Chimeric antigen receptor and use thereof

Abstract

Provided are a chimeric antigen receptor, a nucleic acid, an expression vector, a transgenic immune cell, a pharmaceutical composition, and the use thereof; the chimeric antigen receptor comprises: an extracellular region, which is provided with an amino acid sequence as represented by SEQ ID NO: 1; a transmembrane region, the N end of the transmembrane region being connected to the C end of the extracellular region; and an intracellular region, the N end of the intracellular region being connected to the C end of the transmembrane region. The chimeric antigen receptor targets an NKp30 receptor ligand, has high killing activity on tumor cells, and on the surface of a transgenic immune cell, the NKp30 receptor has high expression abundance. As a consequence, the chimeric antigen receptor or the transgenic immune cell expressing the chimeric antigen receptor can effectively kill B7H6-positive tumor cells and is highly safe.

Description

Chimeric antigen receptors and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor, nucleic acid, expression vector, transgenic immune cell, pharmaceutical composition and application thereof. Background Art

[0002] Chimeric antigen receptor T (CAR-T) cells have achieved remarkable results in the treatment of hematological malignancies. The antigen recognition region of CAR molecules typically utilizes antibody-derived fragment (scFv) sequences, typically from species such as mice, rabbits, and alpacas, and possesses strong immunogenicity. Even with humanized sequences, some immunogenicity persists, generating anti-drug antibodies (ADAs) that impair the persistence of CAR-T cells in vivo. In recent years, numerous new technologies have been applied to address this issue, such as using native receptor protein sequences as receptors to reduce immunogenicity.

[0003] The NKp30 receptor is a natural NK cell activating receptor that is highly expressed on NK cells and can recognize the B7H6 molecule, thereby mediating cytotoxicity. B7H6 is a member of the B7 family. The B7H6 molecule is usually highly expressed on a variety of primary tumor cells, including leukemia, lymphoma, colorectal cancer, gastric cancer, and pancreatic cancer, but is almost not expressed on normal tissue cells. Currently, the function of CAR-T cells constructed based on the NKp30 extracellular segment as a receptor for B7H6 recognition has been verified in research (An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo. J Immunol. 2012 Sep 1; 189(5): 2290-9. doi: 10.4049 / jimmunol.1103495. Patent application numbers US14342060, US16882800). This document or patent uses NKp30 amino acid positions 1-139 as the extracellular segment to construct T cells and other cells expressing chimeric antigen receptors, and observed that the immune cells' killing activity against B7H6-positive tumor cells was enhanced.

[0004] Based on the above research and development status, further research is needed to develop chimeric antigen receptors with higher killing activity and better safety.

[0005] Summary of the Invention

[0006] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides chimeric antigen receptors, nucleic acids, expression vectors, transgenic immune cells, pharmaceutical compositions, and uses thereof. The chimeric antigen receptors of the present invention target the NKp30 receptor ligand and exhibit high tumor cell cytotoxicity. The transgenic immune cells also express a high abundance of NKp30 receptors on their surfaces. Consequently, the chimeric antigen receptors of the present invention or transgenic immune cells expressing the chimeric antigen receptors can effectively kill B7H6-positive tumor cells and are highly safe.

[0007] It should be noted that the present invention is completed based on the following work of the inventors:

[0008] Natural killer (NK) cells are a key component of anti-tumor immunity, participating in anti-tumor immune responses through multiple pathways. NK cell surface expression of NK cytotoxicity receptors (NCRs) such as NKG2D, NKp30, NKp44, and NKp46 is high, and their corresponding ligands are often highly expressed on malignantly transformed or senescent cells. Upon recognition of these ligands, NK cells can mediate potent cytotoxicity, eliminating these malignantly transformed or senescent cells. Recent studies have revealed multiple variants of NKp30, namely NKp30a, NKp30b, NKp30c, NKp30d, NKp30e, and NKp30f. These variants exhibit significant functional differences. Activation of NKp30a mediates the strongest NK cell cytotoxicity and IFN-γ secretion, followed by NKp30b. Activation of NKp30c, however, does not significantly enhance NK cell cytotoxicity or IFN-γ secretion, but instead enhances the secretion of the immunosuppressive factor IL-10.

[0009] Thus, the inventors retained the arginine at position 143 in the natural NKp30a sequence and, through extensive experiments, identified an NKp30a amino acid sequence (1-147). Further experimental results demonstrated that a chimeric antigen receptor constructed based on this NKp30a amino acid sequence significantly enhanced NK cell activation. The resulting genetically modified immune cells were able to effectively kill B7H6-positive tumor cells and were highly safe.

[0010] Therefore, in a first aspect of the present invention, the present invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises:

[0011] an extracellular region having an amino acid sequence as shown in SEQ ID NO: 1;

[0012] a transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region;

[0013] The intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0014] The chimeric antigen receptors according to the embodiments of the present invention have high tumor cell killing activity, and the NKp30 receptor is highly expressed on the surface of the transgenic immune cells. As a result, the chimeric antigen receptors of the present invention significantly enhance the activation of NK cells. The transgenic immune cells obtained based on this can effectively kill B7H6-positive tumor cells and are highly safe.

[0015] In a second aspect, the present invention provides a nucleic acid. The nucleic acid comprises a first nucleic acid molecule encoding the aforementioned chimeric antigen receptor. The nucleic acid according to an embodiment of the present invention may encode a chimeric antigen receptor that significantly enhances NK cell activation.

[0016] In a third aspect, the present invention provides an expression vector carrying the aforementioned nucleic acid. Thus, the constructed vector can effectively express the aforementioned chimeric antigen receptor and / or the aforementioned IL15 / IL15Ra fusion protein.

[0017] In a fourth aspect of the present invention, a transgenic immune cell is provided. The transgenic immune cell comprises:

[0018] Expressing the aforementioned chimeric antigen receptor; or carrying the aforementioned nucleic acid or the aforementioned expression vector.

[0019] The immune cells according to the embodiments of the present invention are obtained by transfecting or transforming the vector or transformant, and the cells can efficiently express the aforementioned chimeric antigen receptor and / or IL15 / IL15Ra fusion protein under appropriate conditions.

[0020] In a fifth aspect of the present invention, a pharmaceutical composition is provided. The pharmaceutical composition comprises:

[0021] The aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned transgenic immune cell.

[0022] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, and transgenic immune cells are also applicable to this use and will not be elaborated upon here.

[0023] In the sixth aspect of the present invention, the present invention proposes the use of the aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned transgenic immune cell or the aforementioned pharmaceutical composition in the preparation of a drug, which is used to prevent and / or treat related diseases mediated by the B7H6 target.

[0024] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, and transgenic immune cells are also applicable to this use and will not be elaborated upon here.

[0025] In a seventh aspect, the present invention provides a method for treating and / or preventing B7H6 target-mediated diseases, comprising: administering a pharmaceutically acceptable amount of the aforementioned transgenic immune cells or the aforementioned pharmaceutical composition to a subject.

[0026] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0028] FIG1 is a schematic diagram of the structures of different chimeric antigen receptors according to Example 1 of the present invention;

[0029] FIG2 is a statistical diagram showing the killing efficiency of NCI-H716 cells by different NKR-NK cells according to Example 2 of the present invention;

[0030] FIG3 is a graph showing the survival rate changes of NK-92 and NKR3-NK-92 cells in Example 2 of the present invention at different IL-2 culture concentrations;

[0031] FIG4 is a statistical graph showing the killing efficiency of NK-92 and NKR3-NK-92 cells against NCI-H716 cells at different IL-2 culture concentrations according to Example 2 of the present invention;

[0032] FIG5 is a diagram showing the tumor inhibition effect of peripheral blood-derived NKR3-NK cells in Example 3 of the present invention on a mouse model bearing human colorectal cancer NCI-H716 cells;

[0033] FIG6 is a graph showing the killing effect of NKR3-NK-92 cells on human ovarian cancer HO8910 cells according to Example 4 of the present invention;

[0034] FIG7 is a diagram showing the in vivo tumor inhibition effect of NKR3-NK-92 cells according to Example 4 of the present invention on a subcutaneous tumor-bearing mouse model of human ovarian cancer cells SK-OV-3;

[0035] FIG8 is a diagram showing the tumor inhibition effect of peripheral blood-derived NKR3-NK cells in Example 5 of the present invention on a mouse model bearing human triple-negative breast cancer cells MDA-MB-231. DETAILED DESCRIPTION

[0036] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0037] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0038] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0039] Terms and Definitions

[0040] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0041] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0042] In this article, the term "(G4S) n " is equivalent to "(Gly4Ser) n ", which means 4 glycines and 1 serine are repeated n times. It is a widely used type of linker peptide and can be located at V H C-terminal and V L N terminal, can also be located at V L C-terminal and V H Between the N-termini. Currently, (G4S)3 is commonly used. Glycine, the amino acid with the smallest molecular weight and shortest side chain, increases side chain flexibility, while serine, the most hydrophilic amino acid, increases the hydrophilicity of the linker peptide. Therefore, (G4S)3 exhibits excellent stability and activity.

[0043] As used herein, "carbon terminus" and "C-terminus" are synonymous; "nitrogen terminus" and "N-terminus" are synonymous.

[0044] The present invention provides a chimeric antigen receptor, nucleic acid, expression vector, transgenic immune cell, pharmaceutical composition and application thereof, which are described in detail below.

[0045] Chimeric antigen receptor

[0046] The present invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises:

[0047] an extracellular region having an amino acid sequence as shown in SEQ ID NO: 1;

[0048] a transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region;

[0049] The intracellular region, wherein the N-terminus of the intracellular region is connected to the C-terminus of the transmembrane region.

[0050] It should be noted that the amino acid sequences involved in the present invention are all shown from N-terminus to C-terminus.

[0051] The chimeric antigen receptors according to the embodiments of the present invention have high tumor cell killing activity, and the NKp30 receptor is highly expressed on the surface of the transgenic immune cells. As a result, the chimeric antigen receptors of the present invention significantly enhance the activation of NK cells. The transgenic immune cells obtained based on this can effectively kill B7H6-positive tumor cells and are highly safe.

[0052] In this article, the term "CSR" is equivalent to "Chimeric Switch Receptor", which is an artificially constructed recombinant receptor that contains an extracellular antigen recognition domain, a transmembrane region, and an intracellular signal transduction domain. Unlike the chimeric antigen receptor "CAR", whose extracellular antigen recognition domain is composed of a single-chain antibody, the extracellular antigen recognition domain of the chimeric switch receptor is mainly composed of inhibitory surface receptors on immune cells.

[0053] As used herein, the term "chimeric antigen receptor-expressing immune cell" is equivalent to "CAR-immune cell," which is a genetically modified immune cell that expresses a specific chimeric antigen receptor and is further used for the prevention and / or treatment of a disease. In some cases, depending on the context, it is equivalent to "chimeric antigen receptor gene-modified immune cell."

[0054] According to an embodiment of the present invention, the intracellular region includes a costimulatory domain and an intracellular signaling domain.

[0055] According to an embodiment of the present invention, the C-terminus of the costimulatory domain is connected to the N-terminus of the intracellular signaling domain.

[0056] According to an embodiment of the present invention, the costimulatory domain is selected from at least one of CD3ζ, CD28, 4-1BB, 2B4, DAP10, DAP12, CD27, CD40, OX40, ICOS intracellular segment or a fragment thereof.

[0057] According to an embodiment of the present invention, the costimulatory domain is the intracellular segment of CD28 or a fragment thereof.

[0058] According to an embodiment of the present invention, the costimulatory domain has an amino acid sequence as shown in SEQ ID NO: 4.

[0059] According to an embodiment of the present invention, the intracellular signaling domain is the CD3ζ intracellular segment or a fragment thereof.

[0060] According to an embodiment of the present invention, the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO:5.

[0061] According to an embodiment of the present invention, the transmembrane region is the transmembrane segment of CD28 or a fragment thereof.

[0062] According to an embodiment of the present invention, the transmembrane region has an amino acid sequence as shown in SEQ ID NO: 30.

[0063] According to an embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 6.

[0064] In this way, transgenic immune cells with further enhanced tumor cell killing activity and higher safety can be obtained.

[0065] Nucleic Acids

[0066] The present invention provides a nucleic acid comprising a first nucleic acid molecule encoding the aforementioned chimeric antigen receptor.

[0067] The nucleic acid according to the embodiments of the present invention can encode a chimeric antigen receptor that significantly enhances the NK cell activation effect.

[0068] According to an embodiment of the present invention, the first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 7. Thus, the expression level of the chimeric antigen receptor on immune cells is further improved.

[0069] According to an embodiment of the present invention, the nucleic acid further includes a second nucleic acid molecule, which is connected to the first nucleic acid molecule, and the second nucleic acid molecule encodes a fusion protein, and the fusion protein includes IL-15Rα and IL-15. According to an embodiment of the present invention, the immune cell containing the above nucleic acid can simultaneously express a chimeric antigen receptor and a fusion protein. Among them, the chimeric antigen receptor enables the immune cell to target the NKp30 receptor ligand and localize to the cell surface expressing the ligand; the fusion protein further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the local microenvironment of the tumor, and maintains its strong tumor-killing activity, which can effectively avoid the toxic side effects caused by high-dose or repeated injections of the whole body.

[0070] According to an embodiment of the present invention, the N-terminus of the IL-15Rα is connected to the C-terminus of the IL-15, or the N-terminus of the IL-15 is connected to the C-terminus of the IL-15Rα.

[0071] According to an embodiment of the present invention, the IL-15Rα has an amino acid sequence as shown in SEQ ID NO: 8.

[0072] According to an embodiment of the present invention, the IL-15 has an amino acid sequence as shown in SEQ ID NO:9.

[0073] According to embodiments of the present invention, immune cells containing the aforementioned nucleic acids can simultaneously express a chimeric antigen receptor and an IL15 / IL15Ra fusion protein on their cell membrane surfaces. Thus, the membrane-expressed IL15 / IL15Ra fusion protein can further enhance the in vivo survival and proliferation of immune cells, reduce IL15-related side effects, and promote immune responses, thereby further enhancing the in vivo anti-tumor activity of the immune cells of the present invention.

[0074] According to an embodiment of the present invention, the fusion protein further includes a connecting peptide.

[0075] According to an embodiment of the present invention, the C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15; or

[0076] The C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15Rα.

[0077] According to an embodiment of the present invention, the signal peptide is selected from one of an IgE signal peptide, a CD8α signal peptide and a GM-CSF signal peptide.

[0078] According to an embodiment of the present invention, the signal peptide is an IgE signal peptide.

[0079] According to an embodiment of the present invention, the IgE signal peptide has an amino acid sequence as shown in SEQ ID NO: 19. Thus, the membrane expression level of the IL15 / IL15Ra fusion protein is further improved.

[0080] According to an embodiment of the present invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 21 or 23.

[0081] According to an embodiment of the present invention, the second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 22 or 24. Thus, the expression level of the aforementioned IL15 / IL15Ra fusion protein on the immune cell membrane is further improved.

[0082] According to an embodiment of the present invention, the nucleic acid further includes a fourth nucleic acid molecule, the fourth nucleic acid molecule encodes a first linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected via the fourth nucleic acid molecule.

[0083] According to an embodiment of the present invention, it further includes a fifth nucleic acid molecule, wherein the fifth nucleic acid molecule encodes a second linker.

[0084] wherein the first nucleic acid molecule and the second nucleic acid molecule are connected via the fourth nucleic acid molecule, and the second nucleic acid molecule and the third nucleic acid molecule are connected via the fifth nucleic acid molecule; or

[0085] The first nucleic acid molecule and the third nucleic acid molecule are connected via the fourth nucleic acid molecule, and the third nucleic acid molecule and the second nucleic acid molecule are connected via the fifth nucleic acid molecule.

[0086] According to an embodiment of the present invention, the first linker or the second linker is independently selected from at least one of P2A, T2A, E2A and F2A. Thus, immune cells can be obtained that uniformly express the chimeric antigen receptor and IL15 / IL15Ra fusion protein on the cell membrane.

[0087] According to an embodiment of the present invention, the first linker and the second linker are both P2A. Thus, immune cells can be obtained that uniformly express the chimeric antigen receptor and IL15 / IL15Ra fusion protein on their cell membranes.

[0088] According to an embodiment of the present invention, the first linker and the second linker both have the amino acid sequence shown in SEQ ID NO: 25. Thus, immune cells are obtained that uniformly express the chimeric antigen receptor and IL15 / IL15Ra fusion protein on their cell membranes.

[0089] According to an embodiment of the present invention, the fourth nucleic acid molecule and the fifth nucleic acid molecule both have the nucleotide sequence shown in SEQ ID NO: 26. Thus, the expression level of the aforementioned IL15 / IL15Ra fusion protein on the immune cell membrane is further improved.

[0090] According to an embodiment of the present invention, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 29. Thus, immune cells expressing the aforementioned chimeric antigen receptor in high abundance can be obtained.

[0091] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0092] expression vector

[0093] The present invention provides an expression vector. The expression vector carries the aforementioned nucleic acid. Thus, the constructed expression vector can express the chimeric antigen receptor of the present invention and / or the aforementioned IL15 / IL15Ra fusion protein in a host cell.

[0094] When the nucleic acid molecule is linked to an expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, that is, not derived from the vector itself. Of course, it is sufficient that the nucleic acid molecule is operably linked to the control elements. According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0095] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0096] According to an embodiment of the present invention, the nucleic acid can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0097] As used herein, the term "operably linked" refers to linking an exogenous gene to a vector so that control elements within the vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, and the like. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable immune cells, they can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby achieving substantial expression of the protein encoded by the nucleic acid molecules on the cell membrane, thereby obtaining transgenic immune cells.

[0098] According to an embodiment of the present invention, the expression vector is a eukaryotic vector or a prokaryotic vector.

[0099] According to an embodiment of the present invention, the expression vector is a non-pathogenic viral vector.

[0100] According to an embodiment of the present invention, the non-pathogenic virus is selected from one of retrovirus, lentivirus and adeno-associated virus.

[0101] According to an embodiment of the present invention, the expression vector is a lentiviral expression vector.

[0102] cell

[0103] The present invention provides a transgenic immune cell. The transgenic immune cell comprises:

[0104] Expressing the aforementioned chimeric antigen receptor; or carrying the aforementioned nucleic acid or the aforementioned expression vector.

[0105] The immune cells according to the embodiments of the present invention are obtained by transfecting or transforming the vector or transformant, and the cells can efficiently express the aforementioned chimeric antigen receptor or the aforementioned chimeric antigen receptor and IL15 / IL15Ra fusion protein under appropriate conditions.

[0106] According to an embodiment of the present invention, the immune cells of the transgenic immune cells are selected from T cells, NK cells, NKT cells, γδT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, peripheral blood NK cells, umbilical cord blood NK cells, NK-92 cells, iPSC-derived or embryonic stem cell cultured and differentiated immune cells. The aforementioned chimeric antigen receptor of the present invention can be transduced into immune cells such as T, NK, NKT, γδT, macrophages, iPSCs, etc. through an expression vector (such as a lentiviral vector), thereby being expressed on the surface of these immune cells.

[0107] According to an embodiment of the present invention, the immune cells of the transgenic immune cells are NK cells.

[0108] The present invention does not impose strict restrictions on the source of NK cells, and NK cells can be derived from peripheral blood NK cells, umbilical cord blood NK cells, iPSCs, or NK-92 cells.

[0109] Pharmaceutical composition

[0110] The present invention provides a pharmaceutical composition. The pharmaceutical composition comprises:

[0111] The aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned expression vector or the aforementioned transgenic immune cell.

[0112] According to an embodiment of the present invention, the pharmaceutical composition further comprises: a pharmaceutically acceptable excipient.

[0113] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with a liquid carrier, a solid carrier, or both.

[0114] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the particular intended dosage form. Except to the extent that any conventional excipient is incompatible with the chimeric antigen receptor, nucleic acid, expression vector, or transgenic immune cell of the present invention, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0115] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, and transgenic immune cells are also applicable to this use and will not be elaborated upon here.

[0116] use

[0117] The present invention provides a use of the aforementioned chimeric antigen receptor, the aforementioned nucleic acid, the aforementioned expression vector, the aforementioned lentiviral vector, the aforementioned transgenic immune cell or the aforementioned pharmaceutical composition in preparing a medicament, wherein the medicament is used to prevent and / or treat related diseases mediated by the B7H6 target.

[0118] According to an embodiment of the present invention, the B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

[0119] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0120] In some specific embodiments of the present invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

[0121] In some specific embodiments of the present invention, the tumor or cancer is colorectal cancer, head and neck tumor, liver cancer, gastric cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, or bile duct cancer.

[0122] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, and transgenic immune cells are also applicable to this use and will not be elaborated upon here.

[0123] Methods for preventing and / or treating tumors

[0124] The present invention provides a method for preventing and / or treating diseases mediated by the B7H6 target. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the above-mentioned transgenic immune cells or the above-mentioned pharmaceutical composition to a subject.

[0125] According to an embodiment of the present invention, the B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

[0126] According to an embodiment of the present invention, the tumor is a solid tumor or a hematological tumor.

[0127] In some specific embodiments of the present invention, the cancer is selected from one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

[0128] In some specific embodiments of the present invention, the tumor or cancer is colorectal cancer, head and neck tumor, liver cancer, gastric cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, or bile duct cancer.

[0129] Those skilled in the art will appreciate that the features and advantages described above for chimeric antigen receptors, nucleic acids, expression vectors, and transgenic immune cells are also applicable to the treatment methods for this disease and will not be elaborated here.

[0130] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient via a suitable means. The transgenic immune cells or pharmaceutical compositions of the present invention may be administered via any common route, provided they reach the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, and subcutaneous injections, but the present invention is not limited to these exemplified modes of administration. Preferably, the compositions of the present invention are administered via intravenous injection.

[0131] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or transgenic immune cell to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing cells containing a chimeric antigen receptor as described herein to an individual in need.

[0132] The effective amount of the transgenic immune cells and pharmaceutical compositions described herein may vary depending on the mode of administration and the severity of the disease to be treated. Preferably, the selection of the effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0133] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0134] The sequences involved in the present invention are detailed in Table 1.

[0135] Table 1: Nucleotide / amino acid sequence description

[0136] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0137] It should be noted that the "plasmid" and "vector" described in the following embodiments have the same meaning and can be used interchangeably.

[0138] Example 1: Preparation of NK cells expressing different NKR structures

[0139] 1. Construction of different NKR structures

[0140] Different CSR sequences were synthesized by whole gene synthesis and cloned into the lentiviral vector pCDG-EF1-MCS-TA2-copGFP through the restriction sites XbaI and BamHI. After sequencing verification, pCDH-EF1a-NKR1, pCDH-EF1a-NKR2 and pCDH-EF1a-NKR3 were obtained.

[0141] The schematic diagram of the genetic element structure of the chimeric antigen receptor of this example is shown in Figure 1, and the nucleotide / amino acid sequences are shown in Table 1. The amino acid sequences of NKp30 1-147, NKp30 1-139, and NKp30 1-201 are shown in SEQ ID NOs: 1-3, respectively, and the nucleotide sequences of NKR1, NKR2, and NKR3 are shown in SEQ ID NOs: 31, 7, and 29, respectively. The NKR1 sequence is based on the sequence disclosed in US Pat. No. 14,342,060.

[0142] 2. Lentivirus packaging

[0143] 5×10 293T cells in the logarithmic growth phase were taken 6Inoculate in a 10cm cell culture dish, add 10mL DMEM medium, and culture overnight in a 37℃, 5% CO2 incubator. When the cell density in the cell culture dish reaches 80-90%, replace with 10mL fresh DMEM medium, and continue to place the cell culture dish in the incubator for standby use. Prepare the lentiviral packaging system, add 6μg psPAX2 and 3μg pMD2.G lentiviral packaging auxiliary plasmids, and 6μg target gene vector plasmids to 250μL serum-free DMEM medium to prepare a plasmid mixture and mix well. Add 15μL Add to 235 μL serum-free DMEM medium and mix well. Add the mixed solution to the above plasmid mixture at one time, mix well, and incubate at room temperature for 15 minutes. Add the mixed solution to the 293T cell culture dish. Change the medium after 24 hours, put the culture dish back into the 37°C, 5% CO2 incubator, collect the cell supernatant after 48 hours, centrifuge at 400×g for 5 minutes, remove cell debris, and filter the supernatant with a 0.45μm filter into a 50mL centrifuge tube. Add 5×PEG8000 solution to concentrate the virus solution, mix evenly by inverting the centrifuge tube, and place it in a 4°C refrigerator overnight. Centrifuge at 4000×g for 20 minutes at 4°C, discard the supernatant, add an appropriate amount of serum-free DMEM to resuspend the virus pellet, transfer it to an EP tube, and store it in a -80°C refrigerator.

[0144] 3. Lentiviral infection of human NK-92 cells

[0145] NK-92 cells in the logarithmic growth phase were harvested by centrifugation at 100 × g for 5 min, and appropriate amount of α-MEM medium was added to resuspend the cells to adjust the cell density to 5 × 10 5 5×10 5NK-92 cells, 0.2 mL of virus concentrate, 0.8 mL of α-MEM medium, and protamine (final concentration 8 μg / mL) were mixed thoroughly. The cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours, the cells were observed, the medium was changed, and the infected cells were transferred to an EP tube. The cells were centrifuged at 100×g for 5 minutes. A small amount of fresh α-MEM medium was added to resuspend the cells. The cells were transferred to a cell culture flask, and 10 mL of fresh α-MEM medium and IL-2 (final concentration 200 IU / mL) were added and cultured for another 48 hours. The cells were transferred to a flow cytometer, 3 mL of 1× PBS solution was added, and the cells were centrifuged at 100×g for 5 minutes. The supernatant was discarded, the cell pellet was flicked, and the cells were washed again with 1× PBS. GFP expression was detected using a flow cytometer. The culture was continued to expand, and the infected NK-92 cells were adjusted for amplification. The infected NK-92 cells were sorted for GFP-positive NKR-NK92 cells by flow cytometry for use in later experiments.

[0146] Example 2: Investigation of biological functions of different NKR-NK cells

[0147] In this example, the inventors investigated the killing activity, survival rate and dependence on IL-2 of different NKR-NK cells obtained in Example 1 in vitro.

[0148] 1. In vitro cytotoxicity of NK-92 cells modified with different NKR receptors

[0149] Colorectal cancer cell line NCI-H716 cells, which highly express the NKp30 ligand B7H6, were selected as target cells, and the in vitro cytotoxic activity of different NKR-NK cells was detected.

[0150] The specific method is as follows: NCI-H716 cells were fluorescently labeled with CFSE and the cells were stained with 2x10 4 NK-92 cells or different NKR-NK-92 cells were plated into 96-well plates and incubated with NCI-H716 cells for 4 hours. Propidium Iodide (PI) was then added to distinguish live and dead cells, and the killing efficiency was measured by flow cytometry. The effector cell to target cell ratio was 2:1.

[0151] The results are shown in Figure 2.

[0152] The results of the in vitro cytotoxicity test showed that: (1) NKR2-NK-92 or NKR3-NK-92 cells modified with the chimeric antigen receptor gene of Example 1 of the present invention had a higher killing efficiency against B7H6-positive NCI-H716 cells than NK-92 cells. (2) The killing efficiency of NKR2-NK-92 and NKR3-NK-92 cells was significantly better than that of NKR1-NK-92 (refer to the sequence disclosed in US14342060) cells.

[0153] 2. NKR3 gene modification promotes NK cell survival

[0154] The inventors further verified the promoting effect of membrane-expressed super IL-15 on the survival of NK-92 cells in vitro.

[0155] The specific method is as follows: NK92 and NKR3-NK92 cells with the same cell number were plated in a 24-well plate, and different IL-2 concentrations (200 IU / mL and 0 IU / mL) were set. The apoptosis rate was detected by flow cytometry every 24 hours. The apoptosis rate was detected by flow cytometry according to the steps in the kit instructions (Lianke Bio, product number AP101). In short, the cells were collected in an EP tube, added 1xPBS solution, centrifuged and washed once, and the cells were resuspended. 5 μL Annexin V-FITC and 10 μL PI were added to each tube. After gently vortexing to mix, incubate at room temperature in the dark for 5 minutes, and resuspend the cells for flow cytometry. The cell viability is the proportion of double-negative Annexin V-FITC and PI staining.

[0156] The results of the NK cell survival rate test after 96 h of culture are shown in FIG3 .

[0157] The results of the in vitro survival rate investigation of NK-92 cells showed that in the absence of IL-2 (IL-2 0 IU / mL), the cell survival rate of the NK92 group decreased significantly from 24 hours, and most cells had undergone apoptosis by 72 hours; while the cells in the NKR3-NK-92 group could still maintain a high cell viability even under the condition of complete withdrawal of IL-2, and few cells underwent apoptosis.

[0158] The above results show that the expression of super IL-15 on the NKR3-NK-92 membrane can further improve the long-term survival and expansion ability of NKR-NK cells in vivo.

[0159] 3. NKR3 gene modification reduces NK cell dependence on IL-2 to maintain high cytotoxic activity

[0160] The inventors further verified that the expression of super IL-15 in the membrane of NKR3 gene can reduce the dependence of NK cells on IL-2 to maintain high killing activity.

[0161] The specific method is as follows: NK-92 and NKR3-NK-92 cells were cultured in the presence of 0 or 200 IU / mL IL-2 for 48 h. NCI-H716 cells were fluorescently labeled with CFSE and the cells were cultured at 2x10 4 Each well was plated into a 96-well plate. Different NK cell lines were then plated separately and incubated with NCI-H716 cells for 4 hours. Propidium Iodide (PI) was then added to distinguish live and dead cells, and the killing efficiency was measured by flow cytometry. The effector cell to target cell ratio was 2:1.

[0162] The test results are shown in Figure 4.

[0163] The results of the NK-92 cell killing activity test showed that: (1) the killing efficiency of NK-92 cells decreased significantly under IL-2-free culture conditions. (2) there was no significant difference in the killing efficiency of the NKR3-NK-92 cells modified with the NKR3 gene under culture conditions with 0 and 200 IU / mL IL-2 concentrations. (3) the killing efficiency of the NKR3-NK-92 cells of the present invention was significantly higher than that of NK-92 cells.

[0164] The above results show that the expression of super IL-15 in the NKR3-NK-92 membrane can reduce the dependence of NK cells on IL-2 to maintain high cytotoxic activity.

[0165] Example 3: Investigation of the in vivo tumor-suppressing activity of NKR3-NK cells

[0166] The inventors established a subcutaneous tumor-bearing model in mice using human colorectal cancer NCI-H716 cells, infected primary human peripheral blood NK cells with NKR3 lentivirus to prepare peripheral blood-derived NKR3-NK cells, and observed the therapeutic effect of primary NKR3-NK cells on the colorectal cancer tumor-bearing model.

[0167] The specific method is as follows: 6-week-old NCG mice were selected for subcutaneous tumor bearing in the armpits, and the tumor volume reached 50 mm. 3 NK cell therapy was initiated at 4 hr. Tumor volume was measured before treatment, and the mice were randomly divided into the untreated group, the NK cell treatment group, and the NKR3-NK cell treatment group according to tumor volume. In the NK cell treatment group, NK cells were infused through the tail vein. Mice were treated once every two days for a total of three times, with a tail vein infusion dose of 8 × 10 6 CD56 + NK cells were injected intraperitoneally every 2 days to maintain NK cell survival; in the NKR3-NK cell treatment group, NKR3-NK cells were reinfused through the tail vein for a single treatment at a dose of 2×10 6NKR3-NK cells / mouse, without IL-2 injection. Tumor volume was measured twice a week, tumor growth curves were drawn, and tumor inhibition rates were calculated.

[0168] The test results are shown in Figure 5.

[0169] The results of in vivo tumor inhibition activity investigation showed that the peripheral blood-derived NKR3-NK cells prepared based on NKR3 of Example 1 of the present invention had a significantly enhanced effect of inhibiting tumor growth compared with the NK cell treatment group (control group, NK cells did not carry any form of CAR or CSR), and the dose of NK cells infused was lower.

[0170] The above results indicate that the NKR3-NK cells of the present invention have a strong anti-tumor effect on colorectal cancer tumors, and can be used at a lower dosage, which is expected to break through the bottleneck of poor therapeutic effect of immune cell therapy on solid tumors.

[0171] Example 4: Inhibitory effect of NKR3-NK cells on ovarian cancer

[0172] In this example, the killing efficiency of NKR3-NK-92 cells modified with the NKR3 gene of the present invention against ovarian cancer cells (human ovarian cancer HO8910 cells) was investigated according to the method of Example 2. The effector cell to target cell ratio (E:T) was 1:1 ( FIG6 ).

[0173] In this example, a subcutaneous tumor-bearing model in mice was established using human ovarian cancer cells SK-OV-3 according to the method of Example 3. NK-92 cells were infected with NKR3 lentivirus to prepare NKR3-NK-92 cells, and the therapeutic effect of NKR3-NK-92 cells on the ovarian cancer tumor-bearing model was observed ( Figure 7 ).

[0174] The results are shown in Figures 6 and 7: Compared with the control NK92 cells, the transgenic immune cells NKR3-NK-92 expressing the chimeric antigen receptor of the present invention have significantly improved killing activity against ovarian cancer cells and have a significantly improved tumor inhibition rate against ovarian cancer.

[0175] Example 5: Inhibitory effect of NKR3-NK cells on B7H6-positive breast cancer

[0176] In this example, according to the method of Example 3, a subcutaneous tumor-bearing model of human breast cancer mice was established using the human triple-negative breast cancer MDA-MB-231 cell line, and human peripheral blood primary NK cells were infected with NKR3 lentivirus to prepare peripheral blood-derived NKR3-NK cells, and the therapeutic effect of primary NKR3-NK cells on the breast cancer tumor-bearing model was observed (Figure 8). The difference from Example 3 is that the NKR3-NK cell treatment group in this example has three infusion dose groups: 2E6, 4E6, and 8E6. Among them, 2E6 means that the infusion dose of NK cells is 2×10 6 CD56 + NKR3-NK cells; 4E6 indicates the infused NK cell dose is 4×10 6 CD56 + NKR3-NK cells; 8E6 indicates the infused NK cell dose is 8×10 6 CD56 + NKR3-NK cells.

[0177] The results are shown in Figure 8: Compared with the control NK cells (NK cell treatment group), the transgenic immune cells NKR3-NK cells expressing the chimeric antigen receptor of the present invention have a significantly improved tumor inhibition rate against B7H6-positive breast cancer, and show a dose-dependent relationship. The high-dose group (8E6) has the best tumor inhibition effect.

[0178] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0179] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that include: An extracellular region, wherein the extracellular region has an amino acid sequence as shown in SEQ ID NO: 1; A transmembrane region, wherein the N-terminus of the transmembrane region is connected to the C-terminus of the extracellular region; The intracellular region has an N-terminus connected to the C-terminus of the transmembrane region.

2. The chimeric antigen receptor according to claim 1, characterized in that The intracellular region includes a costimulatory domain and an intracellular signaling domain; the C-terminus of the costimulatory domain is connected to the N-terminus of the intracellular signaling domain.

3. The chimeric antigen receptor according to claim 2, characterized in that The co-stimulatory domain is selected from at least one of CD3ζ, CD28, 4-1BB, 2B4, DAP10, DAP12, CD27, CD40, OX40, ICOS intracellular segment or a fragment thereof.

4. The chimeric antigen receptor according to claim 2, characterized in that The co-stimulatory domain is the intracellular segment of CD28 or a fragment thereof.

5. The chimeric antigen receptor according to claim 2, characterized in that The costimulatory domain has an amino acid sequence as shown in SEQ ID NO:

4.

6. The chimeric antigen receptor according to claim 2, characterized in that The intracellular signal transduction domain is the CD3ζ intracellular segment or a fragment thereof.

7. The chimeric antigen receptor according to claim 2, characterized in that The intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO:

5.

8. The chimeric antigen receptor according to claim 1, characterized in that The transmembrane region is the transmembrane segment of CD28 or a fragment thereof.

9. The chimeric antigen receptor according to claim 1, characterized in that The transmembrane region has an amino acid sequence as shown in SEQ ID NO:

30.

10. The chimeric antigen receptor according to claim 1, characterized in that The chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:

6.

11. A nucleic acid, characterized in that It comprises a first nucleic acid molecule, wherein the first nucleic acid molecule encodes the chimeric antigen receptor according to any one of claims 1 to 10.

12. The nucleic acid according to claim 11, characterized in that The first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:

7.

13. The nucleic acid according to claim 11, characterized in that It further comprises a second nucleic acid molecule, wherein the second nucleic acid molecule is connected to the first nucleic acid molecule, and the second nucleic acid molecule encodes a fusion protein, wherein the fusion protein comprises IL-15Rα and IL-15.

14. The nucleic acid according to claim 13, characterized in that The N-terminus of the IL-15Rα is connected to the C-terminus of the IL-15, or the N-terminus of the IL-15 is connected to the C-terminus of the IL-15Rα.

15. The nucleic acid according to claim 13, characterized in that The IL-15Rα has the amino acid sequence shown in SEQ ID NO:

8.

16. The nucleic acid according to claim 13, characterized in that The IL-15 has the amino acid sequence shown in SEQ ID NO:

9.

17. The nucleic acid according to claim 13, characterized in that The fusion protein further comprises a connecting peptide; the C-terminus of the IL-15Rα is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15; or The C-terminus of the IL-15 is connected to the N-terminus of the connecting peptide, and the C-terminus of the connecting peptide is connected to the N-terminus of the IL-15Rα.

18. The nucleic acid according to claim 17, characterized in that The connecting peptide is selected from at least one of (G4S)n, ESGRSGGGGSGGGGS, EGKSSGSGSESKST, EGKSSGSGSESKSTQ, GSTSGSGKSSEGKG, KESGSVSSEQLAQFRSLD, ESGSVSSEELAFRSLD, and SGGGSGGGGSGGGGSGGGGSGGGSLQ, and n is a non-zero integer.

19. The nucleic acid according to claim 18, characterized in that The connecting peptide is selected from (G4S)n, where n is any integer between 2 and 6.

20. The nucleic acid according to claim 19, characterized in that The connecting peptide is (G4S)3; or, the connecting peptide is SGGGSG GGGSGGGGSGGGGSGGGSLQ.

21. The nucleic acid according to claim 13, characterized in that The fusion protein further comprises a signal peptide; the C-terminus of the signal peptide is connected to the N-terminus of the IL-15Rα, or the C-terminus of the signal peptide is connected to the N-terminus of the IL-15.

22. The nucleic acid according to claim 21, characterized in that The signal peptide is selected from one of an IgE signal peptide, a CD8α signal peptide and a GM-CSF signal peptide.

23. The nucleic acid according to claim 22, characterized in that The signal peptide is an IgE signal peptide.

24. The nucleic acid according to claim 23, characterized in that The IgE signal peptide has an amino acid sequence as shown in SEQ ID NO:

19.

25. The nucleic acid according to claim 13, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO: 21 or 23.

26. The nucleic acid according to claim 13, characterized in that The second nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 22 or 24.

27. The nucleic acid according to claim 13, characterized in that Further included is a third nucleic acid molecule encoding an FcRγ protein.

28. The nucleic acid according to claim 27, characterized in that The FcRγ protein has the amino acid sequence shown in SEQ ID NO:

27.

29. The nucleic acid according to claim 27, characterized in that The third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:

28.

30. The nucleic acid according to any one of claims 13 to 26 or any one of claims 27 to 29, characterized in that Also included is a fourth nucleic acid molecule, the fourth nucleic acid molecule encodes a first linker, and the first nucleic acid molecule and the second nucleic acid molecule are connected via the fourth nucleic acid molecule; Also included is a fifth nucleic acid molecule encoding a second linker; wherein the first nucleic acid molecule and the second nucleic acid molecule are connected via the fourth nucleic acid molecule, and the second nucleic acid molecule and the third nucleic acid molecule are connected via the fifth nucleic acid molecule; or The first nucleic acid molecule and the third nucleic acid molecule are connected via the fourth nucleic acid molecule, and the third nucleic acid molecule and the second nucleic acid molecule are connected via the fifth nucleic acid molecule.

31. The nucleic acid according to claim 30, characterized in that The first linker or the second linker is each independently selected from at least one of P2A, T2A, E2A and F2A.

32. The nucleic acid according to claim 30, characterized in that The first linker and the second linker are both P2A.

33. The nucleic acid according to claim 30, characterized in that The first linker and the second linker both have the amino acid sequence shown in SEQ ID NO:

25.

34. The nucleic acid according to claim 30, characterized in that The fourth nucleic acid molecule and the fifth nucleic acid molecule both have the nucleotide sequence shown in SEQ ID NO:

26.

35. The nucleic acid according to claim 11, characterized in that The nucleic acid has a nucleotide sequence as shown in SEQ ID NO:

29.

36. An expression vector, characterized in that Carrying the nucleic acid according to any one of claims 11 to 35.

37. The expression vector according to claim 36, characterized in that The expression vector is a non-pathogenic viral vector.

38. The expression vector according to claim 37, characterized in that The non-pathogenic virus is selected from one of a retrovirus, a lentivirus and an adeno-associated virus.

39. The expression vector according to claim 36, characterized in that The expression vector is a lentiviral expression vector.

40. A transgenic immune cell, characterized in that: include: Expressing the chimeric antigen receptor according to any one of claims 1 to 10; or Carrying the nucleic acid according to any one of claims 11 to 35, or the expression vector according to any one of claims 36 to 39.

41. The transgenic immune cell according to claim 40, characterized in that The genetically modified immune cells are selected from at least one of T cells, NK cells, NKT cells, γδT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, peripheral blood NK cells, cord blood NK cells, NK-92 cells, iPSC-derived or embryonic stem cell-cultured and differentiated immune cells.

42. The transgenic immune cell according to claim 41, characterized in that The immune cells of the transgenic immune cells are NK cells.

43. A pharmaceutical composition, characterized in that include: The chimeric antigen receptor according to any one of claims 1 to 10, the nucleic acid according to any one of claims 11 to 35, the expression vector according to any one of claims 36 to 39, or the transgenic immune cell according to any one of claims 40 to 42.

44. The pharmaceutical composition according to claim 43, characterized in that Further including: Pharmaceutically acceptable excipients.

45. Use of the chimeric antigen receptor of any one of claims 1 to 10, the nucleic acid of any one of claims 11 to 35, the expression vector of any one of claims 36 to 39, the transgenic immune cell of any one of claims 40 to 42, or the pharmaceutical composition of claim 43 or 44 in the preparation of a drug for preventing and / or treating B7H6 target-mediated related diseases.

46. ​​The use according to claim 45, characterized in that The B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

47. The use according to claim 46, characterized in that The tumor is a solid tumor or a blood tumor.

48. The use according to claim 46, characterized in that The tumor or cancer is one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

49. The use according to claim 46, characterized in that The tumor or cancer is colorectal cancer, head and neck tumor, liver cancer, gastric cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, bile duct cancer, etc.

50. A method for treating and / or preventing B7H6 target-mediated related diseases, characterized in that: include: A pharmaceutically acceptable amount of the transgenic immune cell according to any one of claims 40 to 42 or the pharmaceutical composition according to any one of claims 43 or 44 is administered to a subject.

51. The method according to claim 49, characterized in that The B7H6 target-mediated related disease is a B7H6-positive tumor or cancer.

52. The method according to claim 51, characterized in that The tumor is a solid tumor or a blood tumor.

53. The method according to claim 51, characterized in that The tumor or cancer is one of leukemia, lymphoma, multiple myeloma, liver cancer, bile duct cancer, esophageal cancer, lung cancer, head and neck cancer, thyroid cancer, glioma, cervical cancer, ovarian cancer, gastric cancer, gastric sarcoma, osteosarcoma, breast cancer, pancreatic cancer, melanoma, colorectal cancer, kidney cancer and prostate cancer.

54. The method according to claim 51, characterized in that The tumor or cancer is colorectal cancer, head and neck tumor, liver cancer, gastric cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, bile duct cancer, etc.

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