B7-H3 Antibodies and Uses Thereof

Abstract

The present application provides a B7-H3 antibody and its use. The heavy chain complementarity determining region of the B7-H3 antibody comprises the amino acid sequences shown in SEQ ID Nos. 6-8, and the light chain complementarity determining region of the B7-H3 antibody comprises the amino acid sequences shown in SEQ ID Nos. 14-16. The B7-H3 antibody binds with high affinity to the B7-H3 antigen and cells expressing the B7-H3 antigen, and can efficiently mediate the killing of B7-H3-positive tumor cells by PBMCs. The B7-H3 antibody is used to construct a B7-H3×CD3 bispecific antibody, and the bispecific antibody has high affinity and stability and can effectively mediate the killing of tumor cells by PBMCs.

Description

[Technical field]

[0001] FIELD OF THE DISCLOSURE The present disclosure is in the technical fields of tumor therapy and molecular immunology and relates to B7-H3 antibodies and uses thereof.

[0002] CROSS REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to a Chinese application bearing application number CN202110817833.7 and entitled "B7-H3 Antibody and Use Thereof," filed with the China Patent Office on July 20, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] B7-H3 (B7 homolog 3 protein, also called CD276) is a member of the B7 / CD28 immunoglobulin superfamily and has a single transmembrane domain (Steinberger P, Majdic O, Derdak SV, et al. Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains[J]. Journal of Immunology, 2004, 172(4):2352-2359.). It consists of 316 amino acids, has a signal peptide at the amino terminus, and contains an immunoglobulin-like variable domain (IgV) in the extracellular domain, a constant domain (IgC), a transmembrane domain, and a cytoplasmic domain consisting of 45 amino acids. B7-H3 is expressed in monocytes, dendritic cells, and activated T cells.

[0004] Currently, the receptor for B7-H3 has not yet been identified, but it has been reported to be TLT-2, a type I transmembrane protein belonging to the triggering receptor expressed on myeloid cells (TREM) family (King RG, Herrin BR, Justement L B. Trem-like transcript 2 is expressed on cells of the myeloid / granuloid and B lymphoid lineage and is up-regulated in response to inflammation.[J]. Journal of Immunology, 2006, 176(10):6012-6021.). The TREM family functions to regulate cellular responses and functions in innate and adaptive immunity (Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and signal integration.[J]. Nature Immunology, 2006, 7(12):1266). TLT-2 protein is constitutively expressed on CD8+ T cells and expression is induced by CD4+ T cell activation.

[0005] B7-H3 is a T cell co-inhibitory molecule with partial co-stimulatory function, which can promote the proliferation of CD4+ and CD8+ T cell populations and selectively stimulate the production of interferon gamma (IFN-γ) in the presence of anti-CD3 antibodies. TLT-2, once transferred to T cells, can promote the production of interleukin (IL)-2 and IFN-γ through interaction with B7-H3, and control CD8+ T cell-mediated hypersensitivity reactions by inhibiting its interaction with B7-H3. On the other hand, human and mouse experiments have demonstrated that B7-H3 has a co-inhibitory effect, allowing tumors to escape from the immune system by suppressing Treg cells, and reducing NK cell function by suppressing NK cell activity in tissue culture.

[0006] B7-H3 is expressed at low levels in normal tissues, but is overexpressed in various cancers, particularly non-small cell lung cancer, renal cancer, urothelial cancer, colon cancer, rectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, and pancreatic cancer. It has been shown that B7-H3 plays an important role in tumor immune evasion through its inhibitory effect on T cells, promoting tumor progression and cancer cell metastasis. B7-H3 is expressed not only in cancer cells, but also in tumors and surrounding blood vessels. In addition, B7-H3 is also associated with autoimmune diseases, playing a key role in the interaction between fibroblast-like synoviocytes and activated T cells in rheumatoid and other autoimmune diseases (see Tran CN, Thacker SG, Louie DM, et al. Interactions of T Cells with Fibroblast-Like Synoviocytes: Role of the B7 Family Costimulatory Ligand B7-H3[J]. Journal of Immunology, 2008, 180(5):2989-2998.). And B7-H3 is involved in the development of sepsis, as it acts as a costimulator in macrophage cytokine release.

[0007] Currently, there is a demand for the development of novel B7-H3 antibodies in the fields of immunotherapy and cancer treatment. Summary of the Invention

[0008] The present disclosure provides a B7-H3 antibody, wherein the heavy chain complementarity determining region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 6 to 8, and the light chain complementarity determining region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 14 to 16.

[0009] In some embodiments, the heavy chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.1, and the light chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.9.

[0010] The present disclosure further provides an affinity matured B7-H3 antibody, the affinity matured B7-H3 antibody being prepared from the B7-H3 antibody by mutational treatment.

[0011] In some embodiments, the affinity matured B7-H3 antibody has a heavy chain complementarity determining region CDR1 that comprises the amino acid sequence shown in SEQ ID No. 6 or any one of SEQ ID Nos. 17 to 25, a heavy chain complementarity determining region CDR2 that comprises the amino acid sequence shown in SEQ ID No. 7 or any one of SEQ ID Nos. 26 to 37, and a heavy chain complementarity determining region CDR3 that comprises the amino acid sequence shown in SEQ ID No. 8 or any one of SEQ ID Nos. 38 to 48, and a light chain complementarity determining region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 14 to 16.

[0012] In some embodiments, the light chain variable region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No. 9, and the heavy chain framework region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No. 2 to 5.

[0013] The present disclosure further provides a bispecific antibody, the bispecific antibody comprising two heavy chains and two light chains, the heavy chain comprising an anti-B7-H3 heavy chain comprising the heavy chain of the B7-H3 antibody or the heavy chain of the B7-H3 antibody by affinity maturation of any one of the above, and an anti-CD3 heavy chain, the light chain comprising an anti-B7-H3 light chain comprising the light chain of the B7-H3 antibody or the light chain of the B7-H3 antibody by affinity maturation of any one of the above, and an anti-CD3 light chain, the heavy chain being linked to the light chain by a disulfide bond, and the anti-B7-H3 heavy chain being linked to the anti-CD3 heavy chain by a disulfide bond.

[0014] In some embodiments, the heavy chain variable region of the anti-CD3 comprises the amino acid sequence set forth in SEQ ID No. 49, and the light chain variable region of the anti-CD3 comprises the amino acid sequence set forth in SEQ ID No. 9.

[0015] In some embodiments, the bispecific antibody comprises a Fab domain of an immunoglobulin that binds to B7-H3, a Fab domain of an immunoglobulin that binds to CD3, and a heterodimeric Fc region, the Fab domain of the immunoglobulin that binds to B7-H3 comprises the light chain of the anti-B7-H3 and the heavy chain variable region VH and constant region CH1 of the anti-B7-H3, the Fab domain of the immunoglobulin that binds to CD3 comprises the light chain of the anti-CD3 and the heavy chain variable region VH and constant region CH1 of the anti-CD3, and the Fc region of the heterodimer comprises an Fc fragment linked to the heavy chain of the anti-B7-H3 and an Fc fragment linked to the heavy chain of the anti-CD3.

[0016] In some embodiments, the Fc fragment linked to the heavy chain of anti-B7-H3 comprises a human Fc fragment or a humanized Fc fragment.

[0017] In some embodiments, the CH3 of the Fc fragment linked to the heavy chain of the anti-B7-H3 comprises a T394D mutation, a P395D mutation, and a P396D mutation.

[0018] In some embodiments, the CH3 of the Fc fragment linked to the heavy chain of the anti-CD3 comprises a P395K mutation, a P396K mutation, and a V397K mutation.

[0019] In some embodiments, the CH2 of the Fc fragment linked to the heavy chain of the anti-B7-H3 comprises the following mutations: L234A, L235A, and P329G.

[0020] The present disclosure further provides a biological material, the biological material comprising: B1): a nucleic acid molecule encoding a heavy chain and / or a light chain of any one of the antibodies (b1) the B7-H3 antibody described above, (b2) the B7-H3 antibody obtained by affinity maturation according to any one of the above, or (b3) the bispecific antibody described above, or a nucleic acid molecule encoding a heavy chain and / or a light chain of an antigen-binding portion of any one of the antibodies (b1) to (b3) described above; B2): an expression cassette comprising the nucleic acid molecule of B1); B3): a recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2); B4): a recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3); B5): a cell line comprising the nucleic acid molecule of B1), a cell line comprising the expression cassette of B2), or a cell line comprising the recombinant vector of B3); B6): The nucleic acid molecule is any one of a nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of any one of the antibodies (b1) to (b3) above, or a nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of the antigen-binding portion of any one of the antibodies (b1) to (b3) above.

[0021] The present disclosure further provides a pharmaceutical composition comprising the B7-H3 antibody, any one of the affinity matured B7-H3 antibodies described above, or a combination of any one or at least two of the bispecific antibodies described above.

[0022] The present disclosure further provides a use of the B7-H3 antibody, the affinity matured B7-H3 antibody of any one of the above, the bispecific antibody of any one of the above, or the pharmaceutical composition in the preparation of an anti-tumor medicament.

[0023] The tumors include any one or a combination of at least two of neural tumors, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer.

[0024] The present disclosure further provides a use of said B7-H3 antibody, the affinity matured B7-H3 antibody of any one of the above, the bispecific antibody of any one of the above, or said pharmaceutical composition for treating a tumor.

[0025] In some embodiments, the tumor comprises any one or a combination of at least two of the following: neural tumor, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer.

[0026] The present disclosure further provides a method for treating a tumor-related disease, comprising administering to a subject in need thereof the B7-H3 antibody, any one of the affinity matured B7-H3 antibodies described above, any one of the bispecific antibodies described above, or the pharmaceutical composition.

[0027] In some embodiments, the tumor comprises any one or a combination of at least two of the following: neural tumor, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows a graph showing the binding activity of 5D9 to the B7-H3 antigen by ELISA. [Figure 2A] FIG. 1 shows the binding activity of affinity matured variants (44F2, 43G11, 46H7, and 45B11) to the B7-H3 antigen detected by ELISA. [Figure 2B] FIG. 1 shows the binding activity of affinity matured variants (43D4, 42F7, and 45F4) to the B7-H3 antigen detected by ELISA. [Figure 2C] FIG. 1 shows the binding activity of affinity matured variants (46H9, 47H4, 46D4, and 43G5) to the B7-H3 antigen detected by ELISA. [Figure 2D]FIG. 1 shows the binding activity of affinity matured variants (44C5, 42A1, and 47C8) to the B7-H3 antigen detected by ELISA. [Figure 2E] FIG. 1 shows the binding activity of affinity matured variants (48E9, 49C3, 49C8, and 49E1) to the B7-H3 antigen detected by ELISA. [Figure 2F] FIG. 1 shows the binding activity of affinity matured variants (49F6, 50A1, 50E3, and 49H5) to the B7-H3 antigen detected by ELISA. [Figure 2G] FIG. 1 shows the binding activity of affinity matured variants (50B10, 48G2, 49D12, and 48D10) to the B7-H3 antigen detected by ELISA. [Figure 2H] FIG. 1 shows the binding activity of affinity matured variants (48A6, 48B12, 49B2, and 49C7) to the B7-H3 antigen detected by ELISA. [Figure 2I] FIG. 1 shows the binding activity of affinity matured variants (50A4, 50A6, and 50A11) to the B7-H3 antigen detected by ELISA. [Figure 3A] FIG. 1 shows the binding activity of affinity matured variants (44F2, 43G11) to cells overexpressing the membrane proximal antigen of B7-H3, detected by FACS. [Figure 3B] FIG. 1 shows the binding activity of affinity matured variants (45B11, 43D4, and 46H7) detected by FACS to cells overexpressing the membrane proximal antigen of B7-H3. [Figure 3C] FIG. 1 shows the binding activity of affinity matured variants (43G5, 42F7, 45F4) to cells overexpressing the membrane proximal antigen of B7-H3, detected by FACS. [Figure 3D] FIG. 1 shows the binding activity of affinity matured variants (46H9, 47H4, 46D4) to cells overexpressing the membrane proximal antigen of B7-H3, detected by FACS. [Figure 3E] FIG. 1 shows the binding activity of affinity matured variants (44C5, 42A1, 47C8) to cells overexpressing the membrane proximal antigen of B7-H3, detected by FACS. [Figure 4A] FIG. 1 shows the killing of B7-H3-positive tumor cells by PBMCs mediated by affinity matured variants (44F2, 43G11, 46H7, 45B11, 43D4, 42F7, 45F4, 46H9). [Figure 4B] FIG. 1 shows the killing of B7-H3-positive tumor cells by PBMCs mediated by affinity matured variants (46D4, 47H4, 48E9, 49C3, 49E1, 50A1, 50A6, 5D9). [Diagram 5] Schematic diagram of the structure of the B7-H3×CD3 bispecific antibody. [Figure 6] FIG. 1 shows the binding activity of the B7-H3×CD3 bispecific antibody. [Figure 7] FIG. 1 shows the effect of T cell activation by B7-H3×CD3 bispecific antibody. [Figure 8A] FIG. 1 shows the killing effect of NCI-N87 cells by PBMCs mediated by B7-H3×CD3 bispecific antibodies (49E1×CD3, 42F7×CD3, 43G11×CD3, 8H9×CD3, 46H7×CD3). [Figure 8B] FIG. 1 shows the killing effect of A498 cells by PBMCs mediated by B7-H3×CD3 bispecific antibodies (42F7×CD3, 43G11×CD3, 49E1×CD3, 8H9×CD3, 46H7×CD3). [Figure 8C] FIG. 1 shows the killing effect of HepG2 cells by PBMCs mediated by B7-H3×CD3 bispecific antibodies (42F7×CD3, 43G11×CD3, 49E1×CD3, 8H9×CD3, 46H7×CD3). [Figure 9A] FIG. 1 shows the results of a stability test on 46H7×CD3 bispecific antibody. [Figure 9B] FIG. 1 shows the results of a stability test on 43G11×CD3 bispecific antibody. [Figure 9C] FIG. 1 shows the results of a stability test on 49E1×CD3 bispecific antibody. [Figure 9D]FIG. 1 shows the results of a stability test on 42F7×CD3 bispecific antibody. [Figure 10] FIG. 1 shows the in vivo antitumor effect of B7-H3×CD3 bispecific antibody in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] In the following, in order to more specifically describe the technical means used in the present disclosure and their effects, the present disclosure will be further described with reference to embodiments, examples, and drawings. Furthermore, the embodiments described herein are merely for the purpose of explaining the present disclosure and do not limit the present disclosure.

[0030] In the embodiments and examples, if no specific techniques or conditions are given, they are carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If no manufacturer is given for the reagents or equipment used, they are all conventional products available on the market through normal channels.

[0031] Unless otherwise defined, scientific and technical terms and their abbreviations used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs, and some of the terms and abbreviations used in this disclosure are listed below.

[0032] Antibody: Antibody, Ab Immunoglobulin, Ig heavy chain, HC Light chain, LC heavy chain variable domain, VH heavy chain constant domain, CH Light chain variable domain, VL light chain constant domain, CL Antigen binding region:antigen binding fragment, Fab Hinge region Fc fragment: fragment crystallizable region, Fc region Monoclonal antibodies, mAbs antibody-dependent cell-mediated cytotoxicity, ADCC complement dependent cytotoxicity, CDC Natural killer cells: NK cells Bispecific antibody, BsAb T cell receptor: T cell receptor, TCR major histocompatibility complex, MHC Complementarity determining region, CDR, antigen-complementary binding region of an antibody Immunoreceptor tyrosine-based activation motif, ITAM Single-chain variable fragment (also called single-chain antibody): single-chain variable fragment, scFv Adoptive cellular immunotherapy, ACI Lymphokine-activated killer cells (LAK cells) Tumor infiltrating lymphocytes: TIL cells Cytokine-induced killer cells (CIK cells)

[0033] The operation steps described in this disclosure, such as molecular cloning, cell culture, protein purification, immunological experiments, microbiological experiments, animal model experiments, etc., are conventional steps widely used in the field. Unless otherwise indicated by the context, singular terms in this disclosure include plurals and plural terms include singulars. Unless otherwise indicated, the nucleotide sequences described in this disclosure are arranged and written from left to right from the 5'-terminus to the 3'-terminus. Unless otherwise indicated, the amino acid sequences described in this disclosure are arranged and written from left to right from the amino-terminus (N-terminus) to the carboxy-terminus (C-terminus). The three-letter abbreviations of amino acids and the one-letter abbreviations of nucleotides referred to in this disclosure are in the format generally accepted in the art, and the one-letter abbreviations of amino acids are in the format recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0034] The term "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a particular given position. As described in this disclosure, "amino acid mutation" refers to amino acid substitution, insertion, deletion, and modification in a polypeptide sequence, as well as any combination of amino acid substitution, insertion, deletion, and modification. A preferred amino acid modification herein is a substitution. In this disclosure, "amino acid substitution" or "substitution" refers to replacing an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. For example, the substitution C220S refers to a mutant polypeptide in which the cysteine ​​at position 220 of the polypeptide is replaced with a serine. Amino acid mutations can be achieved by molecular cloning or chemical methods, including PCR, site-directed mutagenesis, total gene synthesis, and the like.

[0035] The terms "protein," "peptide chain," and "polypeptide chain" refer to molecules in which two or more amino acids are linked by peptide bonds, including naturally occurring proteins, artificial proteins, protein fragments, mutant proteins, fusion proteins, and the like.

[0036] The term "domain" refers to a specific structural region with an independent function in a biopolymer. A domain has an independent tertiary structure and its function does not depend on other parts of the biopolymer. In the present disclosure, a domain refers to a region in a protein such as a heavy chain variable region VH domain or a light chain variable region VL domain, and the domains can be combined with each other to form a larger domain.

[0037] The term "antibody" refers to an immunoglobulin molecule that contains at least one antigen recognition site and specifically binds an antigen. Here, the term "antigen" refers to a substance that can elicit an immune response in vivo and specifically bind to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination thereof. The binding between an antibody and an antigen is mediated by interactions between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the surface of the antigen that binds to the antibody is an "antigenic determinant" or "epitope." Generally, there are multiple determinants per antigen. The term "antibody" referred to in this disclosure encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) containing at least two different epitope-binding domains, human antibodies, humanized antibodies, post-translationally modified antibodies, camelid antibodies, chimeric antibodies, fusion proteins containing antigenic determinants of an antibody, and any other modified immunoglobulin molecule that contains an antigen recognition site, so long as the antibody exhibits the desired biological activity. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain at least one antigen-binding site.

[0038] The term "bispecific antibody" means an antibody that contains two different antigen-binding sites that are capable of simultaneously binding to two different antigen-binding sites.

[0039] The terms "Fab", "Fab region", "Fab fragment" and "Fab molecule" refer to an antigen-binding fragment that includes the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of an immunoglobulin. The first constant region domain CH1 of the heavy chain binds to the constant region domain CL of the light chain, and the variable region domain VH of the heavy chain binds to the variable region domain VL of the light chain.

[0040] The terms "Fc", "Fc region", "Fc fragment" and "Fc molecule" refer to the effector region of an antibody capable of inducing CDC, ADCC, ADCP, cytokine release, and the like. A native antibody Fc is usually formed by the combination of two identical protein fragments containing two or three immunoglobulin constant region domains. In this disclosure, Fc includes native Fc and variant Fc. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues from position C226 or P230 to its carboxy terminus. Under experimental conditions, the fragments generated by cleavage of an immunoglobulin monomer by papaya protease are Fab and Fc, respectively. An antibody "hinge" or "hinge region" refers to a flexible polypeptide comprising the amino acids between the first and second constant domains (CH1 and CH2) of an antibody.

[0041] Unless otherwise specified, the numbering of the amino acids in the variable regions of the antibodies described in this disclosure uses the coding method published by Kabat et al. in 1991, i.e., the "Kabat index" or "Kabat numbering system" (Kabat, EA et al. Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD.:1991). Unless otherwise specified, the numbering of the amino acids in the constant regions of the antibodies described in this disclosure uses the EU index (Edelman GM, et.al. Proc Natl Acad Sci USA 1969, 63:78-85.).

[0042] The term "antigen-binding site" refers to one or more amino acid residues that directly interact with an antigen of an antigen-binding molecule. The antigen-binding site of an antibody consists of the complementarity determining regions (CDRs) of the antigen. A naturally occurring immunoglobulin molecule usually contains two antigen-binding sites, and a Fab molecule usually contains one antigen-binding site.

[0043] The term "T cell activation" refers to one or more immune responses of T lymphocytes, particularly killer T cells, such as proliferation, differentiation, release of cytokines, secretion of effector killer molecules, cell killing, and the like.

[0044] "EC 50 The term "concentration for 50% of maximal effect" refers to the concentration at which an antibody produces 50% of its maximal response.

[0045] As used in this disclosure, "specific binding" refers to the non-random binding of different molecules, such as the reaction of an antibody with its corresponding antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one that binds to an antigen at a concentration of about 10 -5 Less than M, e.g., about 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M, 10 -10 It means binding to this antigen with an affinity (KD) of less than or equal to M. In some embodiments of the present disclosure, the term "target" refers to specific binding.

[0046] As used in this disclosure, "KD" refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, and represents the binding affinity of the antibody to the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity of the antibody to the antigen. Typically, antibodies have a binding affinity of about 10 -5 Less than M, e.g., about 10 -6 Less than M, 10 -7 Less than M, 10 -8 Less than M, 10-9 Less than M, 10 -10 It binds to antigens with an equilibrium dissociation constant (KD) of less than or equal to M.

[0047] The term "single-chain variable fragment" or "scFv" refers to a fusion protein of the heavy and light chain variable regions (VH and VL) of an immunoglobulin, which contains different combinations of the N-terminus of VH or VL and can be prepared by conventional molecular cloning methods to construct recombinant proteins (Sambrook JF, EF et al. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: 2012).

[0048] The term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing some or all of the CDRs of a human immunoglobulin (acceptor antibody) with the CDRs of a non-human antibody (donor antibody). However, the donor antibody may be a non-human (mouse, rat, rabbit, etc.) antibody having the expected specificity, affinity, and reactivity. Some amino acid residues in the framework region (FR) of the acceptor antibody may also be replaced with corresponding amino acid residues of the non-human antibody or amino acid residues of other antibodies to further improve or optimize one or more properties of the antibody.

[0049] The term "host cell" refers to a cell into which an exogenous nucleic acid has been introduced and its progeny, which can express an exogenous polypeptide by transformation or transfection with a nucleotide encoding the polypeptide. Host cells described in this disclosure include, but are not limited to, CHO cells (Chinese hamster ovary cells), HEK293 cells (Human embryonic kidney cells 293), BHK cells (Baby Hamster Kidney cells), myeloma cells, yeast, insect cells, prokaryotic cells such as Escherichia coli, and the like. In addition, the term "host cell" described in this disclosure includes not only a cell into which an exogenous nucleic acid has been introduced, but also the progeny of the cell. Mutations occur in the progeny cells during cell division, but are within the scope of the term described in this disclosure.

[0050] As used herein, an "effective amount" can refer to an amount of a composition or formulation described herein that elicits the desired biological or medical response in a tissue, system, animal, plant, protozoan, bacteria, yeast, or human that is desired by a researcher, veterinarian, physician, or other clinician.

[0051] The term "subject" as used herein refers to a vertebrate, optionally a mammal, optionally a human. Mammals include, but are not limited to, rodents, hominoids, humans, livestock, sport animals, and pet animals. Also included are tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro.

[0052] It should be understood that the terms used in the present specification are intended to describe the context of the present disclosure, such as embodiments and examples, and are not intended to limit the scope of the present invention.

[0053] Some embodiments of the present disclosure provide a B7-H3 antibody, the heavy chain complementarity determining regions (VH-CDR1, VH-CDR2, VH-CDR3) of which comprise the amino acid sequences shown in SEQ ID Nos. 6 to 8, and the light chain complementarity determining regions (VL-CDR1, VL-CDR2, VL-CDR3) of which comprise the amino acid sequences shown in SEQ ID Nos. 14 to 16.

[0054] The B7-H3 antibody disclosed herein can bind to the B7-H3 antigen with high affinity and activate T cells, and thus has important application value in the fields of immunotherapy and cancer treatment.

[0055] In some embodiments, the heavy chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.1.

[0056] In some embodiments, the light chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.9.

[0057] SEQ ID No.1: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAINWVRQAPGQGLEWMGGIIPLFGTANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARDQVVATSGVNFGMDVWGQGTTVTVSS SEQ ID No.6(VH-CDR1):GGTFSNYA SEQ ID No.7(VH-CDR2):IIPLFGTA SEQ ID No.8(VH-CDR3):ARDQVVATSGVNFGMDV SEQ ID No.9: DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIK SEQ ID No.14(VL-CDR1):TGAVTTSNY SEQ ID No.15(VL-CDR2):GTN SEQ ID No.16(VL-CDR3):ALWYSNLWV

[0058] Some embodiments of the present disclosure further provide DNA fragments encoding the B7-H3 antibodies described above.

[0059] In some embodiments, the light chain of the B7-H3 antibody has the nucleotide sequence shown in SEQ ID NO.50, and the heavy chain of the B7-H3 antibody has the nucleotide sequence shown in SEQ ID NO.51.

[0060] SEQ ID NO.50: gatattcagatgacacaagaaccaagtctcactacaagccctggaggcaccgttacactgacctgtcgctcttccactggcgccgtgaccacttccaactatgcaaactgggtgcaggagaaacctggacaggctcctagaggtctgattggaggcactaaca agagagctccagggactcctgccaggttcagcggatctctgatcggtgggaaggcagctctgacaatcactggagtgcaacccgaggatgaggctatctacttctgtgctctctggtactcaaatctgtgggtgttcggaggtggaacaaagctggagatcaag

[0061] SEQ ID NO.51: caggtccagcttgtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcaactatgctatcaactgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctctctttggtacagcaaactacgcacag aagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgacgacggccgtgtattactgtgcgagagaccaggtagtggcaacgagtggcgtcaacttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca

[0062] Some embodiments of the present disclosure further provide an expression vector, said expression vector comprising at least one copy of the DNA fragment as described above.

[0063] Some embodiments of the present disclosure further provide a host cell, the host cell comprising an expression vector as described above.

[0064] Some embodiments of the present disclosure further provide an affinity matured B7-H3 antibody, which is prepared from the above B7-H3 antibody by mutational treatment.

[0065] In some embodiments, the mutation process comprises random mutations to the light chain variable region and / or the heavy chain variable region of the B7-H3 antibody. Without being bound by theory, the mutation process according to the present disclosure can further improve the affinity of the B7-H3 antibody.

[0066] In some embodiments, the affinity matured B7-H3 antibody has a heavy chain complementarity determining region CDR1 that comprises the amino acid sequence shown in SEQ ID No. 6 or any one of SEQ ID Nos. 17 to 25, a heavy chain complementarity determining region CDR2 that comprises the amino acid sequence shown in SEQ ID No. 7 or any one of SEQ ID Nos. 26 to 37, and a heavy chain complementarity determining region CDR3 that comprises the amino acid sequence shown in SEQ ID No. 8 or any one of SEQ ID Nos. 38 to 48.

[0067] In some embodiments, the light chain complementarity determining region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 14 to 16.

[0068] SEQ ID No.17:GGTLRPVL SEQ ID No.18:GGTSGVAL SEQ ID No.19:GGTRPAAL SEQ ID No.20:GGTVRPAL SEQ ID No.21:GGTGGIYL SEQ ID No.22:GGTGGAYL SEQ ID No.23:GGTSRPVL SEQ ID No.24:GGTRPPAL SEQ ID No.25:GGTTGRYL SEQ ID No.26:IIPGFYSL SEQ ID No.27:IIPMWQSV SEQ ID No.28:IIPLFRSS SEQ ID No.29:IIPRFGGV SEQ ID No.30:IIPNFESV SEQ ID No.31:IIPKFRSQ SEQ ID No.32:IIPVFRSV SEQ ID No.33:IIPGFDAV SEQ ID No.34:IIPDFNSA SEQ ID No.35:IIPRFASR SEQ ID No.36:IIPNFHSS SEQ ID No.37:IIPNFYSV SEQ ID No.38:ARDRFTPRSGVNFGMDV SEQ ID No.39:ARDREARESGVNFGMDV SEQ ID No.40:ARDFDAPGSGVNFGMDV SEQ ID No.41:ARDVVVPRSGVNFGMDV SEQ ID No.42:ARDIEVMASGVNFGMDV SEQ ID No.43:ARDQVVPLSGVNFGMDV SEQ ID No.44:ARDVNNPGSGVNFGMDV SEQ ID No.45:ARDVEVFNSGVNFGMDV SEQ ID No.46:ARDEVVVGSGVNFGMDV SEQ ID No.47:ARDAVVPNSGVNFGMDV SEQ ID No.48:ARDQVVPRSGVNFGMDV

[0069] In some embodiments, the affinity matured light chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No.9.

[0070] In some embodiments, the framework region of the heavy chain of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 2-5.

[0071] SEQ ID No.2:QVQLVQSGAEVKKPGSSVKVSCKAS SEQ ID No.3:INWVRQAPGQGLEWMGG SEQ ID No.4:NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYC SEQ ID No.5:WGQGTTVTVSS

[0072] In some embodiments, the framework region of the light chain of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 10-13.

[0073] SEQ ID No.10:DIQMTQEPSLTTSPGGTVTLTCRSS SEQ ID No.11:ANWVQEKPGQAPRGLIG SEQ ID No.12:KRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFC SEQ ID No.13:FGGGTKLEIK

[0074] Without wishing to be bound by theory, the above B7-H3 antibodies and affinity matured B7-H3 antibodies of the present disclosure have the following properties. a.Specifically binds to the human B7-H3 antigen. b. Binds specifically to cells that overexpress the membrane proximal antigen of human B7-H3. c. Mediates the killing of B7-H3 positive tumor cells by PBMCs.

[0075] Some embodiments of the present disclosure further provide a bispecific antibody comprising two heavy chains and two light chains, wherein the heavy chain comprises an anti-B7-H3 heavy chain and an anti-CD3 heavy chain, the anti-B7-H3 heavy chain comprises the heavy chain of the B7-H3 antibody described above or the heavy chain of a B7-H3 antibody obtained by affinity maturation as described above, the light chain comprises an anti-B7-H3 light chain and an anti-CD3 light chain, and the anti-B7-H3 light chain comprises the light chain of the B7-H3 antibody described above or the light chain of a B7-H3 antibody obtained by affinity maturation as described above.

[0076] In some embodiments, the heavy chain is linked to the light chain by a disulfide bond.

[0077] In some embodiments, the heavy chain of anti-B7-H3 is linked to the heavy chain of anti-CD3 by a disulfide bond.

[0078] In some embodiments, the heavy chain variable region of the anti-CD3 comprises the amino acid sequence shown in SEQ ID No.49.

[0079] SEQ ID No.49: EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGTSYVSWFAYWGQGTLVTVSS

[0080] The light chain variable region of the anti-CD3 comprises the amino acid sequence shown in SEQ ID No.9.

[0081] FIG. 5 shows the structure of a bispecific antibody. In some embodiments, the bispecific antibody comprises an immunoglobulin Fab domain that binds to B7-H3, an immunoglobulin Fab domain that binds to CD3, and a heterodimeric Fc region. The immunoglobulin Fab domain that binds to B7-H3 comprises the anti-B7-H3 light chain, the anti-B7-H3 heavy chain variable region VH, and the constant region CH1, and has the property of specifically binding to the B7-H3 antigen. The immunoglobulin Fab domain that binds to CD3 comprises the anti-CD3 light chain, the anti-CD3 heavy chain variable region VH, and the constant region CH1, and has the property of specifically binding to CD3, which is a surface antigen of immune cells. The heterodimeric Fc region comprises an Fc fragment linked to the anti-B7-H3 heavy chain and an Fc fragment linked to the anti-CD3 heavy chain, and the Fc fragment consists of the constant regions CH2 and CH3.

[0082] In some embodiments, the Fc fragment linked to the heavy chain of the anti-B7-H3 comprises a human or humanized Fc fragment.

[0083] In some embodiments, the Fc fragment linked to the heavy chain of the anti-B7-H3 comprises a human IgG1 Fc fragment.

[0084] In some embodiments, the Fc fragment linked to the heavy chain of the anti-CD3 comprises a human or humanized Fc fragment.

[0085] In some embodiments, the Fc fragment linked to the heavy chain of the anti-CD3 comprises a human IgG1 Fc fragment.

[0086] In some embodiments, the CH3 of the Fc fragment linked to the heavy chain of the anti-B7-H3 comprises a T394D mutation, a P395D mutation, and a P396D mutation.

[0087] In some embodiments, the CH3 of the Fc fragment linked to the heavy chain of the anti-CD3 comprises a P395K mutation, a P396K mutation, and a V397K mutation.

[0088] In some embodiments, the CH2 of the Fc fragment linked to the heavy chain of the anti-B7-H3 comprises the following mutations: L234A, L235A, and P329G.

[0089] Bispecific antibodies according to some embodiments of the present disclosure have the following properties: a. Binds to B7-H3 and CD3. b Activate T cells. Mediates the killing of B7-H3-positive tumor cells by cT cells. d. Inhibiting the growth of B7-H3 positive tumor cells in vivo.

[0090] The present disclosure further provides a biological material, which is any one of the following B1) to B6). B1): (b1) the B7-H3 antibody described above, (b2) the B7-H3 antibody obtained by affinity maturation according to any one of the above, (b3) a nucleic acid molecule encoding the heavy chain and / or light chain of any one of the bispecific antibodies described above, or a nucleic acid molecule encoding the heavy chain and / or light chain of the antigen-binding portion of any one of the antibodies (b1) to (b3) above. B2): An expression cassette comprising the nucleic acid molecule of B1). B3): A recombinant vector comprising the nucleic acid molecule of B1), or the expression cassette of B2). B4): A recombinant microorganism comprising the nucleic acid molecule of B1), the expression cassette of B2), or the recombinant vector of B3). B5): A cell line containing the nucleic acid molecule of B1), a cell line containing the expression cassette of B2), or a cell line containing the recombinant vector of B3). B6): A nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of any one of the antibodies (b1) to (b3) above, or a nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of an antigen-binding portion of any one of the antibodies (b1) to (b3) above.

[0091] Some embodiments of the present disclosure provide pharmaceutical compositions comprising any one or a combination of at least two of the above-described B7-H3 antibodies, the above-described affinity matured B7-H3 antibodies, and the above-described bispecific antibodies.

[0092] In some embodiments, the pharmaceutical composition further comprises any one or a combination of at least two of a pharma- ceutically acceptable carrier, diluent, or excipient.

[0093] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising an antibody or antibody fragment, a bispecific antibody or an antibody conjugate according to the present disclosure, and any pharma- ceutically acceptable carrier, surfactant and / or diluent. In some embodiments, the pharmaceutical composition further comprises one or more other therapeutic agents in addition to the antibody, bispecific antibody or antibody conjugate according to the present disclosure. In some embodiments, the other therapeutic agents include, but are not limited to, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, angiogenesis inhibitors, apoptotic agents, microtubule inhibitors and other agents for the treatment of cancer.

[0094] Some embodiments of the present disclosure further provide uses of the above-mentioned B7-H3 antibody, the above-mentioned affinity matured B7-H3 antibody, the above-mentioned bispecific antibody, or the above-mentioned pharmaceutical composition in the preparation of an anti-tumor medicament.

[0095] Some embodiments of the present disclosure further provide for the use of the B7-H3 antibody, any one of the affinity matured B7-H3 antibodies, any one of the bispecific antibodies, or any one of the pharmaceutical compositions in the treatment of tumors.

[0096] Some embodiments of the present disclosure further provide a method for treating a tumor-related disease, comprising administering to a subject in need thereof the B7-H3 antibody, any one of the affinity matured B7-H3 antibodies, any one of the bispecific antibodies, or any one of the pharmaceutical compositions. In some embodiments, the tumor comprises any one or a combination of at least two of the following: neural tumor, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer.

[0097] The present disclosure provides a B7-H3 antibody that binds to B7-H3 antigen with high affinity and uses thereof, which has important use value in the fields of immunotherapy and cancer treatment, overcomes the shortcomings of the prior art, and meets practical needs.

[0098] (1) The B7-H3 antibodies of the present disclosure bind with high affinity to the B7-H3 antigen and cells expressing the B7-H3 antigen, and can efficiently mediate killing of B7-H3-positive tumor cells by PBMCs. (2) The present disclosure further improves the affinity of the B7-H3 antibody by mutating the heavy chain complementarity determining region of the B7-H3 antibody. (3) The present disclosure uses B7-H3 antibody to construct a B7-H3×CD3 bispecific antibody, which has high affinity and stability and can effectively mediate tumor cell killing by PBMCs, thereby significantly suppressing tumor growth.

[0099] The present disclosure further includes nucleic acid sequences encoding these polypeptide chains. In the process of expressing the antibody, the nucleic acid sequence is inserted into a suitable vector. Vectors include, but are not limited to, plasmids, phage vectors, cosmids, artificial chromosomes, phages, and animal viruses. Expression vectors include elements for expression regulation, including, but are not limited to, promoters, transcription initiation sequences, enhancers, signal sequences, and the like. Promoters include, but are not limited to, T7 promoters, T3 promoters, SP6 promoters, β-actin promoters, EF-1α promoters, CMV promoters, and SV40 promoters. To introduce the expression vector into a host cell, any suitable method known in the art may be used, including, but not limited to, calcium phosphate coprecipitation, polyethyleneimine transfection, lipofection, electroporation, and PEI (polyethyleneimine) transfection.

[0100] Working Example Example 1 This embodiment provides a B7-H3 antibody, and the method for preparing the B7-H3 antibody includes the following steps:

[0101] (1) UniProtKB: B7-H3 extracellular domain (29-461) antigen with a sequence derived from Q5ZPR3 (CD276_HUMAN) was prepared, and the antigen sequence was cloned into the eukaryotic expression vector pcDNA3.1-TEV-cFc-His to generate the hB7-H3-TEV-cFc-His recombinant eukaryotic expression plasmid. The gene was introduced into 293F host cells by PEI transfection method, and after 5 days of culture, the cell supernatant was collected and affinity purified with protein A to obtain the B7-H3-TEV-cFc-His fusion protein. The human B7-H3 antigen was then obtained after cleavage with TEV protease and purification with a nickel column.

[0102] (2) Human peripheral blood was collected, peripheral blood mononuclear cells were isolated, total RNA was extracted, and cDNA was obtained using a reverse transcription kit. Using this cDNA as a template, specific primers for PCR were designed with reference to Lim, TS, et al. (Lim TS, Mollova S, Rubelt F, et al. An optimised procedure for amplification of rearranged human antibody genes of different isotypes[J]. New Biotechnology, 2010, 27(2):108-117.), and the VH fragment was amplified by PCR to obtain the human VH gene.

[0103] (3) Using the CD3-binding light chain sequence (SEQ ID No: 9) in the present inventor's published patent (CN110551221A), specific primers were designed and PCR amplified to obtain the CD3 VL gene.

[0104] (4) From the above human VH gene and CD3 VL gene, an scFv library was prepared by overlap extension PCR via G4S-linker, and a human-VH:CD3-VL antibody library was constructed. After cloning the library into a phage vector, host cells (TG1 Electrocompetent Cells: Lucigen.) were transformed by electroporation to obtain a human-VH:CD3-VL antibody library phage library. The phage library was packaged with a helper phage and precipitated by PEG precipitation. An appropriate amount of recombinant human B7-H3 antigen was added to a 96-well plate, incubated overnight, washed several times with PBST (phosphate buffer containing TWEEN20), and then the phage library was added and sieved. The antibody-positive clones enriched by stacking sieves were sequenced to obtain a B7-H3 antibody named 5D9. Table 1 shows the amino acid sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the antibody 5D9.

[0105] [Table 1]

[0106] Example 2 In this example, the antibody 5D9 described in Example 1 was expressed, purified and validated.

[0107] In the present disclosure, all antibodies were transiently expressed using 293F cells, and DNA encoding the heavy and light chains of antibody 5D9 was synthesized and cloned into the expression vector pcDNA3.1. Plasmids expressing the heavy and light chains of antibody 5D9 were transfected into 293F cells by PEI transfection. After culturing for 6 days, the cell culture supernatant was collected by centrifugation, and the antibody in the supernatant was purified by protein A affinity chromatography (GE healthcare).

[0108] In this disclosure, the positive control antibody mimic of Enoblituzumab has a sequence derived from WO2011US26689 and was expressed and purified by the above method. In this disclosure, the positive control antibody mimic of monoclonal antibody 8H9 has a sequence derived from WO2003US07004 and was expressed and purified by the above method.

[0109] The binding of 5D9 to the B7-H3 antigen was detected by ELISA. Human B7-H3 antigen was coated on a 96-well plate and left to stand overnight at 4°C. Antibody 5D9 and control samples were diluted to 10 μg / mL and serially diluted 4-fold to a total of 8 concentrations, which were then added to the antigen-coated wells and incubated. After washing, HRP (horseradish peroxidase)-labeled goat anti-human IgG (H+L) secondary antibody was added and color developed with TMB (3,3',5,5'-tetramethylbenzidine). The wavelength detected by the microplate reader was 450 nm, and the absorbance OD value was read, with the logarithm of the sample concentration on the X-axis and the absorbance OD 450 The EC value is plotted on the Y axis and a dose-response curve is calculated. 50 was calculated. EC 50 represents the ability of the antibody to bind to the B7-H3 antigen. A negative control IgG group and a positive control antibody mimic group of Enoblituzumab were also set up in the experiment, and the results are shown in FIG. 1.

[0110] As shown in FIG. 1, the binding of 5D9 to the B7-H3 antigen is good and slightly weaker than that of the mimic of Enoblituzumab to the B7-H3 antigen.

[0111] Example 3 This example provides affinity matured B7-H3 antibodies.

[0112] As can be seen from the above, the B7-H3 antibody 5D9 according to the present disclosure has a slightly weaker binding activity with the B7-H3 antigen than the mimic of Enoblituzumab. In this embodiment, in order to obtain an antibody with high affinity for the B7-H3 antigen, the heavy chain variable region of the antibody 5D9 is affinity matured in vitro to obtain a B7-H3 antibody by affinity maturation. The preparation method includes the following steps:

[0113] (1) Construction of affinity maturation library Random primers upstream and downstream of the triplet codons of the heavy chain complementarity determining regions HC-CDR1, HC-CDR2, and HC-CDR3 of 5D9 were synthesized, and a random mutation library of the heavy chain complementarity determining region of 5D9 was obtained by SOE-PCR. The random mutation library obtained was then cloned into a phage vector, and host cells (TG1 Electrocompetent Cells: Lucigen.) were transformed by electroporation to obtain a phage library of random mutations in the heavy chain complementarity determining region of 5D9.

[0114] (2) Screening of affinity maturation libraries As in the method of the above example, antibodies were produced by affinity maturation using phage display, the affinity maturation library phage library was packaged, and after three rounds of panning, the enriched antibody positive clones were sequenced to obtain 33 affinity matured variants, in which the light chain variable region (VL) has the amino acid sequence shown in SEQ ID No. 9, and the heavy chain framework regions VH-FR1, VH-FR2, VH-FR3, and VH-FR4 have the amino acid sequences shown in SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, respectively.

[0115] Table 2 shows the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of each variant.

[0116] [Table 2-1]

[0117] [Table 2-2]

[0118] Example 4 In this example, the B7-H3 antibody was expressed and purified by affinity maturation as described in Example 3.

[0119] According to the method described in Example 2, DNA encoding the heavy and light chains of each affinity maturation variant prepared in Example 4 was synthesized and cloned into the expression vector pcDNA3.1. Plasmids expressing the heavy and light chains of each affinity maturation variant were transfected into 293F cells by PEI transfection. After culturing for 5 to 6 days, the cell culture supernatant was collected by centrifugation, and the antibodies in the supernatant were purified by protein A affinity chromatography (GE Healthcare). A total of 33 B7-H3 antibodies were obtained by anti-affinity maturation.

[0120] Example 5 In this example, the functions of each affinity matured variant prepared in Example 4 were verified.

[0121] (1) Measurement of the binding activity of each affinity matured variant to the human B7-H3 antigen by ELISA The binding activity of affinity matured variants to human B7-H3 antigen was detected by ELISA. The logarithm of the sample concentration is plotted on the x-axis and the absorbance OD is plotted on the y-axis. 450 The EC value is plotted on the Y axis and a dose-response curve is calculated. 50 was calculated. EC 50 represents the ability of the sample to bind to human B7-H3 antigen. The experiment set up a positive control group, a mimic of Enoblituzumab, and a negative control group, hIgG, and the results are shown in Figure 2A to Figure 2I.

[0122] As shown in Figures 2A to 2I, each affinity matured variant has high binding activity to the human B7-H3 antigen, and the binding activity is almost similar to that of the mimic of enoblituzumab. Table 3 shows the binding activity (EC 50 ) is shown.

[0123] [Table 3]

[0124] (2) Measurement of the binding activity of each affinity matured variant to cells overexpressing the membrane-proximal antigen of B7-H3 by flow cytometry (FACS). In the present disclosure, in order to further study the binding activity of each of the above affinity matured variants with the functional region of the human B7-H3 antigen, the binding activity of the variants to cells overexpressing the membrane proximal antigen of B7-H3 was detected using FACS. First, a cell line stably expressing the membrane proximal side (243-534 bp) of B7-H3 was constructed and named positive cells. The same type of cells that do not express the membrane proximal side of B7-H3 were named negative cells.

[0125] Positive and negative cells were collected and washed once with 2% FBS / PBS (diluent) pre-cooled to 4°C. Then, the cells were cultured at a cell density of 5.0 × 10 6The cells were resuspended in diluent to a concentration of 100 μL / well, i.e., 5.0 × 105 / well, in a 96-well plate, and the above affinity maturation variants were prepared in diluent to a concentration of 5 μg / mL. 100 μL of the antibody solution was taken and uniformly mixed with the same volume of negative cells or positive cells, respectively, to a final concentration of 2.5 μg / mL. The 96-well plate was incubated at 4 °C for 1 hour, then centrifuged at 500 × g at 4 °C for 5 minutes to remove the supernatant. The cells were then washed twice with 200 μL of diluent, resuspended in PE anti-human IgG FC (Invitrogen) diluent, and incubated at 4 °C for 1 hour in the dark. The 96-well plate was then centrifuged at 500×g for 5 minutes at 4° C. to remove the supernatant, and the plate was washed twice with 200 μL of diluent. The cells were resuspended in 200 μL of diluent and immediately detected by the device. Figures 3A to 3E show the results.

[0126] 3A to 3E show some of the results. Each affinity matured variant bound to positive cells expressing the membrane proximal antigen of B7-H3 (solid line on the right side of the figure), but did not bind to negative cells (solid line on the left side of the figure).

[0127] (3) Measurement of affinity matured variant-mediated killing of B7-H3-positive tumor cells by PBMCs Based on the above ELISA and FACS results, 15 affinity matured variants (44F2, 43G11, 46H7, 45B11, 43D4, 42F7, 45F4, 46H9, 46D4, 47H4, 48E9, 49C3, 49E1, 50A1 and 50A6) with high affinity for human B7-H3 antigen were selected, and the killing activity of NCI-N87 tumor cells mediated by each affinity matured variant was measured using B7-H3 positive human gastric cancer cells NCI-N87 as target cells and human peripheral blood mononuclear cells (PBMC) as effector cells.

[0128] NCI-N87 cells were digested with trypsin to prepare a single-cell suspension, and the cell density was adjusted to 0.40 × 10 in 5% FBS-RPMI 1640 medium without phenol red. 6 The effector cell density of PBMC was adjusted to 4.00 × 10 6 The antibody was adjusted to 100 μL / well and added to a 96-well plate. The antibody to be measured was diluted to 40 μg / mL in 5% FBS-RPMI1640 medium without phenol red, and then diluted 1:4 by volume to obtain 10 concentrations: 40 μg / mL, 10 μg / mL, 2.5 μg / mL, 0.625 μg / mL, 0.1563 μg / mL, 0.0391 μg / mL, 0.0098 μg / mL, 0.0024 μg / mL, 0.0006 μg / mL, and 0.00015 μg / mL. The antibody was then added to a 96-well plate at 50 μL / well. The mixture was mixed homogeneously in a 96-well plate and incubated overnight in a 37°C, 5% CO2 incubator. The next day (about 20 hours later), the cytotoxicity was measured using a lactate dehydrogenase cytotoxicity assay kit (Beyotime). In addition, the killing activity of the antibody was measured, and the formula for the killing rate was as follows: Cytotoxicity%=(OD sample-SR) / (MR-SR)×100%, where SR is the OD spontaneous free well (target cells+effector cells) and MR is the OD maximum free well (target cells). Figures 4A and 4B show the results.

[0129] As shown in Figures 4A and 4B, each of the 15 affinity matured variants can mediate killing of B7-H3-positive human gastric cancer cells NCI-N87 by PBMCs.

[0130] Example 6 This example provides a bispecific antibody that targets CD3 and B7-H3.

[0131] (1) Sequence design of B7-H3×CD3 bispecific antibody To prepare bispecific antibodies targeting CD3 and B7-H3, affinity matured B7-H3 antibodies (42F7, 43G11, 49E1, 46H7) prepared as described above were selected. The bispecific antibody according to the embodiment of the present disclosure is constructed based on a common light chain and two different heavy chains, and has a structure as shown in FIG. 5. The bispecific antibody includes four polypeptide chains named anti-B7-H3 heavy chain (VH-CH1-hinge-CH2-CH3- from the N-terminus to the C-terminus), anti-CD3 heavy chain (VH-CH1-hinge-CH2-CH3- from the N-terminus to the C-terminus), anti-B7-H3 light chain (VL-CL from the N-terminus to the C-terminus), and anti-CD3 light chain (VL-CL from the N-terminus to the C-terminus), and forms one B7-H3-specifically binding immunoglobulin Fab domain, one CD3-specifically binding immunoglobulin Fab domain, and one heterodimeric Fc region. However, the light chains of anti-B7-H3 and anti-CD3 are a common light chain, and the CH3 domains of the two heavy chains contain asymmetric amino acid modifications with opposite charges to form the Fc region of the heterodimer described above.

[0132] The Fab domain of the immunoglobulin that specifically binds to B7-H3 comprises anti-B7-H3 heavy and light chains, the sequences of which are derived from the heavy and light chains of the affinity-matured B7-H3 antibodies (42F7, 43G11, 49E1, 46H7) prepared in the above examples.

[0133] The Fab domain of an immunoglobulin that specifically binds to CD3 comprises the heavy chain (SEQ ID No. 49) and the light chain (SEQ ID No. 9) of anti-CD3.

[0134] SEQ ID No.49: EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAMYYCVRHGNFGTSYVSWFAYWGQGTLVTVSS

[0135] SEQ ID No.9: DIQMTQEPSLTTSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTITGVQPEDEAIYFCALWYSNLWVFGGGTKLEIK

[0136] The Fc portion of the antibody was partially sequence-modified according to the method in the present inventor's published patent PCT (WO2017034770A1). However, the CH3 domain of the Fc portion of the heavy chain of anti-B7-H3 was mutated as T394D, P395D, P396D (EU), and the mutations were marked as OB to have a negative charge. The CH3 domain of the Fc portion of the heavy chain of anti-CD3 was mutated as P395K, P396K, V397K (EU), and the mutations were marked as OA to have a positive charge. In addition, in order to reduce the binding of Fc to its receptor FcγR, the CH2 domains of the Fc portions of the heavy chains of anti-B7-H3 and anti-CD3 both contain L234A, L235A, and P329G (LALA-PG) mutations.

[0137] (2) Molecular cloning of expression plasmids DNAs encoding the heavy and light chains of B7-H3 antibodies 42F7, 43G11, 49E1, and 46H7 were synthesized, respectively, and their Fab gene fragments were constructed into the eukaryotic expression vector pFUSEss-CHIg-hG1-Fc1 (containing OB, L234A, L235A mutations, and P329G mutations), to obtain an expression plasmid named plasmid 1. The heavy chain of anti-CD3 was cloned into the plasmid pFUSE-hIgG1-Fc2 (containing OA, N106T mutations, L234A mutations, L235A mutations, and P329G mutations), to obtain an expression plasmid named plasmid 2. The light chain of anti-CD3 was cloned into pCDNA3.1, to obtain a plasmid named plasmid 3.

[0138] (3) Expression and purification Recombinant plasmid 1, plasmid 2, and plasmid 3 were extracted and co-transfected with PEI and HEK293F cells. The co-transfected cell suspension was cultured in an incubator shaker at 37°C, 5% CO2, and 120 rpm for 6 days in the dark. The culture supernatant was centrifuged at high speed and then packed into a protein A affinity chromatography column for purification. To detect and confirm the size and purity of various BsAbs, SDS-PAGE analysis was performed to obtain B7-H3×CD3 bispecific antibodies named 42F7×CD3, 43G11×CD3, 49E1×CD3, and 46H7×CD3. A similar method was used to construct a 8H9×CD3 bispecific antibody, a mimic of the positive control monoclonal antibody 8H9.

[0139] Example 7 In this example, the function of the B7-H3×CD3 bispecific antibody prepared in Example 6 was verified.

[0140] (1) Binding activity of B7-H3×CD3 bispecific antibody The activity of the B7-H3×CD3 bispecific antibody prepared above to bind to the B7-H3 antigen was measured by indirect ELISA. The operation method is as follows. B7-H3 antigen diluted to 1 μg / mL was added to a microplate at 100 μL / well and left overnight at 4°C while coated. The next day, it was washed with 0.05% PBST and blocked at room temperature for 2 hours. After washing again with 0.05% PBST, serially diluted B7-H3×CD3 bispecific antibody (PBS diluted) was added and incubated at room temperature for 1 hour. After washing the unbound antibody with 0.05% PBST, HRP-labeled goat anti-human IgG (H+L) secondary antibody (Proteintech, Cat.SA0001-17) was added and incubated at room temperature for 1 hour. After washing with 0.05% PBST, TMB color development solution was added, and the plate was incubated at room temperature for 5 minutes in the dark, and the reaction was stopped with H2SO4. The absorbance was measured at 450 nm using a microplate reader, and log (antibody concentration) was plotted as the abscissa and absorbance OD 450The results of plotting dose-response curves with CD4+ / - as the ordinate are shown in Figure 6. As can be seen from Figure 6, all of the tested B7-H3×CD3 bispecific antibodies have high binding activity to the human B7-H3 antigen.

[0141] (2) Measurement of T cell activation by B7-H3×CD3 bispecific antibody T cell activation by B7-H3×CD3 was evaluated using an assay system consisting of Jurkat / NFAT-Luc reporter gene cell line and B7-H3-positive human renal cancer cell line A498. In the Jurkat / NFAT-Luc cell line, the luciferase gene is controlled by the NFAT (nuclear factor of activated T-cell) transcription factor, and these cells were used as effector cells, and B7-H3-positive A498 cells were used as target cells. When Jurkat / NFAT-Luc cells were co-cultured with A498 cells, luciferase was not expressed. However, when B7-H3×CD3 was added, B7-H3 on A498 cells activated the CD3 signal pathway in Jurkat / NFAT-Luc cells via B7-H3×CD3, promoting luciferase expression. Therefore, the strength of Jurkat T cell activation can be determined by detecting the amount of luciferase expression in the cells.

[0142] In the experiment, A498 cells and Jurkat / NFAT-Luc cells were added sequentially to a 96-well cell culture plate, and B7-H3×CD3 was diluted to concentrations of 10 μg / mL, 2.5 μg / mL, 0.625 μg / mL, 0.15625 μg / mL, 0.0390 μg / mL, 0.0097 μg / mL, 0.0024 μg / mL, and 0.0006 μg / mL. Different concentrations of B7-H3×CD3 were added to the cell culture plate and cultured at 37°C for 6 hours. ONE-Glo Luciferase assay reagent was added, and the chemiluminescence value was measured using a multifunctional microplate reader. The logarithm of the antibody concentration was used as the abscissa and the average chemiluminescence value was used as the ordinate. Four-parameter fitting was performed to plot the dose-response curve, and the EC 50 The value was calculated. EC 50indicates the activity of T cell activation by B7-H3×CD3. A positive control antibody 8H9×CD3 group was also included in the experiment.

[0143] The results, as shown in FIG. 7, showed that all four B7-H3×CD3 bispecific antibodies disclosed herein showed excellent activation effects on Jurkat T cells when co-incubated with B7-H3-positive tumor cells A498 and Jurkat / NFAT-Luc cells.

[0144] (3) Measurement of the killing effect of B7-H3×CD3 bispecific antibodies against B7-H3 positive cells The CD3 binding arm of the bispecific antibody B7-H3×CD3 specifically binds to the CD3 complex on the surface of T cells, and the other end of the B7-H3 binding arm specifically binds to the B7-H3 molecule on the surface of tumor cells, forming an immune bridge between T cells and tumor cells, activating T cells and releasing cytotoxic proteins such as perforin and granzyme B, which kill tumor cells. When the cell membrane of a tumor cell is damaged, the permeability of the cell membrane increases and lactate dehydrogenase (LDH) in the cytoplasm is released into the culture supernatant. When a certain amount of the supernatant is taken and lactic acid, a reaction substrate for lactate dehydrogenase, is added, lactate dehydrogenase promotes the dehydrogenation reaction, producing a red product formazan that can produce an absorption peak at 490 nm. The higher the content of lactate dehydrogenase in the cell supernatant, the darker the color and the greater the absorbance. Therefore, the amount of lactate dehydrogenase released from tumor cells can be quantified by measuring the absorbance, and the tumor cell killing activity mediated by B7-H3×CD3 by PBMCs can be calculated. The formula for the killing rate is as follows: Cytotoxicity%=(OD sample-SR) / (MR-SR)×100%, where SR is the OD spontaneous free well (target cells+effector cells) and MR is the OD maximum free well (target cells).

[0145] In this disclosure, human gastric cancer cells NCI-N87, A498, and HepG2, which differentially express B7-H3, were used as target cells, and human peripheral blood mononuclear cells (PBMCs) were used as effector cells, and the tumor cell killing activity of PBMCs mediated by four B7-H3×CD3 bispecific antibodies was measured.

[0146] Freshly isolated human peripheral blood mononuclear cells (PBMCs) were harvested and cultured at a density of 2.00 x 10 6 The cell concentration was adjusted to 2.00 × 10 cells / mL. NCI-N87, A498, and HepG2 cells with good proliferation were harvested and the cell concentration was adjusted to 2.00 × 10 5 The concentration was adjusted to 10 cells / mL. The sample was diluted to 30 μg / mL with 1× PBS buffer (pH 7.4), and then serially diluted 4-fold to obtain a total of 10 concentrations: 30 μg / mL, 7.5 μg / mL, 1.875 μg / mL, 0.4688 μg / mL, 0.1172 μg / mL, 0.0293 μg / mL, 0.0073 μg / mL, 0.0018 μg / mL, 0.0005 μg / mL, and 0.0001 μg / mL. 50 μL / well of target cells, 100 μL / well of PBMC, and 50 μL / well of serially diluted sample were added to a 96-well cell culture plate in that order, and mixed uniformly so that the ratio of the number of cells per unit volume of PBMC and target cells was 20:1. The initial concentration of the samples was 10 μg / mL, and RPMI1640 medium without phenol red was added to the control wells up to 200 μL. The culture plate was placed in a 37°C, 5% CO2 incubator and incubated for 21 ± 1 h, after which the cytotoxicity was measured using a lactate dehydrogenase cytotoxicity assay kit (Biyuntian). The logarithm of the antibody concentration was taken as the abscissa and the killing activity as the ordinate, and a four-parameter fitting was performed. The dose-response curves were plotted, and the EC 50 The value was calculated. EC 50 represents the killing activity of B7-H3×CD3 against target cells. The results are shown in Figures 8A-8C.

[0147] As shown in Figures 8A to 8C, the bispecific antibody B7-H3 x CD3 according to the present disclosure exhibited excellent killing effects against all of NCI-N87, A498, and HepG2 cells in which B7-H3 is differentially expressed.

[0148] (4) Study on the stability of B7-H3×CD3 bispecific antibody To evaluate the stability of the B7-H3×CD3 bispecific antibody after exposure to 40°C for different periods of time, a well-sealed sample (1 mg / mL) was placed in an incubator (BINDER KBF240) at 40°C. At designated time points (baseline (day 0), day 14), 20 μg of the sample was taken and the purity was measured by SEC-HPLC. The conditions for the SEC-HPLC were as follows:

[0149] Size exclusion chromatography column: Superdex200 10 / 300 GL increase Mobile phase: 50mM NaAc, 51mM NaCl, 0.05mM EDTA pH 5.5 Flow rate: 0.5mL / min UV detection wavelength: 280 nm Collection time: 35 minutes

[0150] The equipment used was an AKTA pure 25 L1 chromatograph and the software for recording the graphs and calculating the percentage of residual monomer was UNICORN.

[0151] As shown in Figures 9A to 9D, Figure 9A shows the results of SEC-HPLC purity measurement of 46H7 x CD3 after 14 days at 40 ° C., where the peak retention time of the 46H7 x CD3 bispecific antibody in the liquid phase is 28.5 minutes, the peak area ratio is 100%, and the purity of the 46H7 x CD3 bispecific antibody is higher than 98%. Figure 9B shows the results of SEC-HPLC purity measurement of 43G11 x CD3 after 14 days at 40 ° C., where the peak retention time of the 43G11 x CD3 bispecific antibody in the liquid phase is 28.5 minutes, the peak area ratio is 100%, and the purity of the 43G11 x CD3 bispecific antibody is > 98%. Figure 9C shows the results of SEC-HPLC purity measurement of 49E1 x CD3 after 14 days at 40 ° C. However, the retention time of the peak of the 49E1×CD3 bispecific antibody in the liquid phase is 28.5 minutes, the peak area ratio is 100%, and the purity of the 49E1×CD3 bispecific antibody is >98%. Figure 9D shows the results of the purity measurement by SEC-HPLC of 42F7×CD3 after leaving it at 40°C for 14 days. However, the retention time of the peak of the 42F7×CD3 bispecific antibody in the liquid phase is 28.5 minutes, the peak area ratio is 100%, and the purity of the 42F7×CD3 bispecific antibody is higher than 98%. From the above results, since the proportion of the multimer of the above antibody does not increase over time under the experimental condition of 40°C, it was considered that the above tetravalent bispecific antibody has excellent thermal stability and can be developed.

[0152] (5) Measurement of the antitumor effect of B7-H3×CD3 bispecific antibody in mice in vivo Based on the results of the above examples, one of the B7-H3×CD3 bispecific antibodies (ie, 42F7×CD3 bispecific antibody) was selected, and its antitumor effect in vivo in mice was measured.

[0153] Using 8-week-old female NOD-SCID mice (purchased from Beijing Weitong Lihua Laboratory Animal Co., Ltd.), MC38 / hB7-H3 cells in the logarithmic growth phase and freshly isolated PBMCs were collected and inoculated into the right forelimb of the mice at 100 μL / mouse, and the mice were divided into groups the day after inoculation. The day of grouping was designated as D0, and drug administration began on D0. For the experiment, the mice were divided into two groups, an isotype control hIgG group (5 mg / kg, iv) and a B7-H3 × CD3 group (5 mg / kg, iv), with six mice in each group. Drugs were administered via tail vein injection (iv) on D1, D5, D8, D11, and D15. After the start of drug administration, the tumor size was observed and the mice were weighed. Tumor volume was calculated as follows: Tumor volume (mm 3 ) = 0.5 × (tumor long diameter × tumor short diameter 2 The tumor growth rate (T / C%) and tumor inhibition rate (TGI) were calculated from the tumor volume, and the therapeutic effect was evaluated from the tumor inhibition rate (TGI). Figure 10 shows the results.

[0154] As shown in Figure 10, B7-H3×CD3 significantly suppressed tumor growth in a mouse tumor model in an MC38 / hB7-H3 xenograft model subcutaneously implanted into NOD / SCID mice in which the human immune system had been reconstituted with PBMC.

[0155] As described above, the B7-H3 antibody of the present disclosure binds to the B7-H3 antigen with high affinity, and the affinity of the B7-H3 antibody can be further improved by mutating the heavy chain complementarity determining region, which can effectively mediate the killing of B7-H3-positive tumor cells by PBMCs. Furthermore, the present disclosure uses the B7-H3 antibody to construct a B7-H3×CD3 bispecific antibody, which has high affinity and stability and can effectively mediate the killing of tumor cells by PBMCs, thereby significantly suppressing tumor growth.

[0156] The present applicant has described the detailed method of the present disclosure through the above examples, but the present disclosure is not limited to the above detailed method, that is, the present disclosure can be implemented without relying on the above detailed method. Those skilled in the art should understand that any improvement of the present disclosure, the equivalent replacement of each raw material of the product of the present disclosure, the addition of auxiliary ingredients, the selection of forms, etc., all fall within the protection scope of the present disclosure. (Additional Note) The present disclosure includes the following aspects. Section 1: 1. A B7-H3 antibody comprising: The heavy chain complementarity determining region of the B7-H3 antibody comprises the amino acid sequences shown in SEQ ID Nos. 6 to 8. The light chain complementarity determining region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 14 to 16. A B7-H3 antibody characterized in that Section 2: the heavy chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No. 1, The light chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No. 9. Item 2. The B7-H3 antibody according to item 1, Section 3: Prepared by mutation treatment from the B7-H3 antibody according to item 1 or 2. A B7-H3 antibody obtained by affinity maturation. Section 4: The affinity matured B7-H3 antibody has a heavy chain complementarity determining region CDR1 comprising an amino acid sequence shown in any one of SEQ ID No. 6 or SEQ ID Nos. 17 to 25, a heavy chain complementarity determining region CDR2 comprising an amino acid sequence shown in any one of SEQ ID Nos. 7 or SEQ ID Nos. 26 to 37, and a heavy chain complementarity determining region CDR3 comprising an amino acid sequence shown in any one of SEQ ID Nos. 8 or SEQ ID Nos. 38 to 48; The light chain complementarity determining region of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 14 to 16. Item 4. A B7-H3 antibody obtained by affinity maturation according to item 3. Section 5: The affinity matured light chain variable region of the B7-H3 antibody comprises the amino acid sequence shown in SEQ ID No. 9, The framework region of the heavy chain of the affinity matured B7-H3 antibody comprises the amino acid sequence shown in SEQ ID Nos. 2 to 5. A B7-H3 antibody obtained by affinity maturation according to item 3 or 4. Item 6: Contains two heavy chains and two light chains The heavy chain comprises an anti-B7-H3 heavy chain, which comprises the heavy chain of the B7-H3 antibody according to Aspect 1 or 2 or the heavy chain of the affinity-matured B7-H3 antibody according to any one of Aspects 3 to 5, and an anti-CD3 heavy chain; The light chain comprises an anti-B7-H3 light chain, which comprises the light chain of the B7-H3 antibody described in Aspect 1 or 2 or the light chain of the affinity-matured B7-H3 antibody described in any one of Aspects 3 to 5, and an anti-CD3 light chain; the heavy chain is linked to the light chain by a disulfide bond; The heavy chain of the anti-B7-H3 is linked to the heavy chain of the anti-CD3 by a disulfide bond. A bispecific antibody characterized by: Section 7: the heavy chain variable region of anti-CD3 comprises the amino acid sequence shown in SEQ ID No. 49, The light chain variable region of the anti-CD3 comprises the amino acid sequence shown in SEQ ID No. 9. Item 7. The bispecific antibody according to Item 6, Section 8: The bispecific antibody comprises a Fab domain of an immunoglobulin that binds to B7-H3, a Fab domain of an immunoglobulin that binds to CD3, and a heterodimeric Fc region; the Fab domain of the immunoglobulin binding to B7-H3 comprises the light chain of anti-B7-H3 and the heavy chain variable region VH and constant region CH1 of anti-B7-H3; the Fab domain of the immunoglobulin binding to CD3 comprises the light chain of the anti-CD3 and the heavy chain variable region VH and constant region CH1 of the anti-CD3; The Fc region of the heterodimer comprises an Fc fragment linked to the heavy chain of anti-B7-H3 and an Fc fragment linked to the heavy chain of anti-CD3. Item 8. The bispecific antibody according to Item 6 or 7, Section 9: The Fc fragment linked to the heavy chain of the anti-B7-H3 comprises a human Fc fragment or a humanized Fc fragment. The bispecific antibody according to item 8, characterized in that: Section 10: The CH3 of the Fc fragment linked to the heavy chain of the anti-B7-H3 antibody contains the following mutations: T394D, P395D, and P396D. Item 10. The bispecific antibody according to item 8 or 9, Section 11: The CH3 of the Fc fragment linked to the heavy chain of the anti-CD3 antibody contains the mutations P395K, P396K, and V397K. 11. The bispecific antibody according to any one of items 8 to 10, Section 12: The CH2 of the Fc fragment linked to the heavy chain of the anti-B7-H3 antibody contains the following mutations: L234A, L235A, and P329G. Item 12. The bispecific antibody according to any one of Items 8 to 11, Section 13: B1): (b1) a nucleic acid molecule encoding the heavy chain and / or the light chain of any one of the B7-H3 antibody according to item 1 or 2, (b2) a B7-H3 antibody obtained by affinity maturation according to any one of items 3 to 5, and (b3) a bispecific antibody according to any one of items 6 to 12, or a nucleic acid molecule encoding the heavy chain and / or the light chain of the antigen-binding portion of any one of the antibodies (b1) to (b3); B2): An expression cassette comprising the nucleic acid molecule of B1); B3): A recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2); B4): A recombinant microorganism comprising the nucleic acid molecule of B1), a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3); B5): A cell line comprising the nucleic acid molecule of B1), a cell line comprising the expression cassette of B2), or a cell line comprising the recombinant vector of B3); B6): A nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of any one of the antibodies (b1) to (b3) above, or a nucleic acid molecule encoding a heavy chain variable region and / or a light chain variable region of an antigen-binding portion of any one of the antibodies (b1) to (b3) above. Any one of B1) to B6) above. A biological material characterized by: Section 14: The antibody comprises any one or a combination of at least two of the B7-H3 antibody according to item 1 or 2, the affinity matured B7-H3 antibody according to any one of items 3 to 5, or the bispecific antibody according to any one of items 6 to 12. A pharmaceutical composition comprising: Section 15: For tumors including one or a combination of at least two of the following: neural tumors, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer. Use of the B7-H3 antibody according to item 1 or 2, the B7-H3 antibody obtained by affinity maturation according to any one of items 3 to 5, the bispecific antibody according to any one of items 6 to 12, or the pharmaceutical composition according to item 14 in the preparation of an antitumor drug. Section 16: Preferably, the cancer includes one or a combination of at least two of the following: neural tumor, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer. Use of the B7-H3 antibody according to item 1 or 2, the affinity matured B7-H3 antibody according to any one of items 3 to 5, the bispecific antibody according to any one of items 6 to 12, or the pharmaceutical composition according to item 14, for treating a tumor. Section 17: A method for treating a tumor-related disease, comprising: A method for treating a tumor-related disease comprising administering to a subject in need thereof the B7-H3 antibody according to Aspect 1 or 2, the affinity matured B7-H3 antibody according to Aspect 3 to 5, the bispecific antibody according to Aspect 6 to 12, or the pharmaceutical composition according to Aspect 14; Preferably, the tumor-related disease includes any one or a combination of at least two of the following: neural tumor, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer. A method for treating a tumor-related disease. [Industrial Applicability]

[0157] The present disclosure provides a B7-H3 antibody and its use. The B7-H3 antibody according to the present disclosure can bind to the B7-H3 antigen and cells expressing the B7-H3 antigen with high affinity, and can efficiently mediate the killing of B7-H3-positive tumor cells by PBMCs. The B7-H3 antibody is used to construct a B7-H3×CD3 bispecific antibody, and the bispecific antibody has high affinity and stability, and can effectively mediate the killing of tumor cells by PBMCs, thereby significantly suppressing tumor growth. Therefore, the B7-H3 antibody according to the present disclosure is highly practical.

Claims

1. A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID No. 6, A heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID No. 7; A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID No. 8; A light chain CDR1 comprising the amino acid sequence shown in SEQ ID No. 14; a light chain CDR2 comprising the amino acid sequence shown in SEQ ID No. 15; and A light chain CDR3 comprising the amino acid sequence shown in SEQ ID No.

16. A B7-H3 antibody characterized by:

2. A heavy chain variable region comprising the amino acid sequence shown in SEQ ID No. 1; and a light chain variable region comprising the amino acid sequence shown in SEQ ID No.

9. The B7-H3 antibody according to claim 1.

3. A heavy chain variable region comprising any one of the combinations of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 shown in the table below; a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence shown in SEQ ID No. 14, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID No. 15, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID No. 16; A B7-H3 antibody characterized by: 【Table 1】 【Table 2】

4. A light chain variable region comprising the amino acid sequence shown in SEQ ID No. 9; and a framework region comprising the amino acid sequence shown in SEQ ID No. 2 to 5. The B7-H3 antibody of claim 3.

5. A heavy chain of anti-B7-H3 comprising the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of claim 1, and a light chain of anti-B7-H3 comprising the light chain CDR1, light chain CDR2, and light chain CDR3 of claim 1, which is linked to the heavy chain of anti-B7-H3 by a disulfide bond; an anti-CD3 heavy chain and an anti-CD3 light chain linked to the anti-CD3 heavy chain by a disulfide bond; The heavy chain of the anti-B7-H3 antibody and the heavy chain of the anti-CD3 antibody are bound to each other by a disulfide bond. A bispecific antibody characterized by:

6. A heavy chain comprising the heavy chain variable region of the B7-H3 antibody of claim 3, and a light chain comprising the light chain variable region of the B7-H3 antibody of claim 3, which is bound to the heavy chain via a disulfide bond. an anti-CD3 heavy chain and an anti-CD3 light chain linked to the anti-CD3 heavy chain by a disulfide bond; The heavy chain containing the heavy chain variable region of the B7-H3 antibody is bound to the heavy chain of the anti-CD3 antibody by a disulfide bond. A bispecific antibody characterized by:

7. The heavy chain of the anti-CD3 comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID No. 49, The light chain of the anti-CD3 antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID No.

9. The bispecific antibody according to claim 5 .

8. The heavy chain of the anti-CD3 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 49; The light chain of the anti-CD3 antibody comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID No.

9. The bispecific antibody according to claim 6 .

9. A Fab region of anti-B7-H3, comprising a heavy chain variable region and a CH1 domain, and a light chain variable region and a CL domain; a Fab region of anti-CD3 comprising a heavy chain variable region and a CH1 domain, and a light chain variable region and a CL domain; and a heterodimeric Fc region comprising an Fc region linked to the CH1 domain of the Fab region of the anti-B7-H3 and an Fc region linked to the CH1 domain of the Fab region of the anti-CD3. The bispecific antibody according to claim 6 .

10. The Fc region linked to the CH1 domain in the Fab region of the anti-B7-H3 comprises a human Fc region. The bispecific antibody according to claim 9 .

11. The CH3 domain of the Fc region linked to the CH1 domain of the Fab region of the anti-B7-H3 antibody comprises a T394D mutation, a P395D mutation, or a P396D mutation. The bispecific antibody according to claim 9 .

12. The CH3 domain of the Fc region linked to the CH1 domain of the Fab region of the anti-CD3 comprises a P395K mutation, a P396K mutation, or a V397K mutation. The bispecific antibody according to claim 9 .

13. The CH2 domain of the Fc region linked to the CH1 domain of the Fab region of the anti-B7-H3 antibody comprises a L234A mutation, a L235A mutation, or a P329G mutation. The bispecific antibody according to claim 9 .

14. B1): A combination of a nucleic acid encoding an anti-B7-H3 heavy chain and a nucleic acid encoding a light chain in the B7-H3 antibody according to any one of claims 1 to 4, or the bispecific antibody according to any one of claims 5 to 13; B2): an expression cassette comprising a nucleic acid encoding the heavy chain of anti-B7-H3 and a nucleic acid encoding the light chain of anti-B7-H3, or a combination of an expression cassette comprising a nucleic acid encoding the heavy chain of anti-B7-H3 and an expression cassette comprising a nucleic acid encoding the light chain of anti-B7-H3; B3): A recombinant vector comprising a nucleic acid encoding the heavy chain of anti-B7-H3 and a nucleic acid encoding the light chain of anti-B7-H3, or a combination of a recombinant vector comprising a nucleic acid encoding the heavy chain of anti-B7-H3 and a recombinant vector comprising a nucleic acid encoding the light chain of anti-B7-H3; B4): A recombinant microorganism comprising any one of B1) to B3); B5): A cell line comprising any one of B1) to B3); or B6): A combination of a nucleic acid encoding a heavy chain variable region and a nucleic acid encoding a light chain variable region of anti-B7-H3 in the B7-H3 antibody according to any one of claims 1 to 4 or the bispecific antibody according to any one of claims 5 to 13; Any one of B1) to B6) above. A biological material characterized by:

15. A method for treating a cancer comprising administering to a patient a therapeutically effective amount of a B7-H3 antibody according to any one of claims 1 to 4, or a bispecific antibody according to any one of claims 5 to 13. A pharmaceutical composition comprising:

16. For tumors including one or a combination of at least two of the following: neural tumors, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer. Use of the pharmaceutical composition according to claim 15 in the preparation of an antitumor medicament.

17. Includes one or a combination of at least two of the following: neuronal tumors, colon cancer, rectal cancer, liver cancer, head and neck cancer, lung cancer, malignant melanoma, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, and prostate cancer. The pharmaceutical composition according to claim 15 for treating tumors.

18. A method for producing an affinity matured B7-H3 antibody, comprising: performing a mutation treatment on the B7-H3 antibody described in claim 1 to obtain an affinity matured B7-H3 antibody.

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