Antibodies or antibody fragments targeting B7-H3 and their use in the field of chimeric antigen receptor immune cell therapy

Abstract

The present invention relates to antibodies or antibody fragments that target B7-H3, chimeric antigen receptors and chimeric antigen receptor-immune cells based on said antibodies or antibody fragments, and their use in the treatment of tumors. Specifically, the antibody or antibody fragment comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH comprises VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11, and VL comprises VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:13, and VL-CDR3 shown in SEQ ID NO:14; or wherein VH comprises VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:17, and VL comprises VL-CDR1 shown in SEQ ID NO:18, VL-CDR2 shown in SEQ ID NO:19, and VL-CDR3 shown in SEQ ID NO:20. It contains VL-CDR3 designated NO:20.

Description

Detailed Description of the Invention

[0001] This application claims priority to a prior patent application bearing patent application number 202211681398.0 and entitled "Antibody or antibody fragment targeting B7-H3 and its use in the field of chimeric antigen receptor immune cell therapy," filed with the State Intellectual Property Office of China on December 26, 2022, the entire text of which is incorporated herein by reference.

[0002] [Technical Field] The present invention relates to antibodies or antibody fragments that target B7-H3, chimeric antigen receptors (CARs) based on said antibodies or antibody fragments, and CAR-immune cells, as well as their use in the treatment of tumors.

[0003] [Background technology] As an immunotherapy, monoclonal antibodies have shown significant therapeutic effects in the treatment of malignant diseases and have attracted widespread attention. For example, antibody therapies such as anti-PD-1 and anti-PD-L1 targeting the B7-H1 (PD-L1) / PD-1 signaling pathway have achieved groundbreaking applications in the field of tumor treatment. The key to the success or failure of monoclonal antibody therapy is selecting the appropriate target or immune checkpoint to reactivate and enhance the subject's autoimmune response.

[0004] B7-H3 (also known as CD276) is a type I transmembrane protein consisting of 316 amino acids. In the human body, B7-H3 is divided into two distinct forms, 2Ig-B7-H3 and 4Ig-B7-H3, based on differences in the extramembrane domains. 2Ig-B7-H3 contains a pair of immunoglobulin variable (IgV)-like domains and immunoglobulin constant (IgC)-like domains at the extramembrane end. 4Ig-B7-H3 contains two pairs of IgV- and IgC-domains in the extramembrane segment. B7-H3 belongs to the B7 protein family, and is either absent or expressed at low levels in normal tissues. However, B7-H3 is expressed in a variety of tumor tissues, including pancreatic cancer (positive rate 77.8%), colorectal cancer (positive rate 63.8%), gastric cancer (positive rate 69.2%), lung cancer (positive rate 69.5%), prostate cancer (positive rate 93%), and ovarian cancer (positive rate 73.1%) (more than 60% of tumors express B7-H3), and is also highly expressed in a variety of rare tumors. Therefore, it is also called a "tumor-associated antigen" and is a universal drug target for all tumors with great potential. [1] .

[0005] Like other members of the B7 protein family, such as PD-L1, B7-H3 also functions as an immune checkpoint inhibitor. Blockade of B7-H3 binding with neutralizing monoclonal antibodies (mAbs) enhances the killing function of NK cells and CD8 killer T cells, promotes their infiltration into tumor cells, reduces tumor burden, and prolongs survival in tumor-bearing mice. [2] .

[0006] Therefore, given the widespread high expression of B7-H3 in tumor tissues and low expression levels in healthy tissues, as well as the function of B7-H3 in the tumor microenvironment, the clinical therapeutic potential of B7-H3 has attracted considerable attention. Several clinical trials targeting B7-H3 have been launched, including over 20 clinical trials based on mAbs, including neutralizing antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, antibody-dependent cell-mediated cytotoxicity (ADCC), and NK cell engager-conjugated antibodies.

[0007] Neutralizing antibodies can be used to block ligand-receptor binding and signal transduction, thereby affecting related cellular functions. Blocking B7-H3 signaling via neutralizing antibodies can liberate immune cell functions affected by B7-H3 signaling, potentially improving antitumor activity. Its therapeutic potential has been demonstrated in various solid tumors, including ovarian cancer, melanoma, and colorectal cancer. [3-5] .

[0008] ADCs are drugs that combine an antibody that specifically recognizes an antigen with a cytotoxic small molecule drug, and are characterized by excellent specificity, safety, and high tumor cell killing efficiency. The research team demonstrated the efficacy of ADC drugs targeting B7-H3 in multiple solid tumor models and demonstrated their safety in primates. [6] .

[0009] Bispecific antibodies combine two antibodies that target different targets, and a bispecific antibody targeting both B7-H3 and CD3 has been clinically approved for the treatment of solid tumors (clinical trial number: NCT03406949). This bispecific antibody can recruit activated T cells to B7-H3-positive tumor tissue and help the T cells recognize and kill tumor cells.

[0010] ADCC targeting B7-H3 has also made progress in solid tumors. The research team designed a monoclonal antibody with engineered Fc that targets B7-H3 and can enhance the killing activity of immune cells against B7-H3-positive tumor cells via Fc receptors. Its safety has also been verified in primates. [7] .

[0011] Linking antibodies targeting B7-H3 to NK cell receptor (CD16) antibodies can enhance the ability of NK cells to recognize and kill B7-H3-positive tumor cells. Several teams have further modified the CD16 and B7-H3 antibodies by linking them to the interleukin-15 (IL15) protein. IL15 can further enhance the activity of NK cells. This antibody-protein conjugate has achieved very good results in the treatment of solid tumors in vitro and in animal models. [8] .

[0012] Furthermore, chimeric antigen receptor T cell immunotherapy (CAR-T) targeting B7-H3 has also been shown to be effective in treating brain gliomas. [9] , atypical teratoid rhabdoid tumor

[10] , anaplastic meningioma

[11] Some progress has been made in solid tumors such as

[0013] Several immunotherapies targeting B7-H3 (including B7-H3 monoclonal antibodies and CAR-B7-H3-T therapy) have been deployed worldwide and have shown some initial positive therapeutic effects. However, these targeted immunotherapies still face challenges, such as narrow antibody selectivity and limited clinical efficacy. Further research into new antibodies and new treatments is still needed.

[0014] CAR-T is an innovative immunotherapy based on genetic modification of αβ T cells, and has made significant clinical progress over the past decade. To date, six CAR-T therapeutics have been approved for sale by the FDA, and two have been approved for sale in China. These eight CAR-T drugs all target CD19 or BCMA to treat B-cell-related malignant hematologic tumors, achieving high response rates and disease remission rates. However, CAR-T therapy also has many drawbacks, including little therapeutic efficacy against solid tumors, the inability to produce allogeneic treatments and the lack of off-the-shelf availability, and high cost.

[0015] γδ T cells can solve these problems to some extent. From a cell type perspective, γδ T cells are a type of T cell that expresses the γδ T cell receptor (TCR). They are a special type of immune cell with properties of both innate and adaptive immunity, and their recognition of antigens and pathogens is major histocompatibility complex (MHC)-independent. Therefore, they can be used for allogeneic therapy without modification, without inducing reactions such as GvHD. Many studies have reported that γδ T cells have a broad range of antitumor capabilities and have stronger infiltration and antitumor effects in solid tumors. CAR-modified γδ T cells can further improve the targeting and tumor-killing effects of γδ T cells. [12,13] However, there have been no studies or reports on the application of B7-H3 antibodies in CAR-γδT.

[0016] Reference 1. Kontos, F., et al., B7-H3: An Attractive Target for Antibody-based Immunotherapy. Clin Cancer Res, 2021. 27(5): p. 1227-1235. Citation 2. Lee, Y.-H., et al., Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function. Cell research, 2017. 27(8): p. 1034-1045. Citation 3. Cai, D., et al., Tumor-expressed B7-H3 mediates the inhibition of antitumor T-cell functions in ovarian cancer insensitive to PD-1 blockade therapy. Cell Mol Immunol, 2020. 17(3): p. 227-236. Citation 4. Lee, Y.H., et al., Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function. Cell Res, 2017. 27(8): p. 1034-1045. Citation 5. Lu, H., et al., B7-H3 inhibits the IFN-gamma-dependent cytotoxicity of Vgamma9Vdelta2 T cells against colon cancer cells. Oncoimmunology, 2020. 9(1): p. 1748991. Citation 6. Scribner, J.A., et al., Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer. Mol Cancer Ther, 2020. 19(11): p. 2235-2244. Citation 7. Loo, D., et al., Development of an Fc-enhanced anti-B7-H3 monoclonal antibody with potent antitumor activity. Clin Cancer Res, 2012. 18(14): p. 3834-45. Citation 8. Vallera, D.A., et al., NK-Cell-Mediated Targeting of Various Solid Tumors Using a B7-H3 Tri-Specific Killer Engager In Vitro and In Vivo. Cancers, 2020. 12(9). Citation 9. Tang, X., et al., Administration of B7-H3 targeted chimeric antigen receptor-T cells induce regression of glioblastoma. Signal Transduct Target Ther, 2021. 6(1): p. 125. Citation 10. Theruvath, J., et al., Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid / rhabdoid tumors. Nat Med, 2020. 26(5): p. 712-719. Citation 11. Tang, X., et al., Bioactivity and safety of B7-H3-targeted chimeric antigen receptor T cells against anaplastic meningioma. Clinical & Translational Immunology, 2020. 9(6): p. e1137. Reference 12. Ang, WX, et al., Electroporation of NKG2D RNA CAR Improves Vγ9Vδ2 T Cell Responses against Human Solid Tumor Xenografts. Molecular Therapy - Oncolytics, 2020. 17: p. 421-430. Reference 13. Rozenbaum, M., et al., Gamma-Delta CAR-T Cells Show CAR-Directed and Independent Activity Against Leukemia. Frontiers in Immunology, 2020. 11.

[0017] Summary of the Invention [Problem to be solved by the invention] To solve the above problems, the present invention provides a new B7-H3 antibody or antibody fragment, which can be successfully used in the field of CAR-αβT / CAR-γδT therapy. Furthermore, the present invention provides new options and possibilities for clinical treatment and detection of B7-H3 targets, thereby resolving problems such as little therapeutic effect on solid tumors, the impossibility of allogeneic treatment, the lack of off-the-shelf supply, and high cost.

[0018] [Means for solving the problem] A first aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises VH-CDR1 having the amino acid sequence of SEQ ID NO:9, VH-CDR2 having the amino acid sequence of SEQ ID NO:10, and VH-CDR3 having the amino acid sequence of SEQ ID NO:11, and the light chain variable region (VL) comprises VL-CDR1 having the amino acid sequence of SEQ ID NO:12, VL-CDR2 having the amino acid sequence of SEQ ID NO:13, and VL-CDR3 having the amino acid sequence of SEQ ID NO:14. SEQ ID NO:9:NYWIN SEQ ID NO:10:RIAPGTISTYYNEKFKG SEQ ID NO:11:QDNYFIN SEQ ID NO:12:SASSSISSSDLH SEQ ID NO:13:GTSNLAS SEQ ID NO:14:QQWFSYPFT

[0019] A second aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) comprises VH-CDR1 represented by the amino acid sequence of SEQ ID NO:15, VH-CDR2 represented by the amino acid sequence of SEQ ID NO:16, and VH-CDR3 represented by the amino acid sequence of SEQ ID NO:17, and the light chain variable region (VL) comprises VL-CDR1 represented by the amino acid sequence of SEQ ID NO:18, VL-CDR2 represented by the amino acid sequence of SEQ ID NO:19, and VL-CDR3 represented by the amino acid sequence of SEQ ID NO:20. SEQ ID NO:15:RYDMS SEQ ID NO:16:TISDDGRHTYDRDSVKG SEQ ID NO:17:HRAITTARFDY SEQ ID NO:18:KASQDIYSNIG SEQ ID NO:19:HGTNLED SEQ ID NO:20:LQYVQFPYT

[0020] A third aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3 according to the first and second aspects, wherein the heavy chain variable region VH is any one selected from the amino acid sequence set forth in SEQ ID NO:1, an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO:1 other than the CDR region, an amino acid sequence set forth in SEQ ID NO:2, and an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO:2 other than the CDR region. SEQ ID NO:1 EVQLQQSGAELVKPGASVKLSCKASGYTFTNYWINWIRQRPGQGLEWIGRIAPGTISTYYNEKFKGRATITEDTSTNTAYLQLSSLTSEDTAVYFCARQDNYFINWGQGTLVTVSS SEQ ID NO:2 EFEVQLVESGGGLVKPGGSLKLSCAASGFAFSRYDMSWVRQSPEKRLEWVATISDDGRHTYDRDSVKGRFTISRDNAKNTLYLQMSSLRSEDTALYYCVRHRAITTARFDYWGQGTTVTVSSSR

[0021] A fourth aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3 according to the first or second aspect, wherein the light chain variable region VL is any one selected from the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO:3 outside the CDR region, an amino acid sequence set forth in SEQ ID NO:4, and an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence set forth in SEQ ID NO:4 outside the CDR region. SEQ ID NO:3 DIVLTQSPASLSASLGEKVTITCSASSSISSSDLHWYQQKSGTSPRPWIYGTSNLASGVPPRFSGSGSGTSFSLTISSVEAEDVGTYYCQQWFSYPFTFGTGTKLEIK SEQ ID NO:4 GCADIVMTQSPSSLSVSLGDTVSITCKASQDIYSNIGWLQQLPGQSFKGLIYHGTNLEDGVPSRFSGSGSGTDYSLTISGLESEDFADYYCLQYVQFPYTFGGGTKLEIKASG

[0022] A fifth aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3, the antibody or antibody fragment comprising (i) a heavy chain variable region VH represented by SEQ ID NO:1 and a light chain variable region VL represented by SEQ ID NO:3, or (ii) a heavy chain variable region VH represented by SEQ ID NO:2 and a light chain variable region VL represented by SEQ ID NO:4.

[0023] A sixth aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3 according to any one of the first to fifth aspects, wherein the antibody or antibody fragment is a genetically engineered antibody or antibody fragment.

[0024] A seventh aspect of the present invention provides an isolated antibody or antibody fragment that specifically binds to B7-H3 according to any one of the above first to sixth aspects, wherein the antibody or antibody fragment is an scFv.

[0025] An eighth aspect of the present invention provides a chimeric antigen receptor comprising an antibody or antibody fragment that specifically binds to B7-H3 according to the seventh aspect above.

[0026] A ninth aspect of the present invention provides a chimeric antigen receptor according to the eighth aspect of the present invention, comprising the antibody or antibody fragment according to the seventh aspect above and a transmembrane domain fused to the carboxyl terminus of the antibody or antibody fragment.

[0027] A tenth aspect of the present invention provides a chimeric antigen receptor according to the eighth or ninth aspect, comprising the antibody or antibody fragment according to the seventh aspect, a transmembrane domain fused to the carboxyl terminus of the antibody or antibody fragment, and an immunocompetent cell activation signaling domain fused to the carboxyl terminus of the transmembrane domain.

[0028] An eleventh aspect of the present invention provides a chimeric antigen receptor according to any one of the eighth to tenth aspects, further comprising any one or more of a membrane protein, a secreted protein, an intracellular protein, a small molecule drug, and a cytotoxic drug.

[0029] A twelfth aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antibody fragment according to any one of the first to seventh aspects, or the chimeric antigen receptor according to any one of the eighth to eleventh aspects.

[0030] A thirteenth aspect of the present invention provides a nucleic acid molecule according to the twelfth aspect above, comprising (i) a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO:5 and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:7, or (ii) a nucleotide sequence encoding a heavy chain variable region as shown in SEQ ID NO:6 and a nucleotide sequence encoding a light chain variable region as shown in SEQ ID NO:8. SEQ ID NO:5 GAAGTGCAGCTCCAGCAGAGCGGAGCAGAACTGGTGAAGCCAGGAGCCAGCGTGAAGCTGTCTTGCAAGGCCAGCGGCTACACCTTCACCAACTACTGGATCAATTGGATCAGACAGAGGCCAGGACAGGGACTCGAGTGGATTGGCAGAATCGCCCCAGGCACCATCAGCACCTACTACAACGAGAAGTTCAAGGGCAGGGCCACCATCACCGAGGATACCAGCACCAACACCGCCTACCTCCAGCTGTCTAGCCTGACAAGCGAGGACACAGCCGTGTACTTTTGCGCCAGGCAGGACAACTACTTCATCAATTGGGGCCAGGGAACCCTGGTGACAGTGTCCAGC SEQ ID NO:6 GAGTTCGAGGTGCAGCTGGTGGAGAGCGGAGGTGGGCTAGTTAAACCCGGCGGCTCCTTGAAGCTGTCATGCGCTGCTTCTGGGTTTGCGTTCTCCCGCTACGACATGAGTTGGGTGCGCCAGAGCCCTGAAAAGCGCCTGGAGTGGGTCGCCACCATTAGCGATGACGGCAGACACACCTACGACAGGGATAGCGTAAAGGGTCGCTTCACCATCTCTCGTGACAACGCCAAGAACACGCTTTACCTGCAGATGTCCTCTCTGCGCTCGGAGGACACCGCGCTCTACTACTGCGTGCGACATCGCGCCATCACTACTGCACGGTTCGACTATTGGGGCCAGGGCACCACAGTCACCGTGTCGTCCTCCCGT SEQ ID NO:7 GACATTGTTCTTACACAATCTCCGGCCAGCCTCTCTGCGAGCCTCGGGGAAAAGGTGACAATCACTTGTTCCGCATCATCAAGTATCTCCAGCTCTGACCTGCACTGGTATCAGCAGAAAAGTGGCACCAGTCCGAGGCCGTGGATCTATGGCACTAGCAAT CTTGCGTCAGGAGTCCCACCCCGCTTTAGTGGCAGTGGATCAGGGACATCATTCAGTCTGACAATCTCTAGCGTCGAGGCGGAGGATGTCGGCACATATTACTGCCAACAGTGGTTTTCTTATCCTTTCACATTTGGTACGGGCACGAAACTGGAAATAAAA SEQ ID NO:8 GGGTGCGCGGACATCGTGATGACCCAGAGTCCGTCGTCTCTGAGCGTCTCGCTCGGCGACACCGTGAGCATCACTTGTAAAGCTTCCCAGGACATCTACTCCAACATCGGTTGGCTACAACAGCTGCCCGGACAGTCCTTCAAGGGCCTGATTTACCACGGGACCAACC TGGAGGACGGCGTTCCTTCCCGCTTCAGCGGCTCCGGCTCCGGTACAGATTACTCTCTGACCATTTCTGGCCTTGAGAGCGAAGACTTTGCCGATTACTACTGCCTGCAGTACGTGCAGTTCCCCTATACCTTCGGCGGTGGCACTAAGTTGGAGATCAAGGCCTCAGGC

[0031] A fourteenth aspect of the present invention provides a vector comprising a nucleic acid molecule according to the twelfth or thirteenth aspect above.

[0032] A fifteenth aspect of the present invention provides the vector according to the fourteenth aspect, wherein the vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0033] A sixteenth aspect of the present invention provides a cell comprising a nucleic acid molecule according to the twelfth or thirteenth aspect above, or a vector according to the fourteenth or fifteenth aspect.

[0034] A seventeenth aspect of the present invention provides the cells according to the sixteenth aspect, comprising autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes, wherein the T cells include αβ T cells, γδ T cells, and regulatory T cells.

[0035] An 18th aspect of the present invention provides a method for producing a cell comprising one or more selected from the group consisting of the antibody or antibody fragment according to any one of the above-mentioned first to seventh aspects, the chimeric antigen receptor according to any one of the above-mentioned eighth to eleventh aspects, the nucleic acid molecule according to the 12th or 13th aspect, the vector according to the 14th or 15th aspect, and the cell according to the 16th or 17th aspect, or and a pharmaceutically acceptable carrier; or a pharmaceutical composition comprising one or more selected from the group consisting of the antibody or antibody fragment according to any one of the first to seventh aspects, the chimeric antigen receptor according to any one of the eighth to eleventh aspects, the nucleic acid molecule according to the twelfth or thirteenth aspect, the vector according to the fourteenth or fifteenth aspect, and the cell according to the sixteenth or seventeenth aspect, and another pharmaceutically active reagent or drug.

[0036] A 19th aspect of the present invention provides a method for treating or preventing a B7-H3-positive disease, comprising administering to a subject a therapeutically effective amount of the antibody or antibody fragment according to any one of the above-mentioned first to seventh aspects, the chimeric antigen receptor according to any one of the above-mentioned eighth to 11th aspects, the nucleic acid molecule according to the 12th or 13th aspect, the vector according to the 14th or 15th aspect, the cell according to the 16th or 17th aspect, or the pharmaceutical composition according to the 18th aspect.

[0037] A twentieth aspect of the present invention provides the method according to the nineteenth aspect, wherein the B7-H3-positive disease includes malignant / benign hematologic tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, viral infections, parasitic infections, abnormal bone growth, allografts, transplant rejection, etc., and the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, ankylosing spondylitis, etc.

[0038] A twenty-first aspect of the present invention provides a method according to the twentieth aspect, wherein the disease comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome, etc.

[0039] A 22nd aspect of the present invention provides the antibody or antibody fragment according to any one of the first to seventh aspects, the chimeric antigen receptor according to any one of the eighth to eleventh aspects, the nucleic acid molecule according to the twelfth or thirteenth aspect, the vector according to the fourteenth or fifteenth aspect, the cell according to the sixteenth or seventeenth aspect, or the pharmaceutical composition according to the eighteenth aspect, for treating or preventing a B7-H3-positive disease.

[0040] A twenty-third aspect of the present invention provides an antibody or antibody fragment, a chimeric antigen receptor, a nucleic acid molecule, a vector, a cell, or a pharmaceutical composition for treating or preventing a B7-H3-positive disease according to the twenty-second aspect, wherein the B7-H3-positive disease comprises malignant / benign hematologic tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, viral infections, parasitic infections, abnormal bone growth, allografts, and transplant rejection, and the autoimmune diseases comprise systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, and ankylosing spondylitis.

[0041] A twenty-fourth aspect of the present invention provides an antibody or antibody fragment, chimeric antigen receptor, nucleic acid molecule, vector, cell, or pharmaceutical composition for treating or preventing a B7-H3-positive disease according to the twenty-third aspect, wherein the B7-H3-positive disease comprises acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, gastrointestinal cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.

[0042] A 25th aspect of the present invention provides a method for detecting B7-H3, comprising the step of detecting B7-H3 using the antibody or antibody fragment described in any one of the above-mentioned first to seventh aspects, the chimeric antigen receptor described in any one of the above-mentioned eighth to eleventh aspects, or the nucleic acid molecule described in the above-mentioned twelfth or thirteenth aspect.

[0043] A 26th aspect of the present invention provides a kit for detecting B7-H3, which comprises the antibody or antibody fragment or a labeled product thereof according to any one of the above-mentioned first to seventh aspects, the chimeric antigen receptor or a labeled product thereof according to any one of the above-mentioned eighth to 11th aspects, or the nucleic acid molecule or a labeled product thereof according to the above-mentioned 12th or 13th aspect.

[0044] A 27th aspect of the present invention provides a separation kit for separating B7-H3-positive cells, comprising the antibody or antibody fragment or a labeled product thereof according to any one of the above-mentioned first to seventh aspects, the chimeric antigen receptor or a labeled product thereof according to any one of the above-mentioned eighth to 11th aspects, or the nucleic acid molecule or a labeled product thereof according to the above-mentioned 12th or 13th aspect.

[0045] A 28th aspect of the present invention provides a method for enhancing a cell function in vitro, the method comprising the step of contacting a cell with the antibody or antibody fragment described in any one of the above-mentioned first to seventh aspects, or a labeled product thereof, the chimeric antigen receptor described in any one of the above-mentioned eighth to 11th aspects, or a labeled product thereof, or the nucleic acid molecule described in the above-mentioned 12th or 13th aspect, or a labeled product thereof.

[0046] A twenty-ninth aspect of the present invention provides the use of an antibody or antibody fragment according to any one of the first to seventh aspects above in the manufacture of a product for detecting B7-H3 protein.

[0047] A 30th aspect of the present invention provides the use of an antibody or antibody fragment according to any one of the first to seventh aspects above in the manufacture of a product for blocking B7-H3 protein.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the nucleotide sequences of the heavy chain variable regions of the 1B4 and 2Y31 antibodies, where FIG. 1A shows the nucleotide sequence of the heavy chain variable region of the 1B4 antibody (SEQ ID NO:5), and FIG. 1B shows the nucleotide sequence of the heavy chain variable region of the 2Y31 antibody (SEQ ID NO:6). Figure 2 shows the nucleotide sequences of the light chain variable regions of the 1B4 and 2Y31 antibodies, where Figure 2A shows the nucleotide sequence of the light chain variable region of the 1B4 antibody (SEQ ID NO:7), and Figure 2B shows the nucleotide sequence of the light chain variable region of the 2Y31 antibody (SEQ ID NO:8). Figure 3 shows the amino acid sequences of the heavy chain variable regions of the 1B4 and 2Y31 antibodies, where Figure 3A is the amino acid sequence of the heavy chain variable region of the 1B4 antibody (SEQ ID NO:1), and Figure 3B is the amino acid sequence of the heavy chain variable region of the 2Y31 antibody (SEQ ID NO:2). Figure 4 shows the amino acid sequences of the light chain variable regions of the 1B4 and 2Y31 antibodies, where Figure 4A is the amino acid sequence of the light chain variable region of the 1B4 antibody (SEQ ID NO:3), and Figure 4B is the amino acid sequence of the light chain variable region of the 2Y31 antibody (SEQ ID NO:4). FIG. 5 shows the results of detecting the affinity of the humanized 1B4 antibody. Figure 6 shows the results of binding of the humanized antibody to the glioma cell line U87 and the B7-H3 knockout U87 cell line. In Figure 6A, the gray peak represents the negative control, the solid peak represents the binding of the parent mouse-derived antibody 1B4 to the tumor cell line, and the dotted peak represents the binding of the humanized 1B4 antibody to the tumor cell line. In Figure 6B, m2Y31 represents the binding of the parent mouse-derived antibody 2Y31 to the tumor cell line, and hu2Y31 represents the binding of the humanized antibody 2Y31 to the tumor cell line. 8H9 represents the binding of an antibody recognizing a different B7-H3 antigen epitope to tumor cells, and NC is the negative control. Figure 7 shows a schematic diagram of the CAR structure, which consists of an scFv (VH-(G4S)3-VL), a CD8 hinge region (CD8 hinge), a transmembrane domain (CD8 TM), costimulatory signals CD28 and 4-1BB, and a CD3z signal activation domain. The scFv contains one heavy chain variable region and one light chain variable region linked by a (GGGGS)3-linked peptide. Furthermore, a truncated EGFR structure is linked to the C-terminus of the CAR structure via a P2A cleavage peptide. This truncated EGFR structure can be used as a screening marker for CAR-positive cells, adding a safety switch to clinical studies. Figure 8 shows the binding ability of Jurkat T cells expressing CAR-B7-H3 to B7-H3 protein. Figure 9 shows that wild-type colon cancer tumor cells RKO (dark peak, RKO WT) express high levels of B7-H3, while B7-H3 knockout RKO tumor cells (light peak, RKO B7-H3 KO) do not express B7-H3, as detected with the 1B4 antibody. Figure 10 shows the expression levels of CD69 after stimulation of CAR-Jurkat T cells constructed using the 1B4 antibody with RKO WT or RKO (B7-H3 KO) tumor cells. Figure 11A shows the transduction positivity rate of αβ T cells expressing CAR-B7-H3 (scFv is the light and heavy chain sequence of the 1B4 antibody). Figure 11B shows the expression levels of CD69 after stimulation of αβT or CAR-B7-H3-αβT cells with RKO WT or RKO (B7-H3 KO) tumor cells. Figure 12 shows the in vitro killing ability of RKO tumor cells by CAR-B7-H3-αβ T cells (scFv is the light and heavy chain sequences of the 1B4 antibody). Figure 13 shows the in vitro killing ability of CAR-B7-H3-αβ T cells (scFv is the light and heavy chain sequences of the 1B4 antibody) to glioma cells. Figure 14 shows the transduction positivity of γδ T cells expressing CAR-B7-H3 (scFv is the light and heavy chain sequence of the 1B4 antibody). Figure 15A shows the killing effect of RKO WT tumor cells by CAR-αβT and CAR-γδT expressing CAR-B7-H3 (scFv is the light and heavy chain sequence of the 1B4 antibody). Figure 15B shows the killing effect of CAR-αβT and CAR-γδT expressing CAR-B7-H3 on B7-H3 knockout RKO tumor cells (scFv is the light and heavy chain sequence of the 1B4 antibody). Figure 16 shows the in vitro killing ability of CAR-γδT expressing CAR-B7-H3 to B7-H3-positive tumor cells SKOV3 (scFvs are the light chain and heavy chain sequences of the 1B4 antibody or the 2Y31 antibody, respectively). Figure 17 shows the in vivo antitumor effect of CAR-γδT expressing CAR-B7-H3 (scFv is the light and heavy chain sequences of the 1B4 antibody) on SKOV3. FIG. 18 shows the in vivo growth curve of SKOV3 tumor model mice. FIG. 19 shows the changes in survival curves of SKOV3 tumor model mice.

[0049] [Mode for Carrying Out the Invention] The present invention will be further described below with reference to the description of specific embodiments and drawings, but the present invention is not limited thereto. Those skilled in the art can make various modifications and improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they will all fall within the scope of the present invention.

[0050] Unless otherwise defined, technical or scientific terms used herein have the same meaning as commonly understood by those skilled in the art. It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0051] As used herein, "specifically binds" means that the antigen recognition region of the present invention does not cross-react or does not substantially cross-react with any polypeptides other than the target antigen. The degree of specificity can be determined by immunological techniques, including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, etc.

[0052] As used herein, "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or may be an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies of the present invention may exist in various forms, including, for example, Fab, Fab', F(ab')2, Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, ADC-binding antibodies, multispecific antibodies, single-chain antibodies, and humanized antibodies.

[0053] As used herein, "antibody fragment" refers to a portion of the structure or sequence of an entire antibody.

[0054] As used herein, "heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring conformation in all antibody molecules.

[0055] As used herein, "light chain" refers to the smaller of the two types of polypeptide chains present in the naturally occurring conformation in all antibody molecules.

[0056] As used herein, the term "heavy chain variable region (VH)" refers to approximately 110 amino acid residues near the amino terminus (N-terminus) of the heavy chain, and the composition and sequence of amino acid residues in this region vary greatly.

[0057] As used herein, the term "light chain variable region (VL)" refers to approximately 110 amino acid residues near the amino terminus (N-terminus) of the light chain, and the composition and sequence of amino acid residues in this region vary greatly.

[0058] The term "CDR" as used herein refers to a complementarity-determining region, which is a non-contiguous antigen-binding site found in the variable regions of heavy and light chain polypeptides. The present invention uses the Kabat numbering convention when annotating the CDR and FR regions of the variable regions of an antibody.

[0059] "Homology" as used herein refers to the high degree of amino acid or nucleotide identity shown when a target amino acid sequence or a target nucleotide sequence is compared with a reference sequence. Homology as used herein can be determined using standard software such as BLAST or FASTA.

[0060] As used herein, "homology of 80% or more" means that the homology is 80% or more, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, still more preferably 93% or more, particularly preferably 95% or more, especially more preferably 98% or more, and most preferably 100%.

[0061] As used herein, "genetically engineered antibodies or antibody fragments" refer to antibodies or antibody fragments that have been engineered and modified through genetic engineering. In this context, "genetically engineered" refers to techniques for integrating and expressing genes of different origins using techniques such as gene splicing and DNA recombination. Specific examples include inserting other proteins or protein fragments into antibodies or antibody fragments, replacing portions of antibodies or antibody fragments with other proteins or protein fragments, and linking different antibodies or antibody fragments to other proteins or protein fragments to form larger protein complexes. A "humanized antibody" is also a type of "genetically engineered antibody or antibody fragment" and refers to an antibody that has been modified and re-expressed from a non-human (e.g., mouse, rabbit, etc.) monoclonal antibody through gene cloning or DNA recombination techniques. Its distinctive feature is that it retains the affinity and antigen-binding specificity of the corresponding mouse-derived antibody while reducing the immunogenicity. A basic method for achieving this is for the constant region or entire antibody portion of a non-human-derived antibody to be encoded by human antibody genes. Humanized antibodies include types such as chimeric antibodies, modified antibodies, and fully humanized antibodies, depending on the modification method.

[0062] As used herein, the term "constant region" refers to a relatively stable region of an antibody near its C-terminus. This term includes a "light chain constant region" and a "heavy chain constant region," which refer to a relatively stable region of an antibody light chain near its C-terminus and a relatively stable region of an antibody heavy chain near its C-terminus, respectively.

[0063] As used herein, "scFv" refers to an antibody fragment, a recombinant protein comprising a heavy chain variable region and a light chain variable region connected by a linker, which ultimately forms the antigen-binding site. The size of an scFv is typically one-sixth that of a complete antibody. An scFv preferably has an amino acid sequence encoded by a single nucleotide chain. The scFv used in the present invention can be further modified by conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and / or recombination and / or other modification methods, either alone or in combination. Methods for introducing such modifications into an antibody DNA sequence based on the antibody's amino acid sequence are well known to those skilled in the art (see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989), NY). The modifications are preferably made at the nucleic acid level. The above-mentioned scFv may also include derivatives thereof. An scFv can be expressed as a single polypeptide chain. An scFv retains the specificity of the complete antibody from which it was derived. As long as the specificity of the scFv for its target antigen is maintained, the light chain and heavy chain may be arranged in any order, such as VH-linker-VL or VL-linker-VH. As used herein, an example of a linker is a flexible linker peptide chain rich in glycine and serine.

[0064] As used herein, a "chimeric antigen receptor" (CAR) refers to an artificially engineered receptor that immobilizes a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen on an immune cell (e.g., a T cell), allowing the immune cell to recognize the tumor antigen or viral antigen and kill the tumor cell or virus-infected cell. CARs typically contain, in order, an optional signal peptide, a polypeptide (e.g., a single-chain antibody) that binds to the tumor cell membrane antigen, a hinge region, a transmembrane region, and an immunocompetent cell activation signaling region, wherein the immunocompetent cell activation signaling region further includes a costimulatory domain and a signaling domain.

[0065] The term "hinge region" refers to the hydrophilic region between the antigen-recognition domain and the transmembrane domain. The hinge region may be selected from various antibodies or antigen receptors, particularly from CD molecules. In a specific embodiment, the hinge region may be selected from, for example, CD4, CD8α, CD28, IgG1, and IgG4. In a preferred embodiment of the present invention, the CD8α hinge region is used. Examples of hinge region sequences include the following: CD8 hinge region (SEQ ID NO:21): TTCGTGCCGGTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0066] The "transmembrane domain" may be any domain containing a peptide capable of penetrating a cell membrane. A preferred transmembrane domain is that of a CD molecule. In one embodiment, the transmembrane domain may be selected from, for example, CD4, CD8, CD28, CD3ζ, the α chain or β chain of the T cell receptor, CD3ζ, CD3ε, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, epidermal growth factor receptor (EGFR), NKG2D, or GITR. In one preferred embodiment of the present invention, the CD8α transmembrane domain is used. Examples of transmembrane domain sequences include the following: CD8 transmembrane region (SEQ ID NO:22): ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACAGGAAC CD4 transmembrane domain (SEQ ID NO:23): ATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTC

[0067] The term "costimulatory domain" refers to a portion of a CAR that promotes the proliferation, survival, and / or development of memory cells. A CAR of the present invention may comprise one or more costimulatory domains. Each costimulatory domain may comprise one or more of the costimulatory domains of, for example, CD28, 4-1BB, ICOS, OX40, CD27, CD40, Myd88, HVEM, GITR, and Fc receptor-associated γ chain. In one preferred embodiment of the present invention, a combination of CD28 and 4-1BB is used. The sequences of the costimulatory domains may include the following: 4-1BB (SEQ ID NO: 24): CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAGCTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG CD28 (SEQ ID NO: 25): AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

[0068] The "signaling domain" refers to the portion of the CAR that transmits effector function signals and instructs cells to perform specific functions. Examples of domains that transmit effector function signals include, but are not limited to, CD3ζ and FcεRIγ. In one preferred embodiment of the present invention, the CD3ζ domain is used. The sequence of the signaling domain may include the following: CD3z (SEQ ID NO: 26): AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0069] In the terms "membrane proteins, secreted proteins, intracellular proteins, small molecule drugs, and cytotoxic drugs" used herein, a membrane protein refers to a protein whose entirety or a portion is inserted into various membrane structures (including membrane structures such as the cell membrane, mitochondrial membrane, endoplasmic reticulum membrane, and nuclear membrane). A secreted protein refers to a protein that is secreted outside the cell after protein synthesis. An intracellular protein refers to all proteins present inside the cell membrane, including free proteins in the cytoplasm and membrane proteins on the membranes of various intracellular organelles. A small molecule drug refers to a chemically synthesized drug with a molecular weight of less than 1,000 that can be linked to other proteins or molecular drugs via a linker arm. A cytotoxic drug refers to a drug that has a toxic effect on cells or specific cells, including, but not limited to, alkaloid drugs (e.g., paclitaxel), metabolic drugs (e.g., decitabine), antibiotic drugs (e.g., idarubicin), alkylating agents (e.g., ifosfamide), and platinum agents (e.g., cisplatin). A small molecule drug can also be linked to other proteins or molecular drugs via a linker arm.

[0070] In the chimeric antigen receptor of the present invention, membrane proteins may include, for example, CD40L, CXCR5, CXCR3, 4-1BB, ICOS, OX40, CD27, NKG2D, etc. Secreted proteins may include, for example, IL2, IL15, IL4, IL7, IL10, IL18, IFNγ, IL1β, antibodies (e.g., anti-PD1 antibodies, anti-CTLA4 antibodies, etc.). Small molecule drugs may include, for example, FITC / folic acid, rapamycin, rimiducid, PROTAC compounds, dasatinib, etc. Cytotoxic agents may include, for example, paclitaxel, vinorelbine, docetaxel, hydroxycamptothecin, gemcitabine, cytarabine, tegafur, methotrexate, epirubicin, pirarubicin, idarubicin, mitomycin, mitoxantrone, ifosfamide, dacarbazine, cisplatin, oxaliplatin, and the like.

[0071] As used herein, the term "nucleic acid molecule" refers to a biomolecular compound formed by the polymerization of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), whose building blocks are nucleotides, and nucleotide monomers consist of a pentose sugar, a phosphate group, and a nitrogenous base.

[0072] As used herein, "encode," as applied to a nucleic acid sequence, means that a polynucleotide that "encodes" a polypeptide, either in nature or when manipulated by methods well known to those of skill in the art, can be transcribed and / or translated to produce mRNA for that polypeptide and / or fragment thereof.

[0073] As used herein, the term "vector" refers to a recombinant vector that retains the ability to infect and transduce non-dividing and / or slowly dividing cells and integrate into the genome of target cells. In some preferred embodiments, the vector is derived from or based on a wild-type virus. In further preferred embodiments, the vector is derived from or based on a wild-type lentivirus. Examples may include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, and the like. More specifically, in one preferred embodiment of the present invention, the vector is a lentiviral vector. In another preferred embodiment of the present invention, the vector is a retroviral vector.

[0074] "Autologous or allogeneic" herein refers to "autologous cells" or "allogeneic cells." Here, "autologous cells" refer to cells derived from the same individual and subsequently readministered to that individual. "Allogeneic cells" refer to cells derived from outside the individual.

[0075] The cells in the present invention may include T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes, and among these, T cells may include αβ T cells, γδ T cells, and regulatory T cells. In one preferred embodiment of the present invention, γδ T cells are used.

[0076] The pharmaceutical composition of the present invention may comprise any one of the antibodies, CARs, nucleic acid molecules, vectors, and cells of the present invention, and may further comprise a pharmaceutically acceptable carrier. Alternatively, the pharmaceutical composition of the present invention may comprise two or more of the antibodies, CARs, nucleic acid molecules, vectors, and cells of the present invention, for example, a CAR and a nucleic acid molecule, and may further comprise a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may also comprise other pharmaceutically active agents or drugs, such as chemotherapeutic agents, including asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. In a preferred embodiment, the pharmaceutical composition comprises the cells of the present invention.

[0077] Furthermore, the pharmaceutically acceptable carrier in the pharmaceutical composition may be any pharmaceutically acceptable carrier conventionally used, and a person skilled in the art can select an appropriate pharmaceutically acceptable carrier based on conditions such as chemical and physical conditions and the route of administration. Examples of the pharmaceutically acceptable carrier described herein may include vehicles, adjuvants, excipients, and diluents. It is desirable for the pharmaceutically acceptable carrier to be chemically inert to the active agent and free from adverse side effects or toxicity under the conditions of use.

[0078] As used herein, "treatment or prevention" refers to a method of eliminating a disease by a specific means after the onset of the disease, or a method of avoiding the onset of the disease by a specific means. As used herein, "treatment" and "prevention" do not necessarily mean 100% or complete treatment or prevention, as there are various degrees of treatment or prevention that those skilled in the art recognize as having potential beneficial or therapeutic effects. Furthermore, treatment or prevention by the methods of the present invention may include treatment or prevention of one or more diseases or symptoms of the disease being treated or prevented (e.g., cancer). Similarly, as used for purposes of the present invention, "prevention" may also include delaying the onset of the disease or its symptoms or symptoms.

[0079] As used herein, the term "subject" refers to a mammal. Generally, the subject of the present invention refers to any subject, preferably a human, who has or is at risk of having cancer.

[0080] As used herein, a "therapeutically effective amount" refers to a sufficient amount of an antibody or antibody fragment, chimeric antigen receptor, nucleic acid molecule, vector, cell, or pharmaceutical composition of the present invention to treat a B7-H3-positive disease (e.g., limit the growth or slow or inhibit tumor metastasis) at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily amount of an antibody or antibody fragment, chimeric antigen receptor, nucleic acid molecule, vector, cell, or pharmaceutical composition of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will vary depending on a variety of factors, including the condition and severity of the condition being treated, the activity of the specific dosage used, the specific combination used, the subject's age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion of the specific dosage used, duration of treatment, drugs used in combination with or concomitantly with the specific dosage used, and similar factors well known in the medical arts.

[0081] The term "B7-H3-positive disease" as used herein refers to diseases associated with high expression of B7-H3, including malignant / benign hematologic tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, viral infections, parasitic infections, abnormal bone growth, allogeneic transplants, and transplant rejection, as well as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumors, bladder cancer, digestive cancer, prostate cancer, liver cancer, ovarian cancer, and pancreatic cancer. The autoimmune diseases include pancreatic cancer, endometrial cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome, and the like, wherein the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome, ankylosing spondylitis, etc. The detection kit of the present invention is a kit for detecting B7-H3, and typically includes components commonly used in such kits, such as a pH buffer and stabilizers, as well as accompanying materials such as operating instructions and instructions for detecting B7-H3.

[0082] The isolation kit of the present invention is a isolation kit for isolating B7-H3-positive cells, and typically includes components such as pH buffers and stabilizers that are commonly used in such kits, as well as accompanying materials such as operating instructions and instructions for isolating B7-H3-positive cells.

[0083] Additionally, the term "P2A cleavage peptide" as used herein refers to a short viral peptide (18-25 amino acids in length) that can generate multiple proteins from a single transcript through a process known as "self-cleavage." Its basic principle is that it causes the ribosome to skip synthesis of the C-terminal glycine and proline peptide bonds of the original 2A, resulting in the separation of the 2A sequence end from the downstream product. The P2A polypeptide is one of these polypeptides; the others include T2A, E2A, and F2A. These four 2A peptides are derived from different viruses.

[0084] As used herein, "truncated EGFR," abbreviated as "EGFRt," refers to a truncated version of wild-type EGFR that lacks domains III and IV. This truncated EGFR lacks intracellular signaling and therefore does not transmit other signals to T cells. These two domains are also recognized epitopes by cetuximab. Expression of this epitope on T cells can be used as a screening marker for CAR-T and also adds a safety switch for clinical research. An inducer (e.g., cetuximab) specifically binds to EGFRt, causing CAR-T cells to undergo apoptosis via antibody-dependent cell-mediated cytotoxicity, thereby enabling CAR-T cells to be eliminated from the body at any time.

[0085] The aforementioned "antibody-dependent cell-mediated cytotoxicity," abbreviated as ADCC in English, refers to the process in which the Fab segment of an antibody binds to an antigen epitope on a tumor cell, and its Fc segment binds to FcR on the surface of killer cells (NK cells, macrophages, etc.), mediating the killer cells to directly kill the target cell.

[0086] As used herein, the term "flexible linker peptide chain" refers to an oligopeptide or polypeptide region of approximately 1 to 100 amino acids in length that connects any domain / region of the CAR of the present invention. The linker can be composed of flexible residues (e.g., glycine or serine) to allow adjacent protein domains to move freely relative to each other, and longer linkers can be used if it is desired to prevent steric interference between two adjacent domains. In one preferred embodiment of the present invention, (GGGGS)3 is used.

[0087] As used herein, the term "transduction positivity rate" refers to the rate at which an exogenous gene is transduced into T cells, including the rate at which a CAR is transduced into T cells, and reflects the proportion of CAR-positive T cells among all T cells.

[0088] As used herein, "affinity" or "binding affinity" or "KD" is determined by measuring the equilibrium association constant (ka) and the equilibrium dissociation constant (kd) and calculating the quotient of kd and ka (KD=kd / ka). A smaller KD value indicates a stronger affinity, and a larger KD value indicates a weaker affinity. Experimental Materials and Methods

[0089] 1. Culturing Tumor Cell Lines Six types of adherent tumor cells were used in this study: 293T, U87, HTB15, TJ905, RKO, and SKOV3 cells. 293T cells are a human kidney epithelial cell line commonly used for studies of exogenous gene expression and virus production. U87, HTB15, and TJ905 are all human brain glioma cell lines, RKO is a human colon cancer cell line, and SKOV3 is a human ovarian cancer cell line. All tumor cells except SKOV3 were cultured in DMEM complete medium (DMEM medium + 10% FBS + 1% double antibody). SKOV3 was cultured in McCoy's 5a complete medium (McCoy's 5a medium + 20% FBS + 1% double antibody). Jurkat T cells are a human T lymphocytic leukemia cell line and were cultured in RPMI-1640 complete medium (RPMI-1640 medium + 10% FBS + 1% double antibody).

[0090] 2. Lentivirus Production 1×10 7 293T cells were seeded onto a 10 cm cell culture dish coated with polylysine, and the medium volume was adjusted to 10 mL. The next day, the expression plasmid (backbone derived from pCDH-EF1-MCS-T2A-copGFP Plasmid (Addgene, Plasmid #72263)), auxiliary plasmid pspAx2 (Addgene, #12260), and auxiliary plasmid pCMV-VSV-G (Addgene, Plasmid #8454) were homogenously mixed at 6 μg, 4 μg, and 2 μg, respectively, in 300 μL of opti-MEM medium to prepare the medium containing the plasmid. 30 μg of PEI was also added to 300 μL of opti-MEM medium and mixed thoroughly. This PEI solution was then added to the above medium containing the plasmid, mixed homogenously immediately, and allowed to stand at room temperature for 15 minutes before being homogenously added to the 293T cell culture dish (time 0). After 8 hours, the medium was replaced with 10 mL of fresh DMEM complete medium. Culture supernatants were collected at 48 and 72 hours, respectively. After collecting the supernatant at 48 hours, 8 mL of fresh DMEM complete medium was added. The supernatant was then transferred to Lenti-X TM The virus solution was concentrated using a concentrator (Takara) to obtain a virus solution, and the virus titer of the virus solution was detected using 293T cells. The virus solution was aliquoted and stored for a long period at -80°C.

[0091] 3. Primary Cell Culture

[0092] (1)γδT cells PBMC cells were prepared and washed twice with PBS. Cells were counted using an AO / PI cell counter to detect viability. PBMC cells were cultured in RPMI1640 medium + 10% FBS + 1% dual antibody + 200 U / mL rhIL-2 culture system at an initial cell density of 2 × 10 6 At the same time, 5 μM zoledronic acid (ZOL) was added. After 48 hours, the cells were replenished with an equal volume of liquid (without ZOL), and then the cell density was adjusted every 48 hours (1 × 106 cells / mL).

[0093] (2)αβT cells PBMC cells were prepared and washed twice with PBS. Cells were counted using an AO / PI cell counter to detect viability. Antibody coating treatment for 6-well plates: CD3 antibody and CD28 antibody were diluted to 200 ng / mL in opti-MEM medium, and 2 mL of the antibody dilution was added to each well of the 6-well plate, followed by treatment at 4°C overnight or at 37°C for 2 hours. Immediately before adding PBMC cells to the 6-well plates, the antibody dilutions were aspirated and washed once with 2 mL of PBS. Next, PBMCs were added and resuspended in RPMI 1640 medium + 10% FBS + 1% double antibody + 200 U / mL rhIL-2 culture system to a cell density of 2 × 10 6 The cell density was then adjusted every 48 hours using a culture system of RPMI 1640 medium + 10% FBS + 1% double antibody + 200 U / mL rhIL-2 (1 × 10 6 cells / mL).

[0094] 4. Viral Transduction of αβT and γδT 1×10 7 The lentivirus was diluted with 200 μL of PBS and added to a 24-well plate, which had been previously coated with RetroNectin. The plate was centrifuged at 2000 g and 32°C for 2 hours. After centrifugation, the virus dilution was aspirated and the plate was washed three times with 1 mL of PBS before adding the cells. For transduction of αβT or γδT cells, a total of 1 × 10 αβT or γδT cells were counted after in vitro activation and 24 h of culture. 6 The cells were resuspended in 1 mL of RPMI 1640 medium + 10% FBS + 1% double antibody + 200 U / mL rhIL-2 culture system and added to the virus-treated 24-well plate. After centrifugation at 800 g and 32°C for 10 minutes, the cells were placed in a cell culture incubator and cultured. The cells were then counted every 2–3 days and maintained at a density of 1 × 10.6 The concentration was adjusted to cells / mL.

[0095] 5. Flow Cytometry Staining Analysis Cells for flow cytometry analysis were washed once with PBS. 2 × 10 5 The cells were stained with 50 μL of the staining system. The cells were resuspended in the staining system and incubated at 4°C for 30 minutes. After incubation, the cells were washed twice with flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and then analyzed using a flow cytometer. Prepare the staining system: 50 μL flow cytometry buffer + antibody. Flow cytometry buffer formulation: PBS + 1% FBS + 2.5mM EDTA. The amount of antibody used depends on the antibody concentration and the actual situation. For most antibodies (concentration 0.5 to 1 mg / mL), a 1:50 to 1:100 dilution is sufficient. The antibodies used in this experiment mainly include anti-human CD3, anti-human αβTCR, anti-human γδTCR, and anti-human EGFR.

[0096] 6. Detection of CD69 Expression Effector cells and tumor cells were incubated in a 24-well plate at an effector-target ratio of 1:1. 6 effector cells and 0.5 x 10 6 Tumor cells were added. After 24 hours of incubation, flow cytometry analysis was performed. For Jurkat T cells, the flow cytometry staining system is anti-human EGFR, anti-human CD69. For αβ T cells, the flow cytometry staining system is anti-human CD3, anti-human αβ TCR, anti-human EGFR, anti-human CD69.

[0097] 7. In Vitro Cell Killing Experiments In vitro cell killing experiments were performed using luciferase fluorescence detection. The tumor cells to be detected stably expressed luciferase. Killing tests were performed in 24-well plates, with different effector-target ratios set according to experimental requirements. For an effector-target ratio of 1:1, tumor cells and effector cells were counted separately, and the two types of cells were cultured in their respective media at 0.5 × 10 cells / mL. 6 The cells were resuspended at 1000 μL / mL. 500 μL of tumor cells and 500 μL of effector cells were added to the same well and mixed thoroughly. A control well containing tumor cells alone was also set up, and 500 μL of tumor cell medium and 500 μL of effector cell medium were added. The wells were then placed in a cell culture incubator and cultured for 24 hours, after which luciferase fluorescence signals were detected. Killing efficiency (cytotoxicity%) = (signal value of control well - signal value of test well) / signal value of control well × 100%

[0098] 8. Detection of the binding ability of B7-H3 to CAR-B7-H3 The binding ability of B7-H3 to CAR-B7-H3 was detected by flow cytometry. CAR-B7-H3 was stably expressed in Jurkat T cells. Various concentrations of biotin-B7-H3 antigen (ranging from 0.03125 μg / mL to 16 μg / mL) were added to 1 × 10 cells in 100 μL of flow cytometry buffer staining system. 5The cells were incubated with 100 μL of CAR-B7-H3-Jurkat T cells at 4°C for 45 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes, the supernatant discarded, and resuspended in 500 μL of flow cytometry buffer. The cells were then centrifuged at 500 g for 5 minutes, the supernatant discarded, and resuspended in 100 μL of flow cytometry buffer. At the same time, 0.5 μL of fluorescently labeled streptavidin secondary antibody and 0.5 μL of fluorescently labeled anti-human EGFR antibody were added. The cells were incubated at 4°C for 30 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes, the supernatant discarded, and resuspended in 300 μL of flow cytometry buffer. The fluorescent signal of the streptavidin secondary antibody in the EGFR-positive group was analyzed by flow cytometry.

[0099] 9. Antibody Affinity Detection B7-H3 antigen protein was diluted to seven concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM) and loaded onto a 96-well plate. A human protein G probe was used, with the antigen as the stationary phase and the B7-H3 antibody as the mobile phase. The binding time was set to 180 seconds and the dissociation time to 300 seconds. The binding and dissociation constants of the human monoclonal antibody were determined, and the affinity was calculated.

[0100] 10. Detection of antigen-binding ability of B7-H3 antibody by ELISA The target antigens, including B7-H1, B7-H3, B7-H4, and B7-H5, were diluted in PBS to five concentrations: 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 4 mg / mL. 100 μL of each solution was added to a 96-well ELISA plate and incubated at room temperature for 1 hour. After washing twice with PBST, 100 μL of the solution was added per well and incubated for 5 minutes. B7-H3 antibody was then added at 0 μg / mL, 2 μg / mL, or 4 μg / mL, diluted with PBS, and 100 μL of the solution was added per well and incubated for 1 hour. After washing three times with PBST, 100 μL of the solution was added per well and incubated for 5 minutes. HRP-anti-human Fc antibody was diluted 1:2000 with skim milk (antibody concentration: 0.5 mg / mL), diluted 100 μL per well, and incubated for 1 hour at room temperature. After washing three times with PBST, 100 μL of TMB substrate solution was added per well and allowed to stand for 5 minutes. 100 μL of TMB substrate solution was added per well and allowed to stand in the dark at room temperature for 40 minutes. 100 μL of 2M sulfuric acid was added per well to stop the reaction. The ELISA plate was placed in a microplate reader and the OD value was measured at 450 nm.

[0101] 11. Parental antibody blocking experiments 100 μL of 10 μg / mL B7-H3 protein diluted in PBS was added to a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed twice with PBST, and 100 μL was added per well. Then, 100 μL of parental mouse antibodies diluted in PBS (0.625, 1.25, 2.5, 5, 10, 20, and 40 μg / mL) were added and incubated for 1 hour. The plate was washed twice with PBST, and 100 μL of each antibody was added per well. Then, 100 μL of 2 μg / mL B7-H3 antibody and 2 μg / mL 8H9 antibody, which recognizes a different B7-H3 antigen epitope, were added and incubated. Then, 100 μL of the antibody was detected by ELISA (see Materials and Methods, section 10).

[0102] Example The following specific examples further illustrate the technical solutions provided by the present invention, and are intended to illustrate the present invention, but not to limit the scope of the present invention.

[0103] Unless otherwise specified, all experimental methods in the examples are conventional methods. Unless otherwise specified, all reagents and materials used in the examples are commercially available products.

[0104] Example 1: Affinity studies of humanized 1B4 antibody To determine the affinity of the humanized 1B4 antibody for the B7-H3 antigen, B7-H3 antigen protein was diluted at seven gradient concentrations (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, and 1.56 nM) and loaded onto a 96-well plate. A human protein G probe was used, with the antigen as the stationary phase and 1B4 as the mobile phase. The binding time was 180 seconds and the dissociation time was 300 seconds. The binding and dissociation constants of the human monoclonal antibody were determined, and the affinity was calculated. The results are shown in Figure 5 and show that the association constant ka is 2.81E+05, the dissociation constant kd is 7.38E-05, and the equilibrium dissociation constant KD is 2.625E-10.

[0105] Example 2: Parental antibody blocking experiments 100 μL of 10 μg / mL B7-H3 protein diluted in PBS was added to a 96-well ELISA plate and incubated overnight at 4°C. The plate was washed twice with PBST, and 100 μL was added per well. Then, 100 μL of parental mouse-derived B7-H3 antibodies (m1B4 or m2Y31) diluted in PBS at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 40 μg / mL were added and incubated for 1 hour. The plate was then washed twice with PBST, and 100 μL of parental mouse-derived B7-H3 antibodies (m1B4 or m2Y31) diluted in PBS were added and incubated for 5 minutes. Next, 100 μL of 2 μg / mL humanized B7-H3 antibodies (hu1B4 or hu2Y31) diluted in PBS or 100 μL of 2 μg / mL 8H9 antibody, which recognizes a different B7-H3 antigen epitope, diluted in PBS was added and incubated. Then, ELISA detection was performed. The results are shown in Table 1.

[0106] [Table 1] Experiments showed that the addition of either the 1B4 or 2Y31 parent antibody significantly reduced the binding ability of the corresponding humanized 1B4 or 2Y31 antibody to the B7-H3 antigen, but had no significant effect on the 8H9 antibody, which recognizes a different epitope. The results showed that 0.625 μg / mL of m1B4 was able to block hu1B4 binding to the antigen molecule, suggesting that hu1B4 and m1B4 recognize the same site. m1B4 failed to block 8H9 binding to the antigen molecule at either low or high concentrations, further verifying that 1B4 and 8H9 recognize different sites. Similarly, 0.625 μg / mL of m2Y31 was able to block hu2Y31 binding to the antigen molecule, again suggesting that hu2Y31 recognizes the same site as m2Y31 but a different site from 8H9.

[0107] Example 3: Measurement of B7-H3 expression levels in wild-type U87 tumor cells and B7-H3 knockout U87 tumor cells (U87-B7-H3 CAS9) We used CRISPR-Cas9 technology to construct a B7-H3 knockout U87 tumor cell line. One million human glioma U87 cells were washed once with PBS and resuspended in 100 μL of PBS. Then, 1 μg of reagent was added to each group (NC for negative control, B7-H3 antibody 8H9 for positive control, humanized 1B4 antibody, humanized 2Y31 antibody, parental mouse 1B4 antibody, and parental mouse 2Y31 antibody for experimental groups). The cells were incubated at 4°C for 30 minutes, washed once with PBS, resuspended in 500 μL of PBS, and their binding to the B7-H3 antigen was detected using the instrument. In the above procedure, 1 million human glioma U87 cells were replaced with 1 million B7-H3 knockout U87 cells, except that other procedures were the same as above. The results are shown in Figure 6. As shown in Figure 6A, the binding signal of the humanized 1B4 antibody to the glioma cell line U87 (dashed peak) was significantly higher than the signal of the parent mouse-derived 1B4 antibody (solid peak). Furthermore, after knockout of B7-H3 in the U87 cell line, the two antibodies had no binding effect. This indicates that the 1B4 antibody specifically binds to the B7-H3 antigen. The results in Figure 6B show that the binding signal of the humanized 2Y31 antibody (hu2Y31) to the U87 cell line was significantly higher than that of the parent mouse-derived 2Y31 antibody (m2Y31) and higher than that of the 8H9 antibody. None of the antibodies showed binding signals to the B7-H3 knockout U87 tumor cells. This indicates that the 2Y31 antibody is specific for the B7-H3 antigen and that the binding ability of the humanized antibody to the B7-H3 antigen is higher than that of the parent mouse-derived antibody.

[0108] Example 4: Examination of the binding ability of 1B4 antibody to other proteins of the B7-H3 family To verify the specificity of the 1B4 antibody's binding to the B7-H3 protein and exclude its binding to other members of the B7 family, this patent employed an ELISA assay. The target antigens, B7-H1, B7-H3, B7-H4, and B7-H5, were diluted with PBS at five gradient concentrations: 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 0.5 mg / mL, respectively. 100 μL of each solution was added to a 96-well ELISA plate and incubated at room temperature for 1 hour. After washing twice with PBST, 100 μL of each solution was added per well and incubated for 5 minutes. Next, 0 μg / mL, 2 μg / mL, or 4 μg / mL B7-H3 antibody was added, diluted with PBS, and incubated at room temperature for 1 hour. After washing three times with PBST, 100 μL of each solution was added per well and incubated for 5 minutes. HRP-anti-human Fc antibody was diluted 1:2000 with skim milk (antibody concentration: 0.5 mg / mL), and 100 μL was added per well and left at room temperature for 1 hour. After washing three times with PBST, 100 μL was added per well and left at room temperature for 5 minutes. In the dark, 100 μL of TMB substrate solution was added per well and left at room temperature for 40 minutes. The reaction was stopped by adding 100 μL of 2 M sulfuric acid per well, and the ELISA plate was placed in a microplate reader and the OD value was measured at 450 nm. The results are shown in Table 2.

[0109] [Table 2] As shown in Table 2, 2 μg / mL of 1B4 antibody had very high binding capacity to 0.5 mg / mL of B7-H3 antigen, with an OD450 value of over 3.6, reaching a saturation value. Further increases in the concentration of 1B4 antibody or B7-H3 antigen did not result in a further increase in the OD450 value. On the other hand, 1B4 was unable to bind to B7-H1, B7-H4, or B7-H5, and the OD450 value remained at a basal value of approximately 0.04 even with increasing concentrations of 1B4 antibody or B7-H3 antigen. This indicates that the 1B4 antibody specifically binds to the B7-H3 protein and does not bind to other members of the B7 family.

[0110] Example 5: Construction of CAR structure The CAR structure used in this example is a third-generation CAR structure, as shown in Figure 7. The specific structure is an scFv sequence at the 5' end, followed in order by the CD8 hinge region, CD8 transmembrane domain, CD28 and 4-1BB intracellular costimulatory domains, and CD3ζ signaling domain. From the 5' end, the scFv is composed of an antibody heavy chain variable region, a GGGGS GGGGS GGGG flexible linking polypeptide, and an antibody light chain variable region. A truncated EGFR was linked to the CAR structure via a P2A cleavage peptide. Sequence of the CAR structure constructed with 1B4 antibody (SEQ ID NO: 27): Sequence of the CAR structure constructed with 2Y31 antibody (SEQ ID NO:28):

[0111] Example 6: Experiment on the binding ability of Jurkat T cells expressing CAR-B7-H3 to B7-H3 protein The binding ability of B7-H3 to CAR-B7-H3 was detected by flow cytometry. CAR-B7-H3 was stably expressed in Jurkat T cells. Various concentrations of biotin-B7-H3 antigen (ranging from 0.03125 μg / mL to 16 μg / mL) were added to 1 × 10 cells in 100 μL of flow cytometry buffer staining system. 5 The cells were incubated with 100 μL of CAR-B7-H3-Jurkat T cells at 4°C for 45 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes, the supernatant discarded, and resuspended in 500 μL of flow cytometry buffer. The cells were then centrifuged at 500 g for 5 minutes, the supernatant discarded, and resuspended in 100 μL of flow cytometry buffer. At the same time, 0.5 μL of fluorescently labeled streptavidin secondary antibody and 0.5 μL of fluorescently labeled anti-human EGFR antibody were added. The cells were incubated at 4°C for 30 minutes. After incubation, the cells were centrifuged at 500 g for 5 minutes, the supernatant discarded, and resuspended in 300 μL of flow cytometry buffer. The fluorescent signal of the streptavidin secondary antibody in the EGFR-positive group was analyzed by flow cytometry. The results are shown in Figure 8. The results showed that both CAR-B7-H3(1B4) and CAR-B7-H3(2Y31) had the ability to bind to B7-H3 protein, and among them, CAR-B7-H3(1B4) had a higher binding ability to B7-H3.

[0112] Example 7: Measurement of the function of Jurkat T cells expressing CAR-B7-H3 (1B4) We constructed a B7-H3 knockout RKO tumor cell line using gene editing technology and verified it by flow cytometry analysis using the 1B4 antibody. As shown in Figure 9, wild-type RKO tumor cells (dark peak, RKO WT) expressed high levels of B7-H3, whereas B7-H3 knockout RKO tumor cells did not express B7-H3 (light peak, RKO B7-H3 KO). The CD69 signal activation level of CAR-B7-H3 was then further detected. Effector cells (including Jurkat T-control cells that do not express CAR-B7-H3 and Jurkat T-cells that express CAR-B7-H3) were incubated with tumor cells (RKO WT and RKO B7-H3 KO) at an effector-to-target ratio of 1:1. The specific procedure was as follows: 0.5 × 10 6 effector cells and 0.5 x 10 6 Tumor cells were added. After 24 hours of incubation, flow cytometry analysis was performed. The flow cytometry staining system was anti-human EGFR and anti-human CD69. As can be seen from the results shown in Figure 10, Jurkat T cells expressing CAR-B7-H3 (1B4) (CAR-B7-H3-JurkatT) showed a significant upregulation of their CD69 signal after stimulation with wild-type RKO (RKO WT) tumor cells, but the CD69 signal was not enhanced in RKO tumor cells lacking B7-H3. Furthermore, expression of CAR-B7-H3 (1B4) did not upregulate the background CD69 signal, which eliminated its activating toxic effect on T cells.

[0113] Example 8: Measurement of CAR transduction positivity and function of αβ T cells expressing CAR-B7-H3 (1B4) (1) CAR transduction positivity rate CAR-B7-H3(1B4)-αβ T cells were labeled with a fluorescently labeled EGFR antibody and stained using a flow cytometer. The results shown in Figure 11A indicated that the EGFR positivity rate was 89.4%, which means that the CAR transduction positivity rate was 89.4%. (2) CD69 expression level of CAR-B7-H3(1B4)-αβ T cells To analyze the CD69 expression level of CAR-B7-H3(1B4)-αβ T cells, effector cells (including αβ T-control cells that do not express CAR-B7-H3 and CAR-B7-H3-αβ T cells that express CAR-B7-H3) were incubated with tumor cells (RKO WT and RKO B7-H3 KO) at an effector-target ratio of 1:1. The specific procedure is as follows: 0.5 × 10 cells were incubated in a 24-well plate. 6 effector cells and 0.5 x 10 6 After 24 hours of incubation, flow cytometry analysis was performed using anti-human CD3, anti-human αβ TCR, anti-human EGFR, and anti-human CD69 staining systems. The results are shown in Figure 11B. RKO WT tumor cells significantly enhanced CD69 expression in CAR-B7-H3(1B4)-αβ T cells, whereas RKO tumor cells lacking B7-H3 did not affect CD69 expression in CAR-B7-H3(1B4)-αβ T cells. Furthermore, αβ T cells did not upregulate their own CD69 signal after expressing CAR-B7-H3. (3) In vitro killing ability of CAR-B7-H3(1B4)-αβ T cells against RKO tumor cells In vitro cell killing experiments were performed using luciferase fluorescence detection. Target tumor cells (including RKO WT and B7-H3 knockout RKO cells (RKO B7-H3 KO)) stably expressed luciferase. Killing tests were performed in 24-well plates. Different effector-target ratios were used: 0.5:1, 1:1, and 3:1. For the 0.5:1 effector-target ratio, tumor cells and effector cells were counted and resuspended in their respective media. Effector cells were 0.25 × 10 6 Tumor cells were resuspended at 0.5 x 10 cells / mL. 6 For a 1:1 effector-target ratio, both effector and tumor cells were resuspended at 0.5 x 10 cells / mL. 6 For an effector-target ratio of 3:1, effector cells were resuspended at 1.5 x 10 cells / mL. 6 Tumor cells were resuspended at 0.5 x 10 cells / mL. 6 The cells were resuspended at 1000 μL / mL. 500 μL of tumor cells and 500 μL of effector cells were added to the same well and mixed thoroughly. A control well containing tumor cells alone was also set up, and 500 μL of tumor cell medium and 500 μL of effector cell medium were added. The wells were then placed in a cell culture incubator and cultured for 24 hours, after which luciferase fluorescence signals were detected. Killing efficiency (cytotoxicity%) = (signal value of control well - signal value of test well) / signal value of control well × 100% As can be seen from the results shown in Figure 12, compared to control αβT cells (αβT), CAR-B7-H3-expressing αβT cells exhibited superior in vitro killing of RKO tumor cells, and their killing ability gradually increased with increasing effector-to-target ratio. However, they had no killing effect on RKO tumor cells lacking B7-H3. This further verified that the killing effect of CAR-B7-H3-αβT cells against RKO tumor cells is achieved through recognition of B7-H3. (4) In vitro killing ability of CAR-B7-H3(1B4)-αβ T cells against glioma cells In this patent, using the same experimental method as in (3), we further detected the killing ability of CAR-B7-H3-expressing αβ T cells against glioma tumor cells (including HTB15 and TJ905). As can be seen from the results shown in Figure 13, CAR-B7-H3-expressing αβT cells also had a strong killing effect against glioma tumor cells (including HTB15 and TJ905), and their killing activity gradually increased with increasing effector-to-target ratio, whereas αβT cells that did not express CAR-B7-H3 (αβT) had no killing activity against these two glioma tumor cells. This further verified that the killing activity of CAR-B7-H3-αβT cells against these two glioma tumor cells was achieved through recognition of B7-H3.

[0114] Example 9: Measuring CAR transduction positivity and function of γδ T cells expressing CAR-B7-H3 (1B4) (1) CAR transduction positivity rate CAR-B7-H3(1B4)-γδ T cells were labeled with a fluorescently labeled EGFR antibody, stained, and detected using a flow cytometer. The results shown in Figure 14 indicate that the EGFR positivity rate was 45.9%, meaning that the CAR transduction positivity rate was 45.9%. (2) Killing effect of CAR-αβT and CAR-γδT expressing CAR-B7-H3(1B4) on RKO WT tumor cells Using the experimental method described in Example 8(3), the killing effects of CAR-αβT and CAR-γδT expressing CAR-B7-H3 on RKO WT tumor cells were further analyzed and compared. As can be seen from the results shown in Figure 15, the killing ability of CAR-B7-H3-αβ T cells was significantly higher than that of αβ T cells not expressing CAR-B7-H3 (Figure 15A), the killing ability of CAR-B7-H3-αβ T cells against B7-H3 knockout RKO tumor cells was equivalent to that of αβ T cells not expressing CAR-B7-H3 (Figure 15B), and the killing ability of CAR-B7-H3-γδ T cells against RKO tumor cells was significantly higher than that of γδ T cells not expressing CAR-B7-H3 (Figure 15A), and the tumor-killing ability of CAR-B7-H3-γδ T cells against B7-H3 knockout RKO tumor cells was equivalent to that of γδ T cells not expressing CAR-B7-H3 (Figure 15B). This further validated that CAR-B7-H3-αβ and CAR-B7-H3-γδ T cells enhanced killing of RKO tumor cells through recognition of B7-H3. Furthermore, under the same transduction positivity (adding non-CAR-expressing αβ T cells to CAR-B7-H3-αβ T cells reduced the transduction positivity to the same level as CAR-B7-H3-γδ T cells) and the same effector-to-target ratio, the killing ability of CAR-B7-H3-γδ T cells against RKO tumor cells was significantly higher than that of CAR-B7-H3-αβ T cells. Furthermore, γδ T cells that did not express CAR-B7-H3 also had a certain killing ability against RKO tumor cells (Figure 15A), which was significantly higher than that of αβ T cells that did not express CAR-B7-H3, further demonstrating that the antitumor ability of γδ T cells was higher than that of αβ T cells.

[0115] Example 10: Examination of the functional differences between CAR-γδT derived from two different types of antibodies Using the experimental method described in Example 8(3), the killing effects of γδT expressing CAR-B7-H3(1B4) and CAR-B7-H3(2Y31) on RKO WT tumor cells were further analyzed and compared. As can be seen from the results shown in Figure 16, regardless of whether the scFv was derived from the 1B4 or 2Y31 antibody, CAR-B7-H3-γδT possessed tumor-killing activity against SKOV3, and the killing activity was significantly higher than that of unmodified γδT. There was no significant difference in the killing activity of CAR-B7-H3-γδT derived from the two different antibodies against SKOV3. Under all killing conditions, the antitumor activity of CAR-B7-H3-γδT derived from the two different antibodies improved with increasing effector-to-target ratio.

[0116] Example 11: In vivo antitumor activity experiment In this experiment, an animal model with intraperitoneal tumor formation was used. Each mouse received 1 × 10 6 SKOV3 tumor cells (stably expressing luciferase) were inoculated intraperitoneally, where 1 × 10 6 SKOV3 tumor cells were resuspended in 200 μL of PBS and injected intraperitoneally (the day of inoculation was designated day 0). Body weight measurements and in vivo tumor imaging analysis were performed on day 4 after SKOV3 tumor cell inoculation. Mice were randomly divided into three groups based on body weight and tumor size: control, γδT-treated, and CAR-B7-H3(1B4)-γδT-treated. On day 5 of inoculation, cell intervention therapy was performed, where each mouse in the control group was intraperitoneally injected with 200 μL of PBS, and each mouse in the γδT treatment group was intraperitoneally injected with 1 × 10 6 γδ T cells (resuspended in 200 μL of PBS) were injected intraperitoneally, and 1 × 10 for each mouse in the CAR-B7-H3-γδT treatment group. 6 CAR-B7-H3(1B4)-γδ T cells (resuspended in 200 μL of PBS) were injected intraperitoneally. In vivo tumor imaging analysis was then performed periodically to monitor the survival of the mice and generate survival curves. Procedure for in vivo tumor imaging: Mice were anesthetized with an isoflurane anesthesia machine, and 100 μL (2 mg) of luciferin substrate was aspirated using an insulin syringe and injected intraperitoneally. After 10 minutes, images were captured using an IVIS fluorescence imaging system, and the images were stored and analyzed for fluorescence intensity. Experimental results (1) Tumor growth in the mouse body Referring to Figures 17 and 18, in the SKOV3 tumor model with intraperitoneal tumor formation, intraperitoneal re-injection of CAR-B7-H3-γδT significantly inhibited tumor growth, and it was found that unmodified γδT cells had a certain ability to inhibit tumors, but the effect was not significant. (2) Survival of tumor model mice Referring to Figure 19, all tumor-bearing mice in the control group died around day 85, while the survival time of mice in the γδT treatment group was extended by approximately 10 days to 95 days. The survival time of mice in the CAR-B7-H3-γδT treatment group was significantly extended, with the survival rate remaining at 80% even at day 125. [Brief explanation of the drawings]

[0117] [Figure 1] The nucleotide sequences of the heavy chain variable regions of the 1B4 and 2Y31 antibodies are shown, where FIG. 1A is the nucleotide sequence of the heavy chain variable region of the 1B4 antibody (SEQ ID NO:5), and FIG. 1B is the nucleotide sequence of the heavy chain variable region of the 2Y31 antibody (SEQ ID NO:6). [Figure 2] The nucleotide sequences of the light chain variable regions of the 1B4 and 2Y31 antibodies are shown, where FIG. 2A is the nucleotide sequence of the light chain variable region of the 1B4 antibody (SEQ ID NO:7), and FIG. 2B is the nucleotide sequence of the light chain variable region of the 2Y31 antibody (SEQ ID NO:8). [Figure 3] The amino acid sequences of the heavy chain variable regions of the 1B4 and 2Y31 antibodies are shown, where FIG. 3A is the amino acid sequence of the heavy chain variable region of the 1B4 antibody (SEQ ID NO:1), and FIG. 3B is the amino acid sequence of the heavy chain variable region of the 2Y31 antibody (SEQ ID NO:2). [Figure 4] The amino acid sequences of the light chain variable regions of the 1B4 and 2Y31 antibodies are shown, where FIG. 4A is the amino acid sequence of the light chain variable region of the 1B4 antibody (SEQ ID NO:3), and FIG. 4B is the amino acid sequence of the light chain variable region of the 2Y31 antibody (SEQ ID NO:4). [Figure 5] 1 shows the results of detecting the affinity of the humanized 1B4 antibody. [Figure 6] The results of binding of the humanized antibody to the glioma cell line U87 and the B7-H3 knockout U87 cell line are shown. In Figure 6A, the gray peak represents the negative control, the solid peak represents the binding of the parent mouse-derived antibody 1B4 to the tumor cell line, and the dotted peak represents the binding of the humanized 1B4 antibody to the tumor cell line. In Figure 6B, m2Y31 represents the binding of the parent mouse-derived antibody 2Y31 to the tumor cell line, and hu2Y31 represents the binding of the humanized antibody 2Y31 to the tumor cell line. 8H9 represents an antibody that recognizes a different B7-H3 antigen epitope bound to tumor cells, and NC is the negative control. [Figure 7] This is a schematic diagram of the CAR structure, which includes an scFv (VH-(G4S)3-VL), a CD8 hinge region (CD8 hinge), a transmembrane region (CD8 TM), costimulatory signals CD28 and 4-1BB, and a CD3z signal activation domain. The scFv contains one heavy chain variable region and one light chain variable region linked by a (GGGGS)3-linked peptide. Furthermore, a truncated EGFR structure is linked to the C-terminus of the CAR structure via a P2A cleavage peptide. This truncated EGFR structure can be used as a screening marker for CAR-positive cells, adding a safety switch to clinical studies. [Figure 8] 1 shows the binding ability of Jurkat T cells expressing CAR-B7-H3 to B7-H3 protein. [Figure 9] Detection with the 1B4 antibody shows that wild-type colon cancer tumor cells RKO (dark peak, RKO WT) express high levels of B7-H3, while B7-H3 knockout RKO tumor cells (light peak, RKO B7-H3 KO) do not express B7-H3. [Figure 10] This shows the CD69 expression level after stimulation of CAR-Jurkat T cells constructed using the 1B4 antibody with RKO WT or RKO (B7-H3 KO) tumor cells. [Figure 11]Figure 11A shows the transduction positivity of αβ T cells expressing CAR-B7-H3 (scFv is the light and heavy chain sequence of the 1B4 antibody). Figure 11B shows the CD69 expression levels after stimulation of αβ T or CAR-B7-H3-αβ T cells with RKO WT or RKO (B7-H3 KO) tumor cells. [Figure 12] This shows the in vitro killing ability of RKO tumor cells by CAR-B7-H3-αβ T cells (scFv is the light and heavy chain sequences of the 1B4 antibody). [Figure 13] This shows the in vitro killing ability of CAR-B7-H3-αβ T cells (scFv is the light and heavy chain sequences of the 1B4 antibody) to glioma cells. [Figure 14] The transduction positivity rate of γδ T cells expressing CAR-B7-H3 (scFv is the light and heavy chain sequence of the 1B4 antibody) is shown. [Figure 15] Figure 15A shows the killing effect of CAR-B7-H3-expressing CAR-αβT and CAR-γδT on RKO WT tumor cells (scFv is the light and heavy chain sequences of the 1B4 antibody). Figure 15B shows the killing effect of CAR-B7-H3-expressing CAR-αβT and CAR-γδT on B7-H3 knockout RKO tumor cells (scFv is the light and heavy chain sequences of the 1B4 antibody). [Figure 16] This shows the in vitro killing ability of CAR-γδT expressing CAR-B7-H3 to B7-H3-positive tumor cells SKOV3 (scFvs are the light and heavy chain sequences of the 1B4 antibody or 2Y31 antibody, respectively). [Figure 17] This shows the in vivo antitumor effect of CAR-γδT expressing CAR-B7-H3 (scFv is the light and heavy chain sequences of the 1B4 antibody) on SKOV3. [Figure 18] 1 shows the in vivo growth curve of SKOV3 tumor model mice. [Figure 19] 1 shows changes in survival curves of SKOV3 tumor model mice.

Claims

1. 1. An isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region (VH) and a light chain variable region (VL); The heavy chain variable region VH comprises a VH-CDR1 having the amino acid sequence of SEQ ID NO: 9, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 10, and a VH-CDR3 having the amino acid sequence of SEQ ID NO: 11; The light chain variable region VL comprises a VL-CDR1 represented by the amino acid sequence of SEQ ID NO: 12, a VL-CDR2 represented by the amino acid sequence of SEQ ID NO: 13, and a VL-CDR3 represented by the amino acid sequence of SEQ ID NO: 14; An antibody or antibody fragment.

2. 1. An isolated antibody or antibody fragment that specifically binds to B7-H3, comprising a heavy chain variable region (VH) and a light chain variable region (VL); the heavy chain variable region VH comprises a VH-CDR1 having the amino acid sequence of SEQ ID NO: 15, a VH-CDR2 having the amino acid sequence of SEQ ID NO: 16, and a VH-CDR3 having the amino acid sequence of SEQ ID NO: 17; The light chain variable region VL comprises a VL-CDR1 represented by the amino acid sequence of SEQ ID NO: 18, a VL-CDR2 represented by the amino acid sequence of SEQ ID NO: 19, and a VL-CDR3 represented by the amino acid sequence of SEQ ID NO: 20; An antibody or antibody fragment.

3. the heavy chain variable region VH is any one selected from the amino acid sequence represented by SEQ ID NO: 1, an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 1 other than the CDR region, an amino acid sequence represented by SEQ ID NO: 2, and an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence represented by SEQ ID NO: 2 other than the CDR region; 3. An isolated antibody or antibody fragment that specifically binds to B7-H3 according to claim 1 or 2.

4. the light chain variable region (VL) is any one selected from the amino acid sequence of SEQ ID NO: 3, an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 3 other than the CDR region, an amino acid sequence of SEQ ID NO: 4, and an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 98% or more identity to the amino acid sequence of SEQ ID NO: 4 other than the CDR region; 3. An isolated antibody or antibody fragment that specifically binds to B7-H3 according to claim 1 or 2.

5. 1. An isolated antibody or antibody fragment that specifically binds to B7-H3, comprising: (i) a heavy chain variable region VH as shown in SEQ ID NO: 1 and a light chain variable region VL as shown in SEQ ID NO: 3, or (ii) a heavy chain variable region (VH) represented by SEQ ID NO: 2 and a light chain variable region (VL) represented by SEQ ID NO: 4; An antibody or antibody fragment.

6. The antibody or antibody fragment is a genetically engineered antibody or antibody fragment. An isolated antibody or antibody fragment that specifically binds to B7-H3 according to any one of claims 1-5.

7. the antibody or antibody fragment is an scFv; An isolated antibody or antibody fragment that specifically binds to B7-H3 according to any one of claims 1-6.

8. The antibody or antibody fragment specifically binding to B7-H3 of claim 7. Chimeric antigen receptor.

9. 8. The antibody or antibody fragment of claim 7, and a transmembrane domain fused to the carboxyl terminus of the antibody or antibody fragment. The chimeric antigen receptor of claim 8.

10. 8. The antibody or antibody fragment of claim 7, comprising a transmembrane domain fused to the carboxyl terminus of the antibody or antibody fragment, and an immunocompetent cell activation signaling domain fused to the carboxyl terminus of the transmembrane domain. The chimeric antigen receptor of claim 8 or 9.

11. Further comprising any one or more of a membrane protein, a secreted protein, an intracellular protein, a small molecule drug, and a cytotoxic drug; The chimeric antigen receptor according to any one of claims 8 to 10.

12. A nucleotide sequence encoding the antibody or antibody fragment of any one of claims 1 to 7, or the chimeric antigen receptor of any one of claims 8 to 11. Nucleic acid molecule.

13. (i) a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 5 and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 7; or (ii) comprising a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 6 and a nucleotide sequence encoding a light chain variable region as set forth in SEQ ID NO: 8; The nucleic acid molecule of claim 12.

14. 14. The nucleic acid molecule of claim 12 or 13, vector.

15. The vector is a lentiviral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector; The vector of claim 14.

16. 16. A nucleic acid molecule according to claim 12 or 13, or a vector according to claim 14 or 15. cell.

17. Autologous or allogeneic T cells, B cells, NK cells, macrophages, monocytes, dendritic cells, neutrophils, basophils, eosinophils, mast cells, NK-T cells, MAIT cells, hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, and erythrocytes are included, and the T cells include αβ T cells, γδ T cells, and regulatory T cells. The cell of claim 16.

18. The antibody or antibody fragment according to any one of claims 1 to 7, the chimeric antigen receptor according to any one of claims 8 to 11, the nucleic acid molecule according to claim 12 or 13, the vector according to claim 14 or 15, or the cell according to claim 16 or 17, or The antibody or antibody fragment according to any one of claims 1 to 7, the chimeric antigen receptor according to any one of claims 8 to 11, the nucleic acid molecule according to claim 12 or 13, the vector according to claim 14 or 15, or the cell according to claim 16 or 17, and a pharmaceutically acceptable carrier; or The antibody or antibody fragment according to any one of claims 1 to 7, the chimeric antigen receptor according to any one of claims 8 to 11, the nucleic acid molecule according to claim 12 or 13, the vector according to claim 14 or 15, and the cell according to claim 16 or 17, and any other pharmaceutically active reagent or drug. Pharmaceutical compositions.

19. A method for treating or preventing a B7-H3-positive disease, the method comprising administering to a subject a therapeutically effective amount of the antibody or antibody fragment of any one of claims 1 to 7, the chimeric antigen receptor of any one of claims 8 to 11, the nucleic acid molecule of claim 12 or 13, the vector of claim 14 or 15, the cell of claim 16 or 17, or the pharmaceutical composition of claim 18. method.

20. The B7-H3 positive diseases include malignant / benign hematologic tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, viral infections, parasitic infections, abnormal bone growth, allografts, and transplant rejection, and the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, and ankylosing spondylitis.

20. The method of claim 19.

21. The B7-H3 positive disease includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, digestive cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome; 21. The method of claim 20.

22. The antibody or antibody fragment of any one of claims 1 to 7, the chimeric antigen receptor of any one of claims 8 to 11, the nucleic acid molecule of claim 12 or 13, the vector of claim 14 or 15, the cell of claim 16 or 17, or the pharmaceutical composition of claim 18, for treating or preventing a B7-H3-positive disease.

23. The B7-H3 positive diseases include malignant / benign hematologic tumors, malignant / benign solid tumors, autoimmune diseases, bacterial infections, viral infections, parasitic infections, abnormal bone growth, allografts, and transplant rejection, and the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, Sjogren's syndrome, and ankylosing spondylitis. The antibody or antibody fragment, chimeric antigen receptor, nucleic acid molecule, vector, cell, or pharmaceutical composition for treating or preventing a B7-H3-positive disease according to claim 22.

24. The B7-H3 positive disease includes acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, lung cancer, colorectal cancer, kidney tumor, bladder cancer, digestive cancer, prostate cancer, liver cancer, ovarian cancer, pancreatic cancer, endometrial cancer, stomach cancer, prostate cancer, kidney cancer, cervical cancer, thyroid cancer, uterine cancer, neuroendocrine cancer, head and neck cancer, nasopharyngeal cancer, testicular cancer, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome; The antibody or antibody fragment, chimeric antigen receptor, nucleic acid molecule, vector, cell, or pharmaceutical composition for treating or preventing a B7-H3-positive disease according to claim 23.

25. A method for detecting B7-H3, comprising: Detecting B7-H3 using the antibody or antibody fragment of any one of claims 1 to 7, the chimeric antigen receptor of any one of claims 8 to 11, or the nucleic acid molecule of claim 12 or 13. Methods for detecting B7-H3.

26. A kit for detecting B7-H3, comprising: A method for producing a nucleic acid molecule comprising the antibody or antibody fragment according to any one of claims 1 to 7, or a labeled product thereof, the chimeric antigen receptor according to any one of claims 8 to 11, or a labeled product thereof, or the nucleic acid molecule according to claim 12 or 13, Kit for detecting B7-H3.

27. A separation kit for separating B7-H3-positive cells, comprising: A method for producing a nucleic acid molecule comprising the antibody or antibody fragment according to any one of claims 1 to 7, or a labeled product thereof, the chimeric antigen receptor according to any one of claims 8 to 11, or a labeled product thereof, or the nucleic acid molecule according to claim 12 or 13, Separation kit.

28. 1. A method for enhancing cell function in vitro, comprising: The method comprises a step of contacting a cell with the antibody or antibody fragment or a labeled product thereof according to any one of claims 1 to 7, the chimeric antigen receptor or a labeled product thereof according to any one of claims 8 to 11, or the nucleic acid molecule or a labeled product thereof according to claim 12 or 13. method.

29. Use of an antibody or antibody fragment according to any one of claims 1 to 7 in the manufacture of a product for detecting B7-H3 protein.

30. Use of an antibody or antibody fragment according to any one of claims 1 to 7 in the manufacture of a product for blocking B7-H3 protein.

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