Cells and methods resistant to transplantation rejection

Abstract

To provide cells resistant to transplantation immunological rejection and methods for resisting inhibition or rejection.SOLUTION: The cell expresses a first protein capable of recognizing one or more immune effector cells of a host. Preferably, the cell has a function of inhibiting or killing the immune effector cells of the host. Also provided are a method for preventing or regulating transplantation immunological rejection, and a method for preventing or regulating attacks on exogenous cells by NK cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to cells having a function of resisting transplant immune rejection. The present invention also relates to a method of resisting transplant immune rejection, particularly a method of resisting immune rejection of NK cells.

Background Art

[0002] Due to immunogenetic differences between the donor and the recipient, when a foreign donor is transplanted, as a foreign graft, the donor is recognized and attacked by the immune cells in the recipient's body, thereby inhibiting or eliminating the foreign graft, and host-versus-graft reaction (HVGR) may occur. By knocking out the MHC molecules in the transplanted cells, it is possible to effectively resist the rejection reaction of host T cells against the graft, but it may cause rejection reactions of other immune cells in the host. For example, in allogeneic cell transplantation, if the MHC-I molecules of allogeneic cells are lacking, it will cause a rejection reaction of NK cells in the host and promote the elimination of allogeneic cells (Nat Biotechnol. 2017; 35(8): 765-772. doi: 10.1038 / nbt.3860). Therefore, a method for effectively preventing the immune rejection reaction of host NK cells is very important for the development of allogeneic cell transplantation therapy.

Disclosure of the Invention

[0003] An object of the present invention is to provide cells that are resistant to transplant immune rejection and a method of resisting inhibition and rejection.

[0004] The technical solutions provided by the present invention include the following.

[0005] In a first aspect of the present invention, there is provided a cell that can express a first protein capable of recognizing one or more immune effector cells of a host, and preferably has an inhibitory or killing function against the immune effector cells of the host.

[0006] In a preferred embodiment, the cell is an immune effector cell or an artificially modified cell having the function of an immune effector cell.

[0007] In a preferred embodiment, the cell is selected from immune effector cells derived from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cells, Preferably, the cell is a T cell, More preferably, the first protein is a chimeric receptor.

[0008] In a preferred embodiment, the cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen, and preferably, the second protein is a chimeric receptor or a T cell receptor.

[0009] In a preferred embodiment, the activation of the protein that recognizes the host's immune effector cells is regulated by the second receptor.

[0010] In a preferred embodiment, the activation of the second receptor is regulated by a protein that recognizes the host's immune effector cells.

[0011] In a preferred embodiment, the activation of the protein that recognizes the host's immune effector cells and the second receptor do not affect each other.

[0012] In a preferred embodiment, the cell does not express MHC or the MHC gene endogenously expressed by the cell is silenced, and preferably, the MHC gene is the gene of the MHC class I molecule.

[0013] In a preferred embodiment, the cell does not express HLA or the HLA gene endogenously expressed by the cell is silenced, and preferably, the HLA is the HLA-I gene.

[0014] In a preferred embodiment, the anti-transplant immune rejection is resistant to attack from the host's NK cells, or the first protein can recognize the host's NK cells. Preferably, the first protein can specifically recognize one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxic receptor (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161. More preferably, the first protein can specifically recognize one or more of the NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0015] In a preferred embodiment, the first protein comprises an antibody that can recognize the host's NK cells. Preferably, the antibody can recognize NKG2A. Even more preferably, the antibody comprises HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15. Even more preferably, the antibody comprises the heavy chain variable region described in SEQ ID NO: 1 or the light chain variable region described in SEQ ID NO: 2.

[0016] In a preferred embodiment, the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M. Preferably, the HLA-I gene is B2M.

[0017] In a preferred embodiment, the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

[0018] In a preferred embodiment, the first protein comprises an extracellular domain, a transmembrane domain, and an extracellular signaling domain, Preferably, the cell transmits signals through an intracellular signaling domain and mediates inhibition or killing of host immune effector cells.

[0019] In a preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain, and an extracellular signaling domain, Preferably, the cell transmits signals through an intracellular signaling domain and mediates inhibition or killing of tumors or pathogens.

[0020] In a preferred embodiment, the cell is a T cell in which the HLA-I gene and the endogenous TCR gene are silenced, Preferably, the cell is a T cell in which the B2M and TCR genes are silenced.

[0021] In a preferred embodiment, the second protein can specifically recognize BCMA or CD19, Preferably, the second protein comprises an antibody that can specifically recognize BCMA, More preferably, the antibody that specifically recognizes BCMA comprises HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, and LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21, Even more preferably, the antibody that specifically recognizes BCMA comprises a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO: 23.

[0022] In a preferred embodiment, gene editing technology is used to silence genes.

[0023] Preferably, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector nuclease (transcription activator-like effector, TALE) technology, or TALE-CRISPR / Cas9 technology. More preferably, the gene editing technology is CRISPR / Cas9 technology.

[0024] In a preferred embodiment, the first protein comprises an antibody that recognizes the host's immune effector cells, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain. Preferably, the antibody that recognizes the host's immune effector cells and the antibody that recognizes a tumor antigen or a pathogen antigen are linked by a linker peptide. More preferably, the first protein has the sequence shown in SEQ ID NO: 9.

[0025] In a preferred embodiment, the first protein and the second protein can be in one chimeric receptor. That is, preferably, the chimeric receptor comprises, in order, an antibody that recognizes the host's immune effector cells (the first protein), an antibody that recognizes a tumor antigen or a pathogen antigen (the second protein), a transmembrane domain, and an intracellular domain, or the chimeric receptor comprises, in order, an antibody that recognizes a tumor antigen or a pathogen antigen (the second protein), an antibody that recognizes the host's immune effector cells (the first protein), a transmembrane domain, and an intracellular domain. Preferably, the antibody that recognizes the host's immune effector cells (the first protein) and the antibody that recognizes a tumor antigen or a pathogen antigen (the second protein) are linked by a linker peptide.

[0026] In a second aspect of the present invention, there is provided a cell resistant to transplant immune rejection, wherein the cell is a T cell having a T cell receptor capable of recognizing one or more immune effector cells of a host, and preferably, the cell has an inhibitory or killing function against the host's immune effector cells.

[0027] In a preferred embodiment, the cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen, and preferably, the second protein is a chimeric receptor.

[0028] In a preferred embodiment, the cell does not express MHC, or the MHC gene endogenously expressed by the cell is silenced, and preferably, the MHC gene is a gene of MHC class I molecule.

[0029] In a preferred embodiment, the cell does not express HLA, or the HLA gene endogenously expressed by the cell is silenced, and preferably, the HLA is an HLA-I gene.

[0030] In a preferred embodiment, the T cell receptor is capable of recognizing the host's NK cells, and preferably, the T cell receptor can specifically recognize one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxic receptor (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161. More preferably, the T cell receptor can specifically recognize one or more of NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0031] In a preferred embodiment, the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M, and preferably, the HLA-I gene is B2M.

[0032] In a preferred embodiment, the second protein is a chimeric receptor, and the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC). The chimeric receptor containing the second protein includes the second protein, a transmembrane domain, and an intracellular domain. Preferably, the second protein can specifically recognize BCMA or CD19. Preferably, the second protein includes an antibody that can specifically recognize BCMA. More preferably, the antibody that specifically recognizes BCMA includes HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, as well as LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21. Even more preferably, the antibody that specifically recognizes BCMA includes a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO: 23.

[0033] In a preferred embodiment, gene editing technology is used to silence genes.

[0034] Preferably, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALE) technology, or TALE-CRISPR / Cas9 technology. More preferably, the gene editing technology is CRISPR / Cas9 technology.

[0035] In a third aspect of the present invention, there is provided a method for preventing or regulating transplant immune rejection, which includes administering the cell according to any one of the first or second aspect of the present invention.

[0036] In a fourth aspect of the present invention, there is provided a method for preventing or regulating the attack of NK cells on exogenous cells, which includes administering immune effector cells expressing a first protein that recognizes NK cells. Optionally, the exogenous cells are T cells, NK T cells, stem cells, or engineered T cells, NK T cells, stem cells. Optionally, the immune effector cells are administered before, after, or simultaneously with the administration of the exogenous cells.

[0037] In a preferred embodiment, the exogenous cells are immune effector cells, and preferably, the exogenous cells express a second receptor.

[0038] In a preferred embodiment, the second receptor is a chimeric receptor or a T cell receptor. Preferably, the chimeric receptor is selected from chimeric antigen receptor (CAR), chimeric T cell receptor, and T cell antigen coupler (TAC).

[0039] In a preferred embodiment, the antigen recognized by the first protein that recognizes NK cells is one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxic receptor (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, More preferably, the first protein can specifically recognize one or more of the NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0040] In a preferred embodiment, the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M, and preferably, the HLA-I gene is B2M.

[0041] In a preferred embodiment, the second protein is a chimeric receptor, and the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC). The chimeric receptor containing the second protein includes the second protein, a transmembrane domain, and an intracellular domain. Preferably, the second protein can specifically recognize BCMA or CD19. Preferably, the second protein includes an antibody that can specifically recognize BCMA. More preferably, the antibody that specifically recognizes BCMA includes HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, as well as LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO: 21. More preferably, the antibody that specifically recognizes BCMA comprises a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO: 23.

[0042] In a preferred embodiment, the cell according to any one of the first aspect or the second aspect of the present invention is administered.

[0043] In a fifth aspect of the present invention, there is provided a method for preventing or regulating an attack of NK cells on exogenous cells, comprising administering an immune effector cell expressing a first protein that recognizes NK cells, Optionally, the exogenous cell is a T cell, an NK T cell, a stem cell, or a manipulated T cell, NK T cell, or stem cell, and the method is provided.

[0044] In a preferred embodiment, the exogenous cell is an immune effector cell, and preferably, the exogenous cell expresses a second receptor.

[0045] In a preferred embodiment, the second receptor is a chimeric receptor or a T cell receptor, Preferably, the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC).

[0046] In a preferred embodiment, the antigen recognized by the first protein that recognizes NK cells is one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxic receptor (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161. More preferably, the first protein can specifically recognize one or more of NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0047] In a preferred embodiment, the immune effector cells include T cells, NK cells, NKT cells, macrophages, CIK cells, and immune effector cells derived from stem cells.

[0048] In a sixth aspect of the present invention, there is provided a method for preventing or regulating an attack of NK cells on exogenous immune effector cells expressing a first protein that recognizes NK cells, Preferably, the exogenous immune effector cells are cells that do not contain the HLA-I gene or cells in which the endogenous HLA-I gene is silenced, More preferably, the exogenous immune effector cells are cells that do not contain the B2M gene or cells in which the B2M gene is silenced, and the method is provided.

[0049] In a preferred embodiment, the exogenous immune effector cells are T cells, Preferably, the first protein that recognizes NK cells is a chimeric receptor or a T cell receptor.

[0050] In a preferred embodiment, the antigen recognized by the first protein that recognizes NK cells is one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxic receptor (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, More preferably, the first protein can specifically recognize one or more of the NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0051] In a preferred embodiment, the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC).

[0052] In a preferred embodiment, the exogenous immune effector cells further express a second protein that recognizes a tumor antigen or a pathogen antigen, Preferably, the second protein is a chimeric receptor, and the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

[0053] In a preferred embodiment, the first protein is a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC) that includes an antibody that recognizes NK cells and an antibody that recognizes a tumor antigen or a pathogen antigen.

[0054] In a preferred embodiment, the first protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain. Preferably, the cells transmit signals via an intracellular signal domain and mediate inhibition or killing of host immune effector cells.

[0055] In a preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain. Preferably, the cells transmit signals via an intracellular signal domain and mediate inhibition or killing of tumors or pathogens.

[0056] In a preferred embodiment, the first protein comprises an antibody that recognizes host immune effector cells, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain. Preferably, the antibody that recognizes host immune effector cells and the antibody that recognizes a tumor antigen or a pathogen antigen are linked by a linker peptide. More preferably, the first protein has the sequence shown in SEQ ID NO: 9.

Brief Description of the Drawings

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Summary of the Invention

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, which includes fields such as gene therapy, biochemistry, genetics, and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and appropriate methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Also, unless otherwise specified, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0059] Unless otherwise specified, the practice of the present invention employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which are within the skill of the art. These techniques are well explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrooketal, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J.Gaited., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higginseds. 1984); Transcription An? Translation (B. D. Hames & S. J. Higginseds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells An? Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), particularly Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, e?.); Gene Transfer Vectors For Mammalian Cells (J. H.See Miller and M. P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell An? Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Vol. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0060] In this disclosure, each aspect of the subject matter for which protection is sought is presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the subject matter for which protection is sought. Thus, a description of a range should be considered to specifically disclose every possible subrange and individual value within that range. For example, if a range of values is provided, all intermediate values between the upper and lower limits of that range, as well as any other or intermediate values within that range, are all included within the scope of the subject matter for which protection is sought, and the upper and lower limits of that range are also included within the scope of the subject matter for which protection is sought. The upper and lower limits of those smaller ranges may be independently included within that smaller range and, unless clearly excluded from the range, they also belong to the scope of the subject matter for which protection is sought. If one or two limiting values are included in a set range, the subject matter for which protection is sought also includes the range with one or two of the limiting values excluded. This applies regardless of the breadth of the range.

[0061] As used herein, the term "about" refers to the normal error range of each value that is readily known to those skilled in the art. Values or parameters modified by "about" in this specification include (and describe) embodiments that refer to the value or parameter itself. For example, the description "about X" includes the description of "X". For example, "about" or "comprising" can mean within 1 or exceeding 1 according to the actual standard deviation in the art. Alternatively, "about" or "comprising" can mean a range of up to 10% (i.e., ±10%). For example, about 5 μM can include any number from 4.5 μM to 5.5 μM. When a specific value or composition is provided within the scope of an application and patent application, unless otherwise specified, "about" or "comprising" should be within the tolerance range of the specific value or composition.

[0062] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer within the range and, where appropriate, numerical values of its fractions (e.g., one-tenth and one-hundredth of an integer), unless otherwise specified.

[0063] For a better understanding of the present invention, the related terms are defined as follows.

[0064] The term "transplantation immune rejection" means that after a host transplants a graft such as allogeneic tissue, organ, or cell, the host immune system recognizes the exogenous graft as a "foreign component" and initiates an immunological response such as attacking, destroying, and removing the graft.

[0065] The term "graft" refers to a biological material or preparation that is derived from an individual other than the host and is used for transplantation into the host. The graft can be from any animal source, such as a mammalian source, and preferably from a human. In some embodiments, the graft can be derived from the host; for example, cells from the host are cultured in vitro or modified and then re-transplanted into the host. In some embodiments, the graft can be derived from another allogeneic individual; for example, cells from another human are cultured in vitro or modified and then transplanted into the host. In some embodiments, the graft can be derived from a xenogeneic individual, such as when an organ from another species (such as a mouse, pig, and monkey) is transplanted into a human.

[0066] The term "cell" and other grammatical forms can refer to cells derived from a human or non-human animal.

[0067] The term "host" refers to the recipient of a graft transplantation, and in some embodiments, it can be an individual such as a human who receives a foreign cell transplantation.

[0068] The term "immune effector cell" refers to a cell that is involved in an immune response and produces an immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, cytokine-induced killer (CIK) cells, macrophages, mast cells, and the like. In some embodiments, the immune effector cells are T cells, NK cells, and NKT cells. In some embodiments, the T cells can be autologous T cells, heterologous T cells, or allogeneic T cells. In some embodiments, the NK cells can be allogeneic NK cells.

[0069] The term "artificially modified cells having immune effector cell function" refers to cells that have acquired immune effector cell function after being artificially modified or stimulated by a stimulant. For example, 293T cells are artificially modified to have the function of immune effector cells. For example, stem cells are induced in vitro to differentiate into immune effector cells.

[0070] In some cases, "T cells" are pluripotent stem cells derived from bone marrow and can differentiate and mature into mature T cells with immunological activity in the thymus. In some cases, "T cells" can be a cell population having characteristics of a specific phenotype or a mixed cell population having characteristics of different phenotypes. For example, "T cells" can be cells containing at least one T cell subset from stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and / or effector memory T cells (Tem). In some cases, "T cells" can be a specific subtype of T cells such as γδT cells.

[0071] T cells can be obtained from multiple sources including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumor tissue. In some cases, T cells can be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as FicollTM isolation. In one embodiment, cells from an individual's circulating blood are obtained by apheresis (component collection). Apheresis products typically contain lymphocytes such as T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis collection can be washed to remove plasma molecules and then placed in an appropriate buffer or medium for subsequent processing steps. Alternatively, the cells can be derived from healthy donors or patients diagnosed with cancer.

[0072] The term "MHC" refers to the major histocompatibility complex, which is a collective term for all gene groups encoding antigens of the major histocompatibility complex. MHC antigens are expressed in the tissues of all higher vertebrates and are called HLA antigens in human cells. They play an important role in transplantation reactions, and rejection reactions are mediated by T cells that respond to the major histocompatibility antigens on the surface of the transplanted tissue. MHC proteins play an important role in T cell stimulation. Antigen-presenting cells (usually dendritic cells) display peptides belonging to the degradation products of foreign proteins on the cell surface of MHC, and in the presence of co-stimulatory signals, T cells are activated and act on target cells that display the same peptide / MHC complex. For example, stimulated T helper cells target macrophages that display antigens bound to MHC, or cytotoxic T cells (CTLs) act on virus-infected cells that display foreign viral peptides. MHC antigens are divided into MHC class I antigens and MHC class II antigens.

[0073] The term "Human leukocyte antigen" (HLA) refers to the coding genes of the human major histocompatibility complex, which are located on chromosome 6 (6p21.31) and are closely related to the functions of the human immune system. HLA includes gene segments of class I, class II, and class III. Antigens expressed by HLA class I and class II genes are on the cell membrane, namely MHC-I (encoded by HLA-A, HLA-B, and HLA-C sites) and MHC-II (encoded by the HLA-D region). HLA class I is distributed on the surface of almost all cells in the body and is a heterodimer consisting of a heavy chain (α chain) and β2-microglobulin (B2M). Class II is a glycoprotein mainly on the surface of macrophages and B lymphocytes.

[0074] The term "B2M" refers to β-2 microglobulin, also called B2M, which is the light chain of MHC class I molecules. In humans, B2M is encoded by the b2m gene on chromosome 15 and is opposed to other MHC genes located as a gene cluster on chromosome 6. Mutations in the B2M gene result in hematopoietic grafts from mice lacking normal cell surface MHC I expression being rejected by NK cells of normal mice, indicating that the poor expression of MHC I molecules makes cells more susceptible to rejection by the host immune system (Bix et al. 1991).

[0075] The term "chimeric receptor" refers to a fusion molecule formed by ligating DNA fragments or protein-corresponding cDNAs from different sources by genetic recombination technology and includes an extracellular region, a transmembrane region, and an intracellular region. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs), chimeric T cell receptors (TCRs), and T cell antigen couplers (TACs).

[0076] The term "chimeric antigen receptor" (CAR) includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain includes the functional signaling domains of stimulatory molecules and / or costimulatory molecules. In one aspect, the stimulatory molecule is the ζ chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further includes the functional signaling domains of one or more costimulatory molecules such as 4-1BB (i.e., CD137), CD27, and / or CD28.

[0077] The term "T cell receptor (TCR)" includes classical TCR receptors and optimized TCR receptors, and mediates the recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens by T cells. Classical TCR receptors consist of two peptide chains, α and β, and each peptide chain is further divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. Its antigen specificity resides in the V region. The V regions (Vα, Vβ) each have three hypervariable regions, namely, CDR1, CDR2, and CDR3. In one aspect, T cells expressing classical TCR can induce the specificity of the TCR of T cells for a target antigen by using methods such as antigen stimulation of T cells.

[0078] The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides that make up the TCR, which can bind to surface antigens of target cells and interact with other polypeptides of the complete TCR complex that is usually co-localized on the T cell surface. A chimeric T cell receptor consists of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain. The TCR subunit includes at least a part of the TCR extracellular domain, a transmembrane domain, and a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain. The TCR subunit and the antibody domain are effectively linked, and the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is incorporated into the TCR expressed on T cells.

[0079] The term "T cell antigen coupler (TAC)" includes: 1. an antigen-binding domain containing a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting groups; 2. an extracellular domain domain that brings the TAC receptor close to the TCR receptor by a single-chain antibody that binds to CD3; 3. three functional domains of the transmembrane region and the intracellular region of the CD4 co-receptor. Here, the intracellular region is linked to the protein kinase LCK and catalyzes the phosphorylation of the immunoreceptor tyrosine activation motif (ITAMs) of the TCR complex as the first step in T cell activation.

[0080] The term "signaling domain" refers to the functional part of a protein that functions by transmitting information intracellularly, either by generating a second messenger or acting as an effector in response to such a messenger, in order to regulate the activity of the cell via a defined signaling pathway. The intracellular signaling domain can include all intracellular parts of the molecule, or all natural intracellular signaling domains, or functional fragments or derivatives thereof.

[0081] The term "co-stimulatory molecule" refers to a signal that binds to a cell-stimulating signal molecule such as TCR / CD3 and, in combination, results in the up-regulation or down-regulation of the proliferation and / or important molecules of T cells.

[0082] The terms "activate" and "activate" can refer to the process by which a cell changes from a resting state to an active state. This process can include responses to antigens, transitions, and / or phenotypic or genetic changes in the functional activity state. For example, the term "activation" can refer to the process of stepwise activation of T cells. For example, T cells may require at least one signal to be fully activated.

[0083] The term "gene editing" refers to the ability of humans to "edit" targeted genes to achieve things like knocking out or adding specific DNA fragments.

[0084] The term "gene silencing" refers to the phenomenon of gene non-expression or under-expression for various reasons. Gene silencing can be transcriptional gene silencing caused by DNA methylation, heterochromatinization, position effect, etc., or post-transcriptional gene silencing, that is, inactivating genes at the level of gene transcription by specific inhibition of target RNA, such as antisense RNA, co-inhibition, gene inhibition, RNA interference, and microRNA-mediated translation inhibition.

[0085] "TCR silencing" refers to the non-expression or under-expression of an endogenous TCR.

[0086] "MHC silencing" refers to the non-expression or under-expression of endogenous MHC.

[0087] The term "CRISPR (Clustered regularly interspaced short palindromic repeats)" refers to clustered regularly interspaced short palindromic repeats.

[0088] The term "Cas9 (CRISPR associated nuclease)" refers to a CRISPR-associated nuclease, an RNA-guided technology that uses Cas9 nuclease to edit target genes.

[0089] The "CRISPR / Cas9 system" is collectively referred to as transcripts and other elements involved in the expression of the Cas9 enzyme gene or guiding its activity, and this includes sequences encoding the Cas9 gene, tracr (transactivation CRISPR) sequences (such as tracrRNA or the active part tracrRNA), tracr pairing sequences (including "direct repeats" and partial direct repeats for tracrRNA processing in the context of the endogenous CRISPR system), guide sequences (also called "spacers" in the context of the endogenous CRISPR system, i.e., gRNA), or transcripts with other sequences from the CRISPR locus.

[0090] The term "target sequence" refers to a sequence having complementarity with the guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of the CRISPR complex. The target sequence can include any polynucleotide such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

[0091] Generally speaking, a guide sequence (gRNA) is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize to the target polynucleotide sequence and guide the sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, when performing optimal alignment using an appropriate alignment algorithm, the degree of complementarity between the guide sequence and the corresponding target sequence is about 50% or more, 60% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97.5% or more, 99% or more, or more. Optimal alignment can be determined using any appropriate algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforget.net).

[0092] In some embodiments, the CRISPR enzyme is part of a fusion protein that includes one or more heterologous protein domains (e.g., about 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more domains other than the CRISPR enzyme). The CRISPR enzyme fusion protein can include any additional protein sequences, and linker sequences between any two domains that may be selected. Examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more activities selected from the group consisting of methylase activity, demethylase activity, transcriptional activation activity, transcriptional inhibition activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.

[0093] The term "Cas9 enzyme" may be wild-type Cas9 or artificially modified Cas9.

[0094] The term "sgRNA" refers to a short gRNA.

[0095] When performing gene editing, a specific gRNA, tracr pairing sequence, and tracr sequence can be provided individually, or the complete RNA sequence can be provided.

[0096] Binding of the Cas9 protein to the gRNA can cleave DNA at a specific site. The CRISPR / Cas system recognition sequence derived from Streptococcus pyogenes is 23 bp, and 20 bp can be targeted. The last three NGG sequences of the recognition site are called the PAM (protospacer adjacent motif) sequence.

[0097] The Cas-introduced gene can be delivered by a vector (e.g., AAV, adenovirus, lentivirus), and / or particles and / or nanoparticles, and / or electroporation.

[0098] In one embodiment, the exons of the corresponding coding genes in the constant region of one or both of the α and β chains of the TCR are knocked out using CRISPR / Cas technology to inactivate the endogenous TCR. Preferably, the first exon of the constant region of the endogenous TCR α chain is targeted for knockout.

[0099] To "inhibit" or "suppress" the expression of B2M or TCR means that the expression of B2M or TCR in the cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. More specifically, to "inhibit" or "suppress" the expression of B2M means that the content of B2M in the cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The expression or content of the protein in the cell can be measured by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting, or flow cytometry, using specific antibodies against B2M or TCR.

[0100] The term "modification" as used in the present invention refers to a change in the state or structure of the protein or polypeptide of the present invention. The modification methods can be chemical, structural, and functional.

[0101] The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by a variety of means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0102] The terms "nucleic acid molecule coding", "encoding a DNA sequence", and "encoding DNA" refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid strand. This order of deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0103] The term "individual" refers to any animal, such as a mammal or marsupial. Individuals of the present invention include, but are not limited to, humans, non-human primates (such as monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0104] The term "peripheral blood mononuclear cell" (PBMC) refers to cells having a single nucleus in peripheral blood, including lymphocytes, monocytes, and the like.

[0105] The terms "a T cell is activated" or "activates a T cell" and other grammatical forms can refer to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0106] When used to refer to a nucleotide sequence, the term "sequence" and other grammatical forms used herein may include DNA or RNA and may be single-stranded or double-stranded.

[0107] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0108] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as plasmids and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0109] As used herein, the term "vector" refers to a composition containing an isolated nucleic acid and capable of being used to deliver the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes plasmids or viruses that replicate spontaneously. The term can also include non-plasmid and non-viral compounds, such as polylysine compounds and liposomes, that facilitate the transfer of nucleic acids into cells.

[0110] As used herein, the term "sequence identity" of an array is determined by comparing the percentage of identity by comparing two optimally matched arrays in a comparison window (e.g., at least 20 positions), and a portion of the polynucleotide or polypeptide sequence within the comparison window may include additions or deletions (i.e., indels), e.g., up to 20% indels (e.g., 5-15%, or 10-12%) compared to a reference sequence (without additions or deletions) for the two optimally matched arrays. Usually, the percentage is calculated by determining the number of positions where the same nucleobase or amino acid residue occurs in the two sequences, thereby generating the number of correct matching positions, dividing the number of correct matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to generate the percentage of sequence identity.

[0111] As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide, cell, tissue, etc., which does not have endogenous expression in vivo or has an insufficient expression level to achieve its function when overexpressed.

[0112] The term "endogenous" refers to a nucleic acid molecule or polypeptide, etc., which is derived from the living body itself.

[0113] In some embodiments, the chimeric receptor of the present invention is a chimeric antigen receptor.

[0114] A chimeric antigen receptor usually includes an extracellular antigen-binding region. In some embodiments, the extracellular antigen-binding region may be fully human, or humanized, derived from a mouse, or the chimeric of the extracellular antigen-binding region consists of amino acid sequences from at least two different animals.

[0115] Examples of the extracellular antigen-binding region may include scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain fragments, or natural ligands that bind to their cognate receptors, and any derivatives thereof.

[0116] In some embodiments, the extracellular antigen-binding region (e.g., scFv) can include a light chain CDR specific for the antigen. Optionally, the light chain CDR may include two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc. Optionally, the light chain CDR may include three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively. In one embodiment, a set of CDRs present on a common light chain can be collectively referred to as light chain CDR.

[0117] In some embodiments, the extracellular antigen-binding region (e.g., scFv) may include a heavy chain CDR specific for the antigen. The heavy chain CDR may be the heavy chain complementarity determining region of an antigen-binding unit such as scFv. Optionally, the heavy chain CDR may include two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc. Optionally, the heavy chain CDR may include three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In one embodiment, a set of CDRs present on a common heavy chain can be collectively referred to as heavy chain CDR.

[0118] By using genetic engineering, the extracellular antigen-binding region can be modified in various ways. Optionally, the extracellular antigen-binding region can be mutated and selected to have a higher affinity for its target. Optionally, the affinity of the extracellular antigen-binding region for its target can be optimized for targets that can be expressed at low levels in normal tissue. This optimization can be performed to minimize potential toxicity. In other cases, clones of the extracellular antigen-binding region with higher affinity for the membrane-bound form of the target may be superior to their soluble counterparts. Modification can be performed because different levels of soluble forms of the target can also be detected and those targets may cause unwanted toxicity.

[0119] In some cases, the extracellular antigen-binding region includes a hinge or a spacer. The terms "hinge" and "spacer" can be used interchangeably. The hinge can be considered as part of the CAR used to provide flexibility to the extracellular antigen-binding region. For example, the hinge can be the natural hinge region of the CD8α molecule.

[0120] The term "transmembrane domain" can immobilize the chimeric protein to the plasma membrane of the cell. For example, the transmembrane domains of CD28 and CD8α can be used.

[0121] The term "modulation" refers to positive or negative changes. Examples of modulation include changes of 1%, 2%, 10%, 25%, 50%, 75%, or 100%. In a specific embodiment, it refers to negative changes.

[0122] The term "treatment" refers to an intervention in the process of trying to change a disease, and can also be used for prevention and can intervene in the clinicopathological process. Therapeutic effects include, but are not limited to, prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, delay of disease progression, improvement or alleviation of a condition, alleviation or improvement of prognosis, etc.

[0123] The term "prevention" refers to an intervention carried out before the occurrence of a disease (such as rejection caused by cell transplantation).

[0124] The first protein of the present invention refers to the above-mentioned protein that can recognize one or more immune effector cells of the host.

[0125] The second protein of the present invention refers to the above-mentioned protein that can recognize tumor antigens or pathogen antigens.

[0126] The "second receptor" and the "protein that can recognize one or more immune effector cells of the host" described in the present invention can be expressed in parallel (tandem expression) or separately.

[0127] When the "second receptor" and the "protein capable of recognizing one or more immune effector cells of the host" described in the present invention are expressed separately, they each have an independent transmembrane domain and an intracellular domain. For the expression method, refer to PCT / CN2015 / 095938, Enhancing the specificity of T-cell cultures for adoptive immunotherapy of cancer, Duong CP et al., Immunotherapy 3(1):33-48, etc.

[0128] When the "second receptor" of the present invention is expressed in parallel (tandem expression) with the "protein capable of recognizing one or more immune effector cells of the host", the protein that recognizes one or more immune effector cells of the host can also recognize antigens such as tumor antigens recognized by the "second receptor".

[0129] The "tumor antigen" refers to an antigen that newly appears or is overexpressed during the occurrence and progression of a hyperproliferative disease. In certain embodiments, the hyperproliferative disorder of the present invention refers to cancer.

[0130] The tumor antigen described in the present invention can be a solid tumor antigen or a hematoma antigen.

[0131] The tumor antigens of the present invention are: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Rα); interleukin 11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type-A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); O-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; κ light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; Survivin; TAG72; TEM1; fibronectin; tenascin; carcinoembryonic variant of the tumor necrosis zone; G protein-coupled receptor class C group 5 member? (GPRC5D); X chromosome open reading frame 61 (CXORF61);CD97; CD179a; Anaplastic lymphoma kinase (ALK); Polysialic acid; Placenta-specific 1 (PLAC1); Hexose portion of globoH glycosphingolipid (GloboH); Breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); Adrenergic receptor beta-3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); Sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; P53 variant; Human telomerase reverse transcriptase (hTERT); Sarcoma translocation breakpoint; Melanoma inhibitor of apoptosis (ML-IAP); ERG (membrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); Paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; V-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); Squamous cell carcinoma antigen recognized by T cells 3 (SART3); Paired box protein Pax-5 (PAX5); Proacrosin-binding protein sp32 (OYTES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); Synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79B; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF);C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like peptide 1 (IGLL1), but not limited to these. Preferably, the tumor antigen is BCMA or CD19.;

[0132] The pathogen antigen is selected from antigens of viruses, bacteria, fungi, protozoa, or parasites. The viral antigen is selected from cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.;

[0133] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, experimental methods without specific conditions indicated usually follow conventional conditions such as those described in J. Sambrook et al., eds. Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, 2002, or follow the conditions recommended by the manufacturer.;

Example 1

[0134] Detection of the expression of NK cell surface receptors Monocytes were isolated from peripheral blood using Ficoll-Paque (GE bioscience) by density gradient centrifugation, and negative screening was performed using an NK cell isolation kit (purchased from Miltenyi Biotec) to remove cells such as T cells, B cells, and monocytes. After that, in vitro cell phenotype identification and proliferation were carried out. Flow cytometry was used to identify receptors on the surface of the isolated NK cells, such as markers like NKG2A, NKG2D, NKP30, NKP44, and NKP46. The results of flow cytometry showed that NKG2A, NKP30, NKP44, and NKP46 were expressed in approximately 80% of NK cells, and NKG2D was expressed in more than 90% of NK cells (see Figure 1). Furthermore, the expression of the above surface markers in T cells was also detected. T cells activated by CD3 / CD28 magnetic beads (purchased from Thermo Fisher) were collected, cultured until the 8th day, and stained for flow cytometry. The experimental results showed that NKG2A, NKG2D, NKP30, NKP44, and NKP46 were hardly expressed in T cells, indicating that the above markers can be used as targets for NK cells (see Figure 2).

Example 2

[0135] Preparation and functional verification of CAR-T cells 1. As a representative for preparing CAR-T cells targeting NK cells, NKG2A was selected as the target. Referring to the conventional operation, a vector of a chimeric antigen receptor (amino acid sequence is shown in SEQ ID NO: 5) containing a single-chain antibody against NKG2A (the amino acid sequence of VH is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 2), the CD28 transmembrane domain and intracellular domain (amino acid sequence is shown in SEQ ID NO: 3), and the T cell activation factor CD3ζ (amino acid sequence is shown in SEQ ID NO: 4) was designed and constructed, and the plasmid map is shown in Figure 8. Lentivirus was packaged and named VRRL-NKG2A-28Z(TM). After 48 hours of T cell activation and proliferation, the cell density was adjusted to 2×10^6 / mL, and VRRL-NKG2A-28Z(TM) lentivirus was added at a ratio of MOI = 10 to obtain CAR-T cells targeting NKG2A. For cell proliferation detection, CAR-T cells were collected on day 6, the starting cell number was adjusted to 5×10^5, and the cell numbers were detected at 24 hours, 48 hours, 72 hours, and 96 hours, and the cell diameters were recorded. At the same time, for detecting the expression of the CAR vector, flow cytometry staining was performed using anti-F(ab)’2 antibody. According to the experimental results, NKG2A CAR-T cells and untransfected T cells (UTD) showed similar proliferation curves, there was no significant difference in cell diameter, and about 80% of the CAR-T cells expressed the CAR molecule targeting NKG2A. This indicates that the proliferation characteristics of NKG2A CAR-T cells are normal (see Figure 3). 2. Preparation of CAR-T cells targeting BCMA Referring to the operation in 1, a plasmid targeting the chimeric antigen receptor of BCMA (the amino acid sequence is shown in SEQ ID NO: 6) was constructed, and the plasmid map is shown in Figure 9. Lentivirus was packaged and transfected into T cells to obtain BCMA-CAR T cells targeting BCMA. 3. In vitro killing function experiment Primary NK cells were amplified in vitro using the target cells. The cell density was adjusted to 5*10^5 / mL, and 100 μl was inoculated into a 96-well plate (prepared in parallel with three replicate wells). Corresponding CAR-T cells were inoculated according to three ratios of effector T cell:target cell, namely 1:3, 1:1, and 3:1. MEM-α + 5% FBS was used as the medium and incubated in a 5% CO2 incubator at 37°C for 4 hours and 18 hours respectively. Using a CytoTox-96 non-radioactive cytotoxicity assay kit (purchased from Thermo Fisher), 50 μl of the supernatant was taken to measure the content of lactate dehydrogenase (LDH), and the lysis efficiency of primary NK cells in two groups of UTD and NKG2A CAR-T was calculated. The detection results showed that the LDH value of the NKG2A CAR-T group was significantly higher than that of the UTD group, indicating that NKG2A CAR-T can effectively kill primary NK cells (see Figures 4 and 5).

Example 3

[0136] Preparation of NKG2A UCAR-T cells 1. Knockout of TCR and B2M genes After culturing conventional UTD cells, NKG2A CAR-T cells, and BCMA CAR-T cells (for control) in vitro for 48 hours, the cell density was adjusted to 2×10^7 / mL. Cas9 enzyme (purchased from NEB) and sgRNA were incubated at a ratio of 1:4 for 10 minutes at room temperature to obtain an RNP complex solution. The nucleic acid sequence of TRAC-sgRNA is shown in SEQ ID NO: 7, and the nucleic acid sequence of B2M-sgRNA is shown in SEQ ID NO: 8. 1×10^6 cells were mixed with the RNP complex solution (the final concentration of Cas9 enzyme was 3 μM), and the RNP complex was individually introduced into CAR-T cells using a maxcyte electroporator. On the 7th day after electroporation, flow cytometry was used to detect the knockout of the TCR and B2M genes. The experimental results show that the knockout efficiency of TRAC and B2M is 85% or higher (see Figure 6). 2. Screening of TCR / B2M double-negative cells CAR-T cells and UTD cells with B2M and TCR knocked out were cultured in vitro. On the 8th day, the cell density was adjusted to 1×10^7 / mL. The cells were labeled with anti-HLA-ABC and B2M antibodies and then labeled with a secondary antibody conjugated with phycoerythrin (PE). After the labeled cells were sorted using an anti-PE magnetic bead selection column, TCR and B2M double-negative cells were collected (the sorting kit was purchased from Miltenyi Biotec), and BCMA UCAR-T cells, NKG2A UCAR-T cells, and U-UTD cells lacking TCR and B2M were obtained.

Example 4

[0137] Verification of the resistance function of NKG2A UCAR-T cells against NK cells 1. Detection of the rejection effect of UCAR-T cells against NK cells by LDH experiment UTD cells, BCMA-CAR T cells, NKG2A CAR-T cells, BCMA UCAR-T cells, NKG2A UCAR-T cells, and U-UTD cells were used as target cells. The cell concentration was adjusted to 5*10^5 / mL, 100 μl was inoculated into a 96-well plate, and according to the ratio of 1:1 of the amplified primary NK cells to the target cells, the same amount (volume) and number of NK cells were inoculated and incubated in an incubator for 4 hours and 18 hours respectively. 50 μl of the supernatant was taken to measure the content of lactate dehydrogenase (LDH), and the lysis efficiency of CAR-T and UCAR-T cells was calculated. The detection results show that the LDH values of both the UTD and BCMA CAR-T groups are very low. This indicates that normal CAR-T cells do not cause an attack by NK cells. In contrast, both the U-UTD and BCMA UCAR-T groups show gradually increasing LDH values at 4 hours and 18 hours, indicating that NK cells kill T cells lacking TCR and B2M. NKG2A UCAR-T cells show a lower level of LDH, indicating that NKG2A UCAR-T cells have a resistance effect against NK cells. 2. To further prove the resistance ability of NKG2A UCAR-T cells against NK cells, BCMA UCAR-T cells were selected as a control, the cell concentration was adjusted to 5×10^5 / mL, and 100 μl was inoculated into a 96-well plate. At a ratio of 1:1 of the expanded primary NK cells to the target cells, the same volume and number of NK cells were inoculated and incubated in an incubator for 4 hours, 18 hours, 24 hours, and 42 hours respectively. Flow cytometry was used to label HLA-ABC positive NK cells, and the proportion of UCAR-T cells incubated at different time points was detected. The experimental results, as shown in Figures 7A-7D, indicate that BCMA UCAR-T was at a relatively low proportion of about 20% at 4 hours, and the proportion was always very low with the extension of the detection time, indicating that NK cells significantly inhibited the proliferation of BCMA UCAR-T cells. NKG2A UCAR-T cells were at a relatively low proportion of about 20% at 4 hours, but with the extension of the detection time, the proportion gradually increased and reached nearly 60% at 42 hours. This shows that the proliferation of NKG2A UCAR-T cells was initially inhibited by NK cells, but the proliferation ability gradually recovered over time. The above results indicate that NKG2A UCAR-T can effectively resist the killing ability of NK cells. 3. To further prove the resistance ability of NKG2A UCAR-T cells against primary NK cells, BCMA UCAR-T cells and NKG2A UCAR-T cells expressing GFP were constructed. The amino acid sequence of BCMA-GFP is shown in SEQ ID NO: 24, and the amino acid sequence of NKG2A-GFP is shown in SEQ ID NO: 25. Referring to the operation of Example 2, the plasmid PRRL-BCMA-BBZ-F2A-EGFP expressing BCMA UCAR-T cells that express GFP was constructed, and the plasmid map is shown in Figure 16. The plasmid PRRL-NKG2A-28Z-F2A-EGFP expressing NKG2A UCAR-T cells was constructed, and the plasmid map is shown in Figure 17. Lentivirus was packaged for the constructed plasmids, T cells were transfected, and gene knockout and magnetic bead sorting were performed on the CAR-T cells to obtain BCMA UCAR-T cells expressing GFP and NKG2A UCAR-T cells expressing GFP. The concentration of CAR-T cells was adjusted to 5*10^5 / mL, 100 μl was inoculated into a 96-well plate, and according to the ratio of 1:1 of the proliferated primary NK cells to the target cells, the same volume and number of NK cells were inoculated and cultured in an incubator for 0 hours, 4 hours, 18 hours, 24 hours, and 48 hours respectively. Flow cytometry was used to detect the proportion of GFP cells co-cultured at different time points and was used to track the survival of UCAR-T cells. As shown in Figure 10, the experimental results showed that the proportion of GFP-positive BCMA UCAR-T cells gradually decreased with the extension of time and was almost completely killed by NK cells after 48 hours. However, the proportion of GFP-positive NKG2A UCAR-T cells slightly decreased at 4 hours, significantly increased after 18 hours, and accounted for about 90% after 48 hours, indicating that NKG2A UCAR-T cells can significantly resist the killing of NK cells.

Example 5

[0138] Resistance of NKG2A UCAR-T cells to NK cells in vivo BCMA UCAR-T and NKG2A UCAR-T cells were cultured in vitro, the CAR positive rate was adjusted to 80%, and they were injected into NPG immunodeficient mice via tail vein injection at a dose of 8×10^6 cells / mouse. The mice were divided into two groups: the group administered with BCMA UCAR-T and NK cells (denoted as BCMA UCAR-T+NK), and the NKG2A UCAR-T group and the group administered with NK cells (denoted as NKG2A-UCART+NK). The same amount of NK cells was injected 4 hours after the administration of UCAR T cells. On the 1st, 3rd, and 6th days after the injection of CAR T cells, the survival of human-derived CD4 and CD8 T cells in the peripheral blood of the mice was detected by flow absolute technology (absolute cell counting by flow cytometry). As shown in Figure 11, the experimental results showed that on the 1st day after injection, the number of UCAR-T cells (i.e., human-derived CD4 and CD8 T cells) in the BCMA UCAR-T+NK group and the NKG2A UCAR-T+NK group significantly decreased, indicating that UCAR-T cells were rejected by NK cells. On the 3rd and 6th days after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group was always at a very low level, while the number of UCAR-T cells in the NKG2A UCAR-T+NK group showed a significant increase on the 3rd and 6th days. The above results indicate that in the in vivo model, NK cells significantly inhibit the survival of BCMA UCAR-T cells, while NKG2A UCAR-T cells are effectively resistant to NK cell killing and can recover their proliferation ability.

Example 6

[0139] Construction of CAR T cells targeting BCMA and NKG2A As shown in Figure 12, UCAR-T cells (i.e., BCMA-GS-NKG2A UCAR-T) in which the scFv targeting BCMA and the scFv targeting NKG2A were tandemly linked were prepared. The amino acid sequence of BCMA-GS-NKG2A CAR is shown in SEQ ID NO: 9. Construct the plasmid PRRL-BCMA-GS-NKG2A-BBZ of BCMA-GS-NKG2A UCAR-T, and the plasmid map is shown in Figure 18. Referring to the operations in Examples 2 and 3, perform virus transfection to obtain BCMA-GS-NKG2A UCAR-T cells, perform TRAC and B2M gene knockout on BCMA-GS-NKG2A UCAR-T cells, and then obtain more than 99% TCR- and HLA-ABC-negative BCMA-GS-NKG2A UCAR-T cells by magnetic bead sorting method. Referring to the operations in Examples 2 and 3, prepare BCMA UCAR-T cells and NKG2A UCAR-T cells respectively. Detect the CAR expression of BCMA UCAR-T, NKG2A UCAR-T and BCMA-GS-NKG2A UCAR-T respectively. As shown in Figure 13, the experimental results show that the positive rate reaches more than 60%, indicating that the preparation of BCMA-GS-NKG2A UCAR-T cells was successful.

Example 7

[0140] Verification of in vitro function of BCMA-GS-NKG2A UCAR-T cells Culture BCMA-positive multiple myeloma cell lines RPMI-8226 and NCI-H929 in vitro as target cells, inoculate 1*10^4 tumor cells into 96-well plates, and inoculate the corresponding number of UCAR-T cells according to the ratios of T cells to tumor cells of 3:1, 1:1, and 1:3. After incubating for 18 hours, aspirate 50 μl of supernatant for LDH content detection. As shown in Figure 14, the experimental results show that in the UTD and NKG2A UCAR-T groups, the cell lysis rates of RPMI-8226 and NCI-H929 are very low, and the tumor cell lysis rate of the BCMA-GS-NKG2A UCAR-T group is close to that of the BCMA UCAR-T group, indicating that BCMA-GS-NKG2A UCAR-T cells can effectively kill BCMA-positive tumor cells in vitro.

Example 8

[0141] Verification of the resistance function of BCMA-GS-NKG2A UCAR-T cells against NK cells As negative and positive controls, BCMA UCAR-T and NKG2A UCAR-T cells were selected respectively, the cell concentration was adjusted to 5*10^5 / mL, 100 μl was inoculated into a 96-well plate, and according to the ratio of NK cells to T cells of 1:1, the same volume and number of NK cells were inoculated, and they were cultured in an incubator for 0 hours, 4 hours, 18 hours, 24 hours, and 48 hours respectively. Flow cytometry was used to label HLA-ABC positive NK cells, and the proportion of UCAR-T cells co-cultured at different time points was detected. The experimental results are as shown in Figure 15. The proportion of BCMA UCAR-T cells gradually decreased with the extension of the incubation time and was almost killed by NK cells after 48 hours. However, BCMA-GS-NKG2A UCAR-T and NKG2A UCAR-T cells showed the same change trend, decreased slightly at 4 hours, then gradually increased, reached about 70% or more after 48 hours, and BCMA-GS-NKG2A UCAR-T cells reached 90% after 48 hours, indicating that BCMA-GS-NKG2A UCAR-T cells can significantly resist the killing of NK cells.

Example 9

[0142] Resistance of BCMA-GS-NKG2A UCAR-T cells in vivo against NK cells BCMA UCAR-T and BCMA-GS-NKG2A UCAR-T cells were cultured in vitro, the CAR positive rate was adjusted to 60%, and they were injected into NPG immunodeficient mice via tail vein injection at a dose of 8×10^6 cells / mouse. The mice were divided into two groups: the group administered with BCMA UCAR-T and NK cells (denoted as BCMA UCAR-T+NK), and the group administered with BCMA-GS-NKG2A UCAR-T and NK cells (denoted as BCMA-GS-NKG2A-UCAR-T+NK). The same amount of NK cells was injected 4 hours after the administration of UCAR-T cells. On the 1st, 3rd, and 6th days after the injection of CAR T cells, the survival of human-derived CD45 positive T cells in the peripheral blood of the mice was detected by flow absolute technology. As shown in Fig. 19, the experimental results showed that compared with the 1st day after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group did not show a significant increase on the 3rd and 6th days, indicating that the UCAR-T cells were rejected by the NK cells. The number of UCAR-T cells in the BCMA-GS-NKG2A UCAR-T+NK group significantly increased on both the 3rd and 6th days, and the cell number on the 6th day increased by more than 30 times compared with the cell number on the 1st day. The above results indicate that in the in vivo model, NK cells significantly inhibit the survival of BCMA UCAR-T cells, while BCMA-GS-NKG2A UCAR-T cells are effectively resistant to the killing of NK cells and can recover their proliferation ability.

[0143] The sequences related to this application are shown in the following table.

[0144] JPEG2025111456000001.jpg240170

[0145] JPEG2025111456000002.jpg141170

[0146] JPEG2025111456000003.jpg248170

[0147] JPEG2025111456000004.jpg172170

[0148] JPEG2025111456000005.jpg183170

[0149] JPEG2025111456000006.jpg192170

Claims

1. A cell resistant to transplant immune rejection, which expresses a first protein capable of recognizing one or more immune effector cells of a host, and preferably has an inhibitory or killing function against the immune effector cells of the host.

2. The cell according to claim 1, wherein the cell is an immune effector cell or an artificially modified cell having the function of an immune effector cell.

3. The cell is selected from immune effector cells derived from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cells. Preferably, the cell is a T cell. More preferably, the first protein is a chimeric receptor, the cell according to claim 1 or 2.

4. The cell further expresses a second protein capable of recognizing a tumor antigen or a pathogen antigen, and preferably, the second protein is a chimeric receptor or a T cell receptor, the cell according to any one of claims 1 to 3.

5. The cell does not express MHC, or the MHC gene endogenously expressed by the cell is silenced, and preferably, the MHC gene is a gene of an MHC class I molecule, the cell according to any one of claims 1 to 4.

6. The cell does not express HLA, or the HLA gene endogenously expressed by the cell is silenced, and preferably, the HLA is an HLA-I gene, the cell according to claim 5.

7. Resistance to the transplant immune rejection means resistance to the attack from the host NK cells or the first protein can recognize the host NK cells. Preferably, the first protein can specifically recognize one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, More preferably, the first protein can specifically recognize one or more of the NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46, and the cell according to any one of claims 1 to 6 is characterized in that.

8. The first protein contains an antibody that can recognize the host's NK cells, Preferably, the antibody can recognize NKG2A, More preferably, the antibody contains HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15, Even more preferably, the antibody contains the heavy chain variable region described in SEQ ID NO: 1 or the light chain variable region described in SEQ ID NO: 2, and the cell according to claim 7 is characterized in that.

9. The HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M, and preferably, the HLA-I gene is B2M, and the cell according to claim 8 is characterized in that.

10. The chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC), and the cell according to claim 3 or 4 is characterized in that.

11. The first protein contains an extracellular domain, a transmembrane domain, and an extracellular signal domain, Preferably, the cell according to claim 1 is characterized in that it transmits a signal via an intracellular signal domain and mediates inhibition or killing of host immune effector cells.

12. The second protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain. Preferably, the cell according to claim 4 is characterized in that it transmits a signal via an intracellular signal domain and mediates inhibition or killing of a tumor or a pathogen.

13. The cell is a T cell silenced by an HLA-I gene and an endogenous TCR gene. Preferably, the cell according to claim 6 is characterized in that it is a T cell silenced by a B2M and a TCR gene.

14. The second protein can specifically recognize BCMA or CD19. Preferably, the second protein includes an antibody that can specifically recognize BCMA. More preferably, the antibody that specifically recognizes BCMA includes an HCDR1 shown in SEQ ID NO: 16, an HCDR2 shown in SEQ ID NO: 17, an HCDR3 shown in SEQ ID NO: 18, an LCDR1 shown in SEQ ID NO: 19, an LCDR2 shown in SEQ ID NO: 20, and an LCDR3 shown in SEQ ID NO:

21. Even more preferably, the cell according to claim 4 is characterized in that the antibody that specifically recognizes BCMA includes a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO:

23.

15. The cell according to claim 5, 6 or 13, characterized in that the gene is silenced by gene editing technology.

16. The first protein includes an antibody that recognizes host immune effector cells, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain. Preferably, the antibody that recognizes host immune effector cells and the antibody that recognizes a tumor antigen or a pathogen antigen are linked by a linker peptide. More preferably, the cell according to claim 10 is characterized in that the first protein has a sequence shown in SEQ ID NO:

9.

17. A cell resistant to transplant immune rejection, wherein the cell is a T cell having a T cell receptor capable of recognizing one or more immune effector cells of a host, and preferably, the cell has an inhibitory or killing function against the immune effector cells of the host.

18. The cell according to claim 17, further expressing a second protein that recognizes a tumor antigen or a pathogen antigen, and preferably, the second protein is a chimeric receptor.

19. The cell according to claim 17 or 18, wherein the cell does not express MHC or the MHC gene endogenously expressed by the cell is silenced, and preferably, the MHC gene is a gene of an MHC class I molecule.

20. The cell according to claim 19, wherein the cell does not express HLA or the HLA gene endogenously expressed by the cell is silenced, and preferably, the HLA is an HLA-I gene.

21. The T cell receptor can recognize NK cells of the host. Preferably, the T cell receptor can specifically recognize one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; the natural cytotoxic receptor (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161. More preferably, the T cell receptor can specifically recognize one or more of the NK cell surface antigens NKG2A, NKG2D, NKP30, NKP44, and NKP46, and the cell according to any one of claims 17 to 20.

22. The HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M, and preferably, the HLA-I gene is B2M. The cell according to claim 20 is characterized in that.

23. The second protein is a chimeric receptor, and the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC). The chimeric receptor containing the second protein includes a second protein, a transmembrane domain, and an intracellular domain. Preferably, the second protein can specifically recognize BCMA or CD19. Preferably, the second protein includes an antibody that can specifically recognize BCMA. More preferably, the antibody that specifically recognizes BCMA includes HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, as well as LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, and LCDR3 shown in SEQ ID NO:

21. Even more preferably, the antibody that specifically recognizes BCMA includes a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO:

23. The cell according to claim 18 is characterized in that.

24. A method for preventing or regulating transplant immune rejection, characterized by administering the cell according to any one of claims 1 to 23.

25. A method for preventing or regulating the attack of NK cells on exogenous cells, comprising administering immune effector cells that express a first protein that recognizes NK cells. Optionally, the exogenous cells are T cells, NK T cells, stem cells, or engineered T cells, NK T cells, stem cells. The method is characterized in that.

26. The exogenous cells are immune effector cells, and preferably, the exogenous cells are characterized by expressing a second receptor. The method according to claim 25 is characterized in that.

27. The second receptor is a chimeric receptor or a T cell receptor. Preferably, the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC). The method according to claim 26 is characterized in that.

28. The antigen recognized by the first protein that recognizes NK cells is one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, More preferably, the method according to claim 25, wherein the first protein can specifically recognize one or more of the NK cell surface antigens NKG2A, NKG2D, NKP30, NKP44, and NKP46.

29. The method according to claim 25, wherein the immune effector cells include T cells, NK cells, NKT cells, macrophages, CIK cells, and immune effector cells derived from stem cells.

30. A method for preventing or regulating the attack of NK cells on exogenous immune effector cells, wherein the exogenous immune effector cells express a first protein that recognizes NK cells, Preferably, the exogenous immune effector cells are cells that do not contain the HLA-I gene or cells in which the endogenous HLA-I gene is silenced, More preferably, the method is characterized in that the exogenous immune effector cells are cells that do not contain the B2M gene or cells in which the B2M gene is silenced.

31. The exogenous immune effector cells are T cells, Preferably, the method according to claim 30, wherein the first protein that recognizes NK cells is a chimeric receptor or a T cell receptor.

32. The antigen recognized by the first protein that recognizes NK cells is one or more of the NKG2 receptor family such as NKG2A, NKG2D, NKG2C; the killer immunoglobulin-like receptor (KIR) family such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and other antigens specifically expressed by NK cells such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161, More preferably, the method according to claim 31, wherein the first protein can specifically recognize one or more of the NK cell surface antigens that are NKG2A, NKG2D, NKP30, NKP44, and NKP46.

33. The method according to claim 32, wherein the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC).

34. The exogenous immune effector cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen, Preferably, the second protein is a chimeric receptor, and the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC), according to any one of claims 30 to 33. The method described.

35. The method according to claim 34, wherein the first protein is a chimeric antigen receptor, a chimeric T cell receptor, or a T cell antigen coupler (TAC) comprising an antibody that recognizes NK cells and an antibody that recognizes a tumor antigen or a pathogen antigen.

36. The first protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain, Preferably, the cell transmits a signal through an intracellular signal domain and mediates inhibition or killing of the host's immune effector cells, according to the cell described in claim 30.

37. The second protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain, Preferably, the cell according to claim 34 is characterized in that it transmits a signal through an intracellular signal domain and mediates inhibition or killing against a tumor or a pathogen.

38. The first protein includes an antibody that recognizes an immune effector cell of a host, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain. Preferably, the antibody that recognizes an immune effector cell of the host and the antibody that recognizes a tumor antigen or a pathogen antigen are linked by a linking peptide. More preferably, the cell according to claim 30 is characterized in that the first protein has the sequence shown in SEQ ID NO: 9.

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