Antibodies Targeting B7-H3, Chimeric Antigen Receptors and Their Applications

Abstract

The present invention relates to antibodies targeting B7-H3, chimeric antigen receptors and their applications. Specifically, the present invention provides an antibody targeting B7-H3 or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) fusion protein comprising an antigen-binding domain targeting B7-H3, and an engineered immune cell (such as, CAR-T cell) expressing the CAR fusion protein. The CAR-T cells of the present invention have high affinity and biological activity, and good anti-tumor effects in vivo and in vitro.

Description

[0001] This is a divisional application of a Chinese invention patent application with an application date of July 24, 2024, an application number of CN202410994138.1, and an invention title of "Antibodies, Chimeric Antigen Receptors Targeting B7-H3 and Their Applications". Technical Field

[0002] The present invention belongs to the field of biomedicine, and specifically, it relates to antibodies, chimeric antigen receptors targeting B7-H3 and their applications. Background Art

[0003] B7-H3, also known as CD276, is a type I transmembrane protein belonging to the B7-CD28 superfamily. It is very similar in structure to PD-L1. B7 family molecules can provide stimulatory signals to enhance and maintain T cell immune responses, and can also generate inhibitory signals to limit and weaken T cell immune responses. B7-H3 is widely expressed at the transcriptional level (RNA) in lymphoid tissues and non-lymphoid organs, but the protein expression of B7-H3 is very limited, mainly expressed on activated dendritic cells, monocytes, T lymphocytes, B lymphocytes, NK lymphocytes, and the expression level in other normal tissues is very low.

[0004] Recent studies have found that B7-H3 is highly expressed in a variety of solid tumors, such as highly expressed in lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, glioblastoma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer, gastric cancer, and acute myeloid leukemia. Its overexpression degree is closely related to survival, prognosis, or tumor grading.

[0005] In addition to being highly expressed in tumors, B7-H3 may have a function similar to the T cell inhibitory signal mediated by PD-L1. It has been proposed that B7-H3 has co-stimulatory and co-inhibitory functions, which depend on tumor specificity, microenvironmental factors, and signal intensity. In addition to its role as an immune regulator, B7-H3 is related to enhancing cancer metastasis and angiogenesis.

[0006] Since the expression of B7-H3 is mainly limited to tumor cells and cells related to the tumor microenvironment, B7-H3 is a very important tumor-associated antigen and can be used as a target for potential broad-spectrum immunotherapy.

[0007] However, existing anti-human B7-H3 antibodies have defects such as insufficient affinity and specificity for membrane surface B7-H3, resulting in off-target effects and adverse reactions.

[0008] Therefore, it is of great significance in this field to develop antibodies and antigen chimeric receptors targeting B7-H3 with better affinity and stronger specificity. Summary of the Invention

[0009] The present invention provides antibodies, antigen chimeric receptors targeting B7-H3 with optimized affinity and stronger specificity, and their applications.

[0010] In a first aspect of the present invention, there is provided an antibody targeting B7-H3 or an antigen-binding fragment thereof, the antibody or the antigen-binding fragment thereof having a heavy-chain variable region and a light-chain variable region, and the complementarity-determining regions CDR (HCDR) of the heavy-chain variable region and the complementarity-determining regions CDR (LCDR) of the light-chain variable region are selected from the following groups:

[0011] (1) HCDR1 with the amino acid sequence shown in SEQ ID NO: 40,

[0012] HCDR2 with the amino acid sequence shown in SEQ ID NO: 41,

[0013] HCDR3 with the amino acid sequence shown in SEQ ID NO: 42,

[0014] LCDR1 with the amino acid sequence shown in SEQ ID NO: 44,

[0015] LCDR2 with the amino acid sequence shown in SEQ ID NO: 45, and

[0016] LCDR3 with the amino acid sequence shown in SEQ ID NO: 46;

[0017] (2) HCDR1 with the amino acid sequence shown in SEQ ID NO: 22,

[0018] HCDR2 with the amino acid sequence shown in SEQ ID NO: 23,

[0019] HCDR3 with the amino acid sequence shown in SEQ ID NO: 24,

[0020] LCDR1 with the amino acid sequence shown in SEQ ID NO: 26,

[0021] LCDR2 with the amino acid sequence shown in SEQ ID NO: 27, and

[0022] LCDR3 with the amino acid sequence shown in SEQ ID NO: 28;

[0023] (3) HCDR1 with the amino acid sequence shown in SEQ ID NO: 4,

[0024] HCDR2 with the amino acid sequence shown in SEQ ID NO: 5,

[0025] HCDR3 with the amino acid sequence shown in SEQ ID NO: 6,

[0026] LCDR1 having the amino acid sequence shown in SEQ ID NO:8,

[0027] LCDR2 having the amino acid sequence shown in SEQ ID NO:9, and

[0028] LCDR3 having the amino acid sequence shown in SEQ ID NO:10;

[0029] (4) HCDR1 having the amino acid sequence shown in SEQ ID NO:12,

[0030] HCDR2 having the amino acid sequence shown in SEQ ID NO:13,

[0031] HCDR3 having the amino acid sequence shown in SEQ ID NO:14,

[0032] LCDR1 having the amino acid sequence shown in SEQ ID NO:17,

[0033] LCDR2 having the amino acid sequence shown in SEQ ID NO:18, and

[0034] LCDR3 having the amino acid sequence shown in SEQ ID NO:19;

[0035] (5) HCDR1 having the amino acid sequence shown in SEQ ID NO:31,

[0036] HCDR2 having the amino acid sequence shown in SEQ ID NO:32,

[0037] HCDR3 having the amino acid sequence shown in SEQ ID NO:33,

[0038] LCDR1 having the amino acid sequence shown in SEQ ID NO:35,

[0039] LCDR2 having the amino acid sequence shown in SEQ ID NO:36, and

[0040] LCDR3 having the amino acid sequence shown in SEQ ID NO:37.

[0041] In another preferred embodiment, the antibody or its antigen-binding fragment is murine or humanized.

[0042] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody or a humanized antibody.

[0043] In another preferred embodiment, the antibody or its antigen-binding fragment includes a whole antibody, Fab, single-chain antibody (scFv), or nanobody.

[0044] In another preferred embodiment, the CDR region of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any one of the above sequences.

[0045] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and / or substituted with at least one amino acid and can retain the B7-H3 binding affinity.

[0046] In another preferred embodiment, the number of amino acids added, deleted, modified and / or substituted is 1-3, preferably 1-2, more preferably 1.

[0047] In another preferred embodiment, when the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, the heavy chain variable region and the light chain variable region are selected from the group consisting of:

[0048] (1) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:54, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:55;

[0049] (2) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:57, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:55;

[0050] (3) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:48, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:49.

[0051] In another preferred embodiment, when the antibody is a murine antibody or a chimeric antibody, the heavy chain variable region and the light chain variable region of the antibody are selected from the group consisting of:

[0052] (1) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:43;

[0053] (2) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:21, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:25;

[0054] (3) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:7;

[0055] (4) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:12, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:16;

[0056] (5) A heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 30, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 34.

[0057] In a preferred embodiment, the antibody or its antigen-binding fragment is a single-chain antibody (scFv).

[0058] In another preferred example, the single-chain antibody sequentially comprises a light chain variable region, a linker, and a heavy chain variable region, or sequentially comprises a heavy chain variable region, a linker, and a light chain variable region.

[0059] In another preferred example, the antibody is a monoclonal antibody.

[0060] In another preferred example, the antibody comprises a monospecific, bispecific, or trispecific antibody.

[0061] In a second aspect of the present invention, a chimeric antigen receptor (CAR) fusion protein is provided, and the chimeric antigen receptor fusion protein comprises, from the N-terminus to the C-terminus:

[0062] (i) A single-chain antibody as described in the first aspect of the present invention,

[0063] (ii) A transmembrane domain,

[0064] (iii) At least one co-stimulatory domain, and

[0065] (iv) An activation domain.

[0066] In another preferred example, the chimeric antigen receptor has the structure shown in Formula I below:

[0067] L-scFv-H-TM-C-S (I)

[0068] In the formula,

[0069] Each "-" is independently a linker peptide or a peptide bond;

[0070] L is an optional signal peptide sequence;

[0071] scFv is the single-chain antibody described in the first aspect of the present invention;

[0072] H is an optional hinge region;

[0073] TM is a transmembrane domain;

[0074] C is a co-stimulatory signal molecule;

[0075] S is a cytoplasmic signaling sequence.

[0076] In another preferred example, the signal peptide of L is CD8.

[0077] In another preferred example, L comprises the amino acid sequence shown in SEQ ID NO:58.

[0078] In another preferred example, the hinge region of H is a protein selected from the group consisting of CD8, CD28, or a combination thereof.

[0079] In another preferred example, C is a co-stimulatory signal molecule of a protein selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD30, CD40, CD40L, CD70, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM, SLAMF7, CD7, NKp80, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1(CD11a / CD18), ITGAM, CD11b, ITGAX, CD11c, ITGB1, ITGB2, KLRC2, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TNFRSF18, TNFRSF14, TRANCE / RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, LAT, GADS, SLP-76, PAG / Cbp, HAVCR1, LGALS9, Dap10, DAP12, CDS, ICAM-1, NKG2D, GITR, TLR2, TMIGD2 or a combination thereof.

[0080] In another preferred example, the amino acid sequence of C is as shown in SEQ ID NO:61.

[0081] In another preferred example, the transmembrane region of TM is a protein selected from the group consisting of CD8, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0082] In another preferred embodiment, the S (cytoplasmic transduction sequence) is a cytoplasmic transduction sequence selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, Fc receptor γ chain, FcRβ, CD79a, CD79b, FcγRIIa, DAP10, DAP12, NKp44, NKp30, NKp46, NKG2D.

[0083] In a third aspect of the present invention, there is provided a recombinant protein having:

[0084] (i) an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0085] (ii) optionally, a tag sequence for assisting expression and / or purification.

[0086] In another preferred embodiment, the tag sequence includes a 6His tag.

[0087] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0088] In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

[0089] In a fourth aspect of the present invention, there is provided an antibody-drug conjugate containing:

[0090] (a) an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, or a CAR fusion protein as described in the second aspect of the present invention; and

[0091] (b) a conjugate moiety conjugated to the antibody moiety, the conjugate moiety being selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0092] In another preferred embodiment, the antibody moiety is conjugated to the conjugate moiety through a chemical bond or a linker.

[0093] In a fifth aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

[0094] (1) an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention;

[0095] (2) a chimeric antigen receptor fusion protein as described in the second aspect of the present invention; or

[0096] (3) a recombinant protein as described in the third aspect of the present invention.

[0097] In a sixth aspect of the present invention, there is provided a vector containing the polynucleotide as described in the fifth aspect of the present invention.

[0098] In another preferred embodiment, the vector includes: bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, lentiviruses, exosomes or other vectors.

[0099] In the seventh aspect of the present invention, there is provided a genetically engineered host cell, which contains the vector described in the sixth aspect of the present invention, or polynucleotides integrated into the genome as described in the fifth aspect of the present invention, or expresses the antibody described in the first aspect of the present invention or the CAR fusion protein described in the second aspect of the present invention.

[0100] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0101] In another preferred embodiment, the cell is a mammalian cell.

[0102] In another preferred embodiment, the host cell is an engineered immune cell.

[0103] In another preferred embodiment, the engineered immune cell is a T cell, a macrophage or an NK cell.

[0104] In another preferred embodiment, the engineered immune cell includes a T cell, a macrophage or an NK cell, preferably (i) a chimeric antigen receptor T cell (CAR-T cell); or (ii) a chimeric antigen receptor NK cell (CAR-NK cell).

[0105] In the eighth aspect of the present invention, there is provided a method for preparing an engineered immune cell, the engineered immune cell expressing the CAR fusion protein as described in the second aspect of the present invention, comprising the steps of: transducing the polynucleotide as described in the fifth aspect of the present invention or the vector as described in the sixth aspect of the present invention into a T cell or an NK cell to obtain the engineered immune cell.

[0106] In another preferred embodiment, the method further includes the step of detecting the function and effectiveness of the obtained engineered immune cell.

[0107] In the ninth aspect of the present invention, there is provided the use of the antibody as described in the first aspect of the present invention or its antigen-binding fragment, the CAR fusion protein as described in the second aspect of the present invention, or the host cell as described in the seventh aspect of the present invention, for preparing a drug or preparation for preventing and / or treating B7-H3 positive cancer or tumor.

[0108] In another preferred embodiment, the B7-H3 positive cancer or tumor is a solid tumor or a non-solid tumor.

[0109] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, glioma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer, gastric cancer, and acute myeloid leukemia.

[0110] In a tenth aspect of the present invention, there is provided a preparation, which contains:

[0111] (1) An active ingredient selected from the group consisting of: the antibody or its antigen-binding fragment described in the first aspect of the present invention, the chimeric antigen receptor fusion protein described in the second aspect of the present invention, the vector described in the sixth aspect of the present invention, or the host cell described in the seventh aspect of the present invention; and (2) a pharmaceutically acceptable carrier, diluent, or excipient.

[0112] In an eleventh aspect of the present invention, there is provided a kit for preparing the cell as described in the seventh aspect of the present invention, the kit containing a container, and the polynucleotide described in the fifth aspect of the present invention or the vector described in the sixth aspect of the present invention located within the container.

[0113] In a twelfth aspect of the present invention, there is provided a method for treating a disease, including administering an appropriate amount of the cell described in the seventh aspect of the present invention or the preparation described in the tenth aspect of the present invention to a subject in need.

[0114] In another preferred embodiment, the disease is a B7-H3 positive cancer or tumor.

[0115] In another preferred embodiment, the B7-H3 positive cancer or tumor is a solid tumor or a non-solid tumor.

[0116] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, glioma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer, gastric cancer, and acute myeloid leukemia.

[0117] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 Shows the positive rate of CAR-T cells.

[0119] Figure 2The B7H3 expression in A549 and MDA-MB-231 cells is shown. The left peak is the ISOtype control peak, and the right peak is the B7H3 positive peak: (A) Flow cytometry detection chart of CD276 expression in A549 cells; (B) Flow cytometry detection chart of CD276 expression in MDA-MB-231 cells; (B) Flow cytometry detection chart of CD276 expression in MDA-MB-231 cells.

[0120] Figure 3 The cytotoxic effects of CAR-Ts corresponding to BH001 - BH007 on A549 and MDA-MB-231 cells at an effector-to-target ratio of 1:1 are shown. BM01 is the positive control (benchmark-positive control or benchmark - positive control), and the negative control (or Mock-T) refers to uninfected T cells.

[0121] Figure 4 The cytotoxic effects of CAR-Ts corresponding to BH008 - BH023 on A549 and MDA-MB-231 cells are shown. BM01 is the positive control, and the negative control (or Mock-T) refers to uninfected T cells.

[0122] Figure 5 The cytotoxic effects of CAR-Ts corresponding to BH010, BH012, BH016, BH019, BH025 - BH029 on A549 and MDA-MB-231 cells are shown. BM01 is the positive control, and the negative control (or Mock-T) refers to uninfected T cells.

[0123] Figure 6 The cytotoxic effects of CAR-Ts corresponding to BH001, BH002, BH013, BH016, BH018, BH019, BH023, BH025, BH027, BH028 on A549 and MDA-MB-231 cells are shown. Both BM01 and BM02 are positive controls, originating from different benchmarks of two companies respectively.

[0124] Figure 7 The trend chart of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-Ts corresponding to BH001 - BH007 is shown.

[0125] Figure 8 The trend chart of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-Ts corresponding to BH008 - BH023 is shown.

[0126] Figure 9Shows the trend graph of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-T corresponding to BH010, BH012, BH016, BH019, BH025 - BH029.

[0127] Figure 10 Shows the trend graph of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-T corresponding to BH001, BH002, BH013, BH014, BH015, BH016, BH017, BH018, BH019, BH020, BH023 - BH028.

[0128] Figure 11 Shows the trend graph of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-T corresponding to BH001, BH002, BH013, BH014, BH015, BH016, BH017, BH018, BH019, BH020, BH023 - BH028.

[0129] Figure 12 Shows the long-term killing test results of the preferred CAR-T on A549-3D cell cultures. BM01 and BM02 are both positive controls, respectively from different benchmarks of two companies.

[0130] Figure 13 Shows the short-term killing effect of humanized CAR-T on A549 cells.

[0131] Figure 14 Shows the short-term killing effect of humanized CAR-T on MDA-MB-231 cells.

[0132] Figure 15 Shows the screening of long-term killing of humanized CAR-T (A549 cells).

[0133] Figure 16 Shows the screening of long-term killing of humanized CAR-T (MDA-MB-231 cells).

[0134] Figure 17 Shows the growth trend of tumor bodies in each group of NPG mice after CAR-T cell infusion. BM01 and BM04 are both positive controls, respectively from different benchmarks of two companies.

[0135] Figure 18 Shows the metabolic kinetics curve of CAR-T cells in NPG mice. BM01 and BM04 are both positive controls, respectively from different benchmarks of two companies.

[0136] Figure 19 The weight change curves of each group of NPG mice are shown. BM01 and BM04 are both positive controls, respectively from different benchmarks of two companies. Detailed implementation manners

[0137] Through extensive and in-depth research and a large number of screenings, the present inventors have developed an antibody targeting B7-H3 or an antigen-binding fragment thereof (e.g., single-chain antibody), a chimeric antigen receptor comprising the single-chain antibody thereof, and a CAR-immune cell (such as CAR-T cell) expressing the chimeric antigen receptor. The present invention also provides a humanized CAR-T cell, which reduces immunogenicity.

[0138] Through the screening and careful design of the CAR element structure of the present invention (including but not limited to the screening of scFv suitable for CAR expression and tumor killing effect), the CAR-immune cells of the present invention can specifically kill tumor cells expressing B7-H3 without killing normal cells, thereby improving safety. At the same time, the CAR-immune cells of the present invention have a relatively high killing rate for tumor cells. The present invention provides a new approach for the treatment of B7-H3 positive tumors. The present invention has been completed on this basis.

[0139] Specifically, the experiments of the present invention show that the murine antibodies corresponding to BH001 to BH029 of the present invention all have good affinity. Through short-term killing, long-term killing and in vitro killing experiments on 3D cell cultures, it is found that the murine CAR-Ts corresponding to BH016, BH023, BH015, BH002 and BH025 of the present invention have good killing effects. And based on this, humanized CAR-Ts are constructed. The results show that the humanized B7-H3-targeting CAR-T cells corresponding to BH036, BH037 and BH033 of the present invention have excellent short-term and long-term killing effects on in vitro tumor cells and antitumor effects in vivo.

[0140] Terms

[0141] To more easily understand the present disclosure, certain terms are first defined. As used in this application, unless otherwise clearly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0142] The term "about" may refer to a value or a composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0143] The term "administer" refers to the physical introduction of the product of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0144] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind an antigen and contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain contains a heavy-chain variable region (abbreviated herein as VH) and a heavy-chain constant region. The heavy-chain constant region contains three constant domains CH1, CH2, and CH3. Each L chain contains a light-chain variable region (abbreviated herein as VL) and a light-chain constant region. The light-chain constant region contains one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0145] It should be understood that in this text, amino acid names are identified by a single English letter in the international common use, and their corresponding three-letter abbreviations of amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), Ile(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).

[0146] Chimeric antigen receptor (CAR)-immune cell

[0147] As used herein, the terms "chimeric antigen receptor (CAR)-immune cell", "CAR-immune cell", and "immune cell of the present invention" are used interchangeably and all refer to the specific B7-H3-targeted CAR-immune cells described in the first aspect of the present invention.

[0148] The CAR-immune cells of the present invention have the structure of a conventional chimeric antigen receptor in the art, except for the specific extracellular binding domain.

[0149] The chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain comprises a co-stimulatory signaling region and a ζ-chain moiety. The co-stimulatory signaling region refers to a part of the intracellular domain that comprises a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule required for an effective response of lymphocytes to an antigen, rather than an antigen receptor or their ligands.

[0150] A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to the extracellular domain or the cytoplasmic domain of a polypeptide chain. The linker may comprise from 0 to 300 amino acids, preferably from 2 to 100 amino acids and most preferably from 3 to 50 amino acids.

[0151] As used herein, both "antigen-binding domain" and "single-chain antibody fragment" refer to a Fab fragment, a Fab' fragment, an F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity. An Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of an antibody, but no constant region, and is the smallest antibody fragment having all the antigen-binding sites. Generally, an Fv antibody also comprises a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding. The antigen-binding domain is typically a scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of that of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody that specifically recognizes B7-H3, preferably a single-chain antibody.

[0152] For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to comprise a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding such a domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0153] vector

[0154] Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating it from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be produced synthetically.

[0155] The invention also provides vectors into which the expression cassette of the invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncoviruses such as murine leukemia virus because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0156] Briefly, generally the expression cassette or nucleic acid sequence of the invention is operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0157] The expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the invention provides gene therapy vectors.

[0158] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors such as, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

[0159] Furthermore, the expression vector can be provided to the cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0160] Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0161] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Typically, these are located in the region 30 - 110 bp upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements can act cooperatively or independently to initiate transcription.

[0162] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off the expression when it is not desired. Examples of inducible promoters include but are not limited to the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0163] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cells may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells sought to be transfected or infected with the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0164] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property such as enzyme activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially. Generally, constructs having at least 5 flanking regions that display the highest level of reporter gene expression are identified as promoters. Such promoter regions can be ligated to a reporter gene and used to evaluate the ability of a reagent to modulate promoter-driven transcription.

[0165] Methods for introducing genes into cells and for expressing genes in cells are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell by any method in the art, such as mammalian, bacterial, yeast, or insect cells. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0166] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising a vector and / or exogenous nucleic acid are known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0167] Biological methods of introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0168] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0169] In the case of using non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, included as a suspension within lipids, included within micelles or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include lipid droplets, which occur naturally in the cytoplasm and in such compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0170] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0171] Formulation

[0172] The present invention provides a formulation comprising the CAR-immune cells (e.g., CAR-T cells) described in the seventh aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of the CAR-T cells in the formulation is 1×10 3 -1×10 8cells / ml, more preferably 1×10 4 -1×10 7 cells / ml.

[0173] In one embodiment, the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0174] Therapeutic applications

[0175] The present invention includes therapeutic applications with cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the marker B7-H3 of tumor cells, can be used for autologous and allogeneic tumor treatment, can be prepared on a large scale, with uniform and stable quality, and can be readily available for use in any patient.

[0176] Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, which comprises the step of administering the CAR-T cells of the present invention to the mammal.

[0177] In one embodiment, the present invention includes a class of cell therapies in which T cells are genetically modified to express the CAR of the present invention, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the tumor cells of the recipient. Unlike antibody therapies, CAR-T cells can replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0178] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and for a sustained extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-B7-H3 CAR-T cells elicit a specific immune response against cells expressing B7-H3.

[0179] Although the data disclosed herein specifically disclose a lentiviral vector comprising an anti-B7-H3 scFv, a CD8α hinge region and transmembrane domain, and 4-1BB and CD3ζ signaling domains, the present invention is to be construed as including any number of variations to each of the components of the construct.

[0180] Treatable cancers include tumors that are not vascularized or are substantially non-vascularized, as well as vascularized tumors. Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or can include solid tumors. Cancer types treatable with the CARs of the present invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies such as sarcoma, carcinoma, and melanoma. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0181] A solid tumor is an abnormal mass of tissue that generally does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell type from which they form (such as lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, glioblastoma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer, stomach cancer, and acute myeloid leukemia, etc.).

[0182] The CAR-immune cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0183] For ex vivo immunization, at least one of the following occurs in vitro before the cells are administered into the mammal: i) expanding the cells, ii) introducing a nucleic acid encoding the CAR into the cells, and / or iii) cryopreserving the cells.

[0184] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Optionally, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0185] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0186] The present invention provides a method for treating a tumor, which comprises administering a therapeutically effective amount of the CAR-immune cells of the present invention to a subject in need thereof.

[0187] The CAR-immune cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention can comprise a population of target cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0188] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease - although appropriate dosages can be determined by clinical trials.

[0189] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells / kg body weight, preferably 10 5 to 10 6 cells / kg body weight (including all integer values within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered by infusion techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient's signs of disease and thus adjusting the treatment.

[0190] Administration of the subject composition can be effected in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by i.v. injection. The composition of T cells can be directly injected into the tumor, lymph node or site of infection.

[0191] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously, or after) with any number of relevant treatment modalities, including but not limited to treatment with the following reagents: reagents such as bevacizumab, megestrol acetate dispersible tablets, paclitaxel injection, ifosfamide, and ifosfamide for injection in the treatment of ovarian cancer patients. In further embodiments, the CAR-immune cells of the present invention can be used in combination with: chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination (e.g., before, simultaneously, or after) with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), and cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery.

[0192] The doses of the above treatments administered to a patient will vary depending on the precise nature of the disorder being treated and the recipient of the treatment. Dosage ratios for human administration can be practiced according to accepted practices in the art. Generally, for each treatment or course of treatment, 1×10 6 to 1×10 10 CAR-immune cells of the present invention can be administered to a patient, for example, by intravenous infusion.

[0193] The main advantages of the present invention include:

[0194] (a) The antibodies targeting B7-H3 of the present invention all have optimized affinities (fine-tuned affinity, too high may lead to systemic toxicity, and too low may result in poor effects).

[0195] (b) The antibodies targeting B7-H3 of the present invention all have good targeting specificities.

[0196] (c) The antibodies targeting B7-H3 of the present invention all have species cross-reactivities that are conducive to safety evaluation.

[0197] (d) The murine CAR-T targeting B7-H3 of the present invention has good tumor cell killing effects, including short-term killing and long-term killing (BH016, BH023, BH015, BH002, BH025).

[0198] (e) The humanized B7-H3-targeted CAR-T of the present invention has good tumor killing effects both in vitro and in vivo, and can significantly inhibit tumor growth (BH033, BH036, and BH037).

[0199] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0200] Example 1 Preparation and Screening of Anti-human B7-H3 Antibody Hybridoma Cells

[0201] 1.1 Preparation of Hybridoma Cells

[0202] Immunization: Balb / c mice were immunized with the recombinant protein of the extracellular region of human B7-H3 (Accession No.: UniProtKB-Q5ZPR3, 1aa-461aa), and the serum titer was detected by ELISA using a 96-well enzyme-linked immunosorbent assay plate coated with the recombinant protein of human B7-H3-his (Accession No.: UniProtKB-Q5ZPR3, 1aa-461aa). Mice that met the fusion requirements were used for the next cell fusion.

[0203] Cell fusion and hybridoma preparation: Mice with a titer meeting the requirements were boost-immunized. Three days later, the spleens of the mice were aseptically removed to prepare a suspension of B lymphocytes, which was then fused with SP2 / 0 myeloma cells. The fused cells were resuspended in HAT medium and aliquoted into 96-well cell culture plates. They were cultured in an incubator at 37°C and 5% CO 2 incubator.

[0204] 1.2 Screening of Positive Hybridoma Binding

[0205] Ten to fourteen days after fusion, a 96-well enzyme-linked immunosorbent assay plate was coated with the recombinant protein of the extracellular region of human B7-H3-his (Accession No.: UniProtKB-Q5ZPR3 1aa-461aa) (20 ng / ml) and incubated overnight at 4°C. After washing three times with PBS, it was blocked with 4% skim milk-PBS at room temperature for 1 hour. After washing three times with PBS, the culture supernatant of the hybridoma clones was added and incubated at room temperature for 1 hour.

[0206] Set the following controls:

[0207] (1) Positive control (PC): Mouse serum after immunization (diluted 1:1000 with PBS);

[0208] (2) Negative control (NC): Mouse serum before immunization (diluted 1:1000 with PBS).

[0209] Wash three times with PBST (0.05% Tween-PBS), wash twice with PBS, add HRP goat anti-mouse IgG (FcY), incubate at 37°C for 0.5 hr; then wash 3 times with PBST (0.05% Tween 20-PBS), add TMB chromogenic solution, develop color in the dark for 15 - 30 min, add ELISA stop solution to terminate the reaction; read the A450 value with an enzyme-linked immunosorbent assay reader.

[0210] According to the principle from high to low, select the clones with higher readings and take the clone culture supernatant the next day for secondary ELISA confirmation.

[0211] Example 2 Sequencing of Mouse Anti-Human B7-H3 Antibody

[0212] After expanding the culture of hybridoma clone cells secreting anti-human B7-H3 antibody, extract total cellular RNA according to the steps of the TRIzoI kit (Cat: 15596026, Invitrogen); reverse transcribe the total cellular RNA of hybridoma cells into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB); amplify the variable region of antibody light chain IgVL(x) and the variable region of heavy chain VH sequences using degenerate primers and the Phusion kit (Cat: EO553L, NEB); purify the PCR amplification products using a gel extraction kit (Cat: AP-GX-250, Axygen); ligate the amplified PCR products to the T vector according to the instructions of the T vector cloning kit (Cat: ZC205 Zhuangmeng Biotech) and transform competent Escherichia coli cells, amplify the strain, extract the plasmid, and then perform DNA sequencing to obtain the variable region sequence and CDR sequence of the monoclonal antibody.

[0213] Example 3 Affinity Analysis of Mouse Antibody

[0214] Use the extracellular region of human B7H3 (UniProtKB-Q5ZPR3) fused with mFc protein (B7H3-ECD-mFc) as the antigen. After fully emulsifying it with an equal volume of complete Freund's adjuvant (Sigma, Cat.No.: F5581), immunize 6 - 8-week-old Balb / c mice (purchased from Beijing Sino Biological Inc.) subcutaneously. The antigen immunization dose is 50 μg / mouse. Subsequently, every 2 weeks, fully emulsify the same dose of antigen with incomplete Freund's adjuvant (Sigma, Cat.No.: F5506) and immunize the mice subcutaneously three times. Determine the serum titer of the mice after three immunizations.

[0215] Using PEG Hybri Max (Sigma, Cat. No.: 7181) as the fusogen, mouse spleen cells and SP2 / 0 cells were mixed at a ratio of 4:1, and the fused cells were added to a 96-well plate (1X10 5 cells / well), with each well containing 0.1 mL of 1X HAT (Invitrogen, Cat. No.: 21060-017) medium. On the 3rd day, 0.1 mL of HAT (Invitrogen, Cat. No.: 11067-030) medium was added. On the 7th day, the medium in the 96-well plate was aspirated, and 0.2 mL of fresh HAT medium was added. The supernatant was collected on the 9th day for various screening and tests.

[0216] The B7-H3 antigen protein was prepared into 7 concentrations with gradient dilution of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.5 nM, 3.13 nM, and 1.56 nM, and loaded onto a 96-well plate. Human Protein G probe was selected, with the antigen as the stationary phase and the B7-H3 antibody as the mobile phase. The binding time was set at 180 s, and the dissociation time was set at 300 s. The binding constant and dissociation constant of the human monoclonal antibody were detected, and the affinity was calculated.

[0217] The kinetic constants are shown in Table 1 below. The results in Table 1 indicate that all clones were correctly cloned and could bind to the human B7-H3 recombinant protein.

[0218] Table 1

[0219] ScFv antibody Ka (1 / Ms) Kd (1 / s) KD (M) BH001 1.99E+05 7.44E-05 3.74E-10 BH002 1.38E+05 6.55E-05 4.76E-10 BH003 1.67E+05 1.57E-05 9.42E-11 BH004 2.10E+05 3.58E-05 1.71E-10 BH005 1.25E+05 4.96E-05 3.99E-10 BH006 1.14E+05 1.31E-05 1.14E-10 BH007 1.13E+05 5.08E-05 4.51E-10 BH008 1.34E+05 2.03E-05 1.51E-10 BH009 1.76E+05 1.08E-04 6.14E-10 BH010 2.40E+05 6.82E-05 2.85E-10 BH011 3.38E+05 3.26E-05 9.65E-11 BH012 3.30E+05 3.68E-05 1.12E-10 BH013 1.17E+05 1.70E-05 1.46E-10 BH014 2.87E+05 1.29E-05 4.49E-11 BH015 1.16E+05 1.36E-04 1.17E-09 BH016 1.52E+05 1.63E-04 1.07E-09 BH017 2.65E+05 0.001024 3.87E-09 BH018 7.87E+04 7.97E-04 1.01E-08 BH019 3.65E+05 3.32E-04 9.10E-10 BH020 3.08E+05 7.45E-04 2.42E-09 BH021 2.58E+05 6.11E-04 2.37E-09 BH022 9.81E+04 1.28E-04 1.30E-09 BH023 1.59E+05 1.47E-04 9.26E-10 BH024 3.82E+05 0.001559 4.08E-09 BH025 9.55E+04 5.22E-04 5.46E-09 BH026 3.57E+05 7.07E-04 1.98E-09 BH027 3.67E+05 2.79E-03 7.60E-09 BH028 2.54E+05 6.85E-04 2.69E-09 BH029 1.60E+05 5.56E-04 3.48E-09

[0220] Example 4 Humanization and Mutation Design of Anti-Human B7-H3 Monoclonal Antibody

[0221] 4.1 Humanization of Murine Monoclonal Antibody

[0222] CDR Transplantation

[0223] First, a comprehensive analysis was performed on the heavy chain sequence of the murine antibody to determine the antigen-binding complementarity-determining region (CDR) of the antibody and the framework region that supports the conserved three-dimensional conformation of the antibody. Subsequently, based on the homology alignment results, the most similar human antibody was selected as the basic template, and combined with the results of the full-sequence blast, CDR transplantation was carried out. The CDR region of the murine antibody was retained, and the framework region sequence of the murine antibody was replaced with the framework region sequence of the human germline antibody.

[0224] Secondly, a structural model of the murine antibody was established, and each different amino acid site in the structural model of the human antibody and the corresponding murine antibody was compared one by one. If using the human amino acid sequence at a certain site in the framework region did not cause damage or alteration to the spatial structure of the CDR region, the human amino acid sequence was used at that site; otherwise, the corresponding murine sequence was used at that site (i.e., reverted to the murine sequence).

[0225] According to the structural simulation, some amino acids in the framework region of the humanized antibody were reverted to the murine sequence. Finally, the amino acid sequences of the variable regions of the heavy and light chains of the humanized antibody were obtained.

[0226] Example 5 Cultivation of Tumor Cell Lines

[0227] Four kinds of tumor cells were used in the present invention, namely 293T, A549, MDA-MB-231, and SUP-T1 cells. Among them, 293T cells are a human renal epithelial cell line and are often used to study the expression of foreign genes and virus preparation, etc. A549 is a non-small cell lung cancer cell line, MDA-MB-231 is a breast cancer cell line, and SUP-T1 is a T lymphocyte cell line. 293T cells were cultured in DMEM complete medium (DMEM medium + 10% FBS); A549 cells were cultured in F12K complete medium (F12K + 10% FBS); MDA-MB-231 cells were cultured in complete medium (L15 Leibovitz + 10% FBS); SUP-T1 cells were cultured in complete medium (1640 + 10% FBS).

[0228] Flow cytometry detection of the expression of tumor cell B7H3 target

[0229] The cells to be tested were centrifuged at 300g for 5 minutes to collect the cell pellet; the cells were washed once with PBS. Take 2×10 5 The cells were stained with 100 μL of the staining system. After resuspending the cells in the staining system, they were incubated at 4°C for 30 min. After the incubation, they were washed once with flow cytometry buffer and resuspended with 200 μL of flow cytometry buffer, and then subjected to machine analysis.

[0230] The staining system was configured as: 100 μL of flow cytometry buffer + flow cytometry detection antibody.

[0231] The formula of the flow cytometry buffer was: PBS + 1% FBS + 2.5 mM EDTA.

[0232] The flow cytometry detection results were as Figure 2 shown. The lung cancer cell line A549 and the breast cancer cell line MDA-MB-231 highly expressed the B7-H3 protein.

[0233] Example 6 Construction of CAR Based on scFv of Murine Antibody

[0234] In this example, the CAR structure used is a second-generation CAR structure. The specific structure is that the 5'-end is the CD8 signal peptide, the scFv sequence region, followed by the CD8 hinge region, the CD8 transmembrane region, the 4-1BB intracellular co-stimulatory domain, and the CD3 signal transduction domain. Among them, the scFv starts from the 5'-end with the variable light chain (VL) of the antibody, the G4S (SEQ ID NO:63) flexible linker polypeptide, and the variable heavy chain (VH) of the antibody.

[0235] Example 7 Lentivirus production

[0236] Inoculate 1×10 7 293T cells into a 15-cm cell culture dish with a medium volume of 20 mL. The next day, mix the expression plasmid, the helper plasmid pMDLg-pRRE, the helper plasmid pRSV-Rev, and the helper plasmid pMD2.G in a 2:1:1:1 addition ratio in 2000 μL of opti-MEM medium to prepare a plasmid-containing medium; additionally, add 72.5 μg of PEI to the opti-MEM medium, mix well and let it stand for 10 minutes, then add this plasmid-PEI solution to 20 ml of fresh complete DMEM medium and evenly add it to the 293T cell culture dish. After 8 h, replace with fresh 20 mL of complete DMEM medium. Collect the medium supernatant at 48 h and 72 h respectively. After collecting the supernatant at 48 h, add 15 mL of fresh complete DMEM medium.

[0237] After filtering the supernatant through a 0.45-μm filter membrane, perform ultracentrifugation at 70000g for 3 hours to obtain the virus precipitate. Resuspend the virus precipitate with opti-MEM medium, aliquot the virus solution, and detect the virus titer of the virus solution using SUP-T1 cells. Store the virus solution at -80 °C for long-term preservation.

[0238] Example 8 Activation and virus transduction of T cells

[0239] 8.1 Resuscitation of peripheral blood mononuclear cells (PBMCs)

[0240] Turn on the water bath and preheat the temperature to 37 °C. Take out a vial of PBMCs from the liquid nitrogen tank, quickly put it into the water bath, quickly dissolve the cell cryopreservation solution. After dissolution, spray the cryopreservation tube with 75% alcohol and open it in the biosafety cabinet. Use a pipette to add the PBMC suspension to 14 ml of PBS, mix well, and centrifuge at 500g for 8 minutes.

[0241] 8.2 Activation of PBMC cells

[0242] Resuspend the cells with 10 mL of AIM-V (5% FBS + 300 IU IL-2 / ml), and take a sample for counting. According to the counting results, dilute the cells to 2×10 6 / mL. Add 10 μL of TransAct T cell activator to every 2×10 6 PBMC cells, mix well, and then divide the PBMC cell suspension equally into 24-well plates. Activate and culture in an incubator at 37°C and 5% carbon dioxide for 48 hours.

[0243] 8.3 Collection and viral transduction of activated T cells

[0244] After 48 hours of activation and culture of PBMC, pipette and mix the cells in each well and collect them into a 15 ml centrifuge tube; centrifuge at 500 g for 8 minutes. After centrifugation, resuspend the cells with 10 mL of complete AIM-V medium (5% FBS + 300 IU / ml IL-2). Take a sample for counting.

[0245] Adjust the T cells to 1×10 6 / mL according to the counting results, and divide them equally into 24-well plates at 0.5 mL per well; add the corresponding CAR-T lentivirus at MOI = 10, add Polybrene 8 μg / mL, mix well, and then centrifuge at 1200 g for 90 min in a centrifuge.

[0246] After centrifugation, take out the 24-well plates from the centrifuge, pipette and mix the precipitated cells, and then put them back into the incubator to culture overnight. The next day, replace with fresh complete AIM-V medium (5% FBS + 300 IU / ml IL-2). After culturing for five days, collect some cells and detect the CAR phenotype of the cells.

[0247] Example 9 Flow cytometry staining analysis

[0248] Centrifuge the cells to be tested at 300 g for 5 minutes to collect the cell precipitate; wash the cells once with PBS. Take 2×10 5 cells and stain them with 100 μL of staining system. After resuspending the cells in the staining system, incubate them at 4°C for 30 min. After incubation, wash them once with flow cytometry buffer, resuspend with 200 μL of flow cytometry buffer, and then perform analysis on the machine.

[0249] The staining system is configured as: 100 μL of flow cytometry buffer + antibody.

[0250] The formula of the flow cytometry buffer is: PBS + 0.5% BSA + 2.5 mM EDTA.

[0251] The positive rate of CAR-T cells is as Figure 1 shown. The proportion of CAR + T cells is 53.6%.

[0252] Example 10 Detection of the cytotoxic effect of B7H3-CAR-T cells on tumor cells (short-term killing)

[0253] T cells and the prepared B7H3-CAR-T cells were co-incubated with A549 and MDA-MB-231 cells at effector-to-target ratios of 0.3:1, 1:1, and 3:1 for 24 hours, and then the apoptosis of tumor cells was detected using an LDH kit (Roche, 11644793001).

[0254] The results are as Figures 3 - 6 shown. The results showed that compared with T cells, B7H3-CAR-T cells had a good cytotoxic effect on A549 and MDA-MB-231 tumor cells. This indicates that B7H3-CAR-T cells have a strong cytotoxic effect on solid tumor cells expressing B7H3.

[0255] Figure 3 It is shown that the cytotoxic effects of the CAR-Ts corresponding to BH001, BH002, BH003, and BH004 on A549 and MDA-MB-231 cells are relatively obvious and can enter the final comparison.

[0256] Figure 4 It is shown that the cytotoxic effects of the CAR-Ts corresponding to BH008, BH013, BH018, and BH023 on A549 and MDA-MB-231 cells are relatively obvious and can enter the final comparison.

[0257] Figure 5 It is shown that the cytotoxic effects of the CAR-Ts corresponding to BH016, BH019, BH025, BH027, and BH028 on A549 and MDA-MB-231 cells are relatively obvious and can enter the final comparison.

[0258] Figure 6 What is shown is Figure 3 , 4 , and 5 preferred Binders were finally compared for short-term killing. Those with relatively good performance were: BH002, BH013, BH016, BH025, BH027, and BH028.

[0259] Example 11 Long-term killing experiment

[0260] The prepared CAR-T cells, A549 cells, and MDA-MB-231 cells were collected separately, centrifuged at 300 g for 8 minutes, and then resuspended in AIM-V medium (5% FBS), and samples were taken for counting. The prepared CAR-T cells were adjusted to the same level of CAR% with activated T cells from the same donor; then, according to the effector-to-target ratio E:T = 0.2:1, 2×105 target cells and 0.4×10 5 CAR-T cells were mixed into the same well, and one replicate well was set for each well; in the control group, the same number of activated T cells and the same number of target cells were mixed into the same well.

[0261] The volume of each well was supplemented to 1 mL with AIM-V medium (5% FBS). After co-culturing for 48 hours, the proportion of T cells was detected. At the same time, 2×10 5 target cells were added to continue co-culturing for the next round of detection. The target cells were used to stimulate 5 - 7 times continuously, and the continuous killing ability of CAR-T was analyzed by detecting the proportion of T cells in each well.

[0262] In the last round, the higher the proportion of T cells, the stronger the continuous killing ability of CAR-T and the stronger the ability to resist T cell exhaustion. Similar to the short-term killing group, in the present invention, all CAR-Ts were divided into three batches for long-term killing comparison, and finally the excellent CAR-Ts in each group were collected for a final comparison.

[0263] The results are as Figures 7 - 11 shown. Figure 7 Shown is the trend graph of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-T corresponding to BH001 - BH007. Combining the microscopic observation and the short-term killing effect in the present invention, BH001 and BH002 were selected for the final comparison.

[0264] Figure 8 Shown is the trend graph of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-T corresponding to BH008 - BH023. BH013, BH014, BH017, BH018, and BH023 were selected for the final comparison.

[0265] Figure 9 Shown is the trend graph of the proportion of CD3+ cells in the long-term killing of A549 cells by CAR-T corresponding to BH010, BH012, BH016, BH019, BH025 - BH029. BH016, BH019, BH025, BH027, and BH028 were selected for the final comparison. For the final long-term killing comparison, the present invention selected two cell lines to conduct killing experiments: A549 and MDA-MB-231 cells.

[0266] As Figure 10 , Figure 11 shown, in the case where it is difficult to compare one cell line, the other cell line can be used as a reference for comparison.

[0267] After the final long-term killing comparison, considering the performance of each Binder in A549 and referring to the long-term killing effect on MDA-MB-231, BH002, BH013, BH014, BH015, BH016, BH019, BH023, and BH025 were selected to enter the in vitro killing screening of 3D cell culture.

[0268] In vitro killing of the cell culture in Example 12

[0269] Collect A549 cells by digestion with 0.25% trypsin, wash the cells twice with PBS, take samples for counting, and set aside for use. Matrix GFR, phenol Red-Free, LDEV-Free Matrigel (40186ES08) was placed in a 4°C refrigerator and fully dissolved overnight one day in advance, and then diluted three times with serum-free AIM-V medium. Resuspend A549 cells with the Matrigel medium to a concentration of 50000 cells / mL; place the cell suspension on ice, and use a pre-cooled pipette tip to divide the A549 cell suspension equally into round-bottom 96-well plates, with three replicates in each well. Place the 96-well plates in a 37°C carbon dioxide incubator and let stand for 30 minutes to allow the Matrigel suspension to solidify fully, and then add 100 μL of the complete medium for A549 cells to each well, and culture for 7 - 9 days. During this period, the medium can be replaced semi-quantitatively or quantitatively to maintain cell growth.

[0270] Addition of effector cells: Collect, count, and detect CAR% of CAR-T cells cultured for 7 days after lentiviral infection. Adjust the CAR% of CAR-T to the same level value and adjust the density of CAR-T cells to 4E5 / mL. When A549 cells are cultured for about 8 days, carefully aspirate 100 μL of the supernatant from the culture wells of A549 using a multi-channel pipette; then add 100 μL of the CAR-T cell suspension to it. Replace the medium once every 48 hours, and be careful when aspirating to avoid sucking out the Matrigel.

[0271] Nine days after the addition of effector cells and co-culture, discard the supernatant, and add StemPro TM Accutase TM Cell dissociation reagent and digest for about 30 minutes. During this period, it can be pipetted and mixed repeatedly until there are no gel clumps. Collect the digestion solution and centrifuge at 300g for 8 minutes, resuspend the cells with 100 μL of staining buffer, and add staining antibodies to detect the proportion of T cells. The more T cells there are, the less tumor cells there are, indicating better in vitro killing effect in the corresponding group and stronger infiltration ability of the corresponding T cells.

[0272] The detection results of the continuous killing of the 3D culture for 9 days are as Figure 12As shown. According to the proportion ranking of T cells, the present invention selects BH016, BH023, BH015, BH002, and BH025 to enter the stage of antibody humanization modification and screening. BM01 and BM02 are the positive control groups of this example.

[0273] Example 13 Antibody Humanization and Screening of Short-term and Long-term Killing

[0274] 13.1 Comparison of Short-term Killing

[0275] After obtaining the plasmid of the antibody sequence after humanization modification, lentivirus was prepared and CAR-T cells were prepared; the preparation process was the same as above. Specifically, the scFvs corresponding to the 5 murine CAR-Ts (BH002, BH015, BH016, BH023, and BH025) finally obtained in Example 12 were humanized to obtain eight humanized scFVs and their corresponding humanized CAR-T cells (BH030, BH031, BH032, BH033, BH034, BH035, BH036, and BH037). Their corresponding relationships are shown in Table 2 below.

[0276] Table 2

[0277]

[0278] The technical process of detecting the short-term and long-term killing abilities of each humanized CAR-T by LDH was the same as above. For the results of short-term killing, the present invention shows the results of 1:1 and 3:1 here. After humanization, the present invention mainly compares whether the humanized Binder can maintain the same or better killing effect as the corresponding murine Binder.

[0279] The results are as Figures 13 - 14 shown. Through the short-term killing of A549 and MDA-MB-231 cells, the present inventors found that BH030, BH031, and BH032 after humanization modification were much weaker than the parental clones BH002, BH015, and BH016 and needed to be eliminated after long-term killing confirmation.

[0280] 13.2 Long-term Killing

[0281] The results are as Figures 15 - 16 shown. Through the long-term killing of A549 and MDA-MB-231 cells, the present invention found that the long-term killing abilities of the three humanized CAR-Ts, BH030, BH031, and BH032, were also much weaker than those of the parental clones and could not enter the next animal experiment.

[0282] Example 14 Experiment on Anti-tumor Ability in Vivo - A549 / NPG Animal Model

[0283] The NPG mice were purchased from Beijing Vitalstar Biotechnology Co., Ltd. In this example, a subcutaneous tumor-bearing animal model was used. Each NPG mouse was subcutaneously inoculated with 5×10 6 A549 cells. After modeling, the major axis and minor axis were measured with vernier calipers three times a week. After grouping (5 mice in each group), the tumors were measured twice a week. Then, according to the formula V = a*b 2 / 2, where a is the major axis and b is the minor axis. The body weight was measured every three days after starting the modeling, and twice a week after grouping.

[0284] When the tumor grew to 100 - 130 mm 3 , 3×10 6 CAR-T cells were transfused via the tail vein. Once a week at a fixed time after transfusion, blood was collected to detect the dynamic content ratio of CAR-T cells in the peripheral blood.

[0285] The results are as Figures 17 - 19 shown. Figure 17 Table 3 shows the inhibitory effects of different sequences of CAR-T on the growth of A549 tumors in NPG mice after transfusion. The CAR-T cells corresponding to BH033, BH036, and BH037 had good inhibitory effects on tumor growth. The added positive controls BM01 and BM04 showed no obvious tumor inhibitory effects in this experiment.

[0286] Table 3

[0287] BH036 BH037 BH035 BH032 BH033 BM01 BM04 Negative control Day 0 118 121 117 123 116 119 118 121 Day 7 193 207 231 217 208 197 157 187 Day 11 369 425 420 402 382 334 197 285 Day 14 493 520 429 497 456 513 376 369 Day 18 547 414 587 587 534 601 495 518 Day 22 466 196 628 834 406 819 609 644 Day 25 447 187 932 1,016 532 1,023 882 913 Day 28 496 221 1,164 1,110 590 1,193 967 1,153

[0288] Note: The values in the table are the average volumes of tumors of each group of mice (accurate to the nearest whole number), and the unit is mm 3 .

[0289] Figure 18 shows the metabolic kinetic curves of CAR-T cells in NPG mice after transfusion of different sequences of CAR-T. The CAR-T cells corresponding to BH037 and BH035 showed obvious in vivo amplification.

[0290] Figure 19 shows the change curves of the body weights of each group of NPG mice during the whole experiment. The body weights of each group of mice increased steadily overall, without significant fluctuations.

[0291] The sequence information of the present invention is shown in Table A and Table B below.

[0292] Table A

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] Note: The CDRs in the table are all determined by the Kabat rules.

[0302] CDRs determined by the IMGT rules in Table B

[0303]

[0304] All documents mentioned in the present invention are cited herein by reference as if each individual document was specifically and individually cited by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. An antibody or antigen-binding fragment thereof targeting B7-H3, wherein the antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region, characterized in that: The complementarity determining region CDR (HCDR) of the heavy chain variable region and the complementarity determining region CDR (LCDR) of the light chain variable region are: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 22, The amino acid sequence of HCDR2 is shown in SEQ ID NO: 23, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 24, LCDR1 having an amino acid sequence as shown in SEQ ID NO: 26, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 27, and The amino acid sequence of LCDR3 is shown in SEQ ID NO:

28.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is murine or humanized; Wherein, when the antibody or antigen-binding fragment thereof is of mouse origin, the heavy chain variable region and the light chain variable region include: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:21, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:25; When the antibody or antigen-binding fragment thereof is humanized, the heavy chain variable region and the light chain variable region include: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:48, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

49.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a single-chain antibody (scFv).

4. A chimeric antigen receptor (CAR) fusion protein, characterized in that The chimeric antigen receptor fusion protein comprises from N-terminus to C-terminus: (i) a single-chain antibody, wherein the complementarity determining region CDR (HCDR) of the heavy chain variable region and the complementarity determining region CDR (LCDR) of the light chain variable region of the single-chain antibody are: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 22, The amino acid sequence of HCDR2 is shown in SEQ ID NO: 23, The amino acid sequence of HCDR3 is shown in SEQ ID NO: 24, LCDR1 having an amino acid sequence as shown in SEQ ID NO: 26, LCDR2 having an amino acid sequence as shown in SEQ ID NO: 27, and LCDR3 having an amino acid sequence as shown in SEQ ID NO: 28, (ii) transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) Activation domain.

5. A recombinant protein, the recombinant protein having: (i) the antibody or antigen-binding fragment thereof according to claim 1; and (ii) Tag sequences that facilitate expression and / or purification.

6. An antibody-drug conjugate, characterized in that: The antibody-drug conjugate contains: (a) the antibody or antigen-binding fragment thereof according to claim 1, or the chimeric antigen receptor fusion protein according to claim 4; as well as (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof.

7. The antibody-drug conjugate according to claim 6, characterized in that: The coupling moiety is a radionuclide.

8. A polynucleotide, characterized in that The polynucleotide encodes a polypeptide selected from the group consisting of: (1) the antibody or antigen-binding fragment thereof according to claim 1; (2) the chimeric antigen receptor fusion protein according to claim 4; or (3) The recombinant protein according to claim 5.

9. A carrier, characterized in that The vector contains the polynucleotide according to claim 8.

10. A genetically engineered host cell, characterized in that The host cell contains the vector of claim 9, or has the polynucleotide of claim 8 integrated into its genome, or expresses the antibody or antigen-binding fragment thereof of claim 1 or the chimeric antigen receptor fusion protein of claim 4.

11. Use of the antibody or antigen-binding fragment thereof according to claim 1, the chimeric antigen receptor fusion protein according to claim 4, or the host cell according to claim 10, characterized in that: For preparing a drug or preparation for preventing and / or treating B7-H3-positive cancer or tumor; Wherein, the B7-H3 positive cancer or tumor is selected from the following group: lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, glioma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer and gastric cancer, acute myeloid leukemia.

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