CAR carrying NKG7 and application of CAR in treatment of B7H3 positive tumors

Abstract

The invention discloses a CAR carrying NKG7 and application of the CAR in treatment of B7H3 positive tumors, the CAR comprises a specific antibody targeting B7H3 and the NKG7, in-vivo and in-vitro experimental verification shows that the NKG7 can significantly improve the anti-tumor function of B7H3-CAR-T cells, can be effectively applied to immunotherapy of B7H3 positive related diseases, and is wide in clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically, relates to a CAR carrying NKG7 and its application in treating B7H3-positive tumors. Background Art

[0002] With the development of tumor immunotherapy and clinical biotechnology, chimeric antigen receptor T cell (CAR-T) immunotherapy is one of the most promising tumor immunotherapy methods. Chimeric antigen receptor (CAR) contains three domains, namely, extracellular antigen binding domain, transmembrane domain and intracellular signal transduction domain. The extracellular antigen binding domain contains a single-chain variable region fragment (scFv) targeting tumor antigens. CAR is expressed on the surface of T cells through gene transfection technology. After co-culture with tumor cells, CAR molecules bind to tumor antigens, CAR activates T cells, and exerts specific anti-tumor effects in an antigen-dependent manner, and is not restricted by the major histocompatibility complex (MHC).

[0003] B7H3, also known as CD276, is a type I transmembrane protein belonging to the B7-CD28 superfamily. It is very similar to PD-L1 in structure. 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. B7H3 is expressed at very low levels in normal tissues. Recent studies have found that B7H3 is highly expressed in a variety of solid tumors, such as lung cancer, breast cancer, pancreatic cancer, prostate cancer, bladder cancer, kidney cancer, ovarian cancer, brain glioma, endometrial cancer, peritoneal cancer, colorectal cancer, liver cancer, gastric cancer and acute myeloid leukemia. Its overexpression is closely related to the patient's survival, prognosis or tumor grade.

[0004] Since B7H3 expression is mainly limited to tumor cells and related cells in the tumor microenvironment, B7H3 is a very important tumor-associated antigen and can be used as a potential target for broad-spectrum immunotherapy. However, the chimeric antigen receptors containing anti-human B7H3 antibodies in the prior art have defects such as insufficient specificity for membrane surface B7H3, resulting in off-target effects and adverse reactions. Therefore, the development of chimeric antigen receptors targeting B7H3 with stronger specificity and immune cells expressing the chimeric antigen receptors is of great significance in the field. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a CAR carrying NKG7 and its application in the treatment of B7H3-positive tumors in the art.

[0006] In the present invention, the CAR carrying NKG7 is an enhanced chimeric antigen receptor targeting B7H3, comprising an antibody specifically targeting B7H3 and NKG7. The present invention verifies for the first time through in vivo and in vitro experiments that the combination of antibodies targeting B7H3 and NKG7 can significantly improve the anti-tumor function of B7H3-CAR-T cells. At the same time, overexpression of NKG7 can promote IL-2 secretion of B7H3-CAR-T cells, activate the IL-2 signaling pathway and promote T cell proliferation. In addition, overexpression of NKG7 can inhibit antigen-induced CAR downregulation, maintain membrane surface CAR expression, and significantly reduce the tumor volume in mice, showing better anti-tumor ability than conventional CAR-T.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention provides an enhanced chimeric antigen receptor targeting B7H3.

[0009] Further, the chimeric antigen receptor comprises an antibody targeting B7H3 and NKG7;

[0010] The antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are CDR1, CDR2, and CDR3 in the heavy chain variable region as shown in SEQ ID NO:4; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are CDR1, CDR2, and CDR3 in the light chain variable region as shown in SEQ ID NO:9; the amino acid sequence of NKG7 is as shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:27.

[0011] Further, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are shown as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; optionally, the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are shown as SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; optionally, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:4; optionally, the amino acid sequence of the light chain variable region is shown as SEQ ID NO:9; optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule, or a cytokine; optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, 4-1BB, CD34, IgG4, CD3ε, PD-1, IgG1, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor ; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, CD21, CD22, CD79a, CD79b, TCRζ, CD4, CD5, CD8, CD278, FcεRI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4 -1BB, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signal domain is selected from the costimulatory signal domain of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, NKp30, NKp46, CD40, OX40, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptide of any one of the following molecules: CD 3ζ, α chain and β chain of T cell receptor, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8;Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L; Optionally, the chimeric antigen receptor is a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 costimulatory signal domain, a CD3ζ intracellular signaling domain, a T2A self-cleaving peptide, and the NKG7 are sequentially connected in series;

[0012] Optionally, the Linker is (G4S)3; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:11; optionally, the amino acid sequence of the Linker is as shown in SEQ ID NO:15; optionally, the amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO:19; optionally, the amino acid sequence of the CD28 co-stimulatory signal domain is as shown in SEQ ID NO:21; optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQID NO:23; optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO:25; optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:29.

[0013] In some embodiments, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 corresponding to the heavy chain variable region and light chain variable region of the antibody described in the present invention can be obtained based on the above heavy chain variable region and light chain variable region sequences according to the Kabat, IMGT, Chothia, AbM or Contact numbering system. The CDR sequences defined according to any one or any combination of the Kabat, IMGT, Chothia, AbM, Contact numbering systems or other numbering systems are also included in the protection scope of the present invention.

[0014] A second aspect of the present invention provides a nucleic acid molecule.

[0015] Further, the nucleic acid molecule encodes the chimeric antigen receptor described in the first aspect of the present invention;

[0016] Optionally, the nucleotide sequence encoding the heavy chain variable region of the antibody in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 14; Optionally, the nucleotide sequence encoding the light chain variable region of the antibody in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 18; Optionally, the nucleotide sequence encoding the signal peptide in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 12; Optionally, the nucleotide sequence encoding the Linker in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 16; Optionally, the nucleotide sequence encoding the CD8 hinge region and transmembrane domain in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 20; Optionally, the nucleotide sequence encoding the CD28 co-stimulatory signaling domain in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 22; Optionally, the nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO: 24; Optionally, the nucleotide sequence encoding the T2A self-cleaving peptide in the chimeric antigen receptor according to the first aspect of the present invention is as shown in SEQ ID NO:26; alternatively, the nucleotide sequence encoding NKG7 in the chimeric antigen receptor described in the first aspect of the present invention is shown in SEQ ID NO:28; alternatively, the nucleotide sequence encoding the chimeric antigen receptor described in the first aspect of the present invention is shown in SEQ ID NO:30.

[0017] In some embodiments, those skilled in the art can change the combination category and sequence of signal peptides, hinge regions and transmembrane regions, co-stimulatory signal domains and intracellular signal transduction domains according to actual conditions or needs. Regardless of the form of the change, as long as the chimeric antigen receptor has the sequences of the antibody targeting B7H3 and NKG7 described in the first aspect of the present invention, it is included in the protection scope of the present invention.

[0018] The third aspect of the present invention provides an expression vector.

[0019] Further, the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention;

[0020] Optionally, the vector is a plasmid, a phagemid, a cosmid, an artificial chromosome or a vector of viral origin; optionally, the vector of viral origin is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpesvirus vector, a baculovirus vector, a papillomavirus vector or a papillomasovavirus vector.

[0021] In some embodiments, nucleic acid molecules provided by the invention can be cloned into many types of vectors. For example, this nucleic acid molecule can be cloned into such a vector, which includes but is not limited to: plasmid, phagemid, phage derivative, animal virus and cosmid. Specific vector of interest comprises expression vector, replication vector, probe generation vector and sequencing vector.

[0022] The fourth aspect of the present invention provides a chimeric antigen receptor immune cell.

[0023] Further, the chimeric antigen receptor immune cell comprises the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention and / or the expression vector described in the third aspect of the present invention; optionally, the immune cell is a T lymphocyte, a B lymphocyte, a NK cell, an iNKT cell, a CTL cell, a monocyte, a myeloid cell, a dendritic cell, a macrophage, a mast cell and / or any combination thereof.

[0024] The fifth aspect of the present invention provides a chimeric antigen receptor immune cell population.

[0025] Further, the chimeric antigen receptor immune cell population comprises the chimeric antigen receptor immune cells described in the fourth aspect of the present invention; optionally, the chimeric antigen receptor immune cell population further comprises immune cells that do not comprise the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention and / or the expression vector described in the third aspect of the present invention; optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, mast cells and / or any combination thereof.

[0026] A sixth aspect of the present invention provides a derivative.

[0027] Further, the derivative is a chimeric antigen receptor according to the first aspect of the present invention or a nucleic acid molecule according to the second aspect of the present invention labeled with a detectable marker, a chimeric antigen receptor according to the first aspect of the present invention or a nucleic acid molecule according to the second aspect of the present invention that confers antibiotic resistance, or a chimeric antigen receptor according to the first aspect of the present invention or a nucleic acid molecule according to the second aspect of the present invention that is combined or coupled with a therapeutic agent; optionally, the detectable marker is a fluorescent dye, colloidal gold, a chemiluminescent marker or a chemiluminescent catalyst; optionally, the chemiluminescent marker is luminol and its derivatives, isoluminol and its derivatives, acridinium esters, acridinium ester derivatives, adamantane, a rare earth element or a bipyridine ruthenium complex; optionally, the chemiluminescent catalyst is pyrofluorescent dye, fluorine ... root peroxidase or alkaline phosphatase; optionally, the antibiotic resistance gene is a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene or a kanamycin resistance gene; optionally, the therapeutic agent is a radionuclide, a cytokine, a gold nanoparticle, a viral particle, a liposome, a nanomagnetic particle, a prodrug activating enzyme or a chemotherapeutic agent; optionally, the cytokine is IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF or M-CSF; optionally, the chemotherapeutic agent is cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxaliplatin or eloxadin.

[0028] The seventh aspect of the present invention provides a pharmaceutical composition or biological preparation.

[0029] Further, the pharmaceutical composition comprises the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, the chimeric antigen receptor immune cell described in the fourth aspect of the present invention, the chimeric antigen receptor immune cell population described in the fifth aspect of the present invention and / or the derivative described in the sixth aspect of the present invention; optionally, the pharmaceutical composition further comprises a therapeutic agent for adjuvant treatment and / or prevention of B7H3-positive related diseases;

[0030] In some embodiments, the therapeutic agent comprises an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an antiangiogenic agent, an antiestrogen, an antiandrogen, an immunomodulator, and / or a cytotoxin;

[0031] In some embodiments, the alkylating agent includes chlorambucil, chlorambucil, melphalan, propioperazine, chlorambucil, estramustine, cyclophosphamide, triamidite, carmustine, streptozotocin, cisplatin, carboplatin; the antimetabolite includes methotrexate, 5-fluorouracil, fluoxetine, 5-fluorodeoxyuracil, capecitabine, cytarabine, fludarabine; the anti-tumor antibiotic includes daunorubicin, doxorubicin, methoxy-daunomycin, mithramycin, mitomycin C, valrubicin, mitoxantrone hydrochloride, bleomycin; the mitotic inhibitor includes docetaxel, vinblastine, paclitaxel, vincristine, vinblastine, vinorelbine; the chromatin function inhibitor includes irinotecan, etoposide, topotecan, etoposide phosphate, teniposide; the anti The angiogenic agent includes prinomastat, tanomastat, ilomastat, aprotinin, batimastat; the antiestrogens include toremifene, raloxifene, tamoxifen, anastrozole, exemestane; the antiandrogens include nilutamide, bicalutamide, cimetidine; the immunomodulators include interleukins, tumor necrosis factor, interferon, mushroom polysaccharides, cizosin, roquimek, pidomod, methoxypolyethylene glycol succinamide adenosine deaminase, thymosin preparations; the cytotoxins include DM4, MMAE, DM1, Ozogamicin, Dxd, SN-38, MMAF, PBD, DM2, Amanitin, PNU-159682, IR700, PE-38, PE4E, PE40, PE25, PE35;

[0032] Optionally, the biological preparation comprises the pharmaceutical composition; optionally, the dosage form of the biological preparation is injection, oral preparation, cream, gel, drops, patch or lyophilized powder.

[0033] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may also include a pharmaceutically acceptable carrier and / or excipient. Suitable pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used as needed to help the stability of the formulation or to help improve the biological effectiveness of the active or active substance or to produce an acceptable taste or smell in the case of oral administration. The preparation that can be used in such a pharmaceutical composition or pharmaceutical preparation can be in the form of its original compound itself, or optionally in the form of its pharmaceutically acceptable salt. The pharmaceutical composition or pharmaceutical preparation thus prepared can be administered in any appropriate manner known to those skilled in the art as needed. When using the pharmaceutical composition or pharmaceutical preparation, a safe and effective amount of the pharmaceutical composition or pharmaceutical preparation of the present invention is administered to a human.

[0034] An eighth aspect of the present invention provides any of the following methods:

[0035] (1) A method for preparing the chimeric antigen receptor immune cell of the fourth aspect of the present invention, the method comprising the following steps: providing an immune cell to be modified, introducing the nucleic acid molecule of the second aspect of the present invention or the expression vector of the third aspect of the present invention into the immune cell, to obtain the chimeric antigen receptor immune cell of the fourth aspect of the present invention;

[0036] Optionally, the immune cells to be modified are T lymphocytes, B lymphocytes, NK cells, iNKT cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, mast cells and / or any combination thereof;

[0037] (2) A method for stimulating a target cell population or tissue in a mammal to produce an immune response, the method comprising the following steps: administering to the mammal an effective amount of the chimeric antigen receptor immune cell described in the fourth aspect of the present invention and / or the chimeric antigen receptor immune cell population described in the fifth aspect of the present invention.

[0038] In addition, the present invention also provides a method for treating and / or preventing B7H3-positive related diseases or delaying their progression, the method comprising the following steps: administering to a subject in need thereof an effective amount of the chimeric antigen receptor immune cell described in the fourth aspect of the present invention, the chimeric antigen receptor immune cell population described in the fifth aspect of the present invention, the derivative described in the sixth aspect of the present invention and / or the pharmaceutical composition or biological preparation described in the seventh aspect of the present invention.

[0039] A ninth aspect of the present invention provides any of the following applications:

[0040] (1) Use of the chimeric antigen receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the chimeric antigen receptor immune cell of the fourth aspect of the present invention, the chimeric antigen receptor immune cell population of the fifth aspect of the present invention, the derivative of the sixth aspect of the present invention and / or the pharmaceutical composition or biological preparation of the seventh aspect of the present invention in the preparation of a drug for treating and / or preventing a B7H3-positive related disease;

[0041] (2) Use of the chimeric antigen receptor of the first aspect of the present invention, the nucleic acid molecule of the second aspect of the present invention, the expression vector of the third aspect of the present invention, the chimeric antigen receptor immune cell of the fourth aspect of the present invention, the chimeric antigen receptor immune cell population of the fifth aspect of the present invention and / or the derivative of the sixth aspect of the present invention in the preparation of a biological agent for treating and / or preventing a B7H3-positive related disease;

[0042] (3) Use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in the preparation of chimeric antigen receptor immune cells for treating and / or preventing B7H3-positive related diseases;

[0043] (4) Use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in the preparation of a chimeric antigen receptor immune cell population for treating and / or preventing B7H3-positive related diseases;

[0044] (5) Use of the chimeric antigen receptor described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, and / or the expression vector described in the third aspect of the present invention in the preparation of a kit for preparing chimeric antigen receptor immune cells for treating and / or preventing B7H3-positive related diseases;

[0045] (6) Application of NKG7 in the preparation of enhanced chimeric antigen receptors;

[0046] (7) Application of NKG7 in the preparation of enhanced chimeric antigen receptor immune cells;

[0047] (8) Use of NKG7 in the preparation of a drug for enhancing the killing effect of chimeric antigen receptor immune cells;

[0048] (9) Use of NKG7 in the preparation of a drug for promoting proliferation of chimeric antigen receptor immune cells;

[0049] Optionally, the B7H3-positive related disease is gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, breast cancer, esophageal cancer, oral cancer, endometrial cancer, bladder cancer, osteosarcoma, autoimmune disease, peritoneal cancer, glioma or myeloid leukemia.

[0050] Furthermore, the NKG7 is the NKG7 described in the first aspect of the present invention.

[0051] In the present invention, the B7H3-positive related diseases include solid tumors, hematological tumors, autoimmune diseases or a combination thereof. Exemplarily, the B7H3-positive related diseases include gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, breast cancer, esophageal cancer, oral cancer, endometrial cancer, bladder cancer, osteosarcoma, autoimmune diseases, peritoneal cancer, glioma or myeloid leukemia. It should be noted that the B7H3-positive related diseases described in the present invention are not limited to the above-mentioned diseases, and any diseases and / or symptoms related to the expression of B7H3 are within the scope of protection of the present invention.

[0052] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0053] The present invention provides a chimeric antigen receptor (CAR) overexpressing NKG7 targeting B7H3 and a chimeric antigen receptor immune cell (CAR-T cell), wherein the CAR comprises a specific antibody (AHF23050) targeting B7H3 and NKG7. The present invention verifies through in vivo and in vitro experiments that NKG7 can significantly improve the anti-tumor function of B7H3-CAR-T cells, and can be effectively used in the immunotherapy of B7H3-positive related diseases, and has broad prospects for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : Successfully constructed CAR-T cells overexpressing NKG7 targeting B7H3;

[0055] Figure 2 : Overexpression of NKG7 can enhance the anti-tumor function of B7H3-CAR-T cells (gastric cancer: MKN45, liver cancer: Huh7, colon cancer: SW620, pancreatic cancer: PANC-1);

[0056] Figure 3 : Overexpression of NKG7 can promote IL-2 secretion in B7H3-CAR-T cells, activate the IL-2 signaling pathway and promote T cell proliferation;

[0057] Figure 4 : Overexpression of NKG7 can inhibit antigen-induced CAR downregulation and maintain CAR expression on the membrane surface. After stimulation with four types of tumor cells, flow cytometry was used to detect the expression of CAR on the membrane surface of T cells;

[0058] Figure 5 :Overexpression of NKG7 can inhibit antigen-induced CAR downregulation and maintain CAR expression on the membrane surface. After stimulation with different concentrations of target protein, flow cytometry was used to detect the expression of CAR on the T cell membrane surface.

[0059] Figure 6 : Overexpression of NKG7 can improve the efficacy of B7H3-CAR-T cells in in vivo experiments and maintain the expression of CAR on the surface of intratumoral CAR-T cells. DETAILED DESCRIPTION

[0060] In order to make it easier for those skilled in the art to understand the present application, some terms used in the present application are first defined, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0061] As used herein, the terms “comprise” or “comprising” mean including stated elements or components but not excluding other elements or components.

[0062] As used herein, the term "homology" refers to sequence similarity to an amino acid sequence or nucleotide sequence to be compared. "Homology" includes amino acid sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology to the amino acid sequence provided by the present invention. Homology can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. The 75% or more homology can be 75%, 80%, 85%, 90% or more homology.

[0063] As used herein, the term "chimeric antigen receptor (CAR)" includes an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands. Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect a transmembrane domain to an extracellular domain or a cytoplasmic domain of a polypeptide chain.

[0064] As used herein, the term "chimeric antigen receptor immune cell (CAR-immune cell)" refers to an immune cell modified by a chimeric antigen receptor, which can be used to transplant one or more antigen-specific genetically engineered receptors into immune effector cells (such as T cells), and the chimeric antigen receptor immune cell cell can express a chimeric antigen receptor. The present invention relates to the construction of a chimeric antigen receptor structure targeting B7H3, a method for preparing T cells modified by a chimeric antigen receptor targeting B7H3, and the identification of their effects.

[0065] As used herein, the term "vector" refers to a vector that delivers a nucleic acid sequence encoding a desired molecule. Nucleic acid molecules provided by the invention can be cloned into many types of vectors. For example, the nucleic acid molecules can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0066] Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, 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, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193) that work in at least one organism. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be separated and delivered to the subject cell in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. In one embodiment, a lentiviral vector is used.

[0067] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region 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 when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to initiate transcription.

[0068] Suitable promoters include, but are not limited to, the immediate early cytomegalovirus (CMV) promoter, the elongated growth factor-1α (EF-1α) promoter, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters (such as, but not limited to, the actin promoter, the myosin promoter, the heme promoter, and the creatine kinase promoter).

[0069] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0070] 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 vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Biological methods for 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 methods for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. Chemical means for 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 for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).

[0071] As used herein, the term "effective amount" refers to an amount of the chimeric antigen receptor immune cells, chimeric antigen receptor immune cell populations, derivatives, biologics and / or pharmaceutical compositions described herein that can effectively inhibit, prevent, arrest, delay or treat the symptoms of a particular disease, disorder or side effect. Such diseases, disorders and side effects include, but are not limited to, those pathological conditions associated with B7H3-positive associated diseases (e.g., pathological conditions associated with the treatment and / or prevention of B7H3-positive associated diseases). The therapeutically effective amount can vary depending on the route of administration and dosage form, the age and weight of the subject, and / or the disease or condition being treated.

[0072] As used herein, the term "pharmaceutical composition" or "biological agent" may have any one of the following formulations: tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. In addition, it can be administered once or multiple times. At this time, the pharmaceutical composition and / or biological agent is administered in the form of a liquid preparation, powder, aerosol, capsule, vaginal tablet, capsule or suppository. The route of administration may include, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, intrarectal, etc. When administered orally, it may be formulated into a coating that protects the active ingredient in the pharmaceutical composition to prevent it from degrading in the stomach. In addition, the active ingredient may be administered by any device that can be transferred to the target tissue. In a specific embodiment, the pharmaceutical composition or biological agent provided by the present invention may be prepared into various dosage forms according to actual needs, and may be administered by a clinician according to factors such as the type, age, weight and general disease condition of the subject, and the mode of administration to determine the dosage that is beneficial to the patient. The administration method may be, for example, injection or any other appropriate administration method known to those skilled in the art.

[0073] As used herein, the term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. In some embodiments, the "subject" is preferably a human.

[0074] The present invention is further described below in conjunction with specific embodiments. The specific embodiments are only used to explain the present invention and cannot be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer. The reagents, biomaterials, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0075] Example 1 Monoclonal antibody AHF23050 targeting B7H3

[0076] The present invention constructs a fully human monoclonal antibody AHF23050 that specifically targets B7H3. The sequence information of the AHF23050 antibody is shown in Table 1 below. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 9. In addition, this example demonstrates through experiments that the AHF23050 antibody has good affinity and specificity for B7H3.

[0077] The 50 antibody has a good affinity for B7H3.

[0078] Table 1 Sequence of AHF23050 antibody

[0079]

[0080]

[0081] Example 2 Construction and detection of CAR-T cells overexpressing NKG7 targeting B7H3

[0082] 1. Experimental methods

[0083] (1) Based on the transmembrane domain CD8α, the costimulatory domain CD28ζ, and the signal transduction domain CD3ζ, CAR-T cells targeting the B7H3 target that normally express or overexpress NKG7 were constructed and named B7H3-CAR-T cells and B7H3-CAR-T, respectively. NKG7 cells, the B7H3-CAR-T cells and B7H3-CAR-T NKG7 The schematic diagram of the cell is as follows Figure 1 A. The corresponding coding sequence is shown in Table 2.

[0084] Among them, the CAR in the B7H3-CAR-T is obtained by connecting the signal peptide, AHF23050-VH, Linker, AHF23050-VL, CD8 hinge region and transmembrane domain, CD28 co-stimulatory signal domain, and CD3ζ intracellular signal transduction domain in series in sequence;

[0085] The B7H3-CAR-T NKG7 The CAR is composed of a signal peptide, AHF23050-VH, Linker, AHF23050-VL, CD8 hinge region and transmembrane domain, CD28 co-stimulatory signal domain, CD3ζ intracellular signal transduction domain, T2A self-cleaving peptide, and human NKG7 connected in series.

[0086] (2) Co-incubated with 1 μg / mL Percp-Cy5.5-CD3 and PE-protein L (CAR) antibody at 4°C for 30 min, and the positive rate of CAR-T cells (Percp-Cy5.5+ / PE+) was detected by flow cytometry;

[0087] (3) Total cell protein was collected and subjected to SDS-PAGE electrophoresis and immunoblotting to detect Control T, B7H3-CAR-T, and B7H3-CAR-T NKG7 Expression of NKG7 protein in three cell types, with Actin as the internal reference protein.

[0088] 2. Experimental results

[0089] The results are as follows Figure 1 As shown, the results of flow cytometry showed that B7H3-CAR-T cells and B7H3-CAR-T were successfully constructed in this example. NKG7 Cells; WB validation results showed that compared with Control T and B7H3-CAR-T cells, B7H3-CAR-T NKG7 The cells highly expressed NKG7 protein.

[0090] Table 2 Coding sequence of CAR overexpressing NKG7 targeting B7H3

[0091]

[0092]

[0093] Example 3 Overexpression of NKG7 can enhance the anti-tumor function of B7H3-CAR-T cells

[0094] 1. Experimental methods

[0095] (1) At a ratio of 1:4, 4 × 10 5 B7H3-CAR-T or B7H3-CAR-T NKG7 cells, and then 1.6 × 10 6 Tumor cells (GFP+ gastric cancer cell line MKN45, liver cancer cell line Huh7, colon cancer cell line SW620 and pancreatic cancer cell line PANC-1) were mixed;

[0096] (2) The cells were cultured in a 37°C incubator for 24 h, and then the cell samples were collected, washed twice with PBS, and stained with BV510-Zombie and Percp-Cy5.5-CD3;

[0097] (3) After incubation at room temperature for 15 minutes, the cells were washed twice with PBS and centrifuged at 450 g for 5 minutes to collect the cell pellet. The pellet was resuspended in 300 μL PBS and then analyzed by flow cytometry.

[0098] 2. Experimental results

[0099] The results are as follows Figure 2 As shown in the figure, the results of flow cytometry showed that the ratio of CD3+ / GFP+ cells at 0h was used as a reference to confirm that B7H3-CAR-T and B7H3-CAR-T NKG7 Both cells can effectively kill tumor cells, but B7H3-CAR-T NKG7 The targeted killing effect of cells is significantly better than that of B7H3-CAR-T cells.

[0100] Example 4 Overexpression of NKG7 can promote IL-2 secretion in B7H3-CAR-T cells, activate the IL-2 signaling pathway and promote T cell proliferation

[0101] 1. Experimental methods

[0102] This example uses the following method to detect the intracellular cytokine IL-2:

[0103] (1) At a ratio of 1:1 between efficacy and target, 5 × 10 5 B7H3-CAR-T or B7H3-CAR-T NKG7 Then 5×10 5 tumor cells (MKN45, Huh7, SW620 and PANC-1) and mixed;

[0104] (2) 2 μg / mL BFA was added to inhibit the secretion of cytokines and the cells were cultured in a 37°C incubator for 6 h;

[0105] (3) The cell samples were then collected, washed twice with PBS, and the cells were resuspended with 1 μg / mL PE-CAR and Percp-Cy5.5-CD3 antibodies and stained at 4°C for 30 min;

[0106] (4) Wash twice with PBS, centrifuge at 450 g for 5 min to collect the cell pellet, fix the sample with CytoFix / CytoPerm, and then incubate with 1 μg / mL APC-IL-2 antibody at 4°C overnight;

[0107] (5) Wash the cells with 200 μL 1× Perm / Wash buffer, resuspend in 300 μL PBS and analyze on flow cytometer.

[0108] This example uses the CFSE experiment to detect cell proliferation ability, and the specific experimental steps are as follows:

[0109] (1) Take 1×10 6 B7H3-CAR-T or B7H3-CAR-T NKG7 Cells were incubated with 5 μM CFSE in the dark for 10 min;

[0110] (2) The cells were then washed three times with X-vivo medium containing 10% FBS and centrifuged at 450 g for 5 min to collect the cells;

[0111] (3) The cells were then placed in 4% FBS X-vivo medium and cultured in a 37°C incubator for 72 h;

[0112] (4) After 72 hours, the changes in FITC fluorescence of the cell samples were detected by flow cytometry.

[0113] This example uses WB experiment to detect IL-2 cell pathway, and the specific experimental steps are as follows:

[0114] (1) Take 6×10 6 B7H3-CAR-T or B7H3-CAR-T NKG7 Cells were incubated with 0.25 μg / mL B7H3 protein for 6 h;

[0115] (2) After 6 h, cells were collected and washed twice with PBS, and the cell pellets were collected by centrifugation at 450 g for 5 min;

[0116] (3) After RIPA lysis, total cell protein was extracted and subjected to SDS-PAGE electrophoresis. The expression of NKG7, STAT5, p-STAT5, ERK, p-ERK, AKT, and p-AKT proteins among the groups was detected by immunoblotting, with Actin as the internal reference protein.

[0117] 2. Experimental results

[0118] The results are as follows Figure 3 As shown, the test results showed that after contact with target cells (tumor cells MKN45, Huh7, SW620, PANC-1), B7H3-CAR-T cells were compared with B7H3-CAR-T cells. NKG7 The CFSE experimental results showed that compared with Control T and B7H3-CAR-T cells, B7H3-CAR-T NKG7 Cell proliferation was significantly accelerated. The results of WB detection of IL-2 cell pathway showed that after target protein stimulation, B7H3-CAR-T NKG7 The phosphorylation of IL-2 signaling-related proteins in cells increased significantly. The above results indicate that overexpression of NKG7 can promote IL-2 secretion in B7H3-CAR-T cells, activate the IL-2 signaling pathway and promote T cell proliferation.

[0119] Example 5 Overexpression of NKG7 can inhibit antigen-induced CAR downregulation and maintain membrane surface CAR expression 1. Experimental methods

[0120] This example uses flow cytometry to detect the expression of CAR on the surface of T cell membranes after stimulation of four types of tumor cells. The specific experimental steps are as follows:

[0121] (1) At a ratio of 1:1 between efficacy and target, 5 × 10 5 B7H3-CAR-T or B7H3-CAR-T NKG7 Then 5×105 tumor cells (MKN45, Huh7, SW620 and PANC-1) and mixed;

[0122] (2) Cells were collected at 0, 1, 2, 3, and 4 h, washed twice with PBS, and resuspended with 1 μg / mL PE-CAR and Percp-Cy5.5-CD3 antibodies and stained at 4°C for 30 min;

[0123] (3) Wash twice with PBS, centrifuge at 450 g for 5 min to collect the cell pellet, resuspend in 300 μL PBS and detect the positive rate of CAR-T cells on a flow cytometer.

[0124] This example uses flow cytometry to detect the expression of CAR on the surface of T cell membrane after stimulation with different concentrations of target protein. The specific experimental steps are as follows:

[0125] (1) Take 5×10 5 B7H3-CAR-T or B7H3-CAR-T NKG7 Cells were incubated with 0.25, 0.5, and 1 μg / mL B7H3 protein for 6 h;

[0126] (2) After 6 h, the cells were collected, washed twice with PBS, and resuspended with 1 μg / mL PE-CAR and Percp-Cy5.5-CD3 antibodies and stained at 4°C for 30 min;

[0127] (3) Wash twice with PBS, centrifuge at 450 g for 5 min to collect the cell pellet, resuspend in 300 μL PBS and detect the positive rate of CAR-T cells on a flow cytometer.

[0128] 2. Experimental results

[0129] The results are as follows Figure 4 As shown in the figure, after stimulation with four tumor cells, flow cytometry detection of CAR expression on the surface of T cell membrane showed that compared with B7H3-CAR-T cells, B7H3-CAR-T NKG7 The expression of cell membrane CAR increased significantly. Figure 5 As shown in the figure, after stimulation with different concentrations of target protein, flow cytometry detection of CAR expression on the surface of T cell membrane showed that compared with B7H3-CAR-T cells, B7H3-CAR-T NKG7 The expression of cell membrane CAR increased significantly.

[0130] Example 6 Overexpression of NKG7 can significantly improve the efficacy of B7H3-CAR-T cells in in vivo experiments and maintain the expression of CAR on the surface of CAR-T cell membranes in tumors

[0131] 1. Experimental methods

[0132] (1) In NCG mice, 5×10 6 MKN45 cells were used to construct a gastric cancer MKN45 subcutaneous tumor model;

[0133] (2) When the average tumor volume is 100 mm 3 At 4 × 10 6 CAR-T cells;

[0134] (3) Regularly monitor the volume of mice;

[0135] (4) At the end of the experiment, a single tumor cell suspension was prepared, and the cell samples were stained with BV510-Zombie, APC-CD3, and PE-CAR to examine the positive rate and infiltration number of CAR-T cells in the tumor.

[0136] 2. Experimental results

[0137] The results are as follows Figure 6 As shown, the results showed that overexpression of NKG7 could significantly improve the efficacy of B7H3-CAR-T cells in in vivo experiments and maintain the expression of CAR on the surface of intratumoral CAR-T cell membranes.

Claims

1. An enhanced chimeric antigen receptor targeting B7H3, characterized in that: The chimeric antigen receptor comprises an antibody targeting B7H3 and NKG7; The antibody comprises a heavy chain variable region and a light chain variable region; The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are CDR1, CDR2, and CDR3 in the heavy chain variable region as shown in SEQ ID NO: 4; The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are CDR1, CDR2, and CDR3 in the light chain variable region as shown in SEQ ID NO:9; The amino acid sequence of NKG7 is as shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO:

27.

2. The chimeric antigen receptor according to claim 1, characterized in that The amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively; Optionally, the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4; Optionally, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9; Optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signaling domain, a hinge region, and a co-stimulatory signaling domain; Optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-cleaving peptide, a safety switch, an immunomodulatory molecule or a cytokine; Optionally, the transmembrane domain is selected from the transmembrane domain of any one of the following molecules: CD8, CD28, 4-1BB, CD34, IgG4, CD3ε, PD-1, IgG1, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the intracellular signaling domain is selected from the intracellular signaling domain of any one of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, CD21, CD22, CD79a, CD79b, TCRζ, CD4, CD5, CD8, CD278, FcεRI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge region of any one of the following molecules: CD8, CD28, CD34, OX40, CD3ε, IgG1, IgG4, 4-1BB, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the costimulatory signaling domain is selected from the costimulatory signaling domains of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, NKp30, NKp46, CD40, OX40, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptides of any one of the following molecules: CD3ζ, α chain and β chain of T cell receptor, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF; Optionally, the self-cleaving peptide is selected from any one of the following self-cleaving peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L; Optionally, the chimeric antigen receptor is obtained by sequentially connecting a signal peptide, a heavy chain variable region of the antibody, a linker, a light chain variable region of the antibody, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signal domain, a CD3ζ intracellular signaling domain, a T2A self-cleaving peptide, and the NKG7; Optionally, the Linker is (G4S)3; Optionally, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 11; Optionally, the amino acid sequence of the Linker is shown in SEQ ID NO: 15; Optionally, the amino acid sequence of the CD8 hinge region and transmembrane domain is as shown in SEQ ID NO: 19; Optionally, the amino acid sequence of the CD28 costimulatory signal domain is as shown in SEQ ID NO: 21; Optionally, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO: 23; Optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO: 25; Optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:

29.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the chimeric antigen receptor according to claim 1 or 2; Optionally, the nucleotide sequence encoding the heavy chain variable region of the antibody in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 14; Optionally, the nucleotide sequence encoding the light chain variable region of the antibody in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 18; Optionally, the nucleotide sequence encoding the signal peptide in the chimeric antigen receptor according to claim 1 or 2 is shown in SEQ ID NO: 12; Optionally, the nucleotide sequence encoding the Linker in the chimeric antigen receptor according to claim 1 or 2 is shown in SEQ ID NO: 16; Optionally, the nucleotide sequence encoding the CD8 hinge region and transmembrane domain in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 20; Optionally, the nucleotide sequence encoding the CD28 co-stimulatory signaling domain in the chimeric antigen receptor of claim 1 or 2 is as shown in SEQ ID NO: 22; Optionally, the nucleotide sequence encoding the CD3ζ intracellular signaling domain in the chimeric antigen receptor of claim 1 or 2 is shown in SEQ ID NO: 24; Optionally, the nucleotide sequence encoding the T2A self-cleaving peptide in the chimeric antigen receptor of claim 1 or 2 is shown as SEQ ID NO: 26; Optionally, the nucleotide sequence encoding NKG7 in the chimeric antigen receptor of claim 1 or 2 is shown in SEQ ID NO: 28; Optionally, the nucleotide sequence encoding the chimeric antigen receptor according to claim 1 or 2 is shown as SEQ ID NO:

30.

4. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 3; Optionally, the vector is a plasmid, a phagemid, a cosmid, an artificial chromosome or a vector of viral origin; Optionally, the virus-derived vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpesvirus vector, a baculovirus vector, a papillomavirus vector or a papillomatosis virus vector.

5. A chimeric antigen receptor immune cell, characterized in that: The chimeric antigen receptor immune cell comprises the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3 and / or the expression vector of claim 4; Optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, mast cells and / or any combination thereof.

6. A chimeric antigen receptor immune cell population, characterized in that: The chimeric antigen receptor immune cell population comprises the chimeric antigen receptor immune cell of claim 5; Optionally, the chimeric antigen receptor immune cell population further comprises immune cells that do not comprise the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, and / or the expression vector of claim 4; Optionally, the immune cells are T lymphocytes, B lymphocytes, NK cells, iNKT cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, mast cells and / or any combination thereof.

7. A derivative, characterized in that: The derivative is a chimeric antigen receptor according to claim 1 or 2 or a nucleic acid molecule according to claim 3 labeled with a detectable marker, a chimeric antigen receptor according to claim 1 or 2 or a nucleic acid molecule according to claim 3 conferring antibiotic resistance, or a chimeric antigen receptor according to claim 1 or 2 or a nucleic acid molecule according to claim 3 combined with or coupled to a therapeutic agent; Optionally, the detectable marker is a fluorescent dye, colloidal gold, a chemiluminescent marker or a chemiluminescent catalyst; Optionally, the chemiluminescent marker is luminol and its derivatives, isoluminol and its derivatives, acridinium ester, acridinium ester derivatives, adamantane, rare earth elements or bipyridine ruthenium complex; Optionally, the chemiluminescent catalyst is horseradish peroxidase or alkaline phosphatase; Optionally, the antibiotic resistance gene is a penicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene or a kanamycin resistance gene; Optionally, the therapeutic agent is a radionuclide, a cytokine, a gold nanoparticle, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activated enzyme, or a chemotherapeutic agent; Alternatively, the cytokine is IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN-γ, TNF-β, TNF-α, G-CSF or M-CSF; Optionally, the chemotherapeutic agent is cisplatin, paclitaxel, vincristine, asparaginase, oxaliplatin, oxaliplatin or eloxatin.

8. A pharmaceutical composition or biological preparation, characterized in that: The pharmaceutical composition comprises the chimeric antigen receptor of claim 1 or 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the chimeric antigen receptor immune cell of claim 5, the chimeric antigen receptor immune cell population of claim 6 and / or the derivative of claim 7; Optionally, the pharmaceutical composition further comprises a therapeutic agent for adjuvant treatment and / or prevention of B7H3-positive related diseases; Optionally, the biologic comprises the pharmaceutical composition; Optionally, the biological preparation is in the form of an injection, an oral agent, a cream, a gel, a drop, a patch or a lyophilized powder.

9. Any of the following methods, characterized in that: The method comprises: (1) A method for preparing the chimeric antigen receptor immune cell of claim 5, the method comprising the following steps: providing an immune cell to be modified, introducing the nucleic acid molecule of claim 3 or the expression vector of claim 4 into the immune cell, to obtain the chimeric antigen receptor immune cell of claim 5; Optionally, the immune cells to be modified are T lymphocytes, B lymphocytes, NK cells, iNKT cells, CTL cells, monocytes, myeloid cells, dendritic cells, macrophages, mast cells and / or any combination thereof; (2) A method for stimulating a target cell population or tissue of a mammal to produce an immune response, the method comprising the following steps: administering an effective amount of the chimeric antigen receptor immune cell of claim 5 and / or the chimeric antigen receptor immune cell population of claim 6 to the mammal.

10. Any of the following applications, characterized in that: The applications include: (1) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the chimeric antigen receptor immune cell according to claim 5, the chimeric antigen receptor immune cell population according to claim 6, the derivative according to claim 7 and / or the pharmaceutical composition or biological preparation according to claim 8 in the preparation of a drug for treating and / or preventing a B7H3-positive related disease; (2) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the chimeric antigen receptor immune cell according to claim 5, the chimeric antigen receptor immune cell population according to claim 6 and / or the derivative according to claim 7 in the preparation of a biological agent for treating and / or preventing B7H3-positive related diseases; (3) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3 and / or the expression vector according to claim 4 in the preparation of chimeric antigen receptor immune cells for treating and / or preventing B7H3-positive related diseases; (4) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3 and / or the expression vector according to claim 4 in the preparation of a chimeric antigen receptor immune cell population for treating and / or preventing B7H3-positive related diseases; (5) Use of the chimeric antigen receptor according to claim 1 or 2, the nucleic acid molecule according to claim 3 and / or the expression vector according to claim 4 in the preparation of a kit for preparing chimeric antigen receptor immune cells for treating and / or preventing B7H3-positive related diseases; (6) Application of NKG7 in the preparation of enhanced chimeric antigen receptors; (7) Application of NKG7 in the preparation of enhanced chimeric antigen receptor immune cells; (8) Use of NKG7 in the preparation of a drug for enhancing the killing effect of chimeric antigen receptor immune cells; (9) Use of NKG7 in the preparation of a drug for promoting proliferation of chimeric antigen receptor immune cells; Optionally, the B7H3-positive related disease is gastric cancer, liver cancer, colorectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, breast cancer, esophageal cancer, oral cancer, endometrial cancer, bladder cancer, osteosarcoma, autoimmune disease, peritoneal cancer, glioma or myeloid leukemia.

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