Bispecific antibody targeting MUC16 and CD3, and use thereof

Abstract

Provided is an antibody or an antigen-binding fragment thereof, which is capable of specifically binding to a MUC16 protein, for example, a human MUC16 protein. Also provided is a multispecific binding molecule that specifically binds to MUC16 and CD3, for example, a multispecific antibody. Further provided are a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell, and a preparation method therefor. The antibody can be used for diagnosis and treatment.

Description

Bispecific antibodies targeting MUC16 and CD3 and their applications

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. CN202510311950.4, filed on March 17, 2025, the entire contents of which are incorporated herein by reference.

[0003] This disclosure provides an antibody or an antigen-binding fragment thereof that can specifically bind to the MUC16 protein, such as the human MUC16 protein. This disclosure also provides multispecific binding molecules that specifically bind to both MUC16 and CD3, such as multispecific antibodies. This disclosure further provides a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell, and methods for preparing the same. This disclosure also provides diagnostic and therapeutic methods using the antibodies of this disclosure.

[0004] Background of the Invention

[0005] Bispecific T-cell antibodies (BsAbs) have been used in cancer treatment. These antibodies can form synapses between cytotoxic T lymphocytes and tumor cells, inducing tumor cell destruction. BsAbs typically involve dual targeting of tumor-associated antigens (TAAs) and T-cell surface antigens (also known as T-cell adaptor antigens, TEAs). However, BsAb-based treatment strategies typically rely on the distribution of TAAs on the tumor cells to be treated. This leads to selectivity and limitations in treatment for specific patient populations. Furthermore, studies have shown that treatments targeting a single TAA site may cause disease relapse due to tumor escape mechanisms, thus limiting treatment effectiveness.

[0006] MUC16, also known as CA125, is a member of the mucin family (MUC) and belongs to type I transmembrane mucins. It is aberrantly expressed in various cancers, particularly prominent in ovarian cancer. MUC16 contains approximately 14,000 amino acids with a molecular weight between 1.5 and 5 MDa. It comprises three main domains: an N-terminal domain (MUC16-N), a tandem repeat domain (MUC16-TR), and a C-terminal domain (MUC16-C). Its N-terminal domain contains multiple serine-rich regions within a threonine-rich region approximately 12,000 amino acids long; this region is O-glycosylated only. The TR domain contains 12–60 repeat sequences of 156 amino acids with scattered SEA domains containing O-linked and N-linked glycosylation sites. The C-terminal domain of MUC16 includes an extracellular domain, a short transmembrane region, and a 32-amino acid intracellular domain. MUC16 is overexpressed on the surface of ovarian cancer cells and detaches / is excreted into the bloodstream, making it a recognized serum biomarker for ovarian cancer. MUC16 not only promotes cancer cell proliferation and metastasis but also enhances drug resistance by inhibiting the p53-mediated apoptosis pathway. Ectopic expression of the C-terminal domain of MUC16 induces cisplatin resistance in ovarian cancer cells, an effect mediated by p53 inhibition. MUC16 is also a mediator of EMT (epithelial-mesenchymal transition) in pancreatic cancer; its knockout leads to reduced cancer cell migration in vitro and metastasis in vivo. The recently described interaction between MUC16 and FAK is considered a mechanism of pancreatic cancer metastasis. MUC16, through its large glycosylated extracellular region, blocks the interaction between immune cells and cancer cells, thereby helping cancer cells evade immune system surveillance. Immune cell infiltration analysis of TCGA samples showed a strong negative correlation between mucin mRNA expression and tumor cytotoxic lymphocyte infiltration. MUC16 (CA125) is a common clinical biomarker for monitoring epithelial ovarian cancer. It is frequently mutated in various cancers and is the third most common mutated gene in tumors.

[0007] MUC16 (CA125) is not only an important biomarker for ovarian cancer, but also plays a crucial role in various cancers. Its functions in anti-apoptosis, energy metabolism reprogramming, and evading immune surveillance make it a potential target for cancer therapy.

[0008] Therefore, there is still a need in the field to develop safe anti-MUC16 antibodies suitable as drugs, as well as multispecific antigen-binding molecules based on them that specifically bind to CD3 for therapeutic applications. Summary of the Invention

[0009] One aspect of this disclosure relates to an anti-MUC16 antibody or an antigen-binding fragment thereof.

[0010] In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of the present disclosure specifically binds to MUC16 (e.g., human MUC16 and / or cynomolgus monkey MUC16 and / or mouse MUC16). In some embodiments, the binding affinity K of the anti-MUC16 antibody or its antigen-binding fragment of the present disclosure to MUC16 (e.g., human MUC16) is [not specified]. D Values ​​less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, or 0.2 nM, or between these values, as determined by, for example, bioluminescent interferometry or BLI. In some embodiments, the binding affinity K of the anti-MUC16 antibody or its antigen-binding fragment to cynomolgus monkey MUC16 is... D Values ​​less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, or 0.5 nM, or between these values, are determined, for example, by bioluminescent interferometry or BLI. In some embodiments, the binding affinity K of the anti-MUC16 antibody or its antigen-binding fragment to MUC16 (e.g., human MUC16) disclosed herein is... D The value is greater than or equal to approximately 0.1 nM, as determined by, for example, bioluminescent interferometry or BLI. In some embodiments, the anti-MUC16 antibody of this disclosure or its antigen-binding fragment specifically binds to Domain SEA 15 or Domain SEA 16 of human MUC16.

[0011] Another aspect of this disclosure relates to a multispecific binding molecule (e.g., a multispecific antibody, such as a bispecific antibody) constructed based on the MUC16 antibody of this disclosure, comprising one or more (e.g., two) target binding regions derived from the anti-MUC16 antibody of this disclosure, and optionally one or more other target binding regions (antigen binding regions), such as a CD3-specific target binding region (antigen binding region).

[0012] In one embodiment, this disclosure relates to a multispecific antibody, such as a bispecific antibody, that specifically binds to MUC16 and CD3.

[0013] In one embodiment, this disclosure relates to a bispecific antibody that specifically binds to MUC16 (e.g., human MUC16) and CD3 (e.g., human CD3).

[0014] In some embodiments, the multispecific antibodies disclosed herein, such as bispecific antibodies, can be used to treat tumors.

[0015] In some embodiments, the multispecific antibodies of this disclosure, such as bispecific antibodies, have one or more of the following properties:

[0016] (i) Specific binding to MUC16 (e.g., human MUC16 and / or cynomolgus monkey MUC16 and / or mouse MUC16), for example, the binding affinity K for specific binding to human MUC16. D Values ​​less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, or 0.4 nM; Kbinding affinity for cynomolgus monkey-specific binding. D Values ​​less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, or 0.5 nM; and / or the binding affinity K for mouse-specific binding. D Values ​​less than or equal to approximately 10 nM, 9 nM, 8 nM, or 7 nM, as determined by bio-optical interferometry;

[0017] (ii) It is capable of killing tumor cells in vitro, such as ovarian cancer cells and / or pancreatic cancer cells, preferably with a killing ability superior to control antibodies such as REGN4018;

[0018] (iii) It is capable of specifically binding to tumor cells expressing MUC16, preferably with a binding ability superior to control antibodies such as REGN4018;

[0019] (iv) It can effectively block the binding of antibodies to CD3 antigens on the surface of T cells, but still maintains a strong ability to kill tumor cells in vitro, avoiding cytokine release syndrome (CRS) caused by excessive activation of T cells.

[0020] (v) It has a relatively long half-life of antibody molecules in animals.

[0021] (vi) Inducing T cell activation and causing the killing of tumor cells (e.g., pancreatic cancer cells or ovarian cancer cells), preferably, the ability to induce T cell activation is significantly better than that of the control antibody REGN4018;

[0022] (vii) Inducing lower intensity T cell activation helps reduce toxicity issues;

[0023] (viii) It has better in vitro and / or in vivo antitumor efficacy (e.g., pancreatic cancer cells or ovarian cancer cells), for example, it is superior to the control antibody REGN4018.

[0024] In some embodiments, the bispecific antibody of this disclosure is designed in a 2+1 configuration, thereby reducing cytokine release syndrome (CRS) caused by T cell overactivation and prolonging the half-life of the bispecific antibody molecule in vivo. It also effectively blocks the binding of the antibody to the CD3 antigen on the T cell surface, while still maintaining strong in vitro tumor cell killing ability. Simultaneously, due to the Fab shielding effect, the CD3-binding scFv does not have the ability to bind to T cells when the antibody does not bind to MUC16. Only when the antibody binds to MUC16 does it have the ability to bind to and activate T cells. The resulting bispecific antibody maintains a high binding capacity to MUC16 while reducing antibody waste caused by ineffective binding to T cells when not binding to MUC16, and also avoids the release of inflammatory factors caused by non-target-dependent T cell activation. Furthermore, in the bispecific antibody, the tandem configuration of Fab and scFv effectively prevents light and heavy chain mismatches. Combined with the Innobody platform, which prevents heavy chain mismatches, the bispecific antibody can be correctly paired at a high proportion when expressed in the same cell. Furthermore, combining Innobody in vitro recombinant technology enables a high proportion of correct pairing of multispecific antibodies, such as bispecific antibodies. In contrast, the control antibody REGN4018 is a 1+1 bispecific antibody, with the CD3 antibody fully exposed, exhibiting stronger binding function to the CD3 antigen. Attached Figure Description

[0025] Figure 1 shows the validation of the antibody's binding function to the MUC16 antigen on the surface of OVCAR3 ovarian cancer cells;

[0026] Figure 2 shows the validation of the antibody's binding function to the MUC16 antigen on the surface of CFPAC1 pancreatic cancer cells;

[0027] Figure 3 shows the in vitro killing function of the 1+1 bispecific antibody against different tumor cells.

[0028] Figure 4 shows the validation of the antibody's binding function to the MUC16 antigen on the surface of OVCAR3 ovarian cancer cells;

[0029] Figure 5 shows the single-episode MUC16 / CD3 (HzS2-6xS2-6-sp34.24) dual antibody formulation;

[0030] Figure 6 shows the dual-epitope MUC16 / CD3 (HzS2-193xS2-196-sp34.24 or HzS2-193xS2-6-sp34.24) dual antibody formulation;

[0031] Figure 7 shows the killing effect of the chimeric MUC16 / CD3 bispecific antibody on OVCAR3 ovarian cancer cells;

[0032] Figure 8 shows the binding of the MUC16 / CD3 bispecific antibody to the MUC16 antigen on the surface of OVCAR3 ovarian cancer cells;

[0033] Figure 9 shows the binding of the MUC16 / CD3 bispecific antibody to the MUC16 antigen on the surface of CFPAC1 pancreatic cancer.

[0034] Figure 10 shows the binding of the MUC16 / CD3 bispecific antibody to the CD3 antigen on the surface of T cells;

[0035] Figure 11 shows the killing effect of MUC16 / CD3 bispecific antibody on OVCAR3 ovarian cancer cells;

[0036] Figure 12 shows the killing effect of MUC16 / CD3 bispecific antibody on CFPAC1 pancreatic cancer cells;

[0037] Figure 13 shows the T cell activation induced by the killing of OVCAR3 ovarian cancer cells by the MUC16 / CD3 bispecific antibody;

[0038] Figure 14 shows the T cell activation induced by the killing of CFPAC-1 pancreatic cancer cells by the MUC16 / CD3 bispecific antibody;

[0039] Figure 15 shows the T cell activation induced by the killing of 293 T cells by the MUC16 / CD3 bispecific antibody;

[0040] Figure 16 shows the antitumor effect of MUC16 / CD3 bispecific antibody on OV90-MUC16 tumor-bearing mice;

[0041] Figure 17 shows the antitumor effect of MUC16 / CD3 bispecific antibody on CFPAC-1 tumor-bearing mice.

[0042] Invention Details

[0043] It should be understood that this disclosure is not limited to the specific methods, schemes, embodiments, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure, which is limited only by the appended claims.

[0044] I. Definition

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0046] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0047] The term “about” when used in conjunction with a numeric value means a range of numeric values ​​that have a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) smaller than the specified numeric value and an upper limit of 5% (e.g., 4%, 3%, 2%, or 1%) larger than the specified numeric value.

[0048] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0049] In this article, the use of "first" and "second" is solely for the purpose of distinguishing between two structural domains or two chains, and does not in any way indicate the location of the two structural domains.

[0050] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0051] As used herein, the term "MUC16" refers to a member of the mucin family (MUC) and any naturally occurring MUC16 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice). The term encompasses "full-length" unprocessed MUC16, as well as any form of MUC16 produced by cellular processing. The term also encompasses naturally occurring variants of MUC16, such as splice variants or allele variants. Variants, isotypes, species homologs, and analogs having at least one identical epitope to MUC16 (e.g., human MUC16). Human MUC16 includes, for example, amino acid sequences as shown in NCBI accession number GeneID:94025. In some embodiments, MUC16 is human MUC16, cynomolgus monkey MUC16, or mouse MUC16. The MUC16 protein may also include fragments of MUC16, such as extracellular domains, and fragments of extracellular domains, such as fragments that maintain the ability to bind to any antibody disclosed herein, such as SEA-domain 15 or SEA-domain 16.

[0052] As used herein, "antibody that binds to MUC16," "anti-MUC16 antibody," or "antibody that specifically binds to MUC16" refers to an antibody capable of binding to MUC16 or its domains with appropriate affinity. The antibodies and antigen-binding fragments disclosed herein can bind to MUC16 expressed on the cell surface.

[0053] In some respects, the anti-MUC16 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to MUC16 and other target antigens such as CD3, such as bispecific antibodies.

[0054] As used herein, the term "CD3" refers to the antigen expressed on T cells as a part of the multimolecular T cell receptor (TCR), namely the T cell adaptor antigen T cell surface glycoprotein CD3, which is composed of homodimers or heterodimers formed by two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234. In some embodiments, the CD3 described herein refers to CD3 derived from humans or monkeys (e.g., cynomolgus monkeys).

[0055] As used herein, the terms "CD3-binding antibody" or "anti-CD3 antibody" include an antibody and its antigen-binding fragment that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ, or ζ), and an antibody and its antigen-binding fragment that specifically recognize or bind to two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). The antibodies and antigen-binding fragments of this disclosure 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 lacking a transmembrane region or otherwise not binding to the cell membrane. In one embodiment, the antigen-binding region of the CD3-binding fragment or multispecific antibody of this disclosure may have low binding activity to CD3 or CD3-expressing cells (e.g., T cells). The binding affinity of the antibody to CD3 can be detected by flow cytometry or biofilm layer optical interferometry. In some embodiments, the anti-CD3 antibody of this disclosure, or its antigen-binding fragment or bispecific antibody, binds to the antigen-binding region of CD3 with low binding affinity in human and / or monkey (e.g., cynomolgus monkey) CD3, thereby enabling the activation of human and / or monkey (e.g., cynomolgus monkey) T cells. In some aspects, the anti-CD3 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to CD3 and other target antigens.

[0056] General information on the amino acid and nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0057] The multispecific antibodies disclosed herein may include linkers. As used herein, the term "linker" refers to any molecule that enables direct linkage between different portions of a multispecific binding molecule. Examples of linkers that establish covalent linkages between different molecular portions include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of PEG and polypropylene glycol. In some embodiments, the linker is a peptide linker (also referred to as 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, or a hinge region from an immunoglobulin, for linking the amino acid sequence of a first portion of the binding molecule to a second portion of the binding molecule. 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 linking a light chain variable region to a heavy chain variable region. Preferably, the peptide linker has a length sufficient to link two entities in such a way that they maintain their conformation relative to each other without impeding the desired activity. In one embodiment, the linker peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, or 30 amino acids. In one embodiment, the linker peptide comprises the amino acid sequences (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO: 63), (GGGGS)n (SEQ ID NO: 64), (GGGS)n (SEQ ID NO: 65), and (GGGGS)nG (SEQ ID NO: 66), where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, or 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n, where n = 1, 2, 3, or 4, for example, the sequence shown in SEQ ID NO: 39 or 40. In some embodiments, the peptide linker is GSGGGGS, i.e., the sequence shown in SEQ ID NO: 41.

[0058] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, and two different Fc regions can heterodimerize to form a heterodimeric Fc. In this document, the terms “Fc region,” “Fc portion,” and “dimeric Fc (e.g., heterodimeric Fc)” do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, Cys226, or Asp231 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, when referring to the Fc region, it means the Fc region containing a portion of the hinge region, corresponding to the Asp221 extension of the human IgG1 heavy chain Fc region to the carboxyl terminus of the heavy chain.

[0059] In one embodiment, the human IgG1 Fc region polypeptide (including a portion of the hinge region) comprises or is composed of the following amino acid sequence:

[0060] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:45); or

[0061] In one embodiment, the Fc region is a human-derived Fc region. In one embodiment, the Fc region comprises all or part of the human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass. In one embodiment, the Fc region is an Fc region derived from human IgG1, IgG2, IgG3, or IgG4.

[0062] In this article, "heterodimolecular Fc scaffold" refers to a scaffold containing two different Fc regions or formed by dimerization of two different Fc regions, which can be connected to an antigen-binding domain (e.g., the heavy chain and / or light chain variable region of an antibody that can bind to a target molecule, or the antigen-binding fragment of an antibody, or the soluble portion of a ligand or receptor that can bind to a target molecule) at its N-terminus or C-terminus to form a multispecific binding molecule, such as a multispecific antibody, for example, a bispecific antibody.

[0063] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 215 or EU position 220 (including a portion of the hinge region) (EU numbering system). Unless otherwise specified herein, when referring to the CH1 region, it means the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain includes...

[0064] The amino acid sequence of or composed of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:35).

[0065] In this document, specific amino acid residues in the constant regions of antibody IgG are referred to according to the EU numbering system. For example, “S364” refers to serine at EU position 364. Amino acid mutations at specific positions in the constant regions are represented by (original amino acid, amino acid position, mutant amino acid). For example, “S364R” means that serine (S) at EU position 364 is replaced by arginine (R). The above amino acid mutations can also represent mutations in other constant regions corresponding to that position in a specific constant region, and the original amino acid may not be the amino acid shown. For example, “S364R” can also mean that an amino acid (which can be serine or other amino acids) at position 364 of other constant regions of IgG or IgA corresponding to IgG1 is mutated to R. When referring to combinations of mutations, the combined mutations are connected by a plus sign (+), a negative sign (-), or an AND sign. “S364R+D399K”, “S364R-D399K”, or “S364R and D399K” indicate that the Fc region contains both the mutations S364R and D399K. When multiple mutation possibilities exist at a particular position, this is indicated by the symbol " / ". For example, the mutation "K370T / S" means that the K residue at position 370 can be replaced by either a T or an S residue.

[0066] For polypeptide sequences, "conservative alteration" includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence. For example, a conserved substitution often results in an amino acid being replaced by a chemically similar amino acid. Tables of conserved substitutions of functionally similar amino acids are well known in the art. The following lists eight groups of amino acids containing mutually conserved substitutions: 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 "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the antibody molecule or binding protein molecule of this disclosure containing the amino acid sequence. For example, conserved modified variants maintain at least 80%, 85%, 90%, 95%, 98%, 99%, or higher binding affinity to the target antigen relative to the parent antibody or binding protein, such as 100-110% or higher.

[0067] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0068] The term "binding molecule" refers to any molecule that can specifically bind to a target, such as an antibody or its antigen-binding fragment or fusion protein.

[0069] The term "target" refers to the substance to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen that specifically interacts with an antibody molecule. When the binding molecules of this disclosure involve target-binding regions derived from antibodies, "target" and "antigen" may be used interchangeably.

[0070] As used herein, the term "target-binding region" refers to the portion of a binding molecule, such as a multispecific binding molecule or a bispecific binding molecule, that binds to a specific target or antigen. The target-binding region can be, for example, 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 remaining portion of the binding molecule and can bind to its target as a standalone entity or not. The target-binding region can also be a receptor or ligand, or a ligand-binding domain of a receptor. In the case of multispecific antibodies, such as bispecific antibodies, the "target-binding region" is also referred to as the "antigen-binding region."

[0071] As used herein, the term "antigen-binding region" refers to any portion of an antibody or its antigen-binding fragment, such as a multispecific antibody or a bispecific antibody, that binds to a specific target or antigen. An antigen-binding region can be, for example, the antibody or immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remaining portion of the multispecific or bispecific antibody and may bind or not bind its antigen / epitope as a standalone entity. It should be understood that when this disclosure refers to different antigen-binding regions, it can refer to different antigen-binding regions binding different epitopes of the same antigen. When the binding molecules of this disclosure involve target-binding regions derived from antibodies, "target-binding region" and "antigen-binding region" may be used interchangeably.

[0072] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule containing at least a first target binding region and a second target binding region, wherein the first target binding region binds to one target or antigen and the second target binding region binds to another antigen or target. The multispecific binding molecules according to this disclosure also encompass multispecific binding molecules containing multiple target binding regions / binding sites. In some embodiments, the multispecific binding molecules of this disclosure are multispecific antibodies. In some embodiments, the bispecific binding molecules of this disclosure are bispecific antibodies.

[0073] As used herein, the term “monospecific” refers to a polypeptide / protein molecule having one or more target-binding regions, each of which binds to the same site or structure of the same target or the same epitope of the same antigen.

[0074] As used herein, the term "multispecific" binding molecule, such as an antibody, refers to a molecule having at least two target-binding regions, each of which binds to a different target. A multispecific binding molecule is a binding molecule that has binding specificity to at least two different targets. In one embodiment, this document provides such a bispecific binding molecule having binding specificity against a first target and a second target.

[0075] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigens. In one embodiment, this document provides such a bispecific antibody having binding specificity against a first antigen and a second antigen. In this document, when referring to a bispecific antibody, it also encompasses the specific binding of two antigens, but the bispecific antibody against one of the antigens may bind to two different epitopes. In some embodiments, this disclosure provides a bispecific antibody that specifically binds to CD3 and MUC16, specifically binding one or two epitopes of MUC16 and specifically binding one epitope of CD3, also referred to herein as a "MUC16 / CD3 bispecific antibody".

[0076] When "first antigen-binding region" is mentioned in the context of multispecific or bispecific antibodies, it refers to the region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific or bispecific antibody may contain one or more first antigen-binding regions. Similarly, a bispecific antibody may contain both a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0077] When it is mentioned that "the target or antigen-binding region is derived from the antibody", it means that the binding domains constituting the target / antigen-binding region are or derived from the binding domains of the antibody's specific binding antigen. For example, the Fab or scFv of the antigen-binding region are or derived from the corresponding fragment of the antibody, such as Fab, or the heavy chain variable region and / or light chain variable region of the antigen-binding region are or derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen-binding region are the CDRs of the antibody.

[0078] The term "derived from" means that the fragment in the antigen-binding region is substantially identical to the fragment from the antibody from which it originated, but has mutations at one or more sites, such as substitution, deletion, or addition. In one specific embodiment, the mutation is not in the antibody's CDR.

[0079] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0080] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It contains a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0081] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing the immunoglobulin heavy chain variable region (VH), heavy chain constant region (CH1), light chain variable region (VL), and light chain constant region (CL). One polypeptide chain contains VH and a constant region selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain contains VL and another constant region selected from CL and CH1 from the N-terminus to the C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0082] The complementarity-determining region (CDR) or CDR is a region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable region are called HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable region are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: 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)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (www.imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this disclosure, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing methods or combinations thereof. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this disclosure).In some embodiments, the CDRs in the heavy chain variable region and the light chain variable region of the antibody disclosed herein are determined according to Kabat, Chothia, AbM or IMGT or any combination thereof (e.g., a combination of Kabat and Chothia). For example, HCDR1 of the heavy chain variable region is determined according to the AbM scheme, and HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region are determined according to the Kabat scheme.

[0083] An example CDR definition scheme is as follows:

[0084] The following is a calculation of sequence identity between sequences.

[0085] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0086] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while exhibiting low immunogenicity 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 portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portion of the variable region with the corresponding portion of the human antibody).

[0087] As used herein, the terms “anti,” “binding,” or “specific binding” mean that the binding interaction is selective for the target or antigen and can be distinguished from unwanted or nonspecific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0088] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be determined by the dissociation constant (K).D This indicates that the dissociation constant is the dissociation rate constant and the association rate constant (Kdissociation and Kassociation, respectively). dis and K on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0089] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny.

[0090] The terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated 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 is a human.

[0091] The term "treatment" refers to slowing down, interrupting, blocking, relieving, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indicators of a disease, relieving symptoms, or preventing or inhibiting the further development of the disease, condition, or symptom.

[0092] The term "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0093] The term “therapeutic agent” as used herein encompasses any substance that is effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies (such as immune checkpoint molecules), small molecule drugs, or immunomodulators (such as immunosuppressants or agonists).

[0094] The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0095] The term "drug combination" or "drug combination product" refers to a non-fixed or fixed combination. In some embodiments, the drug combination may be in a kit. The term "non-fixed combination" means that the active ingredients (e.g., (i) antibodies of this disclosure, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the components can produce an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0096] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0097] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0098] "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 can be solid tissue, such as fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; body fluid, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid.

[0099] II. Anti-MUC16 antibody

[0100] In some aspects of this disclosure, the present disclosure relates to an antibody that specifically binds to MUC16 or an antigen-binding fragment thereof. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment of the present disclosure comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment of the present disclosure comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-MUC16 antibody or antigen-binding fragment of the present disclosure comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0101] In some aspects, the anti-MUC16 antibody or its antigen-binding fragment disclosed herein contains a heavy chain variable region (VH, also referred to herein as VH). MUC16 In some aspects, the anti-MUC16 antibody or its antigen-binding fragment disclosed herein contains a light chain variable region (VL, also referred to herein as VL). MUC16 In some aspects, the anti-MUC16 antibody or its antigen-binding fragment of the present disclosure 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 (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0102] In some embodiments, the heavy chain variable region VH of the anti-MUC16 antibody or its antigen-binding fragment disclosed herein

[0103] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-26; or

[0104] (ii) Contains or is composed of any of the amino acid sequences shown in SEQ ID NO: 20-26; or

[0105] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any of SEQ ID NO:20-26, preferably, the amino acid alterations do not occur in the CDR region.

[0106] In some embodiments, the light chain variable region VL contained in the anti-MUC16 antibody or its antigen-binding fragment of this disclosure

[0107] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 27-33; or

[0108] (ii) Contains or is composed of the amino acid sequence shown in any one of SEQ ID NO: 27-33; or

[0109] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any of SEQ ID NO:27-33, preferably, the amino acid alterations do not occur in the CDR region.

[0110] In some embodiments, the three complementarity-determining regions (HCDRs) from the heavy chain variable region of the disclosed anti-MUC16 antibody or its antigen-binding fragment, HCDR1, HCDR2, and HCDR3, are...

[0111] (i) the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in any of SEQ ID NO:20-26, or

[0112] (ii) A sequence that, relative to the sequence in (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions.

[0113] The HCDR can be determined according to any scheme for determining the CDR, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof.

[0114] Preferably, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to the Kabat rule.

[0115] In some embodiments, the three complementarity-determining regions (LCDRs) from the light chain variable region of the disclosed anti-MUC16 antibody or its antigen-binding fragment, LCDR1, LCDR2, and LCDR3, are...

[0116] (i) the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any of SEQ ID NO:27-33, or

[0117] (ii) The sequence relative to (i) contains at least one and no more than 5, 4, 3, 2 or 1 amino acid alteration (preferably amino acid substitution, preferably conservative substitution) in the three LCDR regions.

[0118] The LCDR can be determined according to any scheme for determining the CDR, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof.

[0119] Preferably, LCDR1, LCDR2 and LCDR3 are determined according to Kabat.

[0120] In some embodiments, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, 2 or 3, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the HCDR1, or is composed of the amino acid sequence shown in SEQ ID NO:1, 2 or 3.

[0121] In some embodiments, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:4-9, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the HCDR2, or is composed of the amino acid sequence shown.

[0122] In some embodiments, the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:10-12, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the HCDR3, or is composed of the amino acid sequence shown.

[0123] In some embodiments, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:13, 14 or 15, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the LCDR1, or is composed of the amino acid sequence shown in SEQ ID NO:13, 14 or 15.

[0124] In some embodiments, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:16, 17, 18 or 34, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the LCDR2, or is composed of the amino acid sequence shown in SEQ ID NO:16, 17, 18 or 34.

[0125] In some embodiments, the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:19 or 82, or an amino acid sequence having 1, 2 or 3 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to it, or is composed of said amino acid sequence.

[0126] In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of the present disclosure,

[0127] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4 or 5; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0128] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 7; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:82.

[0129] HCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2; HCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6; HCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:14; LCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:34; and / or LCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:82; or

[0130] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:8 or 9; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0131] In some specific embodiments of this disclosure, the anti-MUC16 antibody or its antigen-binding fragment comprises VH and VL, wherein

[0132] The VH contains the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0133] The VH contains the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0134] The VH contains the amino acid sequence shown in SEQ ID NO:20 or 21, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:27 or 28, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0135] The VH contains the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0136] The VH contains the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0137] The VH contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0138] The VH contains the amino acid sequence shown in SEQ ID NO:22, 23, or 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:29, 30, or 31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0139] The VH contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0140] The VH contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0141] The VH contains the amino acid sequence shown in SEQ ID NO:25 or 26, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:32 or 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0142] In some specific embodiments of this disclosure, the anti-MUC16 antibody or its antigen-binding fragment comprises

[0143] (i) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:20, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27;

[0144] (ii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:21, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:28;

[0145] (iii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:22, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:29;

[0146] (iv) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:23, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:30;

[0147] (v) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:24, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:31;

[0148] (vi) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:32;

[0149] (vii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:26, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:33;

[0150] (viii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:20 or 21, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27 or 28;

[0151] (ix). The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:22, 23, or 24, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:29, 30, or 31; or

[0152] (x) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25 or 26, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:32 or 33;

[0153] Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to Kabat; and LCDR1, LCDR2 and LCDR3 are determined by the Kabat scheme.

[0154] In some specific embodiments of this disclosure, the anti-MUC16 antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0155] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4 or 5; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0156] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 7; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:82.

[0157] HCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:2; HCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:6; HCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:14; LCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:34; and / or LCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:82; or

[0158] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:8 or 9; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0159] In some specific embodiments of this disclosure, the anti-MUC16 antibody or its antigen-binding fragment comprises VH and VL, wherein

[0160] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:20, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:27;

[0161] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:28;

[0162] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 20 or 21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 27 or 28.

[0163] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:22, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:29;

[0164] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:30;

[0165] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:31;

[0166] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 22, 23 or 24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 29, 30 or 31;

[0167] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32;

[0168] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:33; or

[0169] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25 or 26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32 or 33.

[0170] In one embodiment of this disclosure, the amino acid alterations described herein include substitutions, insertions, or deletions of amino acids. In a preferred embodiment, the amino acid alterations described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid alterations described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region. Preferably, the amino acid alterations described herein are amino acid substitutions, preferably conservative substitutions.

[0171] In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment thereof disclosed herein is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, or its antigen-binding fragment thereof, for example, an antibody in the form of IgG1 or its antigen-binding fragment thereof.

[0172] In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure further comprises an antibody heavy chain constant region (HC). In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure further comprises an antibody light chain constant region (LC). In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure further comprises both a heavy chain constant region (HC) and a light chain constant region (LC). In some embodiments, the heavy chain constant region of this disclosure comprises an Fc region, such as an Fc region as defined herein.

[0173] In some embodiments, the heavy chain constant region comprising the anti-MUC16 antibody or its antigen-binding fragment is a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, for example, the heavy chain constant region of human IgG1.

[0174] In some embodiments, the heavy chain constant region applicable to the anti-MUC16 antibody or its antigen-binding fragment of this disclosure is...

[0175] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43;

[0176] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:43; or

[0177] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:43.

[0178] In some embodiments, the heavy chain constant region further includes a mutation that reduces binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in this disclosure has a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation.

[0179] In some embodiments, the heavy chain constant region of the anti-MUC16 antibody or its antigen-binding fragment applicable to this disclosure contains an L234A / L235A mutation, and

[0180] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44;

[0181] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:44; or

[0182] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:44.

[0183] In some embodiments, the light chain constant region comprised of the anti-MUC16 antibody or its antigen-binding fragment is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the light chain constant region is the Kappa light chain constant region or the Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the light chain constant region is the human Kappa light chain constant region.

[0184] In some implementations, the light chain constant region

[0185] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 38 or 81;

[0186] (ii) Containing or consisting of an amino acid sequence of SEQ ID NO: 38 or 81; or

[0187] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 38 or 81.

[0188] In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure comprises an antibody heavy chain. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure comprises an antibody light chain. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure comprises both the heavy chain and the light chain. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment of this disclosure comprises two of the heavy chains and two of the light chains. In some embodiments, the heavy chain comprises the heavy chain variable region and the heavy chain constant region, or is composed of the heavy chain variable region and the heavy chain constant region. In some embodiments, the light chain comprises the light chain variable region and the light chain constant region, or is composed of the light chain variable region and the light chain constant region.

[0189] In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment disclosed herein has one or more of the following characteristics:

[0190] (i) Demonstrates the same or similar binding affinity and / or specificity to MUC16 as any of the anti-MUC16 antibodies listed in the Examples;

[0191] (ii) Inhibit (e.g., competitively inhibit) the binding of any of the anti-MUC16 antibodies listed in the examples to MUC16;

[0192] (iii) Epitopes that bind to the same or overlapping epitopes as any of the anti-MUC16 antibodies listed in the examples;

[0193] (iv) Compete with any of the anti-MUC16 antibodies listed in the examples to bind to MUC16;

[0194] (v) Has one or more biological characteristics of any of the anti-MUC16 antibodies listed in the examples.

[0195] In some implementations, the anti-MUC16 antibody is a monoclonal antibody.

[0196] In some embodiments, the anti-MUC16 antibody is humanized. Humanization can be achieved by replacing one or more amino acid residues, particularly the framework region sequence, in the variable regions of the heavy and light chains of a non-human natural antibody with residues at corresponding positions in the variable regions of a conventional human antibody. Methods for humanizing antibodies are well known in the art. Typically, humanization substitutions are performed in a manner that preserves the favorable binding properties of the antibody. Assays for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art for identifying and selecting suitable mutations or combinations of humanized residues.

[0197] In one embodiment, the anti-MUC16 antibody of this disclosure is a full-length antibody. In one embodiment, the anti-MUC16 antibody of this disclosure also encompasses its antibody fragment (e.g., 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), heavy-chain antibody, or linear antibody. In one embodiment, the anti-MUC16 antibody fragment of this disclosure is Fab, comprising VH and VL as described herein, and CH1 and CL.

[0198] In one embodiment, the anti-MUC16 antibody disclosed herein may also be a bispecific antibody or a multispecific antibody that specifically binds to MUC16, as well as one or more other antigens (e.g., CD3).

[0199] III. Multispecific binding molecules

[0200] In one aspect of this disclosure, therein lies a multispecific binding molecule capable of specifically binding to MUC16 and specifically binding to one or more other targets or antigens. In some embodiments, the other antigen is CD3. In some embodiments, the multispecific binding molecule is a multispecific antibody, such as a bispecific antibody.

[0201] In some embodiments, the antibodies of this disclosure are bispecific antibodies. The term "bispecific antibody" refers to an antibody comprising at least two different antigen-binding regions that specifically bind to two antigens. Therefore, the bispecific antibodies according to this disclosure possess specificity for two different antigens. For example, in some embodiments, the bispecific antibodies of this disclosure may comprise a first antigen-binding region that specifically binds to a first antigen and a second antigen-binding region that specifically binds to a second antigen.

[0202] Bispecific antibody forms include IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8:130). The most common IgG-like antibody type contains two Fab regions and two Fc regions, the heavy and light chains of each Fab may be derived from separate monoclonal antibodies. In some embodiments, in the bispecific antibody, the two Fc regions may be the same or different, preferably different, for example as defined herein; and / or the two Fab light chains may each contain different types of light chain constant regions, for example, one containing a Lambda light chain constant region and the other containing a Kappa light chain constant region. The bispecific antibodies of this disclosure can be prepared using bispecific antibody forms or techniques known in the art. For specific exemplary bispecific forms that may be used in the context of this disclosure, see, for example, Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8):1-24.

[0203] In some embodiments, the IgG-like multispecific antibody of this disclosure, such as a bispecific antibody, refers to a multispecific antibody, such as a bispecific antibody, that comprises an Fc dimer (e.g., a heterodimeric Fc scaffold comprising a first Fc region and a second Fc region). Therefore, in some embodiments, the multispecific antibody of this disclosure comprises an Fc dimer, such as a heterodimeric Fc scaffold composed of distinct first and second Fc regions. In some embodiments, the antibody of this disclosure is a bispecific antibody, preferably an IgG-like antibody comprising two Fc regions.

[0204] Therefore, one aspect of this disclosure relates to a multispecific antibody, such as a bispecific antibody, comprising one or more antigen-binding regions that specifically bind to MUC16 and one or more antigen-binding regions that specifically bind to CD3.

[0205] In some embodiments, this disclosure relates to a bispecific antibody comprising two identical MUC16 antigen-binding regions that specifically bind to the same epitope of MUC16, and an antigen-binding region that specifically binds to CD3.

[0206] In some embodiments, this disclosure relates to a bispecific antibody comprising two distinct MUC16 antigen-binding regions that specifically bind to different epitopes of MUC16, and an antigen-binding region that specifically binds to CD3.

[0207] The antigen-binding region of the multispecific antibody applicable to this disclosure that specifically binds to MUC16 may contain or be composed of the full-length anti-MUC16 antibody of this disclosure or its antigen-binding fragment, as long as it can specifically bind to MUC16, including but not limited to, full-length antibodies, half antibodies, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies that specifically bind to MUC16.

[0208] In some implementations, different antigen-binding regions or antigen-binding regions and Fc regions can be directly connected or connected via a connector.

[0209] This disclosure describes the various components / domains for multispecific binding molecules, such as multispecific antibodies. Those skilled in the art will understand that, unless the context explicitly indicates otherwise, any combination of any technical features of the various components for multispecific binding molecules, such as multispecific antibodies, described herein is within the scope of this disclosure. Furthermore, those skilled in the art will understand that, unless the context explicitly indicates otherwise, the antibodies (including antibodies of any form) disclosed herein may comprise any such combination.

[0210] III-1 specifically binds to the antigen-binding region of MUC16.

[0211] In some implementations, the antigen-binding region that specifically binds to MUC16 is derived from the anti-MUC16 antibody described herein or its antigen-binding fragment, such as the Fab of the anti-MUC16 antibody described herein.

[0212] In some embodiments, the antigen-binding region specifically binding to MUC16 comprises 1, 2, 3, 4, 5, or 6 CDRs of the anti-MUC16 antibody described herein. In some embodiments, the antigen-binding region specifically binding to MUC16 comprises 1, 2, or 3 heavy chain variable region CDRs of the anti-MUC16 antibody described herein, namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region specifically binding to MUC16 comprises 1, 2, or 3 light chain variable region CDRs of the anti-MUC16 antibody described herein, namely LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region specifically binding to MUC16 comprises 3 heavy chain variable region CDRs and 3 light chain variable region CDRs of the anti-MUC16 antibody described herein. In some embodiments, the antigen-binding region specifically binding to MUC16 comprises the heavy chain variable region of the anti-MUC16 antibody described herein. In some embodiments, the antigen-binding region specifically binding to MUC16 comprises the light chain variable region of the anti-MUC16 antibody described herein. In some embodiments, the antigen-binding region that specifically binds to MUC16 comprises the heavy chain variable region and the light chain variable region of the anti-MUC16 antibody described herein, and particularly a combination of the heavy chain variable region and the light chain variable region.

[0213] In some embodiments, the antigen-binding region that specifically binds to MUC16 comprises or is the Fab (Fab group) of the anti-MUC16 antibody described herein. MUC16 ).

[0214] In some implementations, the Fab fragment consists of the VH(VH) MUC16 CH1 of the heavy chain constant region, and the VL (VL) MUC16 The Fab chain consists of two polypeptide chains, VH and CH1, and a light chain constant region CL domain, wherein VH pairs with VL and CH1 pairs with CL to form an antigen-binding region. In some embodiments, in the Fab chain, one chain comprises VH and CH1 (i.e., VH-CH1) or consists of VH and CH1 from the N-terminus to the C-terminus (where the C-terminus of VH is connected to the N-terminus of CH1), and the other chain comprises VL and CL (i.e., VL-CL) or consists of VL and CL from the N-terminus to the C-terminus (where the C-terminus of VL is connected to the N-terminus of CL). In this document, the Fab chain comprising VH-CH1 or consisting of VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain comprising VL-CL or consisting of VL-CL is also referred to as the Fab light chain.

[0215] Therefore, in some embodiments, the antigen-binding region that specifically binds to MUC16 in this disclosure is a Fab that specifically binds to MUC16, said Fab comprising or consisting of the following:

[0216] Fab heavy chain: from N end to C end: VH MUC16 -CH1, where VH MUC16 The C-terminus of CH1 is directly connected to the N-terminus of CH1;

[0217] Fab Light Chain: From N-end to C-end: VL MUC16 -CL, where VL MUC16 The C-terminus of CL is directly connected to the N-terminus of CL;

[0218] Where "-" represents a direct connection, VH MUC16 and VL MUC16 These are VH and VL of antibodies that specifically bind to MUC16, as defined in this article.

[0219] In some embodiments, CH1 is derived from IgG, such as CH1 of (human) IgG1, IgG2, IgG3, or IgG4, preferably CH1 of human IgG1. In some embodiments, CH1

[0220] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:35;

[0221] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:35; or

[0222] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:35.

[0223] In some embodiments, the CL is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the CL is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the CL is a human Kappa light chain constant region. In some embodiments, the CL...

[0224] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 38 or 81;

[0225] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:38 or 81; or

[0226] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 38 or 81.

[0227] III-2 specifically binds to the antigen-binding region of CD3.

[0228] The antigen-binding region of the multispecific antibody applicable to this disclosure that specifically binds to CD3 may contain or be composed of an anti-CD3 antibody or its antigen-binding fragment, as long as it can specifically bind to CD3, including but not limited to, full-length antibodies, haptens, Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy chain antibodies, or linear antibodies, etc.

[0229] In some embodiments, the CD3-specific antigen-binding region comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the CD3-specific antigen-binding region comprises three complementarity-determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the CD3-specific antigen-binding region comprises three HCDRs from the heavy chain variable region and three LCDRs from the light chain variable region.

[0230] In some respects, the antigen-binding region that specifically binds to CD3 includes the heavy chain variable region (VH, also referred to herein as "VH"). CD3 In some respects, the antigen-binding region that specifically binds to CD3 contains a light chain variable region (VL, also referred to herein as "VL"). CD3In some aspects, the antigen-binding region that specifically binds to CD3 includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to Kabat, and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0231] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809A1 (incorporated herein in its entirety), for example, sp34.24 therein. In some embodiments, the antigen-binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD3 (e.g., the CD3 antibody disclosed in WO2022068809A1, for example, sp34.24 therein). In some embodiments, the antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs of a known antibody that specifically binds to CD3 (e.g., the CD3 antibody disclosed in WO2022068809A1, for example, sp34.24 therein), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region comprises one, two, or three light chain variable regions (CDRs) of a known CD3-specific antibody (e.g., the CD3 antibody disclosed in WO2022068809A1, such as sp34.24 therein), namely LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region comprises three heavy chain variable regions and three light chain variable regions of a known CD3-specific antibody (e.g., the CD3 antibody disclosed in WO2022068809A1, such as sp34.24 therein). In some embodiments, the antigen-binding region comprises a heavy chain variable region and a light chain variable region of a known CD3-specific antibody (e.g., the CD3 antibody disclosed in WO2022068809A1, such as sp34.24 therein), particularly a combination of the heavy chain variable regions and light chain variable regions.

[0232] In some embodiments, the heavy chain variable region VH included in the antigen-binding region that specifically binds to CD3

[0233] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:53; or

[0234] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:53; or

[0235] (iii) An amino acid sequence comprising or consisting of the amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:53, preferably, the amino acid changes do not occur in the CDR region.

[0236] In some embodiments, the light chain variable region VL included in the antigen-binding region that specifically binds to CD3

[0237] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:54; or

[0238] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:54; or

[0239] (iii) An amino acid sequence comprising or consisting of the amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:54, preferably, the amino acid changes do not occur in the CDR region.

[0240] In some embodiments, the three complementary determinant regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to CD3, HCDR1, HCDR2, and HCDR3, are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:53. Preferably, the HCDRs can be determined according to any scheme for determining CDRs, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat.

[0241] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to CD3, LCDR1, LCDR2, and LCDR3, are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO:54. Preferably, the LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, LCDR1, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0242] In some embodiments, the antigen-binding region that specifically binds to CD3 in this disclosure,

[0243] HCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:47; HCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:48; HCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:49; LCDR1 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:50; LCDR2 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:51; and / or LCDR3 contains, or is composed of, the amino acid sequence shown in SEQ ID NO:52.

[0244] In some specific embodiments of this disclosure, the antigen-binding region that specifically binds to CD3 includes VH and VL, wherein

[0245] The VH contains the amino acid sequence shown in SEQ ID NO:53 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:54 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0246] In some specific embodiments of this disclosure, the antigen-binding region that specifically binds to CD3 includes the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:53, and the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:54. Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to Kabat; and LCDR, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0247] In some specific embodiments of this disclosure, the antigen-binding region that specifically binds to CD3 includes: HCDR1 as shown in SEQ ID NO:47, HCDR2 as shown in SEQ ID NO:48, HCDR3 as shown in SEQ ID NO:49; LCDR1 as shown in SEQ ID NO:50, LCDR2 as shown in SEQ ID NO:51, and LCDR3 as shown in SEQ ID NO:52.

[0248] In some specific embodiments of this disclosure, the CD3-specific antigen-binding region comprises VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:53, and the VL contains the amino acid sequence shown in SEQ ID NO:54. In some specific embodiments of this disclosure, the CD3-specific antigen-binding region comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:53, and the VL consists of the amino acid sequence shown in SEQ ID NO:54.

[0249] In one embodiment, the antigen-binding region that specifically binds to CD3 is an antigen-binding fragment of an anti-CD3 antibody, selected from antibody fragments such as Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies. Preferably, the antigen-binding region that specifically binds to CD3 is scFv.

[0250] In some embodiments, the scFv fragment comprises a polypeptide chain containing VH and VL domains, wherein the VH and VL are linked (e.g., via a linker) to pair and form an antigen-binding site. In some embodiments, the scFv is in a trans configuration and comprises or consists of VH, a linker, and VL (VH-linker-VL) from the N-terminus to the C-terminus. In other embodiments, the scFv is in a cis configuration and comprises or consists of VL, a linker, and VH (VL-linker-VH) from the N-terminus to the C-terminus. In some embodiments, the linker is a peptide linker consisting of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acid long, for example 5-30 amino acid long, for example 15 or 20 amino acid long. In one embodiment, the linker comprises an amino acid sequence (G4S)n, where n = 1, 2, 3, 4, or 5, preferably n = 3 or 4, more preferably n = 4. In one embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 64 or 40. In some preferred embodiments, the scFv is a disulfide-stabilized scFv.

[0251] In some embodiments, the CD3-specific antigen-binding region included in the multispecific antibody of this disclosure is a CD3-specific scFv. In some embodiments, the CD3-specific scFv fragment included in the multispecific antibody of this disclosure is derived from an anti-CD3 antibody.

[0252] In some embodiments, the CD3-specific antigen-binding region of this disclosure is scFv, which comprises or consists of the following from the N-terminus to the C-terminus: VH CD3 -VL CD3 Where "-" represents a connector or direct connection, and VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via a connector (e.g., connector 2).

[0253] Therefore, in some embodiments, the CD3-specific scFv of this disclosure comprises the amino acid sequence of SEQ ID NO:62, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0254] In some embodiments, the antigen-binding region that specifically binds to CD3 in this disclosure is Fab, the structure of which and the definitions of CH1 and CL can be found in reference to the Fab that specifically binds to MUC16.

[0255] III-3Fc area

[0256] In some embodiments, one or more target-binding regions (antigen-binding regions) of the multispecific binding molecules of this disclosure, such as multispecific antibodies (e.g., bispecific antibodies), further include Fc regions, wherein the included Fc regions may be the same or different.

[0257] In some implementations, the first Fc region and the second Fc region are different, and they are capable of dimerizing to form a heterodimeric Fc scaffold.

[0258] In this document, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass a wide range of 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). In some embodiments, the Fc region of this disclosure comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at its N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region, such as the sequences D221 to P230 according to EU numbers. Mutations may be contained in said hinge region. Unless otherwise specified, when referring to the Fc region in this article, it means the Fc region with a portion of the IgG hinge region (corresponding to the EU numbers D221 to P230 of IgG1) at the N-terminus.

[0259] Unless otherwise noted herein, the amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0260] In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, IgG3, or IgG4, or their subtypes, such as human IgG1, IgG2, IgG3, or IgG4, or their subtypes. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from a human. In some embodiments, the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of an amino acid sequence SEQ ID NO:45 or 46 or an amino acid sequence having at least 90% identity with it, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0261] The Fc region of the binding molecules disclosed herein, such as antibodies, can be mutated to obtain the desired properties. Mutations of the Fc region are known in the art.

[0262] As those skilled in the art will understand, in order to promote the formation of heterodimers in the multispecific antibodies of this disclosure, the Fc regions comprising the multispecific antibodies of this disclosure may contain mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0263] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of the first and second Fc regions are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementary engineered CH3 regions (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). For example, based on the Knob-into-Hole technique, corresponding Knob mutations and Hole mutations are introduced into the first and second Fc regions. This technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).

[0264] Alternatively, based on Innobody technology, corresponding mutations can be introduced into the first and second Fc regions. This technology is illustrated, for example, by PCT / CN2021 / 143141 (the entire contents of which are incorporated herein by reference).

[0265] In a particular implementation scheme

[0266] The first CH3 region contains the S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region contains the S364R mutation, and the second CH3 region contains the K370S mutation. In some embodiments, the first CH3 region contains the S364R mutation, and the second CH3 region contains the K370S mutation.

[0267] In some embodiments, the first CH3 region contains the S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V (preferably K370S) mutation and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region contains S364R / K+D399K / R, and the second CH3 region contains K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region contains S364R+D399K, and the second CH3 region contains K370S+Y349T. In some embodiments, the first CH3 region further contains E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further includes K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0268] In some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D. In some embodiments, the first CH3 region further includes E357N. In some embodiments, the second CH3 region further includes Q347D. In some embodiments, the first CH3 region further includes E357N, and the second CH3 region further includes Q347D. In some embodiments, the first CH3 region and the second CH3 region further each include T350V, or both include T350V.

[0269] Therefore, in some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region includes S364R+D399K+E357N, and the second CH3 region includes K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region includes S364R+D399K+E357N+T350V, and the second CH3 region includes K370S+Y349T+K409D+Q347D+T350V.

[0270] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D+T411R / K, and the second CH3 region comprises D399K / R+K370T / S / A / V. In some implementations, the CH3 region includes K409E+T411R, and the second CH3 region includes D399K+K370T.

[0271] In some specific implementations, the first and second CH3 regions have the following five mutation combinations:

[0272] In one implementation, CH3 in one Fc region contains S364R and D399K mutations, and CH3 in another Fc region contains Y349T, K370S, and K409D mutations.

[0273] The CH3 mutation location described herein refers to the human IgG1 Fc region, for example, the amino acid sequence shown in SEQ ID NO:45 or 46.

[0274] In some implementations, the Fc region also contains other mutations that facilitate heterodimer purification, such as mutations introduced based on the Duobody platform, for example, containing the K409R mutation in one Fc region and the F405L mutation in another Fc region.

[0275] As those skilled in the art will understand, the Fc region can be modified in terms of its effector function (e.g., complement activation function of the Fc region, such as ADCC, ADCP, or CDC effector function) according to the intended use of the molecules described herein, such as antibody molecules. In one embodiment, the effector function has been reduced or eliminated relative to the wild-type Fc region. In one embodiment, the effector function is reduced or eliminated by using a naturally occurring Fc isotype with reduced or eliminated effector function and / or Fc region modification. In a preferred embodiment, the Fc region has reduced Fc region-mediated effector function, such as reduced or eliminated ADCC, ADCP, or CDC effector function, for example, by including mutations that achieve the above-described function.

[0276] In some embodiments, the binding molecules of this disclosure, such as antibody molecules, may also include modifications in the Fc region that alter the binding affinity for one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region contains mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in this disclosure has mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation. In yet another preferred embodiment, the Fc fragment may have mutations that result in an increased serum half-life, such as mutations that improve the binding of the Fc fragment to FcRn.

[0277] Therefore, in a specific embodiment, the multispecific antibody, such as a bispecific antibody, disclosed herein comprises a first Fc region and a second Fc region, wherein the two Fc regions are heterodimerized, wherein

[0278] One Fc-region polypeptide contains the mutants S364R and D399K, while the other Fc-region polypeptide contains the mutants Y349T, K370S, and K409D;

[0279] Optionally, the Fc region may also contain L234A / L235A mutations.

[0280] Therefore, in a specific embodiment, the multispecific antibody of this disclosure, such as a bispecific antibody, comprises a first Fc region and a second Fc region, wherein the two Fc regions are heterodimerized, one Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:36, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:37.

[0281] In one specific embodiment, the multispecific antibody, such as the bispecific antibody, disclosed herein comprises two Fc regions (a first Fc region and a second Fc region), wherein the two Fc regions are heterodimerized, one Fc region comprises an amino acid sequence having at least 85%, 91%, 92%, 93%, 94%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:36, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:37.

[0282] Therefore, in a specific embodiment, the multispecific antibody, such as a bispecific antibody, disclosed herein comprises two Fc regions (a first Fc region and a second Fc region), wherein the two Fc regions are heterodimerized, wherein

[0283] a) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36 and contains the mutations S364R and D399K, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37 and contains the mutations Y349T, K370S, and K409D; or

[0284] b) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36 and contains the mutations S364R and D399K and the L234A / L235A mutation, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37 and contains the mutations Y349T, K370S, and K409D and the L234A / L235A mutation.

[0285] III-4 Exemplary Multispecific Antibody Structures and Sequences

[0286] In some embodiments, the multispecific antibody of this disclosure, such as a bispecific antibody, comprises two antigen-binding regions specifically binding to MUC16 as described herein and one antigen-binding region specifically binding to CD3 as described herein, wherein the two antigen-binding regions specifically binding to MUC16 may bind the same epitope or different epitopes, for example, different epitopes. In some embodiments, the first antigen-binding region specifically binding to MUC16 is the same as or different from the second antigen-binding region specifically binding to MUC16. In some embodiments, the first antigen-binding region specifically binding to MUC16 is different from the second antigen-binding region specifically binding to MUC16 and binds to different epitopes on MUC16. In some embodiments, the first antigen-binding region specifically binding to MUC16 is different from the second antigen-binding region specifically binding to MUC16 and binds to different domains on MUC16, for example, binding to Domain SEA16 and Domain SEA 15, respectively.

[0287] In some implementations, the antigen-binding region that specifically binds to MUC16 is the Fab (Fab group) that specifically binds to MUC16. MUC16 In some implementations, the antigen-binding region that specifically binds to CD3 is the CD3-specific scFv (scFv). CD3 In some implementations, the antigen-binding region that specifically binds to MUC16 is the Fab (Fab group) that specifically binds to MUC16. MUC16 Furthermore, the antigen-binding region that specifically binds to CD3 is the scFv that specifically binds to CD3 (scFv CD3 In some implementations, the first Fab MUC16 The Fab heavy chain is connected directly or via a connector (preferably directly) to the N end of the first Fc region at its CH1 C end, and the second Fab... MUC16 The Fab heavy chain has scFv at the C-terminus of its CH1. CD3 The N-terminus (e.g., the N-terminus of its VH) is connected directly or via a connector (preferably via a connector (connector 1), such as the connector shown in SEQ ID NO:39), and scFv CD3 The C-terminus (e.g., the C-terminus of its VL) is connected directly or via a connector (preferably via a connector (connector 3), such as the connector shown in SEQ ID NO:41) to the N-terminus of the second Fc region. In some embodiments, the first Fab MUC16 With the second Fab MUC16 Same or different. In some implementations, the first Fab MUC16 With the second Fab MUC16 They differ, and are combined with different epitopes on the MUC16. In some implementations, the first Fab... MUC16With the second Fab MUC16 The same, and respectively combined with Domain SEA16 or Domain SEA 15. First Fab MUC16 With the second Fab MUC16 Different, and combined with different structural domains on MUC16, such as combining Domain SEA16 and Domain SEA 15 respectively, for example, the first Fab MUC16 Specifically binds to Domain SEA16 and the second Fab MUC16 Specific Domain SEA 15; or First Fab MUC16 Specifically binds to Domain SEA15 and the second Fab MUC16 Specific Domain SEA 16.

[0288] In some embodiments, the multispecific antibody is a bispecific antibody in which two antigen-binding regions that specifically bind to MUC16 bind to the same MUC16 epitope, preferably the two antigen-binding regions that specifically bind to MUC16 are identical.

[0289] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to different antigen-binding regions of MUC16 and binds to different epitopes or different domains of MUC16.

[0290] In one specific implementation, the multispecific antibody is an IgG-like antibody having the configuration shown in Figure 5 or 6.

[0291] In one specific implementation, the first Fc region and the second Fc region are as defined above, for example, containing innobody mutations, and optionally mutations that reduce the function of effectors mediated by the Fc region or reduce binding to the Fcγ receptor.

[0292] In one embodiment, the multispecific antibody comprises or is composed of the following:

[0293] Heavy chain 1: From the N end to the C end, it includes or consists of the following: First VH MUC16 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0294] Light chain 1: From the N-terminus to the C-terminus, it includes or consists of the following: First VL MUC16 -First CL;

[0295] Heavy chain 2: From the N end to the C end, it includes or consists of the following: Second VH MUC16 -Second CH1-scFv CD3-Second Fc region, where second CH1 and scFv CD3 The N-terminus is connected directly or via connector 1, scFv CD3 The C end is connected directly to the second Fc region or via connector 3;

[0296] Light chain 2: Composed of the following components from the N-terminus to the C-terminus: second VL MUC16 -Second CL;

[0297] Where “-” represents a connector or direct connection;

[0298] First and / or second VH MUC16 It is the heavy chain variable region VH, first and / or second VL of the anti-MUC16 antibody as described in this article. MUC16 It is the light chain variable region (VL) of the anti-MUCH16 antibody as described in this article;

[0299] The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively.

[0300] The first CL and the second CL refer to the constant regions of the light chain, respectively.

[0301] scFv CD3 It is the antigen-binding region scFv that specifically binds to CD3, as described in this article.

[0302] Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different.

[0303] In some embodiments, the first and second VH of the multispecific antibody MUC16 The same and the first and second VL MUC16 The same. In some embodiments, the first VH of the multispecific antibody is... MUC16 - First CH1 and First VL MUC16 - The first CL constitutes the first Fab, which is the antigen-binding region that specifically binds to MUC16. MUC16 , and / or the second VH MUC16 -Second CH1 and Second VL MUC16 - The second CL constitutes the second Fab, which serves as the antigen-binding region specifically binding to MUC16. MUC16 In some embodiments, the first and second Fabs of the multispecific antibody MUC16 One or both are Fab fragments of the anti-MUC16 antibody as described herein. In some embodiments, the first and second Fab fragments of the multispecific antibody... MUC16 They can be the same or different. In some embodiments, the first and second Fabs of the multispecific antibody... MUC16They can be different. In some embodiments, the first and second Fabs of the multispecific antibody... MUC16 They can bind to the same epitope, and preferably the same. In some embodiments, the first and second Fabs of the multispecific antibody... MUC16 Different epitopes or different structural domains of MUC16 can be combined with different components. In some implementations, the first Fab... MUC16 With the second Fab MUC16 The same, and respectively combined with Domain SEA16 or Domain SEA 15. First Fab MUC16 With the second Fab MUC16 Different, and combined with different structural domains on MUC16, such as combining Domain SEA16 and Domain SEA 15 respectively, for example, the first Fab MUC16 Specifically binds to Domain SEA16 and the second Fab MUC16 Specific Domain SEA 15; or First Fab MUC16 Specifically binds to Domain SEA15 and the second Fab MUC16 Specific Domain SEA 16.

[0304] In some embodiments, the first Fc region and the second Fc region of the multispecific antibody are as described herein, for example, as defined in Section III-3. In some embodiments, the first Fc region of the multispecific antibody contains S364R and D399K and the second Fc region contains the mutant Y349T, K370S, and K409D mutations or vice versa; optionally, the first Fc region and the second Fc region also contain the L234A / L235A mutations, respectively.

[0305] In some embodiments, the CH1 and CL of the multispecific antibody are as described herein, for example as defined in Section III-2.

[0306] In some implementations, the second Fab MUC16 Fab heavy chain and scFv CD3 The linker is connected via a linker 1, which may be 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 5-50 amino acid long, for example 5-30 amino acid long, for example 15 or 20 amino acid long. In one embodiment, the linker 1 comprises the amino acid sequence (G4S)n, where n = 1, 2, 3, 4 or 5, preferably n = 2. In one embodiment, the linker 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 64 or 39.

[0307] In some implementations, scFv CD3 The Fc region is connected via a linker 3, wherein the linker 3 may be 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 5-50 amino acid long, for example 5-30 amino acid long, such as 15 or 20 amino acid long. In one embodiment, the linker 3 comprises or consists of the amino acid sequence shown in SEQ ID NO:41.

[0308] In some embodiments, the adapter 1 and adapter 3 of the multispecific antibody are (GGGGS). n Where n = 1, 2, 3, 4, or 5, for example, the amino acid sequence of linker 1 is shown in SEQ ID NO: 39, and / or the amino acid sequence of linker 3 is shown in SEQ ID NO: 41. In some embodiments, the amino acid sequence of linker 2 of the multispecific antibody is shown in SEQ ID NO: 40. In some embodiments, the amino acid sequences of linkers 1, 2, and 3 of the multispecific antibody are shown in SEQ ID NO: 39, 40, and 41, respectively.

[0309] In some specific embodiments, the multispecific antibody comprises or is composed of the following:

[0310] Heavy chain 1: From the N end to the C end, it includes or consists of the following: First VH MUC16 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0311] Light chain 1: From the N-terminus to the C-terminus, it includes or consists of the following: First VL MUC16 -First CL;

[0312] Heavy chain 2: From the N end to the C end, it includes or consists of the following: Second VH MUC16 -Second CH1-scFv CD3 -Second Fc region, where second CH1 and scFv CD3 The N-terminus is connected directly or via connector 1, scFv CD3 The C end is connected directly to the second Fc region or via connector 3;

[0313] Light chain 2: Composed of the following components from the N-terminus to the C-terminus: second VL MUC16 -Second CL;

[0314] Where “-” represents a connector or direct connection;

[0315] First and / or second VH MUC16It is the heavy chain variable region VH, first and / or second VL of the anti-MUC16 antibody as described in this article. MUC16 It is the light chain variable region (VL) of the anti-MUCH16 antibody as described in this article;

[0316] The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively.

[0317] The first CL and the second CL refer to the constant regions of the light chain, respectively.

[0318] First VH MUC16 - First CH1 and First VL MUC16 - The first CL constitutes the first Fab, which is the antigen-binding region that specifically binds to MUC16. MUC16 ;

[0319] Second VH MUC16 -Second CH1 and Second VL MUC16 - The second CL constitutes the second Fab, which serves as the antigen-binding region specifically binding to MUC16. MUC16 ;

[0320] scFv CD3 It is the antigen-binding region scFv that specifically binds to CD3, as described in this article, which is composed of VH CD3 VL CD3 Directly or via connector 2, preferably comprising or consisting of the following from the N end to the C end: VH CD3 -VL CD3 , of which VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via connector 2;

[0321] Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different;

[0322] The respective structural domains are as defined herein; preferably,

[0323] The first Fc region and the second Fc region are as described herein, for example, as defined in Section III-3. In some embodiments, the first Fc region of the multispecific antibody contains S364R and D399K and the second Fc region contains the mutants Y349T, K370S, and K409D, or vice versa; optionally, the first Fc region and the second Fc region also contain the L234A / L235A mutations, respectively.

[0324] The amino acid sequences of linkers 1, 2 and 3 of the multispecific antibody are shown in SEQ ID NO:39, 40 and 41, respectively.

[0325] The Fab MUC16 As defined in Section III-1 and the scFv CD3 As defined in Section III-2.

[0326] In some specific embodiments, the multispecific antibody that specifically binds to MUC16 and CD3 comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, or is composed of said chains, wherein

[0327] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55 or 61, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0328] Light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56 or 60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0329] Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 57 or 59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and / or

[0330] Light chain 2 comprises the amino acid sequence shown in SEQ ID NO:58 or 60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0331] In some specific embodiments, the bispecific antibody that specifically binds to MUC16 and CD3 comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, or is composed of said chains, wherein

[0332] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises 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 with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:57. The amino acid sequence shown in NO:58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence;

[0333] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:59. The amino acid sequence shown in NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence; or

[0334] Heavy chain 1 comprises the amino acid sequence shown in 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 with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in 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 with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:60. The amino acid sequence shown in NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

[0335] In some specific embodiments, the bispecific antibody that specifically binds to MUC16 and CD3 comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, or is composed of said chains, wherein

[0336] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:55; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:56; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:57; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:58.

[0337] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:55; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:56; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:59; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60; or

[0338] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:61; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:59; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60.

[0339] IV. Anti-CD3 antibody or its antigen-binding fragment

[0340] In some embodiments, this disclosure also relates to an anti-CD3 antibody or an antigen-binding fragment thereof, which includes an antigen-binding region that specifically binds to CD3 as defined above.

[0341] In some specific embodiments of this disclosure, the anti-CD3 antibody or its antigen-binding fragment comprises VH and VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:53, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:54.

[0342] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment thereof disclosed herein is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, or its antigen-binding fragment thereof, for example, an antibody in the form of IgG1 or its antigen-binding fragment thereof.

[0343] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of this disclosure further comprises an antibody heavy chain constant region (HC). In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of this disclosure further comprises an antibody light chain constant region (LC). In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of this disclosure further comprises both a heavy chain constant region (HC) and a light chain constant region (LC). In some embodiments, the heavy chain constant region of this disclosure comprises an Fc region, such as an Fc region as defined herein.

[0344] In some embodiments, the heavy chain constant region of the anti-CD3 antibody or its antigen-binding fragment disclosed herein is a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, for example, the heavy chain constant region of human IgG1.

[0345] In some implementations, the heavy chain constant region

[0346] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 or 44;

[0347] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:43 or 44; or

[0348] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:43 or 44.

[0349] In some embodiments, the light chain constant region of the anti-CD3 antibody of this disclosure is the light chain constant region as defined herein. In some embodiments, the light chain constant region is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the light chain constant region is the Kappa light chain constant region or the Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the light chain constant region is the human Kappa light chain constant region.

[0350] In some implementations, the light chain constant region

[0351] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 38 or 81;

[0352] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:38 or 81; or

[0353] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 38 or 81.

[0354] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present disclosure comprises an antibody heavy chain. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present disclosure comprises an antibody light chain. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present disclosure comprises both a heavy chain and a light chain. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present disclosure comprises two heavy chains and two light chains. In some embodiments, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, or is composed of a heavy chain variable region and a heavy chain constant region. In some embodiments, the light chain comprises a light chain variable region and a light chain constant region, or is composed of a light chain variable region and a light chain constant region.

[0355] In some implementations, the anti-CD3 antibody is a monoclonal antibody.

[0356] In some implementations, the anti-CD3 antibody is humanized.

[0357] In one embodiment, the anti-CD3 antibody of this disclosure is a full-length antibody. In another embodiment, the anti-CD3 antibody of this disclosure also encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy-chain antibodies, or linear antibodies.

[0358] In one embodiment, the anti-CD3 antibody fragment of this disclosure is a single-chain antibody comprising an anti-CD3 antibody fragment scFv and a heavy chain constant region Fc (optionally the Fc region includes a hinge region or a partial hinge region), or is composed of scFv and a heavy chain constant region Fc (optionally the Fc region includes a hinge region or a partial hinge region).

[0359] In one embodiment, the anti-CD3 antibody disclosed herein may also be a bispecific antibody or a multispecific antibody that specifically binds to CD3, as well as one or more other antigens.

[0360] IV. Polynucleotides, vectors, and host cells

[0361] This disclosure provides nucleic acids encoding any of the antibody molecules described above (e.g., the anti-MUC16 antibody of this disclosure or its antigen-binding fragment, or multispecific antibodies such as bispecific antibodies).

[0362] In one respect, this disclosure provides nucleic acid molecules that contain or are composed of polynucleotides encoding or being composed of any of the above-mentioned anti-MUC16 antibodies or their antigen-binding fragments or multispecific antibodies such as bispecific antibodies.

[0363] To facilitate production and purification, anti-MUC16 antibodies or their antigen-binding fragments or multispecific antibodies such as bispecific antibodies can be fused to the N-terminus or C-terminus (e.g., C-terminus) with a secretory signal peptide and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label.

[0364] As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0365] In some embodiments, the nucleic acid of this disclosure comprises a polynucleotide encoding an amino acid sequence selected from any one of SEQ ID NO:20-31, 32, 35, 37, 44-47, or a polynucleotide encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:20-31, 32, 35, 37, 44-47, or is composed of said polynucleotide.

[0366] The nucleic acid molecules encoding the molecules disclosed herein can be produced using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0367] In one aspect, this disclosure also provides vectors comprising the nucleic acid molecules of this disclosure. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.1 or pCDNA3.4.

[0368] In one aspect, this disclosure also provides a host cell comprising the nucleic acid molecule or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells, such as (HEK 293 or 293F cells or 293FT cells or Expi293 cells)). In yet another embodiment, the host cell is prokaryotic.

[0369] Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can also be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, and myeloma cell lines such as YO, NSO, P3X63, and Sp2 / 0. Suitable mammalian host cell lines for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO, such as an ExpiCHO cell, or a 293 cell, such as a HEK293 cell, a 293FT cell, or an Expi293 cell.

[0370] V. Production and purification of the molecules disclosed herein

[0371] In another aspect, this disclosure provides a method for producing antibody molecules of the present disclosure (e.g., anti-MUC16 antibody or its antigen-binding fragment or multispecific antibody such as bispecific antibody), the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing a polypeptide chain of the antibody molecule; optionally further comprising assembling the polypeptide chain to produce the molecule under conditions suitable for assembling the polypeptide chain into the antibody molecule.

[0372] For recombinant production, a polynucleotide encoding the polypeptide chain of the disclosed antibody molecule can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the disclosed invention has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0373] The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0374] The purity of the antibody molecules disclosed herein can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0375] VI. Determination Method

[0376] The antibody molecules (e.g., anti-MUC16 antibodies or their antigen-binding fragments, or multispecific antibodies such as bispecific antibodies) provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. Examples of methods for determining the properties of the disclosed anti-MUC16 antibodies or their antigen-binding fragments, or bispecific antibodies, such as thin-layer interferometry (BLI), T-cell reporter gene assays, T-cell activation assays, T-cell mediated killing assays, or antitumor activity assays, are provided.

[0377] VII. Immunoconjugates

[0378] In one aspect, this disclosure provides an immunoconjugate comprising an antibody molecule of this disclosure (e.g., an anti-MUC16 antibody or its antigen-binding fragment or a multispecific antibody such as a bispecific antibody) and one or more other active ingredients (e.g., an active ingredient derived from a medicament or therapeutic agent for treating the disease of this disclosure, such as a small molecule that enhances the therapeutic effect of the molecule of this disclosure).

[0379] In some implementations, the immune conjugate is an antibody-drug conjugate (ADC).

[0380] VIII. Drugs and Uses

[0381] In one aspect, this disclosure also relates to compositions (e.g., pharmaceutical compositions) comprising molecules of the disclosure (e.g., anti-MUC16 antibody or its antigen-binding fragment, or multispecific antibodies such as bispecific antibodies, or immunoconjugates). In some embodiments, the pharmaceutical composition may be a formulation.

[0382] In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. In one embodiment, the composition, such as a pharmaceutical composition, comprises molecules of the present disclosure and a combination of one or more other therapeutic agents.

[0383] The compositions disclosed herein may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used herein, “pharmaceutical carrier” or “pharmaceutical excipient” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, or absorption delay agents. For information on the use and applications of pharmaceutical excipients, see “Handbook of Pharmaceutical Excipients”, 8th edition, R.C. Rowe, P.J. Seskey and S.C. Wen, Pharmaceutical Press, London, Chicago.

[0384] In some embodiments, the composition may be in the form of a formulation; for example, the compositions disclosed herein may be formulations in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0385] A pharmaceutical product comprising the molecules described herein can be prepared, for example in the form of a lyophilized formulation or an aqueous solution, by mixing molecules of the present disclosure having the desired purity (e.g., anti-MUC16 antibody or its antigen-binding fragment or multispecific antibody such as a bispecific antibody or its immunoconjugate) with one or more optional pharmaceutical excipients.

[0386] In one aspect, this disclosure also provides pharmaceutical combinations or pharmaceutical combination products comprising molecules of the disclosure (e.g., anti-MUC16 antibody or its antigen-binding fragment or multispecific antibody such as bispecific antibody or its immunoconjugate), and one or more other therapeutic agents, etc.

[0387] Another aspect of this disclosure is to provide a kit containing the drug combination of this disclosure, preferably in the form of drug dosage units. This allows the dosage units to be provided according to a dosing regimen or drug administration interval.

[0388] In one embodiment, the kit of this disclosure comprises, within the same package:

[0389] - A first container containing a pharmaceutical composition comprising molecules of the present disclosure (e.g., an anti-MUC16 antibody or its antigen-binding fragment, or a multispecific antibody such as a bispecific antibody or its immunoconjugate).

[0390] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0391] In some embodiments, when the molecules of this disclosure (e.g., the anti-MUC16 antibody of this disclosure or its antigen-binding fragment or bispecific antibody or its immunoconjugate) are used to treat tumors, the therapeutic agent is, for example, a variety of therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0392] In one aspect, this disclosure provides methods for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a molecule of this disclosure (e.g., an anti-MUC16 antibody of this disclosure or an antigen-binding fragment thereof, or a multispecific antibody such as a bispecific antibody or an immunoconjugate thereof), a pharmaceutical composition, a combination of drugs, or a kit. In some embodiments, the disease is, for example, an oncology such as cancer.

[0393] In some embodiments, this disclosure relates to molecules of this disclosure (e.g., anti-MUC16 antibody of this disclosure or its antigen-binding fragment or multispecific antibody such as bispecific antibody or its immunoconjugate), pharmaceutical compositions, pharmaceutical combinations or kits for use in therapies, such as for treating tumors such as cancer.

[0394] In some embodiments, this disclosure relates to methods of treating diseases, such as tumors, like cancer, using molecules of this disclosure (e.g., anti-MUC16 antibodies of this disclosure or antigen-binding fragments thereof or multispecific antibodies such as bispecific antibodies or their immunoconjugates), pharmaceutical compositions, drug combinations, or kits.

[0395] In some embodiments, this disclosure relates to the use of the molecules of this disclosure (e.g., the anti-MUC16 antibody of this disclosure or its antigen-binding fragment or multispecific antibody such as a bispecific antibody or its immunoconjugate), pharmaceutical compositions, pharmaceutical combinations or kits for the treatment of diseases such as tumors such as cancer.

[0396] In some embodiments, this disclosure relates to the use of the molecules of this disclosure (e.g., the anti-MUC16 antibody of this disclosure or its antigen-binding fragment or multispecific antibody such as a bispecific antibody or its immunoconjugate), pharmaceutical compositions, pharmaceutical combinations or kits for the preparation of a medicament for the treatment of a disease, such as an oncology, such as cancer.

[0397] In some implementations, the disease is a MUC16-related disease, such as a tumor like cancer.

[0398] In some embodiments, the tumor is a solid tumor or a hematologic malignancy, as well as a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in the early, intermediate, or late stage, or metastatic stage. In some embodiments, the tumor is immune-evading.

[0399] In some embodiments, the tumor is a MUC16-positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or aberrant activity of MUC16.

[0400] In some embodiments, a MUC16-positive tumor or cancer refers to tumor cells expressing MUC16 in a subject suffering from any tumor or cancer. In some embodiments, the subject's tumor cells express MUC16, for example, at low, moderate, or high levels. In some embodiments, the subject possesses (e.g., elevated levels, such as nucleic acid or protein levels or activity) of MUC16 (e.g., compared to healthy subjects). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) contains (e.g., elevated levels, such as nucleic acid or protein levels or activity) of MUC16 (e.g., compared to biological samples from healthy subjects (e.g., corresponding tissues or cells in healthy subjects), or compared to MUC16 in adjacent healthy tissues or cells of the subject).

[0401] In some implementations, the tumor is ovarian cancer or pancreatic cancer, such as MUC16-positive pancreatic cancer or ovarian cancer.

[0402] Depending on their therapeutic use, the antibodies or antigen-binding fragments or immunoconjugates or pharmaceutical compositions described herein may also be administered in combination with one or more other therapies, such as modes of treatment and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0403] In some embodiments, when the molecules of this disclosure (e.g., the anti-MUC16 antibody of this disclosure or its antigen-binding fragment or bispecific antibody or its immunoconjugate) are used to treat tumors, the other therapeutic agents are, for example, various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0404] In some embodiments, when the molecules of this disclosure (e.g., the anti-MUC16 antibody of this disclosure or its antigen-binding fragment or bispecific antibody or its immunoconjugate) are used to treat tumors, the treatment methods include surgery; radiotherapy, local irradiation or focused irradiation, etc.

[0405] In other respects, this disclosure provides for the use of the molecules of this disclosure (e.g., anti-MUC16 antibodies or their antigen-binding fragments or bispecific antibodies or their immunoconjugates, etc.) or compositions or combination products comprising them in the manufacture or preparation of medicaments for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0406] In other respects, this disclosure also provides molecules of this disclosure (e.g., anti-MUC16 antibodies or their antigen-binding fragments or bispecific antibodies or their immunoconjugates), or compositions or pharmaceuticals or formulations or combination products containing them, for use in therapies, such as for treating the related diseases or conditions mentioned herein.

[0407] IX. Diagnosis and Detection

[0408] In some embodiments, the molecules provided herein (e.g., anti-MUC16 antibodies or their antigen-binding fragments or bispecific antibodies or their immunoconjugates) can be used to detect the presence of MUC16 in biological samples.

[0409] When used herein, the term “detection” includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR).

[0410] In some embodiments, the method includes contacting a biological sample with a molecule as described herein (e.g., an anti-MUC16 antibody or its antigen-binding fragment, or a bispecific antibody or its immunoconjugate, etc.) under conditions allowing it to bind to MUC16, and detecting whether a complex is formed between the molecule and MUC16, wherein the formation of the complex indicates the presence of MUC16. This method can be in vitro or in vivo. In one embodiment, the molecules of this disclosure are used to select subjects suitable for treatment with molecules of this disclosure (e.g., an anti-MUC16 antibody or its antigen-binding fragment, or a bispecific antibody or its immunoconjugate, etc.), for example, where MUC16 is a biomarker for selecting the subject.

[0411] In some embodiments, labeled molecules of the present disclosure are provided (e.g., anti-MUC16 antibodies or antigen-binding fragments thereof, or bispecific antibodies or immunoconjugates thereof, etc.). Labeling includes, but is not limited to, labels or portions that are directly detected (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0412] In some embodiments provided herein, the sample is obtained prior to treatment with a molecule of this disclosure (e.g., an anti-MUC16 antibody or its antigen-binding fragment, or a bispecific antibody or its immunoconjugate, etc.) or a composition, pharmaceutical, formulation, or combination product comprising thereof. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0413] In some implementations, MUC16 is detected before treatment, for example, before the start of treatment or before a treatment after a treatment interval.

[0414] In some embodiments, a method for treating the disease of this disclosure is provided, the method comprising: testing a subject (e.g., a sample) for the presence of MUC16, thereby determining a MUC16 value; comparing the MUC16 value with a control value (e.g., a value in a normal individual); and if the MUC16 value is greater than the control value, administering to the subject a therapeutically effective amount of the molecule of this disclosure (e.g., an anti-MUC16 antibody or its antigen-binding fragment or a bispecific antibody or its immunoconjugate, etc.) or a composition, drug, or preparation containing therein, optionally in combination with one or more other therapies, thereby treating the disease.

[0415] X. Specific Implementation Plan

[0416] Implementation scheme A1. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:20, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27.

[0417] Implementation scheme A2. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:21, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:28.

[0418] Implementation scheme A3. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:22, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:29.

[0419] Implementation scheme A4. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:23, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:30.

[0420] Implementation scheme A5. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:24, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:31.

[0421] Implementation scheme A6. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:32.

[0422] Implementation scheme A7. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:26, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:33.

[0423] Implementation scheme A8. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:20 or 21, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27 or 28.

[0424] Implementation scheme A9. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:22 or 23 or 24, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:29 or 30 or 31.

[0425] Implementation Scheme A10. Anti-MUC16 antibody or its antigen-binding fragment, comprising the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25 or 26, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:32 or 33.

[0426] Implementation Scheme A11. An anti-MUC16 antibody or its antigen-binding fragment, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:1; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:4 or 5; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:16; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0427] Implementation Scheme A12. An anti-MUC16 antibody or its antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:2; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6 or 7; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:17; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:82.

[0428] Implementation Scheme A13. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:2; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:82.

[0429] Implementation Scheme A14. An anti-MUC16 antibody or its antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:8 or 9; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:18; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0430] Implementation Scheme A15. The antibody or antigen-binding fragment thereof of any one of Implementation Schemes A1-A14, comprising a heavy chain variable region VH, wherein the heavy chain variable region

[0431] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-26; or

[0432] (ii) Contains or consists of the amino acid sequence shown in any one of SEQ ID NO:20-26.

[0433] Implementation Scheme A16. The antibody or antigen-binding fragment thereof of any one of Implementation Schemes A1-A15, comprising a light chain variable region VL, wherein the light chain variable region

[0434] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 27-33; or

[0435] (ii) Contains or consists of the amino acid sequence shown in any one of SEQ ID NO:27-33.

[0436] Implementation Scheme A17. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0437] Implementation Scheme A18. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0438] Implementation Scheme A19. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 20 or 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 27 or 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0439] Implementation Scheme A20. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0440] Implementation Scheme A21. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0441] Implementation Scheme A22. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0442] Implementation Scheme A23. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 22 or 23 or 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 29 or 30 or 31 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0443] Implementation Scheme A24. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0444] Implementation Scheme A25. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0445] Implementation Scheme A26. An antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A16, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO: 25 or 26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO: 32 or 33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0446] Implementation Scheme A27. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A26, comprising a heavy chain variable region VH and a light chain variable region VL, wherein

[0447] (i) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:20, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:27;

[0448] (ii). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:28;

[0449] (iii) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 20 or 21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 27 or 28;

[0450] (iv) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:22, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:29;

[0451] (v) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:30;

[0452] (vi) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:31;

[0453] (vii). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 22, 23 or 24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 29, 30 or 31;

[0454] (viii) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32;

[0455] (ix). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:33; or

[0456] (x) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25 or 26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32 or 33.

[0457] Implementation Scheme A28. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:20, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:27.

[0458] Implementation Scheme A29. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:28.

[0459] Implementation Scheme A30. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 20 or 21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 27 or 28.

[0460] Implementation Scheme A31. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:22, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:29.

[0461] Implementation Scheme A32. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:30.

[0462] Implementation scheme A33. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:31.

[0463] Implementation Scheme A34. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 22 or 23 or 24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 29 or 30 or 31.

[0464] Implementation scheme A35. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32.

[0465] Implementation Scheme A36. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:33.

[0466] Implementation Scheme A37. An anti-MUC16 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 25 or 26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 32 or 33.

[0467] Implementation Scheme A38. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A37 further comprises a heavy chain constant region, for example, the antibody heavy chain constant region is a heavy chain constant region derived from IgG1, IgG2, IgG3 or IgG4 (e.g., human IgG1, IgG2, IgG3 or IgG4), preferably a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0468] Implementation Scheme A39. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A38 further comprises a heavy chain constant region, wherein the antibody heavy chain constant region is the heavy chain constant region of human IgG1.

[0469] Implementation Scheme A40. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A39 further comprises a heavy chain constant region, wherein the heavy chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43; preferably, it comprises or is composed of an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO:43.

[0470] Implementation Scheme A41. An antibody or antigen-binding fragment thereof of any one of Implementation Schemes A38-A40, wherein the heavy chain constant region comprises a mutation that reduces binding to the Fcγ receptor, such as an LALA mutation, for example, comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44; preferably, comprising or consisting of the amino acid sequence shown in SEQ ID NO:44.

[0471] Implementation Scheme A42. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A41 comprises a light chain constant region, for example, the light chain constant region being a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region).

[0472] Implementation scheme A43. The antibody or antigen-binding fragment thereof described in any one of implementation schemes A1-A41, comprising a light chain constant region, wherein the light chain constant region is the human Lambda light chain constant region.

[0473] Implementation Scheme A44. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A41, comprising a light chain constant region, wherein the light chain constant region is a human Kappa light chain constant region.

[0474] Implementation Scheme A45. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes A1-A41, comprising a light chain constant region, wherein the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38; preferably, comprising or is composed of the amino acid sequence shown in SEQ ID NO:38.

[0475] Implementation Scheme A46. An antibody or antigen-binding fragment thereof according to any one of Implementation Schemes A1-A41, comprising a light chain constant region, wherein the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:81; preferably, comprising or is composed of the amino acid sequence shown in SEQ ID NO:81.

[0476] Implementation scheme A47. The antibody or antigen-binding fragment thereof as described in any one of implementation schemes A1-A46, wherein the antibody is a humanized antibody.

[0477] Implementation scheme A48. The antibody or antigen-binding fragment thereof as described in any one of implementation schemes A1-A46, wherein the antibody is a chimeric antibody.

[0478] Implementation scheme A49. The antibody or antigen-binding fragment thereof described in any one of implementation schemes A1-A48, wherein the antibody is a monoclonal antibody.

[0479] Implementation Scheme A50. The antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A49, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody) or linear antibody.

[0480] Implementation Scheme A51. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes A1-A49, wherein the antigen-binding fragment is a Fab fragment.

[0481] Implementation scheme A52. An antibody or antigen-binding fragment thereof of any one of implementation schemes A-A51, wherein the antibody is a multispecific antibody, such as a bispecific antibody.

[0482] Implementation scheme A53. The antibody or antigen-binding fragment thereof described in implementation scheme A52, wherein the antibody also specifically binds to CD3.

[0483] Implementation scheme B1. A multispecific antibody comprising an antigen-binding region that specifically binds to MUC16 and an antigen-binding region that specifically binds to CD3.

[0484] Implementation Scheme B2. The multispecific antibody described in Implementation Scheme B1 comprises two antigen-binding regions that specifically bind to MUC16 and one antigen-binding region that specifically binds to CD3.

[0485] Implementation Scheme B3. The multispecific antibody described in Implementation Scheme B2, wherein the two antigen-binding regions that specifically bind to MUC16 are the same or different.

[0486] Implementation Scheme B4. The multispecific antibody of any one of Implementation Schemes B1-B3, wherein the two antigen-binding regions that specifically bind to MUC16 respectively include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as defined in any one of Implementation Schemes A1-A14.

[0487] Implementation Scheme B5. The multispecific antibody of any one of Implementation Schemes B1-B4, wherein the two antigen-binding regions that specifically bind to MUC16 respectively comprise VH and VL, wherein VH comprises HCDR1, HCDR2, and HCDR3 as defined in any one of Implementation Schemes A1-A14, and VL comprises LCDR1, LCDR2, and LCDR3 as defined in any one of Implementation Schemes A1-A14.

[0488] Implementation Scheme B6. The multispecific antibody of any one of Implementation Schemes B1-B5, wherein the two antigen-binding regions that specifically bind to MUC16 respectively comprise VH and VL, and the VH comprises VH as defined in Implementation Scheme A15, and the VL comprises VL as defined in Implementation Scheme A16.

[0489] Implementation Scheme B7. The multispecific antibody of any one of Implementation Schemes B1-B6, wherein the two antigen-binding regions that specifically bind to MUC16 respectively include VH and VL as defined in any one of Implementation Schemes A17-A37.

[0490] Implementation Scheme B8. The multispecific antibody of any one of Implementation Schemes B1-B7, wherein the multispecific antibody is a bispecific antibody.

[0491] Implementation Scheme B9. The multispecific antibody of any one of Implementation Schemes B1-B8, wherein the two antigen-binding regions that specifically bind to MUC16 are Fab, respectively.

[0492] Implementation Scheme B10. The multispecific antibody of any one of Implementation Schemes B1-B8, wherein the two antigen-binding regions that specifically bind to MUC16 are respectively the Fab regions of the anti-MUC16 antibody as defined in Implementation Scheme A51.

[0493] Implementation Scheme B11. The multispecific antibody of Implementation Scheme B9 or B10, wherein the Fab of the anti-MUC16 antibody comprises CH1, wherein the CH1 is CH1 derived from human IgG1, IgG2, IgG3 or IgG4.

[0494] Implementation Scheme B12. The multispecific antibody of Implementation Scheme B9 or B10, wherein the Fab of the anti-MUC16 antibody comprises CH1, wherein the CH1 is the CH1 of human IgG1.

[0495] Implementation Scheme B13. The multispecific antibody of Implementation Scheme B11 or B12, wherein CH1 comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:35, preferably comprising or being composed of the amino acid sequence shown in SEQ ID NO:35.

[0496] Implementation Scheme B14. The multispecific antibody of any one of Implementation Schemes B9-B13, wherein the Fab of the anti-MUC16 antibody comprises a light chain constant region, wherein the light chain constant region is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region).

[0497] Implementation Scheme B15. The multispecific antibody of any one of Implementation Schemes B9-B14, wherein the Fab of the anti-MUC16 antibody comprises a light chain constant region, wherein the light chain constant region is the human Lambda light chain constant region.

[0498] Implementation Scheme B16. The multispecific antibody of Implementation Scheme B15, wherein the Fab of the anti-MUC16 antibody comprises a light chain constant region, wherein the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:81; preferably, it comprises or is composed of the amino acid sequence shown in SEQ ID NO:81.

[0499] Implementation Scheme B17. The multispecific antibody of any one of Implementation Schemes B9-B14, wherein the Fab of the anti-MUC16 antibody comprises a light chain constant region, wherein the light chain constant region is a human Kappa light chain constant region.

[0500] Implementation Scheme B18. The multispecific antibody of Implementation Scheme B17, wherein the Fab of the anti-MUC16 antibody comprises a light chain constant region, wherein the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38; preferably, it comprises or is composed of the amino acid sequence shown in SEQ ID NO:38.

[0501] Implementation Scheme B19. The multispecific antibody according to any one of Implementation Schemes B1-B18, wherein the antigen-binding region specifically binding to CD3 includes the three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:53, and the three complementary determinant regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:54.

[0502] Implementation Scheme B20. The multispecific antibody according to any one of Implementation Schemes B1-B19, wherein the antigen-binding region specifically binding to CD3 comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0503] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:47; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:48; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:49; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:50; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:51; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:52.

[0504] Implementation Scheme B21. The multispecific antibody of any one of Implementation Schemes B1-B20, wherein the antigen-binding region specifically binding to CD3 comprises VH and VL, wherein VH comprises or is composed of the sequence shown in SEQ ID NO:53; and VL comprises or is composed of the sequence shown in SEQ ID NO:54.

[0505] Implementation Scheme B22. The multispecific antibody of any one of Implementation Schemes B1-B21, wherein the antigen-binding region that specifically binds to CD3 is scFv.

[0506] Implementation Scheme B23. The multispecific antibody of Implementation Scheme B22, wherein the scFv comprises or consists of the following from the N-terminus to the C-terminus: VH CD3 -VL CD3 Where "-" represents a connector or direct connection, and VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via a connector, preferably via a connector.

[0507] Implementation Scheme B24. The multispecific antibody described in Implementation Scheme B23, wherein the linker is (GGGGS). n , where n = 1, 2, 3, 4 or 5, for example n = 3 or 4, for example containing or consisting of the amino acid sequence shown in SEQ ID NO: 40.

[0508] Implementation Scheme B25. The multispecific antibody of any one of Implementation Schemes B22-B24, wherein the scFv comprises the amino acid sequence shown in SEQ ID NO:62, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0509] Implementation Scheme B26. The multispecific antibody of any one of Implementation Schemes B1-B25, wherein the multispecific antibody is an IgG-like multispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different.

[0510] Implementation Scheme B27. The multispecific antibody described in Implementation Scheme B26, wherein the Fc region is derived from IgG1, IgG2, IgG3 or IgG4, for example, the Fc region of human IgG1, IgG2, IgG3 or IgG4.

[0511] Implementation scheme B28. The multispecific antibody described in implementation scheme B26 or B27, wherein the Fc region is or is derived from the Fc region of human IgG1.

[0512] Implementation Scheme B29. The multispecific antibody according to any one of Implementation Schemes B26-B28, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO:45; preferably, it comprises or is composed of the amino acid sequence shown in SEQ ID NO:45.

[0513] Implementation Scheme B30. The multispecific antibody according to any one of Implementation Schemes B26-B28, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO:46; preferably, it comprises or is composed of the amino acid sequence shown in SEQ ID NO:46.

[0514] Implementation scheme B31. The multispecific antibody of any one of implementation schemes B26-B30, wherein the two Fc regions are different, wherein based on Innobody technology, mutations are introduced into the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region.

[0515] Implementation Scheme B32. The multispecific antibody described in Implementation Scheme B31, wherein the CH3 of one Fc region contains S364R and D399K mutations, and the CH3 mutation of the other Fc region contains Y349T, K370S and K409D mutations.

[0516] Implementation Scheme B33. The multispecific antibody according to any one of Implementation Schemes B26-B32, wherein the first and / or second Fc regions respectively contain mutations that reduce binding to the Fcγ receptor, such as L234A / L235A mutations.

[0517] Implementation Scheme B34. The multispecific antibody according to any one of Implementation Schemes B26-B32, wherein one Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:36, and the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:37.

[0518] Implementation Scheme B35. The multispecific antibody according to any one of Implementation Schemes B26-B32, wherein one Fc region comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36 and comprises the mutations S364R and D399K, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37 and comprises the mutations Y349T, K370S, and K409D; or

[0519] Implementation Scheme B36. The multispecific antibody according to any one of Implementation Schemes B26-B32, wherein one Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36 and contains the mutations S364R and D399K and L234A / L235A mutations, and the other Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37 and contains the mutations Y349T, K370S, and K409D and L234A / L235A mutations.

[0520] Implementation Scheme B37. The multispecific antibody according to any one of Implementation Schemes B1-B36, wherein the antigen-binding region that specifically binds to MUC16 is the Fab (Fab group) that specifically binds to MUC16. MUC16 Furthermore, the antigen-binding region that specifically binds to CD3 is the scFv that specifically binds to CD3 (scFv CD3 );

[0521] The multispecific antibody contains a first Fab MUC16Second Fab MUC16 ,and

[0522] First Fab MUC16 The Fab heavy chain is connected directly or via a connector (preferably directly) to the N end of the first Fc region at its CH1 C end, and the second Fab... MUC16 The Fab heavy chain has scFv at the C-terminus of its CH1. CD3 The N-terminus (e.g., the N-terminus of its VH) is connected directly or via a connector (preferably via a connector, such as the connector shown in SEQ ID NO:39), and scFv CD3 The C-terminus (e.g., the C-terminus of its VL) is connected directly or via a connector (preferably via a connector, such as the connector shown in SEQ ID NO:41) to the N-terminus of the second Fc region.

[0523] Implementation Scheme B38. The multispecific antibody described in Implementation Scheme B37, wherein the first Fab MUC16 With the second Fab MUC16 Combining the same epitopes or the same structural domains on MUC16, preferably, the first Fab MUC16 With the second Fab MUC16 same.

[0524] Implementation Scheme B39. The multispecific antibody described in Implementation Scheme B37, wherein the first Fab MUC16 With the second Fab MUC16 Combining different epitopes or different structural domains on MUC16; preferably, the first Fab MUC16 With the second Fab MUC16 Different and combined different epitopes on MUC16 or different structural domains on MUC16.

[0525] Implementation Scheme B40. The multispecific antibody according to any one of Implementation Schemes B1-B39, comprising or consisting of:

[0526] Heavy chain 1: From the N end to the C end, it includes or consists of the following: First VH MUC16 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected directly or via a connector to the N end of the first Fc region at its C end;

[0527] Light chain 1: From the N-terminus to the C-terminus, it includes or consists of the following: First VL MUC16 -First CL;

[0528] Heavy chain 2: From the N end to the C end, it includes or consists of the following: Second VH MUC16 -Second CH1-scFv CD3 -Second Fc region, where second CH1 and scFv CD3The N-terminus is connected directly or via connector 1, scFv CD3 The C end is connected directly to the second Fc region or via connector 3;

[0529] Light chain 2: Composed of the following components from the N-terminus to the C-terminus: second VL MUC16 -Second CL;

[0530] Where “-” represents a connector or direct connection;

[0531] Among them, the first VH MUC16 - First CH1 and First VL MUC16 -The first CL constitutes the first Fab that specifically binds to MUC16. MUC16 ,

[0532] Second VH MUC16 -Second CH1 and Second VL MUC16 -The second CL constitutes the second Fab that specifically binds to MUC16. MUC16 ;

[0533] First VH MUC16 And the first VL MUC16 Each includes HCDR1, CDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined in any one of embodiments A1-A14; and

[0534] Second VH MUC16 Second VL MUC16 Each includes HCDR1, CDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as defined in any of the embodiments A1-A14;

[0535] scFv CD3 It is scFv as defined in any of the implementation schemes B22-B25;

[0536] The first CH1 and the second CH1 are CH1 as defined in any one of embodiments B11-B13, and optionally the first CH1 and the second CH1 are the same, for example, both are or are CH1 derived from human IgG1;

[0537] The first CL and the second CL are light chain constant regions as defined by any of B14-B18, and optionally the first CL and the second CL are the same, for example, both are or come from the Kappa light chain constant region.

[0538] The first Fc region and the second Fc region are different and are respectively Fc regions as defined in any of the implementation schemes B26-B36;

[0539] Optionally, connector 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:64 or 39; and / or connector 3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:41.

[0540] Implementation Scheme B41. The multispecific antibody described in Implementation Scheme B40, wherein the first VH MUC16 It comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:2, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:7, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; the first VL MUC16 It comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:14, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:17 and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:82.

[0541] Implementation Scheme B42. The multispecific antibody described in Implementation Scheme B40 or B41, wherein the first VH MUC16 Containing or consisting of the amino acid sequence shown in SEQ ID NO:24, the first VL MUC16 It contains or consists of the amino acid sequence shown in SEQ ID NO:31.

[0542] Implementation Scheme B43. The multispecific antibody described in Implementation Scheme B40, wherein the first VH MUC16 It comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:9, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; the first VL MUC16 It comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18 and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0543] Implementation Scheme B44. The multispecific antibody described in Implementation Scheme B40 or B43, wherein the first VH MUC16 Containing or consisting of the amino acid sequence shown in SEQ ID NO:26, the first VLMUC16 It contains or consists of the amino acid sequence shown in SEQ ID NO:33.

[0544] Implementation Scheme B45. The multispecific antibody described in Implementation Scheme B40, wherein the first VH MUC16 It comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; the first VL MUC16 It comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16 and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0545] Implementation Scheme B46. The multispecific antibody described in Implementation Scheme B40 or B45, wherein the first VH MUC16 Containing or consisting of the amino acid sequence shown in SEQ ID NO:21, the first VL MUC16 It contains or consists of the amino acid sequence shown in SEQ ID NO:28.

[0546] Implementation Scheme B47. The multispecific antibody of any one of Implementation Schemes B40-B46, wherein the second VH MUC16 It comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:2, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:7, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; the second VL MUC16 It comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:14, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:17 and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:82.

[0547] Implementation Scheme B48. The multispecific antibody of any one of Implementation Schemes B40-B47, wherein the second VH MUC16 Containing or consisting of the amino acid sequence shown in SEQ ID NO:24, the second VL MUC16It contains or consists of the amino acid sequence shown in SEQ ID NO:31.

[0548] Implementation Scheme B49. The multispecific antibody of any one of Implementation Schemes B40-B46, wherein the second VH MUC16 It comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:9, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; the second VL MUC16 It comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18 and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0549] Implementation scheme B50. The multispecific antibody of any one of implementation schemes B40-B46 and B49, wherein the second VH MUC16 Containing or consisting of the amino acid sequence shown in SEQ ID NO:26, the second VL MUC16 It contains or consists of the amino acid sequence shown in SEQ ID NO:33.

[0550] Implementation Scheme B51. The multispecific antibody described in any one of Implementation Schemes B40-B46, wherein the second VH MUC16 It comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:10; the second VL MUC16 It comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16 and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:19.

[0551] Implementation scheme B52. The multispecific antibody of any one of implementation schemes B40-B46 and B51, wherein the second VH MUC16 Containing or consisting of the amino acid sequence shown in SEQ ID NO:21, the second VL MUC16It contains or consists of the amino acid sequence shown in SEQ ID NO:28.

[0552] Implementation Scheme B53. The multispecific antibody according to any one of Implementation Schemes B40-B52, wherein the antigen-binding region specifically binding to CD3 comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein

[0553] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:47; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:48; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:49; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:50; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:51; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:52.

[0554] Implementation Scheme B54. The multispecific antibody according to any one of Implementation Schemes B40-B53, wherein the antigen-binding region specifically binding to CD3 comprises VH and VL, wherein VH comprises or is composed of the sequence shown in SEQ ID NO:53; and VL comprises or is composed of the sequence shown in SEQ ID NO:54.

[0555] Implementation Scheme B55. The multispecific antibody described in Implementation Schemes B40-B54, wherein

[0556] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises 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 with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:57. The amino acid sequence shown in NO:58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

[0557] Implementation Scheme B56. The multispecific antibody described in Implementation Schemes B40-B54, wherein

[0558] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:59. The amino acid sequence shown in NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence; or

[0559] Implementation Scheme B57. The multispecific antibody described in Implementation Schemes B40-B54, wherein

[0560] Heavy chain 1 comprises the amino acid sequence shown in 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 with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in 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 with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:60. The amino acid sequence shown in NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

[0561] Implementation Scheme B58. The multispecific antibody described in Implementation Scheme B40, wherein...

[0562] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:55; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:56; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:57; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:58.

[0563] Implementation Scheme B59. The multispecific antibody described in Implementation Scheme B40, wherein...

[0564] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:55; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:56; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:59; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60; or

[0565] Implementation Scheme B60. The multispecific antibody described in Implementation Scheme B40, wherein...

[0566] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:61; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:59; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60.

[0567] Implementation Scheme C1. An anti-CD3 antibody or an antigen-binding fragment thereof, comprising VH and VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:53, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:54.

[0568] Implementation Scheme C2. The anti-CD3 antibody or its antigen-binding fragment described in Implementation Scheme C1 further comprises a heavy chain constant region and / or a light chain constant region, preferably,

[0569] The heavy chain constant region is a constant region derived from IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1 or human IgG4; and / or the light chain constant region is derived from the Kappa light chain constant region or the Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambd light chain constant region.

[0570] Implementation Scheme C3. The antibody or antigen-binding fragment thereof described in Implementation Scheme C1 or C2 further comprises a heavy chain constant region, for example, the antibody heavy chain constant region is a heavy chain constant region from IgG1, IgG2, IgG3 or IgG4 (e.g., human IgG1, IgG2, IgG3 or IgG4), preferably a heavy chain constant region from human IgG1, IgG2, IgG3 or IgG4.

[0571] Implementation Scheme C4. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes C1-C3 further comprises a heavy chain constant region, wherein the antibody heavy chain constant region is the heavy chain constant region of human IgG1.

[0572] Implementation Scheme C5. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes C1-C4 further comprises a heavy chain constant region, wherein the heavy chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43; preferably, it comprises or is composed of an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO:43.

[0573] Implementation Scheme C6. An antibody or antigen-binding fragment thereof from any one of Implementation Schemes C3-C5, wherein the heavy chain constant region contains a mutation that reduces binding to the Fcγ receptor, such as an LALA mutation, or similar mutation.

[0574] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44;

[0575] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:44.

[0576] Implementation Scheme C7. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes C1-C6 comprises a light chain constant region, for example, the light chain constant region being a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region).

[0577] Implementation Scheme C8. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes C1-C7, comprising a light chain constant region, wherein the light chain constant region is the human Lambda light chain constant region.

[0578] Implementation Scheme C9. The antibody or antigen-binding fragment thereof described in any one of Implementation Schemes C1-C8, comprising a light chain constant region, wherein the light chain constant region is a human Kappa light chain constant region.

[0579] Implementation Scheme C10. The antibody or antigen-binding fragment thereof of any one of Implementation Schemes C1-C9 comprises a light chain constant region, wherein the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38; preferably, it comprises or is composed of the amino acid sequence shown in SEQ ID NO:38.

[0580] Implementation Scheme C11. The antibody or antigen-binding fragment thereof of any one of Implementation Schemes C1-C10, comprising a light chain constant region, wherein the light chain constant region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:81; preferably, comprising or is composed of the amino acid sequence shown in SEQ ID NO:81.

[0581] Implementation Scheme C12. The antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C11, wherein the antibody is a humanized antibody.

[0582] Implementation Scheme C13. The antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C12, wherein the antibody is a chimeric antibody.

[0583] Implementation Scheme C14. The antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C13, wherein the antibody is a monoclonal antibody.

[0584] Implementation Scheme C15. The antibody or antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C14, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody) or linear antibody.

[0585] Implementation Scheme D1. A nucleic acid molecule comprising, or composed of, any chain encoding any one of the anti-MUC16 antibody or its antigen-binding fragment as described in any one of Implementation Schemes A1-A53, or any one chain of any one of the multispecific antibodies as described in any one of Implementation Schemes B1-B60, or any one chain of any one of the anti-CD3 antibody or its antigen-binding fragment as described in any one of Implementation Schemes C1-C15.

[0586] Implementation scheme D2. An expression vector comprising the nucleic acid molecule described in implementation scheme D1, preferably, the expression vector is pCDNA, such as pCDNA3.1 or pCDNA3.4.

[0587] Implementation Scheme D3. A host cell comprising the nucleic acid molecule described in Implementation Scheme D1 or the expression vector described in Implementation Scheme D2, preferably, the host cell is prokaryotic or eukaryotic.

[0588] Implementation scheme D4. The host cell described in implementation scheme D3 is a 293 cell, such as an Expi293 cell.

[0589] Implementation scheme D5. The host cell described in implementation scheme D3 is a CHO cell, such as an ExpiCHO cell.

[0590] Implementation Scheme D6. A method for preparing an anti-MUC16 antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A53, or a multispecific antibody as described in any one of Implementation Schemes B1-B60, or an anti-CD3 antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C15, the method comprising culturing a host cell containing the nucleic acid molecule described in Implementation Scheme D1 or the expression vector described in Implementation Scheme D2 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0591] Implementation scheme D7. An immunoconjugate comprising any one of the anti-MUC16 antibody or its antigen-binding fragment in any one of implementation schemes A1-A53, or any one of the multispecific antibodies in any one of implementation schemes B1-B60, or any one of the anti-CD3 antibody or its antigen-binding fragment in any one of implementation schemes C1-C15.

[0592] Implementation Scheme D8. A pharmaceutical composition comprising an anti-MUC16 antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A53, or a multispecific antibody as described in any one of Implementation Schemes B1-B60, or an anti-CD3 antibody or an antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C15, or an immunoconjugate as described in Implementation Scheme D7, and optionally pharmaceutical excipients.

[0593] Implementation scheme D9. A pharmaceutical combination or pharmaceutical combination product comprising an anti-MUC16 antibody or its antigen-binding fragment as described in any one of implementation schemes A1-A53, or a multispecific antibody as described in any one of implementation schemes B1-B60, or an anti-CD3 antibody or its antigen-binding fragment as described in any one of implementation schemes C1-C15, or an immunoconjugate as described in implementation scheme D7, and one or more other therapeutic agents.

[0594] Implementation Scheme D10. The drug combination or drug combination product described in Implementation Scheme D9, wherein the therapeutic agent is a variety of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0595] Implementation Scheme D11. A method for preventing or treating tumors in a subject, comprising administering to the subject an effective amount of the anti-MUC16 antibody or its antigen-binding fragment thereof as described in any one of Implementation Schemes A1-A53, or the multispecific antibody as described in any one of Implementation Schemes B1-B60, or the anti-CD3 antibody or its antigen-binding fragment thereof as described in any one of Implementation Schemes C1-C15, or the immune conjugate as described in Implementation Scheme D7, or the pharmaceutical composition as described in Implementation Scheme D8; or the pharmaceutical combination or pharmaceutical combination product as described in Implementation Scheme D9 or D10.

[0596] Implementation scheme D12. The method described in implementation scheme D11, wherein the tumor is a solid tumor or a hematologic tumor.

[0597] Implementation scheme D13. The method described in implementation scheme D11 or D12, wherein the tumor is a MUC16-positive tumor or cancer.

[0598] Implementation scheme D14. The method of any one of implementation schemes D11-D13, wherein the tumor is ovarian cancer or pancreatic cancer.

[0599] Implementation scheme D15. The method of any one of implementation schemes D11-D14, wherein the method further includes administration in combination with other therapies.

[0600] Implementation scheme D16. The method described in implementation scheme D15, wherein the other therapies include treatment methods such as surgery or radiation therapy.

[0601] Implementation Scheme D17. The method described in Implementation Scheme D15 or D16, wherein the other therapy includes other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0602] Implementation scheme D18. The anti-MUC16 antibody or its antigen-binding fragment thereof described in any one of implementation schemes A1-A53, or the multispecific antibody described in any one of implementation schemes B1-B60, or the anti-CD3 antibody or its antigen-binding fragment thereof described in any one of implementation schemes C1-C15, or the immune conjugate described in implementation scheme D7, or the pharmaceutical composition described in implementation scheme D8; or the use of the pharmaceutical combination or pharmaceutical combination product described in implementation scheme D9 or D10 in the preparation of a medicament for the prevention or treatment of tumors.

[0603] The use of the anti-MUC16 antibody or its antigen-binding fragment as described in any one of embodiments A1-A53, or the multispecific antibody as described in any one of embodiments B1-B60, or the anti-CD3 antibody or its antigen-binding fragment as described in any one of embodiments C1-C15, or the immune conjugate as described in embodiment D7, or the pharmaceutical composition as described in embodiment D8; or the use of the pharmaceutical combination or pharmaceutical combination product as described in embodiment D9 or D10 for the prevention or treatment of tumors.

[0604] Implementation scheme D20. Use as described in implementation scheme D18 or D19, wherein the tumor is a solid tumor or a hematologic tumor.

[0605] Implementation scheme D21. Use according to any one of implementation schemes D18-D20, wherein the tumor is a MUC16-positive tumor or cancer.

[0606] Implementation scheme D22. Use according to any one of implementation schemes D18-D21, wherein the tumor is ovarian cancer or pancreatic cancer.

[0607] Implementation scheme D23. Use of any one of implementation schemes D18-D22, wherein the drug or the anti-MUC16 antibody or its antigen-binding fragment or multispecific antibody or anti-CD3 antibody or its antigen-binding fragment or immune conjugate or pharmaceutical composition or combination of drugs or pharmaceutical combination products is administered in combination with other therapies.

[0608] Implementation scheme D24. The use described in implementation scheme D23, wherein the other therapies include treatment methods such as surgery or radiation therapy.

[0609] Implementation scheme D25. The use described in implementation scheme D23 or D24, wherein the other therapy includes other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g. immune checkpoint inhibitors or agonists).

[0610] Implementation scheme D26. The anti-MUC16 antibody or its antigen-binding fragment thereof described in any one of implementation schemes A1-A53, or the multispecific antibody described in any one of implementation schemes B1-B60, or the anti-CD3 antibody or its antigen-binding fragment thereof described in any one of implementation schemes C1-C15, or the immune conjugate described in implementation scheme D7, or the pharmaceutical composition described in implementation scheme D8; or the pharmaceutical combination or pharmaceutical combination product described in implementation scheme D9 or D10, for therapies, such as for the prevention or treatment of tumors.

[0611] Implementation Scheme D27. The anti-MUC16 antibody or its antigen-binding fragment or multispecific antibody or anti-CD3 antibody or its antigen-binding fragment or immune conjugate or pharmaceutical composition or combination of drugs or pharmaceutical combination products described in Implementation Scheme D26, wherein the tumor is a solid tumor or a hematologic tumor.

[0612] Implementation scheme D28. The method described in implementation scheme D26 or D27, wherein the tumor is a MUC16-positive tumor or cancer.

[0613] Implementation scheme D29. The method of any one of implementation schemes D26-28, wherein the tumor is ovarian cancer or pancreatic cancer.

[0614] The method of any one of embodiments D30, D26-29, wherein the anti-MUC16 antibody or its antigen-binding fragment or multispecific antibody or anti-CD3 antibody or its antigen-binding fragment or immunoconjugate or pharmaceutical composition or combination of drugs or pharmaceutical combination products is administered in combination with other therapies.

[0615] Implementation scheme D31. The method described in implementation scheme D30, wherein the other therapies include treatment methods such as surgery or radiation therapy.

[0616] Implementation Scheme D32. The method described in Implementation Scheme D30 or D31, wherein the other therapies include other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists). Example

[0617] Example 1. Development of anti-MUC16 antibody using the Single B technology platform

[0618] 1.1 Immunity

[0619] Balb / c mice (purchased from Vital River) were immunized with human or monkey MUC16 SEA12-16 protein for a total of 3-4 times, with subcutaneous injections every two weeks.

[0620] 1.2 Antibody Screening Based on Single B

[0621] Once the serum titer met the requirements, the spleen of the mouse was harvested to obtain suspended lymphocytes. Next, B cells in the cell suspension were enriched using the EasySep Mouse Pan-B Cell Isolation Kit (Stemcell, 19844A). The enriched B cells were then sorted using an Aria Fusion flow cytometer, and single B cells (Human MUC16 SEA15-16 positive, Human MUC16 SEA16 double positive, and Human MUC16 SEA15-16 positive, Human MUC16 SEA16 negative) were sorted into 96-well PCR plates containing lysis buffer. Amplification products of the antibody heavy and light chain variable regions were obtained through RT-PCR and nested PCR. The paired amplification products were purified, and the purified products were used to construct a linear expression cassette (LEC) using overlapping PCR and transfected into Expi293 cells.

[0622] After 6 days of LEC transfection and culture, the supernatant was collected for initial screening. First, ELISA was used to detect binding to Human MUC16SEA12-16 and Cyno MUC16 SEA12-16 proteins to screen clones with human-monkey cross-activity. Then, flow cytometry was used to detect binding to Human MUC16 SEA15-16, Human MUC16 SEA16 overexpressing cell lines, and OVCAR3 cell lines. Clones specifically binding to Domain SEA15 and Domain SEA16 were classified according to their binding activity. Positive clones were then subjected to affinity assays using thin-layer chromatography (BLI). The PCR products of clones with positive affinity results were used for Sanger sequencing to obtain the antibody heavy and light chain variable region sequences.

[0623] 1.3 Expression and detection of candidate chimeric antibodies

[0624] Antibody expression and preparation:

[0625] After analyzing the sequences based on diversity and PTM sites, 68 pairs of heavy and light chain variable region genes were selected for the construction of chimeric antibody eukaryotic expression plasmids. The light and heavy chain variable region gene fragments were then processed using homologous recombinase (Nanjing Novizan Biotechnology Co., Ltd.). (Catalog No.: C112-01) was ligated into the pcDNA3.1 vector (containing a heavy chain constant region (SEQ ID NO:79) and a light chain constant region (SEQ ID NO:38)) to obtain expression plasmids for light chain and heavy chain antibodies.

[0626] Expi293F cells (purchased from Gibco) were cultured in Expi293F medium (Gibco, REF#A14351-01). Cell density was checked one day before transfection (viability should be greater than 95%), and the medium was adjusted to 3 × 10⁶ cells / day with fresh Expi293F medium. 6 Continue culturing at 100 cells / mL, and adjust the cell density to 3×100 on the day of transfection. 6Cells / mL. Take 1 / 10 of the final transfection volume of Opti-MEM medium (Gibco, REF#31985-070) as transfection buffer, add the DNA to be transfected at a ratio of 1 mg / L, with a light-to-heavy chain plasmid ratio of 1:1, mix well, add PEIMax (Polysciences Inc. Cat#24765-1) at a DNA:PEI mass ratio of 1:3, mix well, incubate at room temperature for 20 minutes, then gently pour the mixture into Expi293F cell suspension while shaking, place the cells in a shaker and culture under the following conditions: 8% CO2, 36.5℃, 120 rpm. After culturing for 16–18 hours, 2% (v / v) of 200 g / L feed (100 g / L Phytone Peptone + 100 g / L Difco Select Phytone), 5 g / L glucose solution, and 2.2 mM Valproic acid sodium salt (Merk, Cat#P4543-100G) were added to the cell suspension. The mixture was gently stirred and incubated for another 7 days at 36.5°C and 120 rpm with 8% CO2. The cells were then harvested. The cell suspension was mixed with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per 1 L of cell suspension) and filtered through a 0.22 μm disposable vacuum filter.

[0627] Affinity chromatography was used to purify the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the column, followed by 10-20 column volumes of distilled water to wash the tubing and column. The column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell sample was then passed through the column, and the column was washed with 10 column volumes of 1×PBS to remove non-specifically bound proteins. The column was then rinsed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2M Tris, and the sample was filtered for sterilization. Size exclusion chromatography (SEC) was used to determine the purity of the sample. Protein samples meeting purity standards were centrifuged in 15ml ultrafiltration centrifuge tubes at 4000 rpm for 10 minutes. The protein was then diluted with PBS and centrifuged again at 4000 rpm for 10 minutes. This process was repeated several times to change the protein storage buffer. After buffer changes, the samples were sterilized by filtration, antibody concentration was measured, and the antibodies were delivered.

[0628] Flow cytometry was used to preliminarily evaluate candidate molecules by measuring their cell-binding activity in different tumor cell lines (see Example 7 for specific experimental steps).

[0629] The control antibodies used were gp120-hIgG1-LALA and 8767P-hIgG1-LALA, with full-length heavy and light chains shown in Table E. The detection antibodies were S2-196, S2-193, and S2-6, with their specific sequences shown in Sequence Information Tables A and B.

[0630] The cells used are as follows:

[0631] OVCAR3 and MUC16 highly expressing ovarian cancer cells: Nanjing Kebai, catalog number CBP60294

[0632] CFPAC1, MUC16 low-expression pancreatic cancer cells: Nanjing Kebai, catalog number CBP60665

[0633] HELA, MUC16 expression of cervical cancer cells: Nanjing Kebai, catalog number CBP60232

[0634] The results of the cell binding assays are shown in Figures 1 and 2. Based on the cell assay results, these three antibodies exhibit strong binding ability to OVCAR3 and CFPAC1 tumor cell lines, making them ideal candidate molecules for further evaluation.

[0635] Simultaneously, the affinity of candidate molecules was determined using BLI to detect their affinity for Human MUC16 SEA12-16 (catalog number CA5-H52H6, ACRO), Cyno MUC16 SEA12-16 (catalog number CA5-C52H8, ACRO), Human MUC16SEA15-16 (SEQ ID NO:67), and Human MUC16 SEA16 (SEQ ID NO:68). The steps are as follows:

[0636] In this study, the binding kinetics (KD) of the antibody-antigen described herein was determined using biofilm thin-layer interferometry (BLI). The BLI affinity assay was performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): 270-8).

[0637] Half an hour before the experiment, according to the sample quantity, take an appropriate number of AHC sensors (18-5060, Sartorius) and soak them in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). Dilute the anti-MUC16 antibody and MUC16 protein mentioned above to 100 nM respectively.

[0638] SD buffer, antibody solution, and each MUC16 protein were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e. The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: baseline equilibration 120s, antibody loading and solidification 100s, baseline equilibration 120s, antigen binding 100s, and dissociation 120s; rotation speed 1000 rpm; temperature 30℃. After the experiment, the KD values ​​were analyzed using ForteBio Octet analysis software. The results are shown in Table 1 below.

[0639] According to the BLI test results, two of the three candidate molecules specifically bind to Domain SEA16 and one specifically binds to Domain SEA15, both exhibiting an affinity of nM or higher for human and monkey antigens.

[0640] Table 1: Affinity Results of MUC16 Candidate Clones

[0641] 1.4 Expression and detection of bispecific antibody molecules

[0642] The candidate molecules screened above were combined with CD3 antibody sp34.24 (sp34.24 in WO2022068809, which has a mutation in the frame region; the light chain variable region is shown in SEQ ID NO:53, and the heavy chain variable region is shown in SEQ ID NO:54) to construct a 1+1 bispecific antibody, which was purified and used to evaluate T cell-mediated in vitro killing activity. The constructed bispecific antibody contains the following chains:

[0643] The bispecific antibody was prepared and constructed as described in Example 1.3, except that the encoding nucleic acids for the heavy chain and light chain were constructed into the vector pcDNA3.1 and transfected into cells for expression.

[0644] The cytotoxic activity of 1+1 MUC16 and CD3 bispecific antibodies and control antibodies (REGN4018 and gp120-hIgG1-LALA) constructed based on S2-6, S2-193, and S2-196 were detected in three cell lines: CFPAC1, HELA (Nanjing Kebai, catalog number CBP60232), and OVCAR3. Specific procedures are detailed in Example 1.3. All three MUC16 and CD3 antibodies exhibited good cytotoxic activity. The cytotoxicity results are shown in Figure 3. These molecules will be used for humanization.

[0645] 1.5 Molecular Optimization

[0646] Total B cell samples enriched from mouse spleen cells used for single B cell sorting were used to extract RNA using the Trizol method, followed by library construction and NGS sequencing. Using the chimeric antibody S2-193 light and heavy chain sequences as target sequences, similar sequences were searched in the B cell sequencing library, and mutation site patterns were summarized. Finally, six light and heavy chain combinations were selected for expression validation. As shown in Figure 4, cell binding assays confirmed that the chimeric antibody S2-193-H0L1 was used for subsequent humanization.

[0647] Example 2. Antibody Humanization

[0648] 2.1 Human-centered design

[0649] Based on Discovery Studio and PyMOL software, the chimeric antibodies S2-193, S2-196, and S2-6 obtained from hybridoma screening were humanized through the following steps:

[0650] (1) Determine the CDR region of the chimeric antibody;

[0651] (2) Screen the homologous sequences that are closest to the V / J regions of the heavy and light chains of the chimeric antibody in the human pedigree sequence database;

[0652] (3) The CDR region of the chimeric antibody was constructed onto the human Germline backbone;

[0653] (4) Based on sequence and structural features, determine the amino acid positions in the backbone region that maintain the CDR function, and perform reverse mutations (return to the input amino acid type) at the sequence positions that are identified as important.

[0654] (5) After preparing the antibody according to the synthetic sequence shown in Example 1.3, the affinity of the humanized molecule to human MUC16.SEA12-16 was determined by thin-layer biofilm interferometry (BLI).

[0655] Based on the affinity data in Table 2, it can be concluded that the humanized molecule maintained an affinity comparable to that of the chimeric antibody. The amino acid sequences of the chimeric antibody and the humanized molecule are shown in Tables A and B.

[0656] Table 2. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0657] Chimeric antibodies and humanized antibodies were prepared as described in Example 1.3.

[0658] Example 3. Structural design of a bispecific antibody molecule targeting the T-cell adaptor MUC16 and CD3.

[0659] Based on humanized antibodies targeting the MUC16 SEA15 domain, the MUC16 SEA16 domain, and CD3, bispecific antibody molecules were designed. The format is shown in Figures 5 and 6.

[0660] The antibody has the following composition:

[0661] Heavy chain 1: Contains the heavy chain variable region VH and the heavy chain constant regions CH1 and Fc.

[0662] Light chain 1: Contains the variable region VL and the constant region CL of the light chain.

[0663] Heavy chain 2: includes the heavy chain variable region VH, the heavy chain constant region CH1, linker 1, the single-chain antibody scFv linked by linker 2, linker 3, the Fc region, and

[0664] Light chain 1: includes the light chain variable region VL and the light chain constant region CL.

[0665] The antibody targeting MUC16 is in Fab form, and the antibody targeting CD3 is in scFv form. Fc is the constant region of the heavy chain of IgG1. S364R and D399K mutations (SEQ ID NO:36, Fc-Innobody A) are introduced into the first Fc region, and Y349T, K370 and K409D mutations (Innobody platform, EU number) (SEQ ID NO:37, Fc-Innobody B) are introduced into the second Fc region. Through charge complementarity and spatial complementarity, the pairing of heterologous heavy chains is increased. At the same time, amino acid mutations L234A and L235A (EU number) that weaken the effector function are introduced into the Fc region.

[0666] Because the tandem arrangement of Fab and scFv effectively prevents light and heavy chain mismatches, the Innobody platform, which also prevents heavy chain mismatches, enables a high proportion of correct pairing of bispecific antibodies when expressed in the same cell. Furthermore, combining Innobody in vitro recombinant technology can achieve a high proportion of correct pairing of bispecific antibodies.

[0667] The full-length amino acid sequences of the bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 are shown in Sequence Information Table C, and the amino acid sequences of the negative antibody and the control antibody are shown in Sequence Information Table E.

[0668] 3.1 Preparation of Bispecific Antibody Protein

[0669] 1) Plasmid construction: Plasmids were synthesized by Baiying, and the DNA encoding each strand was constructed into pcDNA3.4 respectively.

[0670] 2) Protein expression: Protein was produced using the ExpiCHO™ Expression system (Gibco, A29133). Specifically, ExpiCHO-STM cells (Gibco) were passaged according to the required transfection volume, and the cell density was adjusted to 3.5 × 10⁶ cells / day before transfection. 6 Cells / mL. The cell density was adjusted to 6 × 10⁶ cells / mL on the day of transfection. 6 Cells / mL. Take a 50 mL centrifuge tube and add OptiPRO™ SFM (Gibco, 12309019) transfection buffer to 8% of the cell volume. Calculate the total required plasmid amount based on 0.8 μg / mL of transfection. Premix all parental antibodies at a light chain:heavy chain plasmid mass ratio of 1:1. Filter the transfection buffer containing DNA plasmids through a 0.22 μm filter into a new 50 mL centrifuge tube. Add DNA:Reagent to the filtered mixture at a ratio of 1:4. (Polyplus, 101000019) Mix thoroughly and incubate at room temperature for 10 minutes. Then, immediately and slowly add the transfection reagent and plasmid DNA mixture to the cells while gently shaking the flask. Incubate the transfected cells at 37°C in a shaker with 8% CO2. After 18-22 hours, add 6 μL / mL Enhancer (Gibco, 100033019) and 300 μL / mL Feed (Gibco, A29101-01) to the cells. Continue culturing at 37°C, 120 rpm, and 8% CO2. Collect the cell culture medium on day 7 or when cell viability is ≤70%. Mix the cell culture medium with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L of cell culture medium) and filter using a 0.22 μm disposable vacuum filter. Use the supernatant for subsequent affinity purification.

[0671] 3) Affinity chromatography purification of the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the column, followed by 10-20 column volumes of distilled water to wash the tubing and column. The column was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was then passed through the column, and the column was washed with 10 column volumes of 1×PBS to remove non-specifically binding proteins. The column was then washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2M Tris, and the solution was filtered for sterilization. The two parental antibodies obtained were named Innobody A antibody (Fc region: Innobody A) and Innobody B antibody (Fc region: Innobody B).

[0672] 4) In vitro reduction and oxidation: The purified innobody A and innobody B were mixed in a 1:1 molar ratio, and an appropriate amount of GSH was added. The pH of the reaction was adjusted to 8.0 with 1M arginine. The mixture was incubated overnight at room temperature. The reaction mixture was then transferred to PBS and stored at 4°C for later use.

[0673] 5) Ion exchange chromatography purification of bispecific antibodies: A Mono S 5 / 50GL ion exchange chromatography column (from GE Healthcare) was used and placed in an AKTApure system (from GE Healthcare). Endotoxins were removed from the AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column using 0.5M NaOH for 2 hours. The system and column were then washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20mM phosphate, pH 6.6) until conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was reequilibrated with 5 column volumes of loading buffer. Linear elution was performed using a gradient of 0-40% elution buffer (20mM phosphate, 1M NaCl, pH 6.6) for a total of 30 column volumes. Samples were collected based on UV absorbance.

[0674] The purity of samples collected from each fraction was determined using size exclusion chromatography (SEC). Samples from fractions with a purity greater than 95% were pooled based on the SEC results. Antibody concentration was then determined. Further qualitative and quantitative analysis of antibody purity and impurities was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0675] Similarly, control antibodies gp120 x gp120-sp34.24, REGN4018, 8767P-hIgG1-LALA, and gp120-hIgG1-LALA were prepared.

[0676] Example 4. ForteBio determined the binding kinetics of the MUC16 / CD3 bispecific antibody and control antibody to the antigen.

[0677] The equilibrium dissociation constant (KD) of the antibody bound to human, cynomolgus monkey, and mouse MUC16 was determined using ForteBio biointerferometry. ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody antigen affinity and epitope binning. MAbs, 2013.5(2)).

[0678] Half an hour before the start of the experiment, according to the number of samples, take an appropriate number of AHC (18-5060, Sartorius) sensors and immerse them in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%).

[0679] 100 μL of SD buffer, antibody, and antigen [including human MUC16 (CA5-H52H6, Acro biosystems), cynomolgus monkey MUC16 (CA5-C52H8, Acro biosystems), and mouse MUC16 (CA5-M52H6, Acro biosystems)] were added to 96-well black polystyrene semi-mass microplates (Greiner, 675076). The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample binding and dissociation rates, the rotation speed was 1000 rpm, and the temperature was 30℃. KD values ​​were analyzed using ForteBio analysis software.

[0680] Based on the results in Tables 3 and 4, compared with the control antibody REGN4018, the bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 exhibited higher affinity for human and cynomolgus monkey MUC16. Based on the results in Table 5, compared with the control antibody REGN4018, the bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 exhibited affinity for mouse MUC16.

[0681] Table 3. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0682] Table 4. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0683] Table 5. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0684] Example 5. Detection of the in vitro killing function of chimeric antibodies against tumor cells

[0685] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS, catalog number PB050C-W) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a laminar flow hood. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0686] Viability assays: 1) CD8T cell sorting: After PBMC cell counting, CD8T cells were isolated from PBMCs using a CD8T cell isolation kit (Stemcell, catalog number 19053) for subsequent in vitro experiments. 2) CD8T cell plating: CD8T cells were resuspended in phenol red-free 1640 medium (Gibco, 11835-030) containing 1% serum, and the cell density was adjusted to 2 x 10⁻⁶ cells / mL. 6 100 μL per well was added to a 96-well flat-bottom cell culture plate (NEST, catalog number 701001). 3) Tumor cell plating: After digestion of tumor cells, the cells were resuspended in phenol red-free 1640 medium containing 1% serum and the cell density was adjusted to 4 x 10⁻⁶ cells / mL. 54) Antibody preparation: Antibody was prepared using phenol red-free 1640 medium containing 1% serum. The antibody was diluted 10-fold with 100 nM in a sterile 96-well V-bottom plate (Beyotime, FPT019), resulting in 8 gradients. 50 μL of the antibody was added to each well of the cell culture plate and incubated at 37°C with 5% CO2 for 16 hours. 5) Control group setup: IgG group (gp120 x gp120-sp34.24 antibody administration group), target cell spontaneous LDH release wells, target cell maximum LDH release wells (with lysis buffer added), and culture medium background control wells (PBMCs + tumor cells). Each group had 3-4 replicates. Finally, the total medium was added to 200 μL / well and incubated at 37°C with 5% CO2 for 16 hours. 6) LDH detection: After 16 hours of incubation, at 400g for 5 minutes, transfer 50 μL of supernatant suspension per well into a new 96-well V plate. Add an equal volume of LDH detection reagent (Promega, catalog number G1780), gently shake, and incubate at room temperature in the dark for 10 minutes before instrument detection.

[0687] The experimental results are shown in Figure 7. The bispecific antibodies S2-193xS2-196-sp34.24, S2-193xS2-6-sp34.24 and S2-6 x S2-6-sp34.24 can kill OVCAR3 ovarian cancer cells in vitro, and their killing ability is significantly better than that of the control antibody REGN4018.

[0688] Example 6. Detection of the in vitro binding effect of humanized antibodies on MUC16 antigen of different tumor cells.

[0689] Cell resuscitation: 1) Remove tumor cells OVCAR3 (Nanjing Kebai, catalog number CBP60294), CFPAC-1 (Nanjing Kebai, CBP60665), and COV362 (Nanjing Kebai, CBP60835) from the liquid nitrogen tank and gently shake them in a 37°C water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination. 2) After thawing, immediately disinfect with 75% alcohol and transfer to a laminar flow hood. Strictly follow aseptic techniques thereafter. 3) Prepare complete culture medium in advance: IMDM (Gibco, 12440-061), 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0690] Activity assay: 1) Digest cultured tumor cells, centrifuge at 1000 rpm for 5 minutes, resuspend in FACS buffer (PBS + 5% FBS + 2 nM EDTA), adjust cell concentration to 50k-100k cells / well, mix well, and seed cells into 96-well plates. 2) Centrifuge at 600g for 2 minutes, resuspend in FACS buffer containing Fc blocker (1:200), and incubate at room temperature in the dark for 10 minutes. 3) Centrifuge at 600g for 2 minutes, resuspend in 200 μL of FACS buffer, and wash twice. 4) Prepare the disclosed antibody and control antibody (300 nM, 5-fold dilution). After incubation, centrifuge at 600g for 2 minutes, discard unbound antibody in the plate, add 200 μL of FACS wash buffer to each well, and wash twice. 5) Add secondary antibody, PE Goat anti-human IgG (catalog number 2040-09, Biolegend) (1:5000), 50 μL per well, and incubate at 4°C for 30 minutes. 6) After incubation, centrifuge at 600g for 2 minutes, discard the supernatant, and add 200 μL of FACS wash buffer to each well, washing three times. 7) After washing, resuspend the cells in 100 μL of FACS buffer to each well and analyze using flow cytometry.

[0691] The experimental results are shown in Figures 8 and 9. The bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 can bind to the MUC16 antigen on the surface of OVCAR3 ovarian cancer and CFPAC-1 pancreatic cancer cells, and their binding ability is superior to that of the control antibody REGN4018.

[0692] Example 7. Detection of the in vitro CD3 antigen binding effect of antibody on T cells.

[0693] Cell resuscitation: 1) Remove Jurkat tumor cells (Nanjing Kebai, catalog number CBP60942) from the liquid nitrogen tank and gently agitate them in a 37℃ water bath until thawed, keeping the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench, then strictly follow aseptic techniques thereafter; 3) Prepare complete culture medium in advance: 1640 (Gibco, 22400-071), 10%... FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010); 3) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 4) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0694] Activity assay: 1) Digest cultured Jurkat cells, centrifuge at 1000 rpm for 5 minutes, resuspend in FACS buffer (PBS + 5% FBS + 2 nM EDTA), adjust cell concentration to (50k-100k cells / well), mix well, and seed cells into 96-well plates. 2) Centrifuge at 600g for 2 minutes, resuspend in FACS buffer containing Fc blocker (1:200), and incubate at room temperature in the dark for 10 minutes. 3) Centrifuge at 600g for 2 minutes, resuspend in 200 μL of FACS buffer, and wash twice. 4) Prepare the disclosed antibody and control antibody (300 nM, 5-fold dilution). After incubation, centrifuge at 600g for 2 minutes, discard unbound antibody in the plate, add 200 μL of FACS wash buffer to each well, and wash twice. 5) Add secondary antibody (1:5000), 50 μL per well, and incubate at 4℃ for 30 minutes. 6) After incubation, centrifuge at 600g for 2 minutes, discard the supernatant, add 200μL of FACS washing buffer to each well, and wash three times. 7) After washing, resuspend the cells in 100μL of FACS buffer to each well and analyze with flow cytometry.

[0695] The experimental results are shown in Figure 10. To reduce cytokine release syndrome (CRS) caused by excessive T cell activation and prolong the half-life of the bispecific antibody molecules in animals, HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 were designed as a 2+1 bispecific antibody, which can effectively block the binding of the antibody to the CD3 antigen on the surface of T cells, but still maintains a strong in vitro tumor cell killing ability. In contrast, the control antibody REGN4018 is a 1+1 bispecific antibody, in which the CD3 antibody is fully exposed and has a stronger CD3 antigen binding function.

[0696] Example 8. Detection of the in vitro killing effect of antibodies on different tumor cells

[0697] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS, catalog number PB050C-W) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a laminar flow hood. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0698] Viability assays: 1) CD8T cell sorting: After PBMC cell counting, CD8T cells were isolated from PBMCs using a CD8T cell isolation kit (Stemcell, catalog number 19053) for subsequent in vitro experiments. 2) CD8T cell plating: CD8T cells were resuspended in phenol red-free 1640 medium (Gibco, 11835-030) containing 1% serum, and the cell density was adjusted to 2 x 10⁻⁶ cells / mL. 6100 μL per well was added to a 96-well flat-bottom cell culture plate (NEST, catalog number 701001). 3) Tumor cell plating: After digestion of tumor cells, the cells were resuspended in phenol red-free 1640 medium containing 1% serum and the cell density was adjusted to 4 x 10⁻⁶ cells / mL. 5 4) Antibody preparation: Antibody was prepared using phenol red-free 1640 medium containing 1% serum. The antibody was diluted 10-fold with 100 nM in a sterile 96-well V-bottom plate (Beyotime, FPT019), resulting in 8 gradients. 50 μL of the antibody was added to each well of the cell culture plate and incubated at 37°C with 5% CO2 for 16 hours. 5) Control group setup: negative control gp120-hIgG1-LALA and gp120 x gp120-sp34.24 antibody administration groups, target cell spontaneous LDH release wells, target cell maximum LDH release wells (with lysis buffer added), and culture medium background control wells (PBMCs + tumor cells). Each group had 3-4 replicates. Finally, the total medium was added to 200 μL / well and incubated at 37°C with 5% CO2 for 16 hours. 6) LDH detection: After 16 hours of incubation, at 400g for 5 minutes, transfer 50 μL of supernatant suspension per well into a new 96-well V plate. Add an equal volume of LDH detection reagent (Promega, catalog number G1780), gently shake, and incubate at room temperature in the dark for 10 minutes before instrument detection.

[0699] The experimental results are shown in Figures 11 and 12. The bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 can kill OVCAR3 ovarian cancer and CFPAC-1 pancreatic cancer cells in vitro, and their killing ability is significantly better than that of the control antibody REGN4018.

[0700] Example 9. Detection of the effect of antibody on T cell activation induced by tumor killing.

[0701] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS, catalog number PB050C-W) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a laminar flow hood. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0702] Viability assays: 1) CD8T cell sorting: After PBMC cell counting, CD8T cells were isolated from PBMCs using a CD8T cell isolation kit (Stemcell, catalog number 19053) for subsequent in vitro experiments. 2) CD8T cell plating: CD8T cells were resuspended in phenol red-free 1640 medium (Gibco, 11835-030) containing 1% serum, and the cell density was adjusted to 2 x 10⁻⁶ cells / mL. 6 100 μL per well was added to a 96-well flat-bottom cell culture plate (NEST, catalog number 701001). 3) Tumor cell plating: After digestion of tumor cells, the cells were resuspended in phenol red-free 1640 medium containing 1% serum and the cell density was adjusted to 4 x 10⁻⁶ cells / mL. 54) Antibody preparation: Antibody was prepared using phenol red-free 1640 medium containing 1% serum. The antibody was diluted 10-fold with 100 nM in a sterile 96-well V-bottom plate (Beyotime, FPT019), resulting in 8 gradients. 50 μL of the antibody was added to each well of the cell culture plate and incubated at 37°C with 5% CO2 for 16 hours. 5) Control group setup: IgG group, target cell spontaneous LDH release wells, target cell maximum LDH release wells (with lysis buffer added), and culture medium background control wells (PBMCs + tumor cells). Each group had 3-4 replicates. Finally, the total volume was brought to 200 μL / well with complete culture medium and incubated at 37°C with 5% CO2 for 16 hours. 6) Staining panel preparation: Prepare flow cytometry dyes related to T cell activation maker in advance, including Live Dead (L34968A, Invitrogen), CD45 (304014, Biolegend), CD3 (560835, BD), CD8 (344724, Biolegend), CD25 (356116, Biolegend), CD69 (310910, Biolegend), and PD-1 (379210, Biolegend). 7) T cell activation maker detection: After 16 hours of culture, incubate at 400g for 5 minutes, remove the supernatant, and place the suspended T cells in a sterile 96-well V-plate (Beyotime, FPT019). After staining for 1 hour, wash away unbound and non-specifically bound antibodies, and perform flow cytometry analysis.

[0703] The experimental results are shown in Figures 13 and 14. The bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 can induce T cell activation in vitro and kill OVCAR3 ovarian cancer cells and CFPAC-1 pancreatic cancer cells. Moreover, their ability to induce T cell activation is significantly better than that of the control antibody REGN4018.

[0704] As shown in Figure 15, in vitro T cell activation induced by killing 293T cells without expressing the MUC16 antigen, the REGN4018 control antibody induced stronger T cell activation, including upregulation of CD25 and CD69 expression. In contrast, the bispecific antibodies HzS2-193xS2-196-sp34.24, HzS2-193xS2-6-sp34.24, and HzS2-6xS2-6-sp34.24 induced weaker T cell activation, which is beneficial in reducing toxicity issues.

[0705] Example 11. Detection of the antitumor effect of antibody in a human ovarian cancer model with OV90 overexpression MUC16

[0706] To demonstrate the in vivo efficacy of HzS2-193xS2-196-sp34.24 and HzS2-6xS2-6-sp34.24 molecules, we used OV90-MUC16 cells (catalog number CTCC-001-0363, Mason cells) to inoculate NOG mice and determine the antitumor efficacy of the molecules disclosed herein. SPF-grade female mice (16-19g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment. Certificate number: 110011241107373367. After arrival, the mice were acclimatized and fed for 3 days before the experiment began.

[0707] Subcutaneous inoculation in mice: Day-3, NOG mice were intravenously injected with PBMC cell suspension, 4 × 10⁶ cells / day. 6 On Day 0, NOG mice were shaved on the right dorsoventral side and inoculated with OV90-MUC16 cells at a dose of 3 × 10⁶ cells / mouse. 6 / mouse, inoculation volume 200μL / mouse (PBS:Matrigel-1:1). On day 7 after cell inoculation, the maximum width and maximum length axes of mouse tumors were measured with calipers to calculate tumor volume. Based on tumor volume, mice were divided into four groups (n=6 per group) for drug administration: gp120-hIgG1-LALA, 0.5mg / kg; REGN4018, 0.5mg / kg; HzS2-6xS2-6-sp34.24, 0.6mg / kg; and HzS2-193xS2-196-sp34.24, 0.6mg / kg). Drug administration was performed once on day 7 and once on day 14. Monitoring continued until day 29 after intraperitoneal injection.

[0708] The relative tumor inhibition rate (TGI%) was calculated on day 29 post-vaccination using the following formula: TGI% = 100% * (Tumor volume in the control group - Tumor volume in the treatment group) / (Tumor volume in the control group - Tumor volume in the control group before administration). Tumor volume was measured using calipers, measuring the maximum long axis (L) and maximum wide axis (W). The tumor volume was calculated using the following formula: V = L * W² / 2. Body weight was measured using an electronic balance.

[0709] The results are shown in Table 6 and Figure 16. At the same dose, the bispecific antibodies HzS2-193xS2-196-sp34.24 and HzS2-6xS2-6-sp34.24 had better antitumor efficacy than the control antibody REGN4018.

[0710] Table 6. Tumor inhibition rate of the OV90-MUC16 model

[0711] Example 12. Detection of the antitumor effect of antibody in the CFPAC-1 human pancreatic cancer model

[0712] To demonstrate the in vivo efficacy of HzS2-193xS2-196-sp34.24 and HzS2-6xS2-6-sp34.24 molecules, we used CFPAC-1 cells to inoculate NOG mice and determined the antitumor efficacy of the molecules disclosed herein. SPF-grade female mice (16-19g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment. Certificate number: 110011241107373367. After arrival, the mice were acclimatized and fed for 3 days before the experiment began.

[0713] Subcutaneous inoculation in mice: Day-4, NOG mice were intravenously injected with PBMC cell suspension, 4 × 10⁶ cells / day. 6 On Day 0, NOG mice were shaved on the right dorsal and ventral side and inoculated with CFPAC-1 cells at a dose of 3 × 10⁶ cells / mouse. 6 / mouse, inoculation volume 200μL / mouse (PBS:Matrigel-1:1). On day 6 after cell inoculation, the maximum width and maximum length axes of mouse tumors were measured with calipers to calculate tumor volume. Based on tumor volume, mice were divided into four groups (n=6 per group) for serpentine drug administration: gp120-hIgG1-LALA, 0.2mg / kg; REGN4018, 0.2mg / kg; HzS2-6xS2-6-sp34.24, 0.24mg / kg; and HzS2-193xS2-196-sp34.24, 0.24mg / kg). Administered once on day 6. Monitoring continued until day 29 after intraperitoneal injection.

[0714] The relative tumor inhibition rate (TGI%) was calculated on day 29 post-vaccination using the following formula: TGI% = 100% * (Tumor volume in the control group - Tumor volume in the treatment group) / (Tumor volume in the control group - Tumor volume in the control group before administration). Tumor volume was measured using calipers, measuring the maximum long axis (L) and maximum wide axis (W). The tumor volume was calculated using the following formula: V = L * W² / 2. Body weight was measured using an electronic balance.

[0715] The results are shown in Table 7 and Figure 17. At the same dose, the bispecific antibodies HzS2-193xS2-196-sp34.24 and HzS2-6xS2-6-sp34.24 had better antitumor efficacy than the control antibody REGN4018.

[0716] Table 7. Tumor inhibition rate of CFPAC-1 model

Claims

1. Anti-MUC16 antibody or its antigen-binding fragment, comprising (i) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:20, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27; (ii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:21, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:28; (iii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:22, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:29; (iv) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:23, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:30; (v) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:24, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:31; (vi) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:32; (vii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:26, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:33; (viii) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:20 or 21, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:27 or 28; (ix). The three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:22, 23, or 24, and the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:29, 30, or 31; or (x) The three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:25 or 26, and the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:32 or 33.

2. An anti-MUC16 antibody or its antigen-binding fragment, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein... (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:1; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4 or 5; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:10; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

19. (ii) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 7; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

82. (iii) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:6; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:34; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:82; or (iv) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:8 or 9; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

19.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region VH, wherein the heavy chain variable region (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 20-26; or (ii) Contains or consists of the amino acid sequence shown in any one of SEQ ID NO:20-26.

4. The antibody or antigen-binding fragment of any one of claims 1-3, comprising a light chain variable region VL, wherein the light chain variable region... (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 27-33; or (ii) Contains or consists of the amino acid sequence shown in any one of SEQ ID NO:27-33.

5. The antibody or antigen-binding fragment of any one of claims 1-4, comprising a heavy chain variable region VH and a light chain variable region VL, wherein... (i) The VH contains the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (ii) The VH contains the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (iii) The VH contains the amino acid sequence shown in SEQ ID NO:20 or 21 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:27 or 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it or is composed of said amino acid sequence; (iv) The VH contains the amino acid sequence shown in SEQ ID NO:22 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (v) The VH contains the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (vi) The VH contains the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (vii) The VH contains the amino acid sequence shown in SEQ ID NO:22, 23, or 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and the VL contains the amino acid sequence shown in SEQ ID NO:29, 30, or 31, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (viii) The VH contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (ix). The VH contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; (x) The VH contains the amino acid sequence shown in SEQ ID NO:25 or 26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:32 or 33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

6. The antibody or antigen-binding fragment of any one of claims 1-5, comprising a heavy chain variable region VH and a light chain variable region VL, wherein... (i) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:20, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:27; (ii). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:28; (iii) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 20 or 21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 27 or 28; (iv) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:22, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:29; (v) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:30; (vi) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:31; (vii). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 22, 23 or 24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 29, 30 or 31; (viii) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32; (ix). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:33; or (x) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25 or 26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32 or 33.

7. Anti-MUC16 antibody or its antigen-binding fragment, comprising the heavy chain variable region VH and the light chain variable region VL. (i) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:20, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:27; (ii). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:28; (iii) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 20 or 21, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 27 or 28; (iv) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:22, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:29; (v) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:23, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:30; (vi) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:31; (vii). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO: 22, 23 or 24, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO: 29, 30 or 31; (viii) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32; (ix). The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:33; or (x) The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25 or 26, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:32 or 33.

8. The antibody or antigen-binding fragment of any one of claims 1-7, further comprising a heavy chain constant region, for example, the antibody heavy chain constant region being a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1, IgG2, IgG3, or IgG4), preferably a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, and preferably, the heavy chain constant region... (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43; or (ii) Contains or consists of an amino acid sequence selected from or composed of the amino acid sequence shown in SEQ ID NO:

43.

9. The antibody of claim 8 or its antigen-binding fragment, wherein the heavy chain constant region comprises a mutation that reduces binding to the Fcγ receptor, such as the LALA mutation, etc. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

44.

10. The antibody or antigen-binding fragment of any one of claims 1-9, comprising a light chain constant region, for example, the light chain constant region being a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region), for example, the light chain constant region being the human lambda light chain constant region or the human kappa light chain constant region.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the light chain constant region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 38 or 81; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:38 or 81.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody is a humanized antibody or a chimeric antibody.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody is a monoclonal antibody.

14. The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody) or linear antibody.

15. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is a multispecific antibody, such as a bispecific antibody.

16. The antibody of claim 15 or an antigen-binding fragment thereof, wherein the antibody further specifically binds to CD3.

17. A multispecific antibody comprising an antigen-binding region specifically binding to MUC16 and an antigen-binding region specifically binding to CD3; optionally, comprising two antigen-binding regions specifically binding to MUC16 and one antigen-binding region specifically binding to CD3. Two antigen-binding regions that specifically bind to MUC16 may be identical or different, and each contains VH and VL respectively. The VH includes HCDR1, HCDR2, and HCDR3 as defined in claim 1 or 2, and the VL includes LCDR1, LCDR2, and LCDR3 as defined in claim 1 or 2; or The VH and VL are as defined in any one of claims 3-7; Preferably, the multispecific antibody is a bispecific antibody.

18. The multispecific antibody of claim 17, wherein the antigen-binding regions that specifically bind to MUC16 are the Fab regions of the anti-MUC16 antibody as defined in any one of claims 1-14.

19. The multispecific antibody of claim 17 or 18, wherein the Fab of the anti-MUC16 antibody comprises CH1, wherein the CH1 is CH1 derived from human IgG1, IgG2, IgG3 or IgG4, preferably CH1 derived from human IgG1.

20. The multispecific antibody of claim 19, wherein CH1 (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:35; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

35.

21. The multispecific antibody of any one of claims 18-20, wherein the Fab of the anti-MUC16 antibody comprises a light chain constant region, wherein the light chain constant region is a light chain constant region derived from a Kappa light chain constant region or a Lambda light chain constant region (e.g., a human Kappa light chain constant region or a human Lambda light chain constant region), for example, the light chain constant region is a human lambda light chain constant region or a human kappa light chain constant region.

22. The multispecific antibody of claim 21, wherein the Kappa light chain constant region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38; or (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:38; or The Lambda light chain constant region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 81; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

81.

23. The multispecific antibody of any one of claims 17-22, wherein the antigen-binding region specifically binding to CD3 comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:47; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:48; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:49; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:50; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:51; and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

52.

24. The multispecific antibody of any one of claims 17-23, wherein the antigen-binding region specifically binding to CD3 comprises VH and VL, wherein VH comprises or is composed of the sequence shown in SEQ ID NO:53; and VL comprises or is composed of the sequence shown in SEQ ID NO:

54.

25. The multispecific antibody according to any one of claims 17-24, wherein the antigen-binding region that specifically binds to CD3 is scFv. Preferably, the scFv comprises or consists of the following from the N-terminus to the C-terminus: VH CD3 -VL CD3 Where "-" represents a connector or direct connection, and VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via a connector; Optionally, the connector is (GGGGS). n , where n = 1, 2, 3, 4 or 5, for example n = 3 or 4, for example containing or consisting of the amino acid sequence shown in SEQ ID NO: 40; Optionally, the scFv comprises the amino acid sequence shown in SEQ ID NO:62, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

26. The multispecific antibody of any one of claims 17-25, wherein the multispecific antibody is an IgG-like multispecific antibody comprising an Fc dimer, wherein the two Fc regions constituting the Fc dimer are the same or different, optionally, the Fc regions are derived from IgG1, IgG2, IgG3 or IgG4, for example, the Fc regions of human IgG1, IgG2, IgG3 or IgG4, optionally, the Fc regions (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO: 45 or 46; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:45 or 46.

27. The multispecific antibody of claim 26, wherein the two Fc regions are different, wherein mutations are introduced into the first Fc region and the second Fc region based on Innobody technology to promote heterodimerization of the first Fc region and the second Fc region, preferably one Fc region contains S364R and D399K mutations in CH3, and the other Fc region contains Y349T, K370S and K409D mutations in CH3, and optionally the first and / or the second Fc region contains mutations that reduce binding to the Fcγ receptor, such as L234A / L235A mutations.

28. The multispecific antibody according to claim 26 or 27, wherein... a) One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:36, and the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:37; b) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36 and contains the mutations S364R and D399K, and another Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37 and contains the mutations Y349T, K370S, and K409D; or c) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36 and contains the mutations S364R, D399K, and L234A / L235A, and the other Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37 and contains the mutations Y349T, K370S, K409D, and L234A / L235A.

29. The multispecific antibody according to any one of claims 17-28, wherein The antigen-binding region that specifically binds to MUC16 is the Fab (Fab) region that specifically binds to MUC16. MUC16 Furthermore, the antigen-binding region that specifically binds to CD3 is the scFv that specifically binds to CD3 (scFv CD3 ); The multispecific antibody contains a first Fab MUC16 Second Fab MUC16 ,and First Fab MUC16 The Fab heavy chain is connected directly or via a connector (preferably directly) to the N end of the first Fc region at its CH1 C end, and the second Fab... MUC16 The Fab heavy chain has scFv at the C-terminus of its CH1. CD3 The N-terminus (e.g., the N-terminus of its VH) is connected directly or via a connector (preferably via a connector, such as the connector shown in SEQ ID NO:39), and scFv CD3 The C-terminus (e.g., the C-terminus of its VL) is connected directly or via a connector (preferably via a connector, such as the connector shown in SEQ ID NO:41) to the N-terminus of the second Fc region; in, First Fab MUC16 With the second Fab MUC16 Combining the same epitopes or the same structural domains on MUC16, or combining different epitopes or different structural domains on MUC16; for example, the first Fab MUC16 With the second Fab MUC16 Same, or first Fab MUC16 With the second Fab MUC16 Different and combined different epitopes on MUC16 or different structural domains on MUC16.

30. The multispecific antibody according to any one of claims 17-29, comprising or consisting of: Heavy chain 1: From the N end to the C end, it includes or consists of the following: First VH MUC16 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected directly or via a connector to the N end of the first Fc region at its C end; Light chain 1: From the N-terminus to the C-terminus, it includes or consists of the following: First VL MUC16 -First CL; Heavy chain 2: From the N end to the C end, it includes or consists of the following: Second VH MUC16 -Second CH1-scFv CD3 -Second Fc region, where second CH1 and scFv CD3 The N-terminus is connected directly or via connector 1, scFv CD3 The C end is connected directly to the second Fc region or via connector 3; Light chain 2: Composed of the following components from the N-terminus to the C-terminus: second VL MUC16 -Second CL; Where "-" represents a connector or direct connection; Among them, the first VH MUC16 - First CH1 and First VL MUC16 -The first CL constitutes the first Fab that specifically binds to MUC16. MUC16 , and / or the second VH MUC16 -Second CH1 and Second VL MUC16 -The second CL constitutes the second Fab that specifically binds to MUC16. MUC16 ; First and / or second VH MUC16 It is the VH, first and / or second VL of the anti-MUC16 antibody as defined in any one of claims 1-7. MUC16 It is the VL,scFv of the anti-MUC16 antibody as defined in any one of claims 1-7. CD3 It is the scFv as defined in claim 25; The first CH1 and the second CH1 are CH1 as defined in claim 19 or 20, and optionally the first CH1 and the second CH1 are the same; The first CL and the second CL are light chain constant regions as defined in claim 21 or 22, and optionally the first CL and the second CL are the same, for example, both are from the Kappa light chain constant region; The first Fc region and the second Fc region are different and are respectively the Fc regions as defined in claim 27 or 28; Optionally, connector 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:64 or 39; and / or connector 3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

41.

31. The multispecific antibody of claim 30, wherein... Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises 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 with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:

57. The amino acid sequence shown in NO:58, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence; Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:55, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:

59. The amino acid sequence shown in NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence; or Heavy chain 1 comprises the amino acid sequence shown in 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 with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in 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 with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:59, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:

60. The amino acid sequence shown in NO:60, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

32. The multispecific antibody of claim 31, wherein... Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:55; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:56; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:57; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

58. Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:55; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:56; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:59; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60; or Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:61; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:60; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:59; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

60.

33. A nucleic acid molecule comprising, or composed of, any chain encoding any one of the anti-MUC16 antibodies or antigen-binding fragments thereof as described in any one of claims 1-16, or any one chain of any one of the multispecific antibodies as described in any one of claims 17-32.

34. An expression vector comprising the nucleic acid molecule of claim 33, preferably, the expression vector being pCDNA, such as pCDNA3.1 or pCDNA3.

4.

35. A host cell comprising the nucleic acid molecule of claim 33 or the expression vector of claim 34, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as Expi293 cells or ExpiCHO cells.

36. A method for preparing an anti-MUC16 antibody or an antigen-binding fragment thereof as described in any one of claims 1-16 or a multispecific antibody as described in any one of claims 17-32, the method comprising culturing a host cell containing a nucleic acid molecule as described in claim 34 or an expression vector as described in claim 35 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

37. An immunoconjugate comprising the anti-MUC16 antibody of any one of claims 1-16 or its antigen-binding fragment, or the multispecific antibody of any one of claims 17-32.

38. A pharmaceutical composition comprising the anti-MUC16 antibody of any one of claims 1-16 or its antigen-binding fragment, or the multispecific antibody of any one of claims 17-32, or the immunoconjugate of claim 37, and optionally a pharmaceutical excipient.

39. A pharmaceutical combination comprising the anti-MUC16 antibody of any one of claims 1-16 or its antigen-binding fragment, or the multispecific antibody of any one of claims 17-32, or the immunoconjugate of claim 37, and one or more other therapeutic agents, preferably, the therapeutic agents being various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

40. A method for preventing or treating tumors in a subject, comprising administering to the subject an effective amount of the anti-MUC16 antibody or its antigen-binding fragment as described in any one of claims 1-16, or the multispecific antibody as described in any one of claims 17-32, or the immunoconjugate as described in claim 37, or the pharmaceutical composition as described in claim 38; or the pharmaceutical composition as described in claim 39.

41. The method of claim 40, wherein the tumor is a solid tumor or a hematologic malignancy, such as a MUC16-positive tumor or cancer, such as ovarian cancer or pancreatic cancer.

42. The method of claim 40 or 41, wherein the method further comprises administration in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents, preferably, the therapeutic agents being various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

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