID NO: 2). The VH region (hu34H1) and VL region (hu34L2) synthesized at the same time were inserted into the expression vector pcDNA3.1(+) containing a gene encoding a human IgG1 heavy chain constant region Fc region (amino acid sequence of SEQ ID NO: 46) via the single chain antibody 23L2 (23L2ScFv) (SEQ ID NO: 11) consisting of the (G4S)3 linked peptide SEQ ID NO: 12, to obtain a plasmid expressing DA023AH23L2 (scFv-Fc) (SEQ ID NO: 13). The heavy chain constant regions used all contained mutations at L234A, L235A, D265A and P329A (according to Eu numbering) sites to eliminate effector functions.

[0423] The single chain antibody (23L2) was named 23L2ScFv and its amino acid sequence is shown in SEQ ID NO: 11. The synthesized anti-CD3 single chain antibody containing a constant region Fc domain was named DA023AH23L2 (VH-linked peptide-VL-Fc) and its sequence is SEQ ID NO: 13, of which the amino acid sequence of VH-linked peptide-VL is SEQ ID NO: 11 and the amino acid sequence of the Fc region is SEQ ID NO: 46.

[0424] ExpiCHO was incubated according to the manufacturer's instructions. TM The plasmids obtained above were co-transfected into ExpiCHO-S cells using an expression system (ThermoFisher, Cat#A29133) to express the control antibodies Roche-CD3, 017-2 and DA023AH23L2. After transfection, the cells were cultured for 10-12 days, and the supernatant was collected when the cell viability dropped to 60%-70%. The antibodies expressed in the supernatant were purified using the MabSelect Sure Protein A affinity chromatography system (GE healthcare). The purified antibodies were concentrated and sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and molecular exclusion. The results showed that the purity of the antibodies met the requirements and could be used for the next experiment.

[0425] 1.2 Detection of the activation effect of humanized CD3 antibody The activation effect of CD3 antibody was detected by Jurkat / NFAT-luc (luciferase) reporter gene system. According to the manufacturer's instructions, the reporter gene element in NFAT-luc2P (plasmid: Promega, Cat#E8481) was transfected into Jurkat (Cell Bank of the Committee for the Preservation of Typical Cultures, Chinese Academy of Sciences, Cat#SCSP-513) cells by Gene Pulser X Cell TM (BIO-RAD) electroporation, and NFAT-luc2P polyclonal cell line was obtained by screening with hygromycin B (Invitrogen, Cat#10687010), which was then subjected to finite dilution to obtain the monoclonal cell line Jurkat / NFAT-luc, which was stable and highly expressed NFAT-luc2P.

[0426] The cells were harvested and centrifuged, the supernatant was removed, and the cell density was adjusted to 5 x 10 5 The cells were resuspended in test buffer (99% 1640 medium + 1% FBS) to a volume of 100 μL / mL. The cells were seeded into a 384-well plate (Corning, Cat#3570), with 40 μL per well. Then, gradient-diluted humanized anti-CD3 antibodies DA023AH23L2, Roche-CD3 (final concentration 20 nM, 3-fold dilution, 8 gradients) were added at 10 μL / well. The 384-well plate was placed in a 37°C incubator, and after 6 hours, 30 μL of One-Glo (Promega, Cat#E6130) reagent was added to each well, and finally the luminescence signal was detected by a multifunctional microplate reader (Tecan Infinite F200PRO), and the expression status of luciferase directly reflected the activation effect of the CD3 antibody. The results showed that 017-2 and DA023AH23L2 had activation effect, as shown in Figure 1.

[0427] Example 2: Immunogen Because of the difficulty in producing recombinant GPRC5D antigen, GPRC5D [human and cyno] transfected cell lines were generated using standard methods and used as whole cell antigens for antibody production and characterization studies (Table 2).

[0428] The human GPRC5D protein (NP_061124.1 (NCBI sequence number), sp|Q9NZD1 (Uniprot sequence number), sequence number 57) gene sequence was codon optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd. Lipofectamine TM The gene sequence encoding human GPRC5D protein was transfected into CHO-K1 (ACTT, Cat#CCL-61) cells and HEK293T (Cell Bank of the Committee for the Preservation of Typical Cultures, Chinese Academy of Sciences, Cat#SCSP-502) cells using Invitrogen 2000 (Cat#11668019) transfection reagent, respectively. The gene sequence of cynomolgus monkey GPRC5D protein (XP_005570249.2 (NCBI sequence number), SEQ ID NO:58) was codon-optimized and gene-synthesized by General Biological Systems (Anhui) Co., Ltd. Lipofectamine TM The gene sequence encoding the cynomolgus monkey GPRC5D protein was transfected into HEK293T cells using 2000. The transfected cells were screened with 0.3 μg / mL puromycin (Gibco, Cat#A1113802) and monoclonal seeded to obtain CHO-K1 (CHO-K1-HuGPRC5D) and HEK293T artificial monoclonal cells (HEK293T-HuGPRC5D) highly expressing human GPRC5D, and HEK293T polyclonal cells (HEK293T-CynoGPRC5D) highly expressing cynomolgus monkey GPRC5D (Table 2). The amino acid sequences encoding the human GPRC5D protein and the cynomolgus monkey GPRC5D protein are shown in the sequence table.

[0429] [Table 3]

[0430] Example 3. Production of anti-human GPRC5D antibodies 3.1 Hybridoma 3.1.1 Animal immunity Five BALB / c mice (Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.) were immunized with CHO-K1-HuGPRC5D cell line (Table 2). The mice were injected twice a week for four times (Table 3). Seven days after the first immunization and seven days after the second booster immunization, the mice were venously bled, respectively, and two mice were selected for animal fusion by performing flow cytometry (FACS) assays on the mouse immune serum using HEK293T-HuGPRC5D cell line and HEK293T-CynoGPRC5D cell line (Table 2). Four days after the final booster immunization, the spleens of the mice were harvested, and the lymphocytes isolated from the spleens were used for fusion to generate hybridomas.

[0431] [Table 4]

[0432] 3.1.2 Hybridoma screening Tissue culture supernatants from 249 hybridomas were subjected to a primary screening test by FACS assay, in which 31 clones that bound to HEK293T-HuGPRC5D or HEK293T-CynoGPRC5D cell lines were selected for a next round of confirmation testing. Positive cultures were retested with the screened antigen to confirm secretion.

[0433] 3.1.3 Subclones The selected hybridoma cell line (parent clone) was subcloned to ensure monoclonality. Subcloning was performed by reseeding the parent clone using a single-step cloning system. 24 subclones were transferred to a 96-well culture plate. The subclones were screened by FACS method to screen for antibodies that bind to both HuGPRC5D and CynoGPRC5D.

[0434] 3.1.4 Hybridoma sequencing The mouse anti-GPRC5D antibody light and heavy chain variable region sequences were obtained by polymerase chain reaction (PCR) amplification technology. The total RNA of the 24 antibodies was isolated using RNAprep pure Cell Kit (TIANGEN, Cat#DP430) and reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo, K1622). The heavy and light chain variable regions were cloned using PCR method, and all PCRs were performed using high fidelity polymerase. The PCR amplified antibody heavy and light chain variable region sequence fragments were sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The amino acid sequences encoding the light and heavy chain variable regions of the produced antibodies were measured, and the Kabat light and heavy chain variable region sequences of the antibodies are shown in Table 4.

[0435] [Table 5]

[0436] Example 4: Characterization of GPRC5D antibodies obtained by hybridoma technology 4.1 Synthesis and expression of anti-GPRC5D chimeric antibodies The DNA sequences of the three mouse antibodies in Table 4 were codon optimized and gene synthesized by General Biosystems (Anhui) Co., Ltd. The mouse chimeric antibodies were obtained by sequentially inserting the genes encoding the VH and VL regions of the antibodies into the expression vector pcDNA3.1(+) containing the genes encoding the human IgG1 heavy chain constant region (SEQ ID NO: 48) and the kappa light chain constant region (SEQ ID NO: 50).

[0437] The sequences of the humanized control antibodies GPRC5D (clone number GC5B596, the heavy chain amino acid sequence is SEQ ID NO: 55 and the light chain amino acid sequence is SEQ ID NO: 56) and CD3 (clone number CD3B219, the heavy chain amino acid sequence is SEQ ID NO: 53 and the light chain amino acid sequence is SEQ ID NO: 54) are derived from patent WO2018017786A2.

[0438] Plasmids encoding the heavy and light chains of the anti-GPRC5D antibody were co-transfected into ExpiCHO-S cells to express the mouse chimeric antibodies shown in Table 5, as well as the control antibodies GC5B596 and CD3B219, and the corresponding purification was performed. ExpiCHO-S cells were transfected according to the manufacturer's instructions to express the anti-GPRC5D chimeric antibodies, as well as the control antibodies GC5B596 and CD3B219 in a similar manner. TM Plasmids encoding the heavy and light chains of the anti-GPRC5D antibodies shown in Table 5 were co-transfected into ExpiCHO-S cells using an expression system (ThermoFisher, Cat#A29133). After transfection, the cells were cultured for 10-12 days, and the supernatant was collected when cell viability dropped to 60%-70%, and the antibody expressed in the supernatant was purified using the MabSelect Sure Protein A affinity chromatography system (GE Healthcare). The purified antibody was concentrated, sterile filtered, and the purity of the antibody protein was detected by SDS-PAGE and molecular exclusion. The results showed that the purity of the antibody was over 90% and could be used for the next experiment.

[0439] [Table 6]

[0440] 4.2 Anti-GPRC5D chimeric antibodies bind to cells expressing human GPRC5D protein or cynomolgus monkey GPRC5D protein Whether or not the anti-GPRC5D chimeric antibody of the present invention can bind to human GPRC5D protein or cynomolgus monkey GPRC5D protein stably expressed on the cell surface was determined by a cell binding experiment.

[0441] The HEK293T-HuGPRC5D cell line and HEK293T-CynoGPRC5D shown in Table 2 were enzymatically digested to obtain single cell suspensions of the two cells, which were centrifuged at room temperature at 400×g, and the medium was discarded. The resulting cell pellets were washed once with PBS, then resuspended in gradient-diluted anti-GPRC5D chimeric antibodies and control antibody GC5B596 shown in Table 5 (initial concentration 133.3 nM, 4-fold equal-fold gradient dilution, total of 8 concentration points), and incubated at 4°C for 30 minutes. The cells were washed once with PBS, and then fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098) diluted 1:200 was added, and incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS, the cells were resuspended, and finally, the two-channel fluorescent signals were detected by a BD Accuri C5 (BD Bioscience) flow cytometer.

[0442] The results shown in Figure 2 indicate that the mouse chimeric anti-GPRC5D antibody can bind to cells expressing human GPRC5D protein (Figure 2A) and cynomolgus monkey GPRC5D protein (Figure 2B), and that the binding ability of chimeric antibody 29H12C9 to the chimeric antibody 39A9F1 clone is superior to the binding ability of the control antibody (GC5B596) to the cells.

[0443] Example 5: Humanization of a murine anti-GPRC5D antibody and its characterization 5.1 Humanization of mouse anti-GPRC5D antibody The antibody 29H12C9 was humanized by the method described in Example 1. The sequences of 29H12C9 were searched and aligned in the IMGT database to obtain the human germline gene sequence IGHV1-46*01, which has a relatively high homology with the 29H12C9 heavy chain variable region, as the heavy chain variable region humanized framework, and the human germline gene sequence IGKV4-1*01, which has a relatively high homology with the light chain variable region, as the light chain variable region humanized framework. The CDRs of the 29H12C9 heavy and light chain variable regions were grafted into the corresponding humanized framework to form the humanized 29H12C9 antibody variable region. In order to maintain the affinity of the antibody GPRC5D, it is necessary to perform back mutation on the obtained humanized antibody variable region. This resulted in the humanized heavy chain variable region and humanized light chain variable region sequences, respectively, and the amino acid sequence of the humanized variable region of mouse antibody 29H12C9 is shown in Table 6. The amino acid sequences of the variable regions were sent to General Biosystems (Anhui) Co., Ltd. for codon optimization and gene synthesis. Genes encoding the VH and VL regions of the antibody were sequentially inserted into the expression vector pcDNA3.1(+) containing a gene encoding the human IgG1 heavy chain constant region (SEQ ID NO: 48) and a gene encoding the kappa light chain constant region (SEQ ID NO: 50), to obtain a plasmid expressing the full-length heavy chain of the anti-GPRC5D humanized antibody and a plasmid encoding the full-length light chain. The name, variable region sequence, and heavy and light chain sequences of each antibody are shown in Table 6.

[0444] [Table 7] Alternatively, the amino acids at positions 55 and 56 of the antibody CDR2 can be mutated to obtain the following antibody: [Table 8]

[0445] 5.2 Expression and purification of humanized anti-GPRC5D antibodies The combination of plasmids encoding heavy and light chains (Table 6) was co-transfected into ExpiCHO-S cells to express anti-GPRC5D humanized antibodies as described in Example 4.1, and the corresponding purification was performed as described in Example 1.1. The purified antibodies were concentrated, sterile filtered, and the purity of the antibodies was detected by SDS-PAGE and molecular exclusion chromatography (SEC).

[0446] 5.3 Physicochemical analysis of humanized anti-GPRC5D antibodies The purity of the obtained humanized anti-GPRC5D antibody was confirmed by molecular exclusion chromatography. Specifically, 20 μg of the antibody sample was injected into a TSK G3000SWXL column using 100 mM sodium phosphate + 100 mM Na2SO4 (pH 7.0) as the electrophoresis buffer. Electrophoresis was performed for 30 minutes. The collected effluent was measured by Agilent 1220 HPLC, and the data was analyzed by OpenLAB software. The results showed that the purity of the antibody was over 90% and could be used for the next experiment.

[0447] 5.4 Humanized anti-GPRC5D antibodies bind to engineered cells expressing human GPRC5D protein Whether or not the anti-GPRC5D humanized antibody of the present invention can bind to human GPRC5D protein stably expressed on the cell surface was determined by a cell binding experiment. The HEK293T-HuGPRC5D cell line shown in Table 2 was enzymatically digested to obtain a single cell suspension of HEK293T-huGPRC5D cells, which was centrifuged at 400×g at room temperature and the medium was discarded. The cell pellet was washed once with PBS, then resuspended in gradient-diluted anti-GPRC5D humanized antibody and mouse chimeric antibody 29H12C9 (initial concentration 166.67 nM, 4-fold equal-fold gradient dilution, total of 8 concentration points) and incubated at 4°C for 30 minutes. The cells were washed once with PBS, and then fluorescent secondary antibody R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch, Cat#109-116-098) diluted 1:200 was added and incubated at 4°C in the dark for 30 minutes. After washing the cells twice with PBS, the cells were resuspended, and finally, the two-channel fluorescent signals were detected by a Beckman Cytoflex flow cytometer. The results shown in FIG. 3 demonstrated that the anti-GPRC5D humanized antibody was capable of binding to cells expressing human GPRC5D protein, and that the antibody binding ability was close to that of the mouse anti-control antibody (29H12C9).

[0448] Example 6: Anti-CD3 / GPRC5D bispecific antibody 6.1 Construction of bispecific antibodies The present invention constructed bispecific antibodies with three different asymmetric structures as shown in FIG. 4, in which the anti-GPRC5D antigen-binding region of each bispecific antibody was derived from the humanized GPRC5D antibody produced in Example 5 above, the anti-CD3 antigen-binding region was derived from the humanized CD3 antibody produced in Example 1, and the bispecific antibody constant regions all contained a knob-in-hole (KIH) structure (Merchant, AM, et al. (1998). "An efficient route to human bispecific IgG." Nat Biotechnol 16(7):677-681.) to eliminate effector functions, and contained mutations at L234A, L235A, D265A, and P329A (according to EU numbering) sites. Such bispecific antibodies are also referred to herein as "anti-CD3 / GPRC5D bispecific antibodies" or "anti-CD3 / anti-GPRC5D bispecific antibodies", sometimes abbreviated as "bispecific antibody molecules" or "bispecific antibodies". The anti-GPRC5D portion of the anti-CD3 / GPRC5D bispecific antibody targets cells expressing GPRC5D, while the anti-CD3 portion activates T cells. The bispecific antibody binds to both GPRC5D on tumor cells and CD3 on T cells, thereby aiding in the targeted killing of tumor cells by activated T cells. The present invention uses standard construction methods to obtain three anti-CD3 / GPRC5D bispecific antibodies with the structures shown in FIG. 4, and their amino acid sequences are shown in Table 7 below. Among them, the heavy chains of the bispecific antibodies are named sequentially in the source sequence from N-terminus to C-terminus, for example, 23L2-Rknob (also called Hu34Scfv-Rknob) indicates that this sequence contains ScFv from 23L2 and a constant region containing a knob mutation in N-terminus to C-terminus, and 29H6-Rhole indicates that this sequence contains a constant region containing a sequence from 29H6 and a hole mutation in N-terminus to C-terminus. In order to eliminate the post-translational modification (PTM) site of the 29H6 heavy chain molecule, the 55th asparagine (N) or the 56th glycine (G) of the 29H6 heavy chain is mutated to serine (S) or glutamic acid (E), respectively, and after the mutation, it is abbreviated as NS or GE (Kabat) in the molecule. Table 7 below summarizes the constructs and associated sequences of each bispecific antibody:

[0449] [Table 9] Each chain of each bispecific antibody molecule was constructed as follows: 23L2-Rknob (also called Hu34Scfv-Rknob): The nucleotide sequence encoding the anti-CD3 single chain antibody 23L2 was cloned into the pcDNA3.1(+) vector with Fc(Knob) and expressed to obtain the 23L2-Knob molecule. 29H6-23L2-Rknob: Using the humanized anti-GPRC5D antibody heavy chain coding sequence as a template, the nucleotide sequence encoding the heavy chain variable region and the heavy chain constant region CH1 segment was amplified, and using overlap PCR technology, the sequence was directly joined to the nucleotide sequence encoding the complete 23L2-Rknob (without a linking peptide between the two sequences), and the joined nucleotide sequence was cloned into the expression vector pcDNA3.1(+) vector and expressed to obtain the 29H6-23L2-Rknob molecule. 29H6NS-23L2-Rknob: Using the constructed 29H6-23L2-Knob as a template, the 55th asparagine (N) was mutated to serine (S) by overlapping PCR technique, cloned into the expression vector pcDNA3.1(+) vector, and expressed to obtain the 29H6NS-23L2-Rknob molecule. 29H6GE-23L2-Rknob: Using the constructed 29H6-23L2-Rknob as a template, the 56th glycine (G) was mutated to glutamic acid (E) by overlapping PCR technique, cloned into the expression vector pcDNA3.1(+) vector, and expressed to obtain the 29H6GE-23L2-Rknob molecule. 29H6-Rhole: The nucleotide sequences encoding the humanized anti-GPRC5D antibody heavy chain variable region and heavy chain constant region CH1 segment were cloned into the pcDNA3.1(+) vector with Fc(Hole) and expressed to obtain the 29H6-Rhole molecule. 29H6NS-Rhole: Using the constructed 29H6-Rhole as a template, the 55th asparagine (N) was mutated to serine (S) by overlapping PCR technique, cloned into the expression vector pcDNA3.1(+) vector, an...

Claims

1. A bispecific antibody, comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to a tumor-associated antigen and / or the second antigen-binding region specifically binds to CD3; wherein the second antigen-binding region is an scFv of an anti-CD3 antibody comprising a VH and a VL, and optionally the VH and VL are connected via a linker comprising the amino acid sequence (GS)n, e.g., where n=1, 2, 3, 4, or 5, preferably n=3 or 4, more preferably n=3.

2. The VH contained in the scFv contains three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL contained in the scFv contains three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, among which (i) HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH shown in SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 are selected from the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 4, respectively; or (ii) HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:5, HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:7, LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO:8, LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO:9, and LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO:

10. The bispecific antibody of claim 1.

3. the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and / or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; The bispecific antibody of claim 2.

4. the first antigen-binding region is a Fab that specifically binds to the first antigen; The bispecific antibody according to any one of claims 1 to 3.

5. The Fab comprises a CH1, wherein the CH1 is derived from IgG1, IgG2, IgG3, or IgG4, preferably from IgG1; The bispecific antibody of claim 4.

6. the CH1 (i) comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:44; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 44; The bispecific antibody of claim 5.

7. The bispecific antibody is an IgG-like bispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are homologous or different. The bispecific antibody of claim 1.

8. one or both of the Fc regions comprises a mutation that reduces binding to an Fcγ receptor, e.g., one or more of an L234A / L235A mutation, a D265A mutation, and a P329A mutation, e.g., an L234A / L235A mutation, a D265A mutation, and a P329A mutation; The bispecific antibody of claim 7.

9. One or both of the Fc regions comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46 or 49, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity thereto; The bispecific antibody of claim 8.

10. the two Fc regions are different, preferably by introducing corresponding knob and hole mutations into the first and second monomer Fc regions, respectively; The bispecific antibody according to any one of claims 7 to 9.

11. a) one Fc-region polypeptide comprises the knob mutation T366W and the other Fc-region polypeptide comprises the hole mutations T366S, L368A and Y407V; or b) one Fc-region polypeptide comprises the knob mutations T366W and Y349C and the other Fc-region polypeptide comprises the hole mutations T366S, L368A, Y407V and S354C; or c) one Fc-region polypeptide comprises knob mutations T366W and S354C, and the other Fc-region polypeptide comprises hole mutations T366S, L368A, Y407V, and Y349C; Optionally, the Fc region further comprises a mutation that reduces binding to an Fcγ receptor, e.g., one or more of an L234A / L235A mutation, a D265A mutation, or a P329A mutation, e.g., an L234A / L235A mutation, a D265A mutation, and a P329A mutation. The bispecific antibody of claim 10.

12. one or both of the Fc regions comprises a hinge region, e.g., EPKSS or EPKSC; The bispecific antibody of claim 7.

13. (i) the first Fc region comprises a knob mutation, and said knob mutation a) comprising or consisting of the amino acid sequence of SEQ ID NO: 43 or 89; or b) comprising or consisting of an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 43 or 89 and comprising the L234A / L235A mutations, the D265A mutation and the P329A mutation, and knob mutations (e.g., S354C and T366W); and / or (ii) the second Fc region comprises a hole mutation, and said hole mutation a) comprising or consisting of the amino acid sequence of SEQ ID NO: 30, 42 or 59; or b) comprising or consisting of an amino acid sequence having at least 90% identity, e.g., 95%, 96%, 97%, 99% or more identity, to SEQ ID NO: 30, 42 or 59, and comprising the L234A / L235A mutation, the D265A mutation and the P329A mutation, and the hole mutation (e.g., Y349C, T366S, L368A and Y407V); The bispecific antibody of claim 12.

14. The bispecific antibody is an IgG-like bispecific antibody comprising one Fab fragment that specifically binds to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the Fc region containing the hole mutation constitutes the first heavy chain; the C-terminus of the CH1 of the Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc region comprising a knob mutation, thereby forming a second heavy chain; and VL-CL of the Fab fragment constitutes the light chain; The bispecific antibody of claim 1.

15. The bispecific antibody is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of CH1 of the first Fab fragment is fused to CH2 or a hinge region of an Fc region containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the CH1 of a second Fab fragment is fused to the N-terminus of the scFv fragment, and the C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc region comprising a knob mutation, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; wherein the first Fab fragment and the second Fab fragment are homologous or different; The bispecific antibody of claim 1.

16. The bispecific antibody is an IgG-like bispecific antibody comprising one Fab fragment that specifically binds to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of CH1 of the Fab fragment is fused to CH2 or a hinge region of an Fc region containing a hole mutation, thereby forming a first heavy chain; the C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc region containing a knob mutation, thereby forming a second heavy chain; and VL-CL of the Fab fragment constitutes the light chain; The bispecific antibody of claim 1.

17. The bispecific antibody is an IgG-like bispecific antibody comprising two Fab fragments that specifically bind to a first antigen, one scFv that specifically binds to a second antigen, and an Fc heterodimer, the Fab fragment comprises VH-CH1 and VL-CL, and the scFv comprises VH-linker-VL or VL-linker-VH; the C-terminus of the CH1 of the first Fab fragment is fused to the N-terminus of the VH of the second Fab fragment, and the C-terminus of the CH1 of the second Fab fragment is fused to the CH2 or hinge region of an Fc region containing a hole mutation, thereby forming a first heavy chain; The C-terminus of the scFv fragment is fused to the CH2 or hinge region of an Fc region containing a knob mutation, thereby forming a second heavy chain; and the VL-CL of the first and second Fab fragments constitute two light chains; wherein the first Fab fragment and the second Fab fragment are homologous or different; The bispecific antibody of claim 1.

18. the first antigen is a tumor-associated antigen, MUC16, human MUC16, GPRC5D, or human GPRC5D; The bispecific antibody of claim 1.

19. The first antigen is human MUC16, and the first antigen-binding region includes three CDRs of the heavy chain variable region VH, namely HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region VL, namely LCDR1, LCDR2, and LCDR3, among which the HCDR1, HCDR2 and HCDR3 are selected from HCDR1, HCDR2 and HCDR3, which are three complementarity determining regions contained in VH shown in SEQ ID NO: 113 or 92, respectively, and the LCDR1, LCDR2 and LCDR3 are LCDR1, LCDR2 and LCDR3, which are three complementarity determining regions contained in VL shown in SEQ ID NO: 114, 102, 100 or 96, respectively; 19. The bispecific antibody of claim 18.

20. The first antigen-binding region an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 93, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 94, an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 95, an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 103, 101 or 97, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 98, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 99, 20. The bispecific antibody of claim 19, comprising:

21. The first antigen-binding region comprises a heavy chain variable region VH, wherein the heavy chain variable region is comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 113 or 92, or comprising or consisting of the amino acid sequence of SEQ ID NO: 113 or 92; 21. A bispecific antibody according to claim 19 or 20.

22. the first antigen-binding region comprises a light chain variable region VL, wherein said light chain variable region comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 114, 102, 100 or 96, or comprises or consists of the amino acid sequence of SEQ ID NO: 114, 102, 100 or 96; 21. A bispecific antibody according to claim 19 or 20.

23. The first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 113 or 92, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 114, 102, 100, or 96, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; 21. A bispecific antibody according to claim 19 or 20.

24. the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 92, and VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 96, 100, or 102; or The first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 113, and VL comprises or consists of the amino acid sequence set forth in SEQ ID NO:

114.

21. A bispecific antibody according to claim 19 or 20.

25. The first antigen is human GPRC5D, and the first antigen-binding region includes three CDRs of the heavy chain variable region VH, namely HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region VL, namely LCDR1, LCDR2, and LCDR3, among which (i) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH shown in any one of SEQ ID NOs: 62, 38, or 40, respectively, and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 63, respectively; (ii) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH set forth in SEQ ID NO: 72, respectively, and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL set forth in SEQ ID NO: 73, respectively; or (iii) the HCDR1, HCDR2, and HCDR3 are selected from the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH shown in SEQ ID NO: 82, respectively, and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 83, respectively; 19. The bispecific antibody of claim 18.

26. The first antigen-binding region (i) an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 64; an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 65, 39, or 41; an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 66; an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 67; an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 68; and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 69; (ii) an HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 74, an HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 75, an HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 76, an LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 77, an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78, and an LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79, or (iii) HCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 84, HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 85, HCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 86, LCDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 87, LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 78, and LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 88, 26. The bispecific antibody of claim 25, comprising:

27. The first antigen-binding region comprises a heavy chain variable region VH, wherein the heavy chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 62, 72, 82, 16, 23, 25, 27, 29, 38 or 40; 27. A bispecific antibody according to claim 25 or 26.

28. The first antigen-binding region comprises a light chain variable region VL, wherein the light chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 63, 73, 83, 17, 19 or 21; 27. A bispecific antibody according to claim 25 or 26.

29. The first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, (i) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (ii) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; (iii) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 82, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 83, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or (iv) the heavy chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, 23, 25, 27, 29, 38, or 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, 19, or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; 27. A bispecific antibody according to claim 25 or 26.

30. The first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, of which VH and VL have the amino acid sequences shown below, respectively: (i). SEQ ID NO: 62 and SEQ ID NO: 63; (ii). SEQ ID NO: 72 and SEQ ID NO: 73; (iii). SEQ ID NO: 82 and SEQ ID NO: 83; (iv) SEQ ID NO: 16, 23, 25, 27, 29, 38 or 40 and SEQ ID NO: 21; (v) SEQ ID NO: 16 or 25 and SEQ ID NO: 17, or (vi). SEQ ID NO: 16 or 27 and SEQ ID NO: 19; comprising or consisting of 27. A bispecific antibody according to claim 25 or 26.

31. A nucleic acid molecule encoding or consisting of any chain of the bispecific antibody of claim 1.

32. 32. An expression vector comprising the nucleic acid molecule of claim 31, wherein the expression vector is preferably pCDNA, e.g. pCDNA3.

1.

33. 33. A host cell comprising the nucleic acid molecule of claim 31 or the expression vector of claim 32, preferably the host cell is prokaryotic or eukaryotic, such as a 293 cell or a CHO cell, such as a 293F cell or a 293T cell or a CHO-S cell.

34. 33. A method for preparing a bispecific antibody according to claim 1, said method comprising culturing a host cell for the nucleic acid molecule of claim 31 or the expression vector of claim 32 under conditions suitable for expression of the chains of the antibody, and optionally recovering the antibody from the host cell (or host cell medium).

35. An immunoconjugate comprising the bispecific antibody of claim 1.

36. A pharmaceutical composition or medicament or formulation comprising the bispecific antibody of claim 1 and optionally pharmaceutical auxiliary materials.

37. 10. A pharmaceutical combination product comprising the bispecific antibody of claim 1 and one or more additional therapeutic agents (e.g., a chemotherapeutic agent, a cytokine, a cytotoxic agent, an additional antibody, a small molecule drug, or an immunomodulatory agent).

38. 38. The bispecific antibody of claim 1, or the immunoconjugate of claim 35, or the pharmaceutical composition or formulation of claim 36, or the pharmaceutical combination product of claim 37 for preventing or treating cancer in a subject.

39. tumor cells of the cancer have elevated protein and / or nucleic acid levels (e.g., elevated expression) of the first antigen; 39. The bispecific antibody, immunoconjugate, pharmaceutical composition or formulation, or pharmaceutical combination product of claim 38.

40. The first antigen is selected from GPRC5D or MUC16.

39. The bispecific antibody, immunoconjugate, pharmaceutical composition or formulation, or pharmaceutical combination product of claim 38.

41. The cancer is a solid tumor or a hematological tumor, for example, ovarian cancer (e.g., serous ovarian cancer, e.g., ovarian serous cystadenocarcinoma, ovarian serous adenocarcinoma, ovarian mucinous adenocarcinoma, endometrial adenocarcinoma, ovarian clear cell adenocarcinoma), myeloma, colon cancer, rectal cancer or colorectal cancer; 39. The bispecific antibody, immunoconjugate, pharmaceutical composition or formulation, or pharmaceutical combination product of claim 38.

42. further comprising administering in combination with an additional therapy, e.g., a form of treatment (e.g., surgical therapy or radiation therapy) and / or an additional therapeutic agent (e.g., a chemotherapeutic agent, cytokine, cytotoxic agent, additional antibody, small molecule drug, or immunomodulatory agent); 39. The bispecific antibody, immunoconjugate, pharmaceutical composition or formulation, or pharmaceutical combination product of claim 38.

43. 1. A method for detecting the presence of a first antigen in a biological sample, comprising: (i) contacting a biological sample with the bispecific antibody of claim 1 under conditions that allow its binding to the first antigen; and (ii) detecting whether a complex is formed between the antibody or bispecific antibody and a first antigen; Including, wherein the formation of a complex indicates the presence of the first antigen.

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