Anti-b7h3 antibodies and application thereof

Abstract

The present invention provides anti-B7H3 antibodies and their application thereof A group of murine anti-B7H3 monoclonal antibodies are prepared by hybridoma technology, using recombinant B7H3 protein as immunogen. Human-murine chimeric antibodies generated from the aforementioned murine anti-B7H3 hybridomas are capable of specifically binding to recombinant B7H3 protein and membrane-bound B7H3 on tumor cell lines. The derived B7H3 scFv-CD3ε-engineered T cells exhibit a significant cytotoxic effect on B7H3-positive tumor cell lines. Therefore, the anti-B7H3 antibodies and B7H3 scFv-CD3ε-engineered T cells have great application prospects in treating or ameliorating B7H3-associated diseases.

Description

[0001] Anti-B7H3 antibodies and application thereof

[0002] Field Of Invention

[0003] The present invention relates to the field of biotechnology, specifically to a group of anti -human B7H3 antibodies, anti-B7H3 scFv-CD3e fusion protein engineered immune cells, and their preparation and application.

[0004] Background

[0005] B7H3 (also known as CD276) is a type I transmembrane glycoprotein, belonging to the B7-CD28 family of immune checkpoint molecules. It consists of an N-terminal signal peptide, an extracellular domain composed of immunoglobulin constant (IgC) and variable (IgV) subunit, a transmembrane domain, and 45 amino acids of the cytoplasmic domain. Based on the structural characteristics of the extracellular domain, it is classified into 2Ig-B7H3 and 4Ig-B7H3 isoforms, whereas the extracellular of 21g- B7H3 comprises an IgV-IgC structure, expressed in mouse and human cells; the extracellular of 4Ig-B7H3 comprises a tandemly repeated IgV-IgC-IgV-IgC structure, specifically expressed in human cells (Collins M, 2005; Hofmeyer K, 2008). Previous studies indicate that 2Ig-B7H3 in mice and 4Ig-B7H3 in humans exhibit similar functionalities without differences (Ling V, 2003; Hofmeyer K, 2008). Crystallization analysis reveals that the FG loop of the IgV region is one of the key epitopes for its functional activity (Vigdorovich, 2013).

[0006] B7H3 is a potential bifunctional molecule with already-known T cell co-stimulatory / co-inhibitory effects. Early studies find that B7H3 promotes the proliferation of CD4+ and CD8+ T cells, with enhancement of IFN-y secretion and T cell cytotoxicity (Chapoval Al, 2001). Recently, numerous studies have demonstrated that B7H3 exhibited significant immunosuppressive effects, potentially inhibiting T cell activation and proliferation through NFAT (nuclear factor activating T cells), NF-KB (nuclear factor-KB), and AP-1 factors (activator protein 1) pathways (Suh WK, 2003; Veenstra RG, 2015, Hofmeyer K, 2008). Additionally, B7H3 can also inhibit the function of natural killer (NK) cells (Castriconi R, 2004). B7H3 primarily exists in the form of membrane-bound and soluble protein The membrane-bound B7H3 is mainly on the surface of tumor cells, occasionally in exosomes and other extracellular vesicles; soluble B7H3 is derived from membrane protein through metalloproteinase cleavage and abundantly distributed in the serum of tumor patients, indicating that B7H3 can serve as a biomarker (Xie, 2016). B7H3 is widely expressed at the mRNA level, with detectable signals in various tissues and organs, including heart, liver, placenta, kidney, prostate, testis, uterus, pancreas, small intestine, and colon (Collins M, 2005). However, B7H3 protein expression is strictly regulated and limited to non-immune cells such as resting fibroblasts, endothelial cells, osteoblasts, and amniotic stem cells, as well as activated dendritic cells, monocytes, T cells, B cells, andNK cells, as reported (Yi KH, 2009) Clinical investigations find that B7H3 is abnormally highly expressed in various cancer cells or tissues, including gastric cancer, lung cancer, prostate cancer, renal cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, liver cancer, colorectal cancer, oral cancer, bladder cancer, osteosarcoma, and hematological malignancies. B7H3 is closely associated with tumor growth, metastasis, recurrence, and poor prognosis in the aforementioned malignant tumors. It can downregulate the helper type-1 T cell- mediated immune response, inhibiting the activation of CD4+ T cells and suppressing cytokine production, thereby promoting the immune escape of tumor cells. Additionally, other studies have reported that elevated B7H3 expression in tumor cells leads to increased adhesion capacity of cell adhesion molecules, thereby enhancing tumor cell migration and invasion capabilities, further supporting its involvement in tumor immune escape. In summary, expression levels of B7H3 are closely associated with poor patient prognosis and clinical outcomes, making it a promising target for tumor immunotherapy.

[0007] Adoptive T-cell therapy (ACT), referring to allogenic / autologous T cell product that has been genetically modified using viral / non-viral vectors encoding tumor- associated / specific antigen / antigenic peptide recognizable antigen binding fragment / T cell receptor (TCR), is infused into the patient to directly kill tumor cells or stimulate the body's immune response to kill tumor cells. It’s such an attractive approach of tumor immunotherapy, including CAR-T, TCR-T, and TIL therapies, that it has achieved great breakthroughs in tumor immunotherapy Till now, over ten CAR-T products and one TCR-T product have been approved for commercialization, with over 300 ACT products undergoing clinical investigation. This therapeutic approach not only brings curative benefits to patients with hematological tumors, but also exerts encouraging clinical efficacy in solid tumors. Decades of B7H3 -targeted T cell products exhibited promising antitumor activity in preclinical studies, including pancreatic cancer, gastric cancer, liver cancer, and osteosarcoma.

[0008] Given this, the present invention is hereby proposed.

[0009] Summary Of the Invention

[0010] The first objective of the present invention is to provide an antibody or antigen-binding fragment that specifically binds to B7H3.

[0011] The second objective of the present invention is to provide a B7H3 scFv-CD3e fusion protein prepared from the aforementioned antibody or antigen-binding fragment.

[0012] The third objective of the present invention is to provide an immune cell that express the aforementioned B7H3 scFv-CD3s fusion protein.

[0013] The fourth objective of the present invention is to provide a method for preparing and using the aforementioned immune cells.

[0014] Specifically, according to the first aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof specifically binding to B7H3, and the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein:

[0015] (1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group:

[0016] (a) Amino acid sequences are set forth in SEQ ID NO:1 , 2, 3;

[0017] (b) Amino acid sequences are set forth in SEQ ID NO: 7, 8, 9; or CDRs having at least 85% sequence identity with the amino acid sequences set forth in (a) or (b); and

[0018] (2) The light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the group:

[0019] (a) Amino acid sequences are set forth in SEQ ID NO:4, 5, 6;

[0020] (b) Amino acid sequences are set forth in SEQ ID NO: 10, 11, 12; or CDRs having at least 85% sequence identity with the amino acid sequences set forth in (a) or (b).

[0021] Preferably, the CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region are selected from (a) or (b):

[0022] (a). HCDR1 sequence of the heavy chain variable region set forth in SEQ ID NO:1, HCDR2 sequence of the heavy chain variable region set forth in SEQ ID NO:2, HCDR3 sequence of the heavy chain variable region set forth in SEQ ID NO:3, LCDR1 sequence of the light chain variable region set forth in SEQ ID NO:4, LCDR2 sequence of the light chain variable region set forth in SEQ ID NO:5, and LCDR3 sequence of the light chain variable region set forth in SEQ ID NO: 6;

[0023] (b). HCDR1 sequence of the heavy chain variable region set forth in SEQ ID NO:7, HCDR2 sequence of the heavy chain variable region set forth in SEQ ID NO:8, HCDR3 sequence of the heavy chain variable region set forth in SEQ ID NO:9, LCDR1 sequence of the light chain variable region set forth in SEQ ID NO: 10, LCDR2 sequence of the light chain variable region set forth in SEQ ID NO: 11, and LCDR3 sequence of the light chain variable region set forth in SEQ ID NO: 12.

[0024] Preferably, the antibody or antigen-binding fragment thereof specifically binding to B7H3 comprises an amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 13 or 14, and an amino acid sequence of the light chain variable region set forth in SEQ ID NO: 15 or 16.

[0025] Preferably, the amino acid of the heavy chain variable region and the light chain variable region are selected from (a) or (b):

[0026] (a) The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 15;

[0027] (b) The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16.

[0028] In one embodiment, the present invention provides B7H3-targeting antibody or antigenbinding fragment 38D8F5, wherein the heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 are respectively set forth in SEQ ID NO: 1, 2, 3 or CDRs having at least 85% sequence identity with the amino acid sequences set forth in SEQ ID NO: 1, 2, 3; and wherein the light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NO:4, 5, 6 or CDRs having at least 85% sequence identity with the amino acid sequences set forth in SEQ ID NO:4, 5, 6. The heavy chain variable region of 38D8F5 comprises the amino acid sequence set forth in SEQ ID NO: 13, and the light chain variable region of 38D8F5 comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0029] In one embodiment, the present invention provides a B7H -targeting antibody or antigen-binding fragment 49F2D11, wherein the heavy chain variable region comprising the amino acid sequences of HCDR1 , HCDR2 and HCDR3 are respectively set forth in SEQ ID NO:7, 8, 9 or CDRs having at least 85% sequence identity with the amino acid sequences set forth in SEQ ID NO:7, 8, 9; and wherein the light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3 are respectively set forth in SEQ ID NQ:10, 11, 12 or CDRs having at least 85% sequence identity with the amino acid sequences set forth in SEQ ID NO: 10, 11, 12. The heavy chain variable region of 49F2D11 comprises the amino acid sequence set forth in SEQ ID NO: 14, and the light chain variable region of 49F2D11 comprised the amino acid sequence set forth in SEQ ID NO: 16.

[0030] In several other embodiments, the present invention also provides a group of anti-B7H3 antibodies or antigen-binding fragments, which are 16C7B5, 31F6G6, 41E9D9, 46E1C5, 47H2H11, 48H5F5, 49F7H4, and 55F3E5, respectively.

[0031] Preferably, the antibody comprises at least one selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a multi-specific antibody. In some specific embodiments, the antibody is a chimeric antibody.

[0032] Preferably, the chimeric antibody comprises a constant region.

[0033] Preferably, the heavy chain constant region of the chimeric antibody is selected from IgGl, IgG2, IgG3, or IgG4 or any variant thereof, preferably IgGl; the light chain constant region of the chimeric antibody is selected from K chain, chain, or any variant thereof, preferably K chain.

[0034] Preferably, the B7H3 -targeting antigen-binding fragment comprises at least one selected from Fab, F(ab’), F(ab’)2, Fd, single-chain antibody scFv, disulfide-linked Fv (sdFv), and single-domain antibody.

[0035] In some embodiments, the B7H3-targeting antigen-binding fragment is scFv.

[0036] Preferably, the amino acid sequence of the heavy chain variable region of the scFv is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 15, or the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:14, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16.

[0037] In some embodiments, the B7H3 -targeting scFv is organized in the orientation of VH- linker-VL or VL-linker-VH.

[0038] In some embodiments, the sequence of the linker can be an existing linker sequence. Preferably, the sequence of the linker can be GGGGSGGGGSGGGGS, or (GGGGS)n, where n=l -6; preferably, n=3.

[0039] Preferably, the amino acid sequences of the scFv are set forth in SEQ ID NO: 19 and SEQ ID NO:20.

[0040] The present invention also provides a nucleic acid encoding the aforementioned antibody or antigen-binding fragment that specifically binds to B7H3.

[0041] The present invention also provides a vector comprising the aforementioned nucleic acid.

[0042] The present invention also provides a host cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0043] The present invention also provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof that specifically binds to B7H3, which comprises the aforementioned host cell. Preferably, suitable methods for preparing antibodies are known in the art. For example, standard hybridoma methods. In addition, other methods can also be used, such as phage display systems or yeast display systems, which are known in the art. Suitable methods for preparing antigen-binding fragments include, but are not limited to, hydrolytic digestion of intact antibody and expression by host cells containing antigen-binding fragments.

[0044] The present invention also provides a B7H3 scFv-CD3e fusion protein, which comprises an antigen-binding region capable of specifically binding to B7H3, the antigen-binding region comprises the aforementioned antibody or antigen-binding fragment thereof specifically binding to B7H3.

[0045] Preferably, the B7H3 scFv-CD3e fusion protein comprises the amino acid sequence set forth in SEQ ID NO:23 or 24.

[0046] The present invention also provides an isolated nucleic acid encoding the aforementioned B7H3 scFv-CD3e fusion protein.

[0047] Preferably, the nucleotide sequences of the aforementioned isolated nucleic acid are set forth in SEQ ID NO:25 or 26.

[0048] The present invention also provides a vector comprising the aforementioned isolated nucleic acid.

[0049] The present invention also provides a host cell comprising the aforementioned vector The present invention also provides a method for preparing the B7H3 scFv-CD3s fusion protein, which comprises culturing the aforementioned host cell.

[0050] According to the third aspect of the present invention, there is also provided an immune cell that expresses the aforementioned antibody or antigen-binding fragment thereof that specifically binds to B7H3, or the aforementioned B7H3 scFv-CD3e fusion protein. Preferably, the immune cell comprises at least one selected from pluripotent stem cells, embryonic stem cells, T lymphocytes, NKT cells, NK cells, macrophages, dendritic cells, monocytes, and hematopoietic stem cells.

[0051] The present invention also provides a method for preparing the aforementioned immune cell, which comprises the aforementioned isolated nucleic acid or is transduced with the aforementioned vector.

[0052] Preferably, the present invention also provides a method for preparing an engineered T cell expressing the B7H3 scFv-CD3e fusion protein , comprising the following steps:

[0053] (i). Artificially synthesis the DNA sequences of B7H3 scFv-CD3e fusion protein set forth in SEQ ID NO:25 or 26, or wherein the B7H3 scFv-CD3e gene sequence encodes an amino acid sequence set forth in SEQ ID NO:23 or 24;

[0054] (ii). Insertion of the B7H3 scFv-CD3e gene sequence into the pCCL vector to obtain the recombinant plasmid pCCL-B7H3 SCFV-CD3E,

[0055] Preferably, the pCCL-B7H3 scFv-CD3e comprises pCCL-ml6C7B5-CD3s, pCCL- m31F6G6-CD3e, pCCL-m38D8F5-CD3e, pCCL-m41E9D9 -CD3e, pCCL-m46ElC5- CD3s, pCCL-m47H2Hl l-CD3s, pCCL-m48H5F5-CD3e, pCCL-m49F2Dll-CD3e, m49F7H4-CD3e, or pCCL-m55F3E5-CD3e;

[0056] (iii). Co-transfection of the recombinant plasmid pCCL-B7H3 scFv-CD3e with the envelope plasmid and packaging plasmid into host cells to obtain B7H3 SCFV-CD3E encoded lentiviral vectors;

[0057] (iv). Transduction of CD3-positive T lymphocytes with the aforementioned lentivirus, obtaining the B7H3 scFv-CD3e engineered T cells after isolation.

[0058] Preferably, the envelope plasmid is VSVG, the packaging plasmids are RRE and REV, and the host cells are HEK293T cells.

[0059] Preferably, in the step (iv), the CD3-positive T lymphocytes are isolated from human peripheral blood mononuclear cells.

[0060] Preferably, the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. The present invention also provides a pharmaceutical composition, comprising the aforementioned antibody or antigen-binding fragment thereof specifically binding to B7H3, the aforementioned nucleic acid, the aforementioned B7H3 scFv-CD3e fusion protein, the aforementioned isolated nucleic acid, the aforementioned vector, or the aforementioned immune cell The present invention also provides the use of a reagent in the preparation of a drug for treating or ameliorating cancer, wherein the reagent comprises at least one selected from the antibody or antigen-binding fragment thereof specifically binding to B7H3, the aforementioned nucleic acid, the aforementioned vector, the aforementioned host cell, the aforementioned antibody or antigen-binding fragment thereof that specifically binds to B7H3 prepared by the method for preparing the antibody or antigen -binding fragment thereof specifically binding to B7H3, the aforementioned B7H3 scFv-CD3e fusion protein, the aforementioned isolated nucleic acid, the aforementioned vector, the aforementioned host cell, the aforementioned B7H3 scFv-CD3s fusion protein prepared by the method for preparing the B7H3 scFv-CD3e fusion protein, the aforementioned immune cell, or the aforementioned immune cell prepared by the method for preparing the immune cell.

[0061] The present invention also provides that in the use of the reagent in the preparation of a drug for treating or ameliorating cancer, wherein the cancer comprises at least one selected from brain glioma, kidney cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer, intestinal cancer, prostate cancer, and pancreatic cancer.

[0062] The present invention has the following beneficial effects:

[0063] (i). The antibody or antigen-binding fragment provided by the present invention can specifically bind to B7H3 proteins from various species (including human, rhesus and mice), providing more options for subsequent pharmacodynamic and toxicological studies. The binding capacity of the antibody to recombinant B7H3 protein and membrane-bound B7H3 on the surface of tumor cells is significantly superior to that of positive control molecules such as MGA271 and 8H9;

[0064] (ii) The antibodies of the present invention can be used to prepare B7H3 scFv-CD3e engineered T cells, which exhibit significant cytotoxicity toward cells stably expressing B7H3. Therefore, they have significant therapeutic value for B7H3-positive cancers, such as gastric cancer, pancreatic cancer, lung cancer, liver cancer, and breast cancer, etc. Brief Description Of the Drawings

[0065] Figure 1. ELISA binding of the anti-B7H3 antibodies towards human 4IgB7H3 protein. The x-axis represents antibody concentration (nM), and the y-axis represents OD450 value.

[0066] Figure 2. ELISA binding of anti-B7H3 antibodies towards human 2IgB7H3 protein. The x-axis represents antibody concentration (nM), and the y-axis represents OD450 value.

[0067] Figure 3. ELISA binding of anti-B7H3 antibodies towards rhesus B7H3 protein. The x-axis represents antibody concentration (nM), and the y-axis represents OD450 value. Figure 4. ELISA binding of anti-B7H3 antibodies towards mouse B7H3 protein. The x-axis represents antibody concentration (nM), and the y-axis represents OD450 value. Figure 5. Binding activity of anti-B7H3 antibodies with membrane-bound B7H3 on the surface of tumor cell lines. The x-axis represents concentration (nM), and the y-axis represents MFI value.

[0068] Figure 6 Schematic diagram of B7H3 scFv-CD3s fusion protein.

[0069] Figure 7. Positivity ratio of B7H3 scFv-CD3e engineered T cell.

[0070] Figure 8. Cytotoxicity of B7H3 scFv-CD3e engineered T cells against PanC-1 cells. 8A. Real-time cytotoxicity curve of B7H3 scFv-CD3e engineered T cells against PanC- 1 cells; SB Killing rate of B7H3 scFv-CD3s engineered T cells against PanC-1 cells; 8C. Results of cytokine release detection in the co-cultured supernatants.

[0071] Figure 9. Cytotoxicity of B7H3 scFv-CD3e engineered T cells against AGS cells. 9A. Real-time cytotoxicity curve of B7H3 scFv-CD3e engineered T cells against AGS cells; 9B. Killing rate ofB7H3 scFv-CD3e engineered T cells against AGS cells; 9C. Results of cytokine release detection in the co-cultured supernatants.

[0072] Embodiments Of the Present Invention

[0073] Definitions

[0074] Unless otherwise specified, all technical and scientific terms used in the present invention are consistent with the common understanding of a person skilled in the art to which the present invention pertains The following terms are used in accordance with the definitions below when describing and claiming protection for this disclosure. In the present invention, the term “anti-B7H3 antibodies” has the same meaning as "antibody specifically binding to B7H3" and the two can be used interchangeably.

[0075] The term “at least 85% sequence identity” refers to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some preferred embodiments, the sequence identity described in the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sequence comparison and determination of identical percentage between two sequences may be performed using the BLASTN / BLASTP algorithm available on the National Center for Biotechnology Information (NCB1) website.

[0076] The term “antibody” is synonymous with immunoglobulin and refers to a tetramer composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. Based on differences in the amino acid composition and arrangement of the constant regions of the immunoglobulin heavy chain, immunoglobulins can be classified into five classes: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains p, 8, y, a, and e, respectively. Within the same class of immunoglobulin, further subclasses can be distinguished based on differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chains. For example, IgG can be subdivided into IgGl, IgG2, lgG3, and IgG4. The constant regions of light chains are classified into either K chain or Z chain. Each of the five Ig classes can have either K chain or chain.

[0077] The term “antigen-binding fragment” refers to one or more fragments of an antibody that retain the ability to bind to an antigen Examples of binding fragments included in the “antigen-binding fragment” include: (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) F(ab')2 fragment, a bivalent fragment consisting of two Fab fragments connected by disulfide bonds across the hinge region; (iii). Fd fragment consisting of the VH and CHI domains; (iv). Fv fragment consisting of the VH and VL domains of the antibody's single arm; (v). A single-domain or dAb fragment consisting of the VH domain; (vi) Isolated complementarity-determining regions (CDRs); or (vii). A combination of two or more isolated CDRs connected by a linker; (viii). Additionally, single-chain Fv (scFv) molecules produced by connecting VL and VH via a linker are also included in the term “antigen-binding fragment”

[0078] The term “CDR” refers to the complementarity-determining region, which is the binding site of the antibody or antigen-binding fragment to the antigen. The heavy chain variable region and light chain variable region of known antibodies typically contain three complementarity-determining regions (CDRs) and four framework regions (FRs). The CDRs are connected by the backbone region, and the FR molecule curls to bring the CDR molecules closer to each other when recognizing the antigen. The primary structure of the CDRs contains highly variable sites, which are adjacent to each other in the tertiary structure and determine the specificity of antibody binding to the antigen. In the present invention, “HCDR1”, “HCDR2”, and “HCDR3” refer to the three complementarity-determining regions of the heavy chain variable region, while “LCDR1”, “LCDR2”, and “LCDR3” refer to the three complementarity-determining regions of the light chain variable region.

[0079] The term “hybridoma” or “hybridoma cell” refers to a cell or cell line derived from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells (typically myeloma or lymphoma cells). As is known to those skilled in the art, hybridomas can proliferate and continuously produce specific monoclonal antibodies. Methods for producing hybridomas are well -described in the art (Harlow L, 1988). When referring to the terms “hybridoma” or “hybridoma cell”, these also include subclones and descendant cells of the hybridoma.

[0080] The term “chimeric antibody” refers to an antibody in which part of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remaining part of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies For example, the variable regions of the heavy chain and light chain of a human-mouse chimeric antibody are derived from a first antibody (e.g., a mouse-derived antibody), while the constant regions of the heavy chain and light chain of the antibody are derived from a second antibody (e g , a human antibody). Antibodies created by fusing the variable region of a mouse-derived antibody with the constant region of a human antibody can reduce the immune response induced by the mouse-derived antibody. To construct chimeric antibodies, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region gene from mouse hybridoma cells and subsequently clone the constant region gene of the human antibody as needed. The mouse variable region gene is then fused with the human constant region gene to form a chimeric gene, which is inserted into a vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic expression system or a prokaryotic expression system.

[0081] The term “vector” refers to a nucleic acid carrier into which a nucleic acid sequence encoding a desired molecule is inserted. The nucleic acid sequence encoding the desired molecule may be obtained using known recombinant methods in the art, including screening a library from cells expressing the gene, obtaining the gene from a known vector containing the gene, or directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest may be synthetically produced. The vector may be introduced into host cells via transformation, transduction, or transfection, enabling the genetic material it carries to be expressed in the host cells. The vector of the present invention may be any vector used for propagation, amplification, and expression, and may be used to transform or transfect any suitable host cells. It includes but is not limited to, plasmids, bacteriophages, Kos plasmids, artificial chromosomes, bacteriophages, viruses or viral vectors, plant vectors, and animal vectors, etc. Viruses that can be used as vectors include, but are not limited to, reverse transcriptase viruses (including lentiviruses), adenoviruses, adeno- associated viruses, retroviral vectors, herpesviruses (such as herpes simplex virus), poxviruses, baculovi ruses, papillomaviruses, and papillomatous vacuolar viruses (such as SV40), etc.

[0082] The following description provides further details of the present invention with reference to the accompanying drawings and examples. The examples are provided solely for the purpose of explaining the present invention and are not intended to limit the scope of protection of the present invention.

[0083] Example 1: Construction of Benchmark Antibodies and Tool Cell Lines

[0084] 1. Preparation of Benchmark Antibodies

[0085] MGA271 (Enoblituzumab), disclosed in patent application WO2021 / 231309A1, and 8H9 (Omburtamab), disclosed in patent application PCT / US2008 / 058030, were used as benchmark antibodies. The amino acid sequences of the heavy chain and light chain of MGA271 are cited from SEQ ID NO: 18 and SEQ ID NO: 17 disclosed in WO2021 / 231309A1. The amino acid sequence of 8H9 scfv is cited from SEQ ID NO: 11 disclosed in PCT / US2008 / 058030.

[0086] According to the preparation method disclosed in the prior art, the DNA sequences encoding the heavy chain variable regions ofMGA271 and 8H9 were codon optimized, synthesized, and then subcloned into a eukaryotic vector containing the human IgGl heavy chain constant region (HC-pcDNA3.1) via restriction enzyme sites Notl and Nhel, respectively. Similarly, DNA sequences encoding the light chain variable regions of MGA271 and 8H9 were synthesized and subcloned into a eukaryotic expression vector containing the light chain constant region (hx-pcDNA3. 1) via restriction enzyme sites Notl and BsiWI, respectively, to prepare the recombinant MGA271 and 8H9 hlgGlx as benchmark antibodies.

[0087] 2. Construction of B7H3 over-expressing stable cell lines

[0088] The B7H3 encoding plasmid pCMV3-SP-His-CD276-tl (Sino Biology, catalog number: HG11188-NH) was transfected into CH0K1 cells (Cobioer Biology, catalog number: CBP60296) via liposome transfection and cultured at 37°C, 5% CO2. After 48 hrs, the cells were transferred into culture medium containing 600pg / mL hygromycin. After 16 days of pressure screening, the positivity ratio of the transfected cell pool was detected by flow cytometry after incubation with CD276 antibody (Biointron, catalog number: B137901) and secondary anti-human Fc antibody. The B7H3-positive cell pool was plated into a 96-well plate (at a density of 1E6 cells / ml, lOOpL / well), and the B7H3 highly expressed cell clone was picked and named as hB7H3-CHOKl.

[0089] Example 2: Hybridoma 1. Animal immunization

[0090] Commercially available 4IgB7H3 antigen and 2IgB7H3 antigen (AcroBiosystem, catalog numbers: B7B-HP2H9 and B73-H52E2) were used as immunogen to immunize several mouse strains, including SJL strain mice, Balb / c strain mice, and mB7H3 KO- C57 strain mice (ModelORG). As shown in Table 1, each animal was administered 50 pg of antigen for the first immunization and 25 pg of immunogen per animal for subsequent immunizations. IMMU-PLUS adjuvant (SATYA Pharmaceutical, Catalog Number: SP580IP001) was used to emulsify the antigen following the manufacturer's instructions. Specifically, both 41g-B7H3 and 21g-B7H3 proteins were dropped into the adjuvant solution with sufficient vortexing to form an oil-in-water emulsion, then injected into the mice.

[0091] Table 1: Immunization schedule

[0092] 2. Hybridoma fusion and selection

[0093] Splenic cell isolation and preparation: After booster immunization, mice were euthanized, immersed in 75% ethanol and dissected. The spleen was ground with a grinding rod, and filtered through a cell sieve to prepare a single-cell suspension. Splenic cells were centrifuged at 2000 rpm for 5 minutes, discarding the supernatant. With the addition of 2 mL of red blood cell lysis buffer, 2 minutes lysis at room temperature, and termination by adding PBS to a total volume of 20 mL, live cells were centrifuged at 1500 rpm for 7 minutes, resuspended, and counted. Sp2 / 0 cells were collected by centrifugation at 1000 rpm for 5 minutes, resuspended, and counted. Splenic cells and Sp2 / 0 cells were mixed at a ratio of 1 :1, centrifuged at 1500 rpm for 7 minutes, resuspended in 20 mL of electroporation buffer, and centrifuged. After repetition of the above step, the cells were resuspended in the electroporation buffer and adjusted to a density of approximately 2zIO7cells / mL. The cell suspension was added to the electroporation fusion chamber for fusion. Afterwards, the cell suspension was transferred to 15 mL of RPMI 1640 complete medium containing 20% FBS and incubated at room temperature for 20 minutes. Finally, the fused cells were resuspended in RPMI 1640 medium (containing IX HAT, IX BI0MY3, and 20% FBS) and seeded lOOpL of the cell suspension into each well of several 96-well cell culture plates, ensuring approximately 4 x 104cells per well, and incubated at 37°C. After 5 days, the culturing medium (RPMI 1640 medium containing IX HAT, IX BI0MY3, and 20% FBS) was supplemented.

[0094] 3. Subcloning and screening

[0095] One week after fusion, the cell culture supernatant was collected and screened for the hybridoma parental clones that bind to human 4IgB7H3 antigen, human 2IgB7H3, and rhesus B7H3 protein using ELISA. Further screening using flow cytometry was conducted to identify the parental clones that can bind to hB7H3-CHOKl cell lines. Subcloning of the positive parental clones was performed using limited dilution. After one week of culture, screen for monoclonal hybridomas secreting B7H3 antibodies was conducted using ELISA protein binding assays and flow cytometry binding assays.

[0096] 4. Hybridoma sequencing

[0097] The positive hybridoma cells were expanded for RNA extraction using the RNeasy Plus Mini Kit (Qiagen, catalog number: 74134) according to the manufacturer’s instructions, and cDNA was reverse transcribed using the Prime Script 1st strand cDNA synthesis Kit (Takara, catalog number: 6110A).

[0098] Antibody subtyping was performed using the SB A Clonotyping System-C57BL / 6- HRP (Southern Biotech, catalog number: 53OO-O5B) according to the instructions, with results shown in Table 2:

[0099] Table 2 Isotype characterization of hybridoma IgG

[0100] Specific primers based on the variable regions of mouse antibodies were designed as reported, and PCR amplification was performed using cDNA as a template to obtain gene fragments of the variable regions of the light chain and heavy chain of hybridomas. Primer design references (Anke Kreber, et al, Journal of Immunological Methods, 1997; Simon Koren, et al, Appl Microbiol Biotechnol, 2008) were used for DNA sequencing, with the sequencing results shown in Table 3

[0101] Tabic 3 Sequence of Hybridoma IgG

[0102] Example 3: Preparation of the chimeric anti-B7H3 specific antibodies (hereinafter shortened as the chimeric antibodies)

[0103] 1. Vector construction

[0104] DNA sequences of the heavy chain variable region and the light chain variable region obtained in Example 2 were codon-optimized, synthesized, and subcloned into a eukaryotic expression vector containing the human IgGl heavy chain constant region (HC-pcDNA3.1) / or the human IgG K constant region as described in Example 1.

[0105] 2. Expi293 cell expression system

[0106] The chimeric antibodies were expressed by transient co-transfection of the respective plasmids (encoding the heavy chain and light chain, respectively) using the Expi293 system (Invitrogen, #A14635CN) according to the manufacturer’s instructions. Briefly, 100 mL of Expi293 cells at a density of 1E6 cells in serum-free Expi293 expression medium was inoculated into a 1 L Erlenmeyer shake flask (Corning) the day before transfection and cultured at 125 rpm under 37°C, 5% CO2 atmosphere. On the next day, when the cell density reached 2.5-' IO cells / mL with a viability of >95%, lOOpg of total plasmid DNA (at a concentration of Ipg / mL, including 50pg of heavy chain plasmid and 50pg of light chain plasmid) was added into 5 mL of Opti-MEM medium (Invitrogen), labeled as Solution A; 260pL of Expi293 fectin (at a concentration of 2.6pL / mL) was added into another 5 mL of Opti-MEM medium, labeled as Solution B. Solution A was dropped into Solution B and incubated at room temperature for 20 minutes, then slowly added into the Expi293 cell culture medium. Following the manufacturer’s instructions, Enhancer 1 and Enhancer 2 were added on the day after transfection. After 5 days, the supernatant containing the secreted antibody was collected, then either purified or stored at -20°C.

[0107] 3. Purification

[0108] Proteins were purified from filtered cell culture supernatants, referring to standard protocols Tn brief, supernatant of the chimeric antibodies was applied to a one-step Protein A-affinity chromatography (equilibrating buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0 followed by immediate pH neutralization of the sample. Aggregated protein was separated from monomeric antibodies by size exclusion chromatography (Superdex200, GE Healthcare) in PBS or 20 mM Histidine, 50 mM NaCl at pH 5.5. Monomeric molecule fractions can be pooled, concentrated (if required) using MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen and stored at -20°C or -80°C.

[0109] 4. Protein measurement

[0110] The concentration of purified antibodies was determined by the optical density (OD) at 280 nm, using the molar extinction coefficient calculated based on the amino acid sequence, according to Pace et al., Protein Science, 1995, 4, 2411-1423. Example 4: Binding activity assessment of chimeric antibodies towards human / monkey / mouse CD276

[0111] The binding activity of the chimeric antibodies from Example 3 towards human 4IgB7H3, human 2IgB7H3, rhesus B7H3, and mouse B7H3 recombinant proteins was detected by ELISA. As shown in Figure 1, all the chimeric antibodies exhibited strong binding capacity with human 4IgB7H3, compared to benchmark antibodies MGA291 and 8H9. Figure 2 showed that all the chimeric antibodies exhibited strong binding capacity with human 2IgB7H3, compared to benchmark antibodies MGA291 and 8H9. Similarly, Figure 3 showed that all the chimeric antibodies exhibited strong binding capacity with rhesus B7H3. Figure 4 showed that the chimeric antibodies 16C7B5, 31F6G6, m41E9D9, 47H2H11, 49F7H4, and 55F3E5 exhibited weak binding capacity with mouse B7H3, while 38D8F5, 46E1C5, 48H5F5, 49F2D11, and benchmark antibodies did not bind to mouse B7H3.

[0112] Example 5: Binding activity assessment of chimeric antibodies to CD276-positive tumor cell lines

[0113] The binding activity of the chimeric antibodies from Example 3 to CD276-positive tumor cell lines A549 and PanC- 1 was analyzed by flow cytometry. Specifically, the two cell lines were collected in FACS buffer (PBS containing 2% FBS), counted, and adjusted to a cell density of 2 * 106cells / mL. The cell suspensions were then aliquoted into U-bottom 96-well plates (50pL per well), and 50pL of FACS buffer-diluted chimeric antibody or human IgG isotype (as a negative control) was added (concentration range: 300nMto 0.017nM). After incubation at room temperature for 60 minutes, the cell pellet was centrifuged (3 minutes, 300*g) and washed twice with 150pL / well of FACS buffer. Afterwards, lOOpL of 5000-fold diluted AF647-labeled anti-human Fc secondary antibody (Jackson, catalog number: 109-605-008) was added to resuspend the cells and incubated at room temperature for 60 minutes. Finally, the cells were washed three times with 150pL / well of FACS buffer, collected in lOOpL of FACS buffer, and analyzed by flow cytometry (Beckman). The data was processed by FlowJo and GraphPad Prism

[0114] Figure 5A showed that all the chimeric antibodies exhibited stronger binding affinity to B7H3 on the surface of PanC-1 cells, compared to benchmark antibody MGA271. Consistently, Figure 5B showed that all the chimeric antibodies exhibited stronger binding affinity to B7H3 on the surface of A549 cells, compared to the benchmark antibody MGA271.

[0115] Example 6 Construction of B7H3 scFv-CD3c Engineered T Cells

[0116] 1. Construction of B7H3 SCFV-CD3E vector

[0117] DNA sequences of B7H3 scFv-CD3s fusion protein, containing the anti-B7H3 singlechain fragment (scFv) derived from Example 2, the linker, the extracellular domain, the transmembrane region, and the intracellular signal segment of CD3s, as shown in Figure 6, were codon-optimized, synthesized, and subcloned into the lentiviral vector pCCLby GENEWIZ. The derived B7H3 scFv-CD3e constructs are sequentially named as follows: ml6C7B5-CD3s, m31F6G6-CD3s, m38D8F5-CD3s, m41E9D9-CD3s, m46El C5-CD3s, m47H2H1 l -CD3s, m48H5F5-CD3s, m49F2Dl l-CD3e, m49F7H4- CD3s, and m55F3E5-CD3s. The amino acid sequences of m38D8F5-CD3s and m49F2Dll-CD3e are shown in SEQ ID NO: 23 and 24, respectively.

[0118] B7H3 scFv-CD3s constructs with correct sequencing were inoculated into 200 mL LB medium, incubated overnight, and purified according to the kit instructions

[0119] 2. B7H3 SCFV-CD3E lentiviral production and detection

[0120] Lenti viruses were packaged using the cationic polymer PEI (Polyplus, catalog number: 101000033) according to the following procedure: PEI and the lentiviral packaging plasmids (viral backbone plasmid, VSVG, RRE, REV) were diluted separately in serum-free Opti-MEM; then, the PEI / Opti-MEM mixture was added to the plasmid / Opti-MEM mixture and mixed thoroughly, and incubated for 15 minutes. Afterwards, the plasmid-PEI mixture was added to HEK293T cells and incubated at 37°C in 5% CO2 incubator for 60-72 hours. Finally, viral supernatant was collected by centrifugation at 3000g, 4°C for 10 minutes to remove debris, purified with Pre-sterilize the Centricon Plus-70 (Millipore, catalog number: UFC710008), aliquoted, and stored at -80°C.

[0121] 3. Production of B7H3 scFv-CD3s-engineered T cells and positivity rate detection of B7H3 a. PBMC isolation and activation

[0122] CD3+T cells were isolated from human peripheral blood of volunteers using magnetic beads, and stored in CS10 cry opreservation solution (2E7M / vial).

[0123] DO: The cryopreserved CD3+T cells were thawed and resuspended in T complete medium (1000 mL Optimizer medium + 26 mL Supplement + 50 mL SR + 20 mL Glutamax + 400 lU / mL rIL-2), adjusting the cell density to 1.43 x 106 / mL. T cells were activated using Transact (Miltenyi, catalog number: 200-076-204), with the volume ratio of Transact to T cells =1 :50. b. Lentiviral transduction

[0124] DI: After 24 hours of activation, T cells were counted, adjusted the cell density to 5-7 x 105 / mL, and added the lentiviral solution based on MOI calculation.

[0125] D2-D6: After infection, T cell culture medium with 400 TU / mL IL-2 was supplemented every two days to maintain T cell density at 5 * 105 / mL for efficient cell expansion. c. B7H3 positivity rate detection

[0126] D8-D12: Once the cells proliferation meets the desired amount, T cells were harvested to detect the positivity rate of B7H3 scFv-CD3s fusion protein on lentiviral -transduced T cells. Specifically, 1 x10sT cells from the B7H3 scFv-CD3e lentiviral transduced group and the un-transduced group were centrifuged, washed once with FACS buffer, and stained with FITC-CD276 (Aero Biosystem, catalog number: B7B-HF2E7) at room temperature for 60 minutes. Afterwards, the cells were washed three times with FACS buffer, resuspended and analyzed by flow cytometry. The data was processed with FlowJo.

[0127] As shown in Figure 7, the positivity ratio of m38D8F5-CD3e and m49F2Dl l-CD3e transduced T cells was approximately 74% and 81%, respectively, indicating the successful generation of B7H3 scFv-CD3s engineered T cells. Example 7: Cytotoxicity assessment of B7H3 scFv-CD3a engineered T cells against B7H3-positive tumor cells

[0128] The cytotoxicity of B7H3 SCFV-CD3E engineered T cells generated in Example 6 was assessed on B7H3-positive PanC-1 and AGS cells. Specifically, 50pL 10% FBS- RPMI1640 medium was added into each well of the 96-well E-Plate, and the baseline impedance value was measured. PanC-l / AGS All cells were collected by 0.25% trypsin digestion, resuspended in 10% FBS-RPMI1640 medium. For PANC-1 cells, the cell density was adjusted to 2 x io5cells / mL, and 50pL / well, 1 - 104cells / per well were seeded into each well. For AGS-A11 cells, the cell density was adjusted to 4 x 105cells / mL, and 50pL / well, 2 x 104cells / well were seeded into each well. After incubation at 37°C for 30 minutes, the 96-well E-Plate was placed in the RTCA and measured every 15 minutes. On the next day, B7H3 scFv-CD3e engineered T cells were added into each well at the target cell: effector cell (E:T) ratio of 2: 1 and 1 :2. Each group had at least two replicate wells. Target cells co-cultured with un-transduced T cells served as negative control groups (UT) The E-plate was placed on the detection devices for real-time cell killing monitoring for 72 hrs. The supernatant was collected after 24 hrs and IFN-r secretion was detected by ELISA. The killing rate of B7H3 scFv-CD3e-T cells at a specified time point was calculated using the following formula.

[0129] Mock-T killing rate (%) = (cell index control - cell index MOCR-T) / cell indexconlroi* 100 B7H3 scFv-CD3s engineered T cells killing rate (%) = (cell index control - cell index B7H3 scFv-CD3s -T) / Cell index control * 100

[0130] As shown in Figures 8A and 8B, both m38D8F5-CD3e-T cells and m49F2Dl l-CD3e- T cells exhibited strong killing activity against PanC-1 cells at the E:T ratio of 2:1, with the corresponding killing rates of 97% and 95%, respectively; at the E:T ratio of 1:2, the killing rates of these two were 95% and 91%, respectively. Consistently, the supernatant of PanC-1 co-cultured with these two B7H3 scFv-CD3e -T cells showed a significant increase of IFN-r levels (Figure 8C).

[0131] As shown in Figures 9 and 9B, m38D8F5-CD3s-T cells and m49F2Dll-CD3e-T cells exhibited extremely strong killing activity against AGS cells at E:T ratios of 2:1 and 1 :2, with corresponding killing rates of 100%. Consistently, the co-culture supernatant of AGS with these two B7H3 scFv-CD3s -T cells showed a significant increase of IFN-r levels (Figure 9C).

[0132] The above results indicate that the anti-B7H3 antibodies provided by the present invention can specifically bind to soluble and membrane-bound B7H3 proteins. The derived B7H3 scFv-CD3e engineered T cells can efficiently kill B7H3-positive tumor cells. It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art to the present invention fall within the scope defined by the appended claims of the present invention

Claims

Claims1. An antibody or antigen-binding fragment thereof specifically binding to B7H3, the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the CDR sequences of the heavy chain variable region and the CDR sequences of the light chain variable region are selected from (a) or (b):(a) HCDR1 sequence of the heavy chain variable region set forth in SEQ ID NO:1, HCDR2 sequence of the heavy chain variable region set forth in SEQ ID NO:2, HCDR3 sequence of the heavy chain variable region set forth in SEQ ID NO:3, LCDR1 sequence of the light chain variable region set forth in SEQ ID NO:4, LCDR2 sequence of the light chain variable region set forth in SEQ ID NO:5, and LCDR3 sequence of the light chain variable region set forth in SEQ ID NO:6;(b) HCDRI sequence of the heavy chain variable region set forth in SEQ ID NO:7, HCDR2 sequence of the heavy chain variable region set forth in SEQ ID NO:8, HCDR3 sequence of the heavy chain variable region set forth in SEQ ID NO:9, LCDR1 sequence of the light chain variable region set forth in SEQ ID NO: 10, LCDR2 sequence of the light chain variable region set forth in in SEQ ID NO: 11, and LCDR3 sequence of the light chain variable region set forth in SEQ ID NO: 12.

2. The antibody or antigen-binding fragment thereof specifically binding to B7H3 of claim 1, wherein the antibody or antigen-binding fragment comprises an amino acid sequence of the heavy chain variable region set forth in SEQ ID NO: 13 or 14, and an amino acid sequence of the light chain variable region set forth in SEQ ID NO: 15 or 16, preferably,(a) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 15;(b) the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 16.

3. The antibody or antigen-binding fragment thereof specifically binding to B7H3 of claim 1 or 2, wherein the antibody comprises at least one selected from a monoclonal antibody, a chimeric antibody, a humanized antibody, and a multi-specific antibody; the antigen-binding fragment comprises at least one selected from Fab, F(ab’), F(ab’)z, Fd, single-chain antibody scFv, disulfide-linked Fv (sdFv), and single-domain antibody.

4. A nucleic acid encoding the antibody or antigen-binding fragment thereof specifically binding to B7H3 of any one of claims 1 to 3.

5. Avector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4 or the vector of claim 5.

7. A method for preparing the antibody or antigen-binding fragment thereof specifically binding to B7H3, comprising culturing the host cell of claim 6.

8. AB7H3 scFv-CD3s fusion protein, wherein the fusion protein comprises an antigenbinding region capable of specifically binding the B7H3, the antigen-binding region comprises the antibody or antigen-binding fragment thereof specifically binding to B7H3 of any one of claims 1 to 3.

9. The B7H3 scFv-CD3e fusion protein of claim 8, wherein the fusion protein comprises an amino acid sequence set forth in SEQ ID NO:23 or 24.

10. An isolated nucleic acid encoding the B7H3 scFv-CD3e fusion protein of claims 8 or 9, wherein the fusion protein comprises a nucleotide sequence set forth in SEQ ID NO:25 or 26.

11. Avector comprising the isolated nucleic acid of claim 10.

12. A host cell comprising the vector of claim 11.

13. A method for preparing the B7H3 scFv-CD3e fusion protein, comprising culturing the host cell of claim 12.

14. An immune cell, wherein the immune cell expresses the antibody or antigen-binding fragment thereof specifically binding to B7H3 of any one of claims 1 to 3, or the B7H3 scFv-CD3e fusion protein of claims 8 or 9.

15. The immune cell of claim 14, wherein the immune cell comprises at least one selected from pluripotent stem cells, embryonic stem cells, T lymphocytes, NKT cells,NK cells, macrophages, dendritic cells, monocytes, and hematopoietic stem cells.

16. A method for preparing an immune cell of claim 15, wherein the immune cell comprises the isolated nucleic acid of claim 10 or is transduced with the vector of claim17. A pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof specifically binding to B7H3 of any one of claims 1 to 3, the nucleic acid of claim 4, the B7H3 scFv-CD3e fusion protein of claim 8 or 9, the isolated nucleic acid of claim 10, the vector of claim 11, or the immune cell of claim 14 or 15.

18. Use of a reagent in the preparation of a drug for treating or ameliorating cancer, wherein the reagent comprises at least one selected from the antibody or antigenbinding fragment thereof specifically binding to B7H3 of any one of claims 1 to 3, the nucleic acid of claim 4, the vector of claim 5, the host cell of claim 6, the antibody or antigen-binding fragment thereof that specifically binds to B7H3 prepared by the method for preparing the antibody or antigen-binding fragment thereof specifically binding to B7H3 of claim 7, the B7H3 scFv-CD3e fusion protein of claim 8 or 9, the isolated nucleic acid of claim 10, the vector of claim 11, the host cell of claim 12, the B7H3 scFv-CD3e fusion protein prepared by the method for preparing the B7H3 scFv- CD3s fusion protein of claim 13, the immune cell of claim 14 or 15, and the immune cell prepared by the method for preparing the immune cell of claim 16.

19. In the use of the reagent of claim 18 in the preparation of a drug for treating or ameliorating cancer, wherein the cancer comprises at least one selected from brain glioma, kidney cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer, intestinal cancer, prostate cancer, and pancreatic cancer.

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