Cells and methods against transplantation response

Abstract

The invention relates to a cell for resisting transplantation immunological rejection, which is characterized in that 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 immune effector cells of a host. The invention also relates to a method for preventing or regulating transplantation immunological rejection and a method for preventing or regulating exogenous cells from being attacked by NK cells.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2020800437591, invention name “Cells and methods for anti-transplantation reaction” and application date June 29, 2020 (PCT application number PCT / CN2020 / 098930). Technical Field

[0002] The present invention relates to a cell with anti-transplant rejection function, and also relates to a method for resisting transplant immune rejection, in particular to a method for resisting NK cell immune rejection. Background Art

[0003] Due to the immunogenetic differences between the donor and the recipient, when an exogenous donor is transplanted, the donor, as an exogenous graft, may also be recognized and attacked by the immune cells in the recipient's body, thereby inhibiting or eliminating the exogenous graft, resulting in a host-versus-graft reaction (HVGR). By knocking out the MHC molecules in the graft cells, the host T cell rejection of the graft can be effectively resisted, but it may cause rejection reactions by other immune cells in the host. For example, in allogeneic cell transplantation, when the MHC-I class molecules of allogeneic cells are missing, it will lead to NK cell rejection in the host body and enhance the clearance of allogeneic cells (Nat Biotechnol. 2017; 35(8): 765-772. doi: 10.1038 / nbt.3860). Therefore, how to effectively prevent the immune rejection reaction of the host NK cells is crucial to the development of allogeneic cell transplantation therapy. Summary of the Invention

[0004] The purpose of the present invention is to provide a cell for resisting transplantation immune rejection and a method for resisting transplantation rejection.

[0005] The technical solution provided by the present invention includes:

[0006] In a first aspect of the present invention, a cell is provided, wherein the cell expresses a first protein capable of recognizing one or more immune effector cells of the host; preferably, the cell has an inhibitory or killing function on the immune effector cells of the host.

[0007] In a preferred embodiment, the cells are immune effector cells or artificially modified cells having immune effector cell functions.

[0008] In a preferred embodiment, the cells are selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells;

[0009] Preferably, the cell is a T cell,

[0010] More preferably, the first protein is a chimeric receptor.

[0011] In a preferred embodiment, the cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen, preferably a chimeric receptor or a T cell receptor.

[0012] In a preferred embodiment, the activation of the protein that recognizes host immune effector cells is regulated by the second receptor.

[0013] In a preferred embodiment, activation of the second receptor is regulated by a protein that recognizes the host's immune effector cells.

[0014] In a preferred embodiment, the protein that recognizes the host's immune effector cells and the activation of the second receptor do not affect each other.

[0015] In a preferred embodiment, the cell does not express MHC, or the MHC gene endogenously expressed in the cell is silenced; preferably, the MHC gene is a gene of MHC class I molecule.

[0016] In a preferred embodiment, the cell does not express HLA, or the HLA gene endogenously expressed in the cell is silenced; preferably, the HLA is an HLA-I class gene.

[0017] In a preferred embodiment, the anti-transplant immune rejection is against the attack of the host's NK cells, or the first protein can recognize the host's NK cells.

[0018] Preferably, the first protein can specifically recognize one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161,

[0019] More preferably, the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0020] In a preferred embodiment, the first protein contains antibodies capable of recognizing host NK cells;

[0021] Preferably, the antibody is capable of recognizing NKG2A;

[0022] Further 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;

[0023] More preferably, the antibody comprises the heavy chain variable region of SEQ ID NO: 1 or the light chain variable region of SEQ ID NO: 2.

[0024] 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.

[0025] 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).

[0026] In a preferred embodiment, the first protein comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain;

[0027] Preferably, the cells transmit signals through intracellular signaling domains to mediate the inhibition or killing of the host's immune effector cells.

[0028] In a preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain;

[0029] Preferably, the cells transmit signals through intracellular signaling domains to mediate inhibition or killing of tumors or pathogens.

[0030] In a preferred embodiment, the cells are T cells with HLA-I gene and endogenous TCR gene silenced;

[0031] Preferably, the cells are B2M and TCR gene-silenced T cells.

[0032] In a preferred embodiment, the second protein can specifically recognize BCMA or CD19;

[0033] Preferably, the second protein contains an antibody that can specifically recognize BCMA;

[0034] Further 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;

[0035] More preferably, the antibody that specifically recognizes BCMA contains the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23.

[0036] In a preferred embodiment, gene silencing is achieved using gene editing technology.

[0037] Preferably, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas9 technology;

[0038] More preferably, the gene editing technology is CRISPR / Cas9 technology.

[0039] In a preferred embodiment, the first protein comprises an antibody that recognizes host immune effector cells, an antibody that recognizes tumor antigens or pathogen antigens, a transmembrane domain, and an intracellular domain;

[0040] Preferably, the antibody that recognizes host immune effector cells and the antibody that recognizes tumor antigens or pathogen antigens are connected via a connecting peptide;

[0041] More preferably, the first protein has the sequence shown in SEQ ID NO:9.

[0042] In a preferred embodiment, the first protein and the second protein may be in a chimeric receptor, that is, preferably, the chimeric receptor contains an antibody (first protein) that recognizes host immune effector cells, an antibody (second protein) that recognizes tumor antigens or pathogen antigens, a transmembrane domain, and an intracellular domain that are sequentially linked; or

[0043] The chimeric receptor contains an antibody (second protein) that recognizes a tumor antigen or pathogen antigen, an antibody (first protein) that recognizes a host immune effector cell, a transmembrane domain, and an intracellular domain that are sequentially linked;

[0044] Preferably, the antibody that recognizes host immune effector cells (first protein) and the antibody that recognizes tumor antigens or pathogen antigens (second protein) are connected via a connecting peptide.

[0045] In the second aspect of the present invention, a cell for resisting transplant immune rejection is provided, characterized in that the cell is a T cell having a T cell receptor capable of recognizing one or more immune effector cells of the host, and preferably the cell has an inhibitory or killing function on the host's immune effector cells.

[0046] 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.

[0047] In a preferred embodiment, the cell does not express MHC, or the MHC gene endogenously expressed in the cell is silenced; preferably, the MHC gene is a gene of MHC class I molecule.

[0048] In a preferred embodiment, the cell does not express HLA, or the HLA gene endogenously expressed in the cell is silenced; preferably, the HLA is an HLA-I class gene.

[0049] In a preferred embodiment, the T cell receptor is capable of recognizing the host's NK cells,

[0050] Preferably, the T cell receptor can specifically recognize one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptor (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161,

[0051] More preferably, the T cell receptor can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP46.

[0052] 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.

[0053] 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), and the chimeric receptor comprising the second protein includes the second protein, a transmembrane domain, and an intracellular domain.

[0054] Preferably, the second protein can specifically recognize BCMA or CD19;

[0055] Preferably, the second protein contains an antibody that can specifically recognize BCMA;

[0056] Further 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;

[0057] More preferably, the antibody that specifically recognizes BCMA contains the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23.

[0058] In a preferred embodiment, gene silencing is achieved using gene editing technology.

[0059] Preferably, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas9 technology;

[0060] More preferably, the gene editing technology is CRISPR / Cas9 technology.

[0061] In a third aspect of the present invention, a method for preventing or regulating transplant immune rejection comprises administering the cells described in any one of the first or second aspects of the present invention.

[0062] In a fourth aspect of the present invention, a method for preventing or regulating the attack of exogenous cells by NK cells is provided, characterized in that immune effector cells expressing a first protein that recognizes NK cells are administered;

[0063] Optionally, the exogenous cells are T cells, NK T cells, stem cells, or engineered T cells, NK T cells, stem cells;

[0064] Optionally, the immune effector cells are administered before, after, or simultaneously with the exogenous cells.

[0065] In a preferred embodiment, the exogenous cells are immune effector cells. Preferably, the exogenous cells express a second receptor.

[0066] In a preferred embodiment, the second receptor is a chimeric receptor or a T cell receptor;

[0067] Preferably, the chimeric receptor is selected from the group consisting of: chimeric antigen receptor (CAR), chimeric T cell receptor, and T cell antigen coupler (TAC).

[0068] In a preferred embodiment, the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptor (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161,

[0069] More preferably, the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0070] 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.

[0071] 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), and the chimeric receptor comprising the second protein includes the second protein, a transmembrane domain, and an intracellular domain.

[0072] Preferably, the second protein can specifically recognize BCMA or CD19;

[0073] Preferably, the second protein contains an antibody that can specifically recognize BCMA;

[0074] Further 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;

[0075] More preferably, the antibody that specifically recognizes BCMA contains the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23.

[0076] In a preferred embodiment, the cells according to any one of the first aspect or the second aspect of the present invention are administered.

[0077] In a fifth aspect of the present invention, a method for preventing or regulating the attack of exogenous cells by NK cells is provided, comprising administering immune effector cells expressing a first protein that recognizes NK cells;

[0078] Optionally, the exogenous cells are T cells, NK T cells, stem cells, or engineered T cells, NK T cells, stem cells.

[0079] In a preferred embodiment, the exogenous cells are immune effector cells. Preferably, the exogenous cells express a second receptor.

[0080] In a preferred embodiment, the second receptor is a chimeric receptor or a T cell receptor;

[0081] Preferably, the chimeric receptor is selected from the group consisting of: chimeric antigen receptor (CAR), chimeric T cell receptor, and T cell antigen coupler (TAC).

[0082] In a preferred embodiment, the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptor (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161,

[0083] More preferably, the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0084] In a preferred embodiment, the immune effector cells include T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells.

[0085] In a sixth aspect of the present invention, a method for preventing or regulating exogenous immune effector cells from being attacked by NK cells is provided, characterized in that the exogenous immune effector cells express a first protein that recognizes NK cells;

[0086] Preferably, the exogenous immune effector cells are cells that do not contain the HLA-I gene or have endogenous HLA-I gene silenced;

[0087] More preferably, the exogenous immune effector cells are cells that do not contain the B2M gene or whose B2M gene is silenced.

[0088] In a preferred embodiment, the exogenous immune effector cells are T cells,

[0089] Preferably, the first protein that recognizes NK cells is a chimeric receptor or a T cell receptor.

[0090] In a preferred embodiment, the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptor (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specifically expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161,

[0091] More preferably, the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, and NKP46.

[0092] 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).

[0093] In a preferred embodiment, the exogenous immune effector cells further express a second protein that recognizes tumor antigens or pathogen antigens;

[0094] Preferably, the second protein is a chimeric receptor selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

[0095] In a preferred embodiment, the first protein is a chimeric antigen receptor, a chimeric T cell receptor, or a T cell antigen coupler (TAC) containing antibodies that recognize NK cells and tumor antigens or pathogen antigens.

[0096] In a preferred embodiment, the first protein comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain;

[0097] Preferably, the cells transmit signals through intracellular signaling domains to mediate the inhibition or killing of the host's immune effector cells.

[0098] In a preferred embodiment, the two proteins comprise an extracellular domain, a transmembrane domain and an intracellular signaling domain;

[0099] Preferably, the cells transmit signals through intracellular signaling domains to mediate inhibition or killing of tumors or pathogens.

[0100] In a preferred embodiment, the first protein comprises an antibody that recognizes host immune effector cells, an antibody that recognizes tumor antigens or pathogen antigens, a transmembrane domain, and an intracellular domain;

[0101] Preferably, the antibody that recognizes host immune effector cells and the antibody that recognizes tumor antigens or pathogen antigens are connected via a connecting peptide;

[0102] More preferably, the first protein has the sequence shown in SEQ ID NO:9. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 : Expression of NK cell surface markers;

[0104] Figure 2 :Expression of NK cell surface markers in T cells. ;

[0105] Figure 3 : Growth characteristics of NKG2A CAR-T cells. A, cell proliferation curve; B, cell diameter; C, CAR positive rate;

[0106] Figure 4 : In vitro killing ability of NKG2A CAR-T cells on NK cells after co-incubation for 4 hours;

[0107] Figure 5 : In vitro killing ability of NKG2A CAR-T cells on NK cells after co-incubation for 18 hours;

[0108] Figure 6 : Efficiently knock out TCR and B2M in CAR-T cells;

[0109] Figure 7A 、 Figure 7B 、 Figure 7C and Figure 7D :FACS detection of the resistance of NKG2A UCAR-T cells to NK cells;

[0110] Figure 8 : Plasmid map of CAR targeting NKG2A;

[0111] Figure 9 : Plasmid map of CAR targeting BCMA;

[0112] Figure 10 :FACS detection of the resistance of NKG2A UCAR-T cells to NK cells;

[0113] Figure 11 :FACS detection of UCAR-T cell survival in mouse peripheral blood;

[0114] Figure 12 :Schematic diagram of the structure of BCMA-GS-NKG2A UCAR-T;

[0115] Figure 13 : Preparation of BCMA-GS-NKG2A UCAR-T cells;

[0116] Figure 14 :In vitro anti-tumor effect of BCMA-GS-NKG2A UCAR-T cells;

[0117] Figure 15 :The resistance of BCMA-GS-NKG2A UCAR-T cells to NK cells;

[0118] Figure 16 : The plasmid map of PRRL-BCMA-BBZ-F2A-EGFP;

[0119] Figure 17 : The plasmid map of PRRL-NKG2A-28Z-F2A-EGFP;

[0120] Figure 18 : The plasmid map of PRRL-BCMA-GS-NKG2A-BBZ;

[0121] Figure 19 : Shows the results of BCMA-GS-NKG2A UCAR-T cells' resistance to NK cells in vivo. DETAILED DESCRIPTION

[0122] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art in the fields of 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, where 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 the event of conflict, the present specification, including definitions, will prevail. In addition, unless otherwise specified, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0123] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art and are fully explained in the literature. See, 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, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic AcidHybridization (BD Harries&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, APractical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols.154 and 155 (Wuetal.eds.) and Vol.185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MPCaloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volume I-IV (DMWeir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0124] In the disclosure, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description in range form is merely for convenience and brevity and should not be interpreted as a hard limit to the scope of the claimed subject matter. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, where a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated or intermediate values ​​within the range are included in the claimed subject matter, and the upper and lower limits of the range also fall within the scope of the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller range, and they also fall within the scope of the claimed subject matter unless the upper and lower limits of the range are explicitly excluded. When a range is set to include one or two limits, the claimed subject matter also includes a range that excludes one or both of the limits. This applies regardless of the width of the range.

[0125] As used herein, the term "about" refers to the usual error range of each value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments directed to the value or parameter itself. For example, a description of "about X" includes a description of "X." For example, "about" or "including" may mean within 1 or more than 1 according to the actual standard deviation in the field. Or "about" or "including" may mean a range of up to 10% (i.e., ±10%). For example, about 5uM may include any number between 4.5uM and 5.5uM. When a specific value or composition is provided in an application or patent scope, unless otherwise noted, "about" or "including" should be assumed to be within the acceptable error range of the specific value or composition.

[0126] Unless otherwise indicated, 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, fractions thereof (e.g., tenths and hundredths of an integer).

[0127] To facilitate a better understanding of the present invention, the following definitions are given for relevant terms:

[0128] The term "transplant rejection" refers to the situation in which, after a host receives an allogeneic tissue, organ, or cell transplant, the host's immune system recognizes the foreign transplant as a "foreign component" and initiates an immunological response to attack, destroy, and eliminate the transplant.

[0129] The term "graft" refers to a biological material or preparation derived from an individual other than a host, which is used to be implanted into a host. The graft may be from any animal source, such as a mammalian source, preferably from a human. In some embodiments, the graft may be from a host, such as cells from a host that are cultured in vitro or transformed and then implanted into the host. In some embodiments, the graft may be from another allogeneic individual, such as cells from another person that are cultured in vitro or transformed and then implanted into the host. In some embodiments, the graft may be from a xenogeneic individual, such as an organ from another species (such as a mouse, pig, or monkey) that is implanted into a human.

[0130] The term "cell" and its grammatical alternatives may refer to cells of human or non-human animal origin.

[0131] The term "host" refers to a recipient of a transplant, and in some embodiments, may be an individual, such as a human, into whom exogenous cells are implanted.

[0132] The term "immune effector cell" refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, etc. 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, xenogeneic T cells, or allogeneic T cells. In some embodiments, the NK cells can be allogeneic NK cells.

[0133] The term "artificially modified cells with immune effector cell function" refers to cells or cell lines that lack immune effector function that have been artificially modified or stimulated to acquire immune effector cell function. For example, 293T cells have been artificially modified to acquire immune effector cell function; for example, stem cells have been induced to differentiate into immune effector cells in vitro.

[0134] In some cases, "T cells" can be pluripotent stem cells from the bone marrow, which differentiate and mature into mature T cells with immune activity in the thymus. In some cases, "T cells" can be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as "T cells" can be cells comprising at least one T cell subset: memory stem cell-like T cells (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 certain subtype of T cells, such as γδ T cells.

[0135] T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. In some cases, any number of techniques known to those skilled in the art, such as Ficoll™ separation, can be used to obtain T cells from blood collected from an individual. In one embodiment, cells from the circulating blood of an individual are obtained by single blood sampling. Single blood sampling products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, the cells collected by single blood sampling can be washed to remove plasma molecules and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. Alternatively, cells can be derived from healthy donors or from patients diagnosed with cancer.

[0136] The term "MHC" stands for histocompatibility complex, a general term for all gene groups that encode antigens of biocompatible complexes. 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 transplant reactions, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue. MHC proteins play a vital role in T cell stimulation. Antigen-presenting cells (usually dendritic cells) display peptides that are degradation products of foreign proteins on the cell surface on MHC. In the presence of co-stimulatory signals, T cells are activated and act on target cells that also display the same peptide / MHC complex. For example, stimulated T helper cells will target macrophages that display antigens bound to their MHC, or cytotoxic T cells (CTLs) will act on virus-infected cells that display foreign viral peptides. MHC antigens are divided into NHC class I antigens and MHC class II antigens.

[0137] The term "human leukocyte antigen" (HLA) refers to the genes encoding the human major histocompatibility complex, located on chromosome 6 (6p21.31) and closely related to the human immune system. HLA comprises class I, class II, and class III genes. The antigens expressed by HLA class I and class II genes are located on the cell membrane, representing MHC-I (encoded by the HLA-A, HLA-B, and HLA-C loci) 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 composed of a heavy chain (α chain) and β2 microglobulin (B2M). Class II is a glycoprotein primarily located on the surface of macrophages and B lymphocytes.

[0138] The term "B2M" stands for beta-2 microglobulin, also known as B2M, which is the light chain of MHC class I molecules. In humans, B2M is encoded by the b2m gene located on chromosome 15, opposite the other MHC genes located as a gene cluster on chromosome 6. Studies have shown that when the B2M gene is mutated, hematopoietic transplants from mice lacking normal cell-surface MHC class I expression are rejected by NK cells in normal mice, indicating that defective expression of MHC class I molecules predisposes cells to rejection by the host immune system (Bix et al. 1991).

[0139] The term "chimeric receptor" refers to a fusion molecule composed of extracellular, transmembrane, and intracellular domains, formed by connecting DNA fragments or protein-specific cDNAs from different sources using genetic recombination technology. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs), chimeric T cell receptors (TCRs), and T cell antigen couplers (TACs).

[0140] 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 a functional signaling domain of a stimulatory molecule and / or a costimulatory molecule, in one aspect, the stimulatory molecule is a ζ chain bound to a T cell receptor complex; in one aspect, the cytoplasmic signaling domain further includes a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD137), CD27, and / or CD28.

[0141] The term "T cell receptor (TCR)" mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, and includes classical TCR receptors and optimized TCR receptors. Classical TCR receptors are composed of two peptide chains, α and β. Each peptide chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. Its antigen specificity lies in the V region, and the V region (Vα and Vβ) each has three hypervariable regions, CDR1, CDR2, and CDR3. In one aspect, T cells expressing classical TCRs can be induced to have TCR specificity for target antigens by, for example, antigen stimulation of the T cells.

[0142] The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides that constitute the TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the complete TCR complex, usually co-localized on the surface of T cells. A chimeric T cell receptor is composed of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least a portion 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 operatively linked, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the chimeric T cell receptor is integrated into the TCR expressed on the T cell.

[0143] The term "T cell antigen coupler (TAC)" includes three functional domains: 1. The antigen-binding domain, which can include a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting groups; 2. The extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and 3. The transmembrane region and the intracellular region of the CD4 co-receptor, which is connected to the protein kinase LCK and catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the initial step in T cell activation.

[0144] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. The intracellular signaling domain can include the entire intracellular portion of a molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0145] The term "co-stimulatory molecule" refers to a signal that, in combination with a cell-stimulatory signaling molecule, such as TCR / CD3, results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0146] The terms "activation" and "activation" are used interchangeably and may refer to the process by which a cell transitions from a quiescent state to an active state. This process may include response to an antigen, migration, and / or phenotypic or genetic changes in functional activity. For example, the term "activation" may refer to the step-by-step process by which a T cell becomes activated. For example, a T cell may require at least one signal to fully activate.

[0147] The term "gene editing" refers to the ability for humans to "edit" target genes to achieve the knockout, addition, etc. of specific DNA fragments.

[0148] The term "gene silencing" refers to the phenomenon of gene non-expression or low expression due to various reasons. Gene silencing can occur at the transcriptional level due to factors such as DNA methylation, heterochromatinization, and position effects. It can also occur post-transcriptionally, where the gene is inactivated by specific inhibition of target RNA after transcription. This includes antisense RNA, co-suppression, gene repression, RNA interference, and microRNA-mediated translational inhibition.

[0149] The "TCR silencing" refers to the non-expression or low expression of endogenous TCR.

[0150] The "MHC silencing" refers to the non-expression or low expression of endogenous MHC.

[0151] The term "CRISPR (Clustered regularly interspaced short palindromic repeats)" refers to clustered regularly interspaced short palindromic repeats.

[0152] The term "Cas9 (CRISPRassociated nuclease)" refers to CRISPR-associated nuclease, which is an RNA-guided technology that uses the Cas9 nuclease to edit targeted genes.

[0153] The "CRISPER / Cas9 system" refers collectively to transcripts and other elements involved in the expression of the Cas9 enzyme gene or directing its activity, including sequences encoding the Cas9 gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (encompassing "direct repeats" and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" (i.e., gRNA) in the context of endogenous CRISPR systems), or other sequences and transcripts from the CRISPR locus.

[0154] The term "target sequence" refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of the CRISPR complex. A target sequence can comprise 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.

[0155] In general, a guide sequence (gRNA) is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0156] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may comprise any other protein, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression 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.

[0157] The term "Cas9 enzyme" may refer to wild-type Cas9 or artificially modified Cas9.

[0158] The term "sgRNA" refers to short gRNA.

[0159] During gene editing, the gRNA, tracr pairing sequence, and tracr sequence can be given separately or as a complete RNA sequence.

[0160] The combination of Cas9 protein and gRNA can achieve DNA cutting at specific sites. The CRISPR / Cas system derived from Streptococcus pyogenes recognizes a 23bp sequence and can target 20bp. The last three NGG sequences of its recognition site are called PAM (protospacer adjacent motif) sequences.

[0161] The Cas transgene can be delivered via vectors (e.g., AAV, adenovirus, lentivirus), and / or particles and / or nanoparticles, and / or electroporation.

[0162] In one embodiment, the exons of the corresponding coding genes of the constant regions of one or both of the α and β chains of the TCR are knocked out using CRISPER / Cas technology to render the endogenous TCR inactive, preferably by site-directed knockout of the first exon of the constant region of the endogenous TCR α chain.

[0163] "Inhibition" or "suppression" of B2M or TCR expression refers to a reduction in the expression of B2M or TCR in a cell 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, "inhibition" or "suppression" of B2M expression refers to a reduction in the level of B2M in a cell 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 level of a protein in a cell can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western blotting or flow cytometry using a specific antibody against B2M or TCR.

[0164] As used herein, the term "modification" refers to a change in the state or structure of a protein or polypeptide of the present invention. The modification may be chemical, structural, or functional.

[0165] The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various 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.

[0166] The terms "nucleic acid molecule encoding," "coding DNA sequence," and "coding DNA" refer to the sequence or order of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0167] The term "subject" refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

[0168] The term "peripheral blood mononuclear cell" (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc.

[0169] The term "T cell activation" or "T cell activation" and its grammatical alternatives may refer to a state of T cells that are sufficiently stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0170] As used herein, the term "sequence" and its grammatical alternatives when used to refer to a nucleotide sequence may include DNA or RNA and may be single-stranded or double-stranded.

[0171] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0172] The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as plasmids, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0173] The term "vector" as used herein is a composition comprising an isolated nucleic acid and useful for delivering the isolated nucleic acid to 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 autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells may also be included, such as polylysine compounds, liposomes, and the like.

[0174] The term sequence "identity" as used herein is determined by comparing two optimally matched sequences over a comparison window (e.g., at least 20 positions) to determine the percentage of identity, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps), such as 20% or less gaps (e.g., 5 to 15%, or 10 to 12%) for the two optimally matched sequences compared to the reference sequence (which does not contain additions or deletions). Percentages are typically calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0175] The term "exogenous" as used herein refers to a nucleic acid molecule, polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or that is expressed at a level insufficient to achieve the function it would have when overexpressed.

[0176] The term "endogenous" refers to a nucleic acid molecule, polypeptide, etc. that originates from the organism itself.

[0177] In some embodiments, the chimeric receptor of the invention is a chimeric antigen receptor.

[0178] A chimeric antigen receptor typically comprises an extracellular antigen-binding region. In some embodiments, the extracellular antigen-binding region can be fully human, humanized, murine, or a chimera in which the extracellular antigen-binding region is composed of amino acid sequences from at least two different animals.

[0179] Examples of extracellular antigen-binding regions may be 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.

[0180] In some aspects, the extracellular antigen binding region (e.g., scFv) may comprise a light chain CDR specific for an antigen. In some cases, the light chain CDR may comprise two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc. In some cases, the light chain CDR may comprise 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 group of CDRs present in a common light chain may be collectively referred to as light chain CDRs.

[0181] In some aspects, the extracellular antigen binding region (such as scFv) can include heavy chain CDRs that are specific for the antigen. Heavy chain CDRs can be the heavy chain complementary determining regions of antigen binding units such as scFv. In some cases, the heavy chain CDRs can include two or more heavy chain CDRs, which can be referred to as heavy chain CDR-1, CDR-2, etc. In some cases, the heavy chain CDRs can include three heavy chain CDRs, which can be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In one embodiment, a group of CDRs present in a common heavy chain can be collectively referred to as heavy chain CDRs.

[0182] By using genetic engineering, the extracellular antigen binding region can be modified in various ways. In some cases, the extracellular antigen binding region can be mutated so that the extracellular antigen binding region can be selected to have higher affinity for its target. In some cases, the affinity of the extracellular antigen binding region to its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be carried out to minimize potential toxicity. In other cases, the clone of the extracellular antigen binding region with higher affinity for the membrane-bound form of the target can be superior to its soluble counterpart. This modification can be carried out because different levels of soluble forms of the target can also be detected, and their targeting can cause undesirable toxicity.

[0183] In some cases, the extracellular antigen binding region also includes a hinge or spacer, and the terms hinge and spacer can be used interchangeably. The hinge can be considered to be a part of the CAR that provides flexibility to the extracellular antigen binding region. For example, the hinge can be the natural hinge region of the CD8α molecule.

[0184] The term "transmembrane domain" can anchor the chimeric protein to the plasma membrane of the cell. For example, the transmembrane domain of CD28 or CD8α can be used.

[0185] The term "modulate" refers to a positive or negative change. Examples of modulation include a 1%, 2%, 10%, 25%, 50%, 75%, or 100% change. In one embodiment, it refers to a negative change.

[0186] The term "treatment" refers to interventions that attempt to alter the course of a disease, including both preventative and clinical pathological interventions. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of a disease, improving or relieving symptoms, and alleviating or improving prognosis.

[0187] The term "prevention" refers to interventions that attempt to prevent disease (such as rejection of a cell transplant) before it develops.

[0188] The first protein described in the present invention refers to the above-mentioned protein capable of recognizing one or more immune effector cells of the host.

[0189] The second protein described in the present invention refers to the above-mentioned protein that recognizes tumor antigens or pathogen antigens.

[0190] The "second receptor" and the "protein capable of recognizing one or more immune effector cells of the host" described in the present invention can be expressed in tandem or separately.

[0191] When the "second receptor" and the "protein capable of recognizing one or more immune effector cells of the host" are expressed separately, the two have independent transmembrane domains and intracellular domains. The expression methods can refer to PCT / CN2015 / 095938, Enhancing the specificity of T-cell cultures for adoptive immunotherapy of cancer, Duong CP et al., Immnuotherapy 3(1):33-48, etc.

[0192] When the "second receptor" described in the present invention is expressed in tandem 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 the antigen recognized by the "second receptor", such as a tumor antigen.

[0193] "Tumor antigen" refers to an antigen that appears or is overexpressed during the development and progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present invention is cancer.

[0194] The tumor antigen of the present invention may be a solid tumor antigen or a hematological tumor antigen.

[0195] The tumor antigens of the present invention include but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD 138; 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; proteinase 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 Segment-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (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); claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa 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; oncofetal variant of tumor necrosis zone; G protein-coupled receptor class C group 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);Hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (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 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 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytic leukemia viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (Rho C); cytochrome P4501B1 (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 anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAI R1); Fc fragment of the 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); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). Preferably, the tumor antigen is BCMA or CD19.

[0196] 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.

[0197] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning Laboratory Manual, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0198] Example 1: Detection of NK cell surface receptor expression

[0199] Monocytes were isolated from peripheral blood using density gradient centrifugation using Ficoll-Paque (GE bioscience). Negative screening was performed using an NK cell isolation kit (purchased from Miltenyi Biotec) to remove T cells, B cells, and monocytes. Cell phenotype identification and expansion were then performed in vitro. Flow cytometry was used to identify surface receptors on isolated NK cells, such as NKG2A, NKG2D, NKP30, NKP44, and NKP46. Flow cytometry results showed that NKG2A, NKP30, NKP44, and NKP46 were expressed in approximately 80% of NK cells, while NKG2D was expressed in more than 90% of NK cells (see Figure 1 ).

[0200] Furthermore, we also detected the expression of the above surface markers in T cells. T cells activated by CD3 / CD28 magnetic beads (purchased from Thermo Fisher) were cultured for flow cytometry staining on day 8. The experimental results showed that NKG2A, NKG2D, NKP30, NKP44, and NKP46 were almost not expressed in T cells, indicating that the above markers can be used as targets for NK cells (see Figure 2 ).

[0201] Example 2. Preparation and functional verification of CAR-T cells

[0202] 1. Select NKG2A as a representative target to prepare CAR-T cells targeting NK cells. According to conventional procedures, a vector 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), a CD28 transmembrane domain and intracellular domain (the amino acid sequence is shown in SEQ ID NO: 3), and a chimeric antigen receptor (amino acid sequence is shown in SEQ ID NO: 5) of the T cell activating factor CD3ζ (the amino acid sequence is shown in SEQ ID NO: 4) was designed and constructed. The plasmid is shown in the figure below. Figure 8 As shown, the lentivirus was packaged and named VRRL-NKG2A-28Z(TM).

[0203] After 48 hours of T cell activation and expansion, the cell density was adjusted to 2*10^6 / mL, and VRRL-NKG2A-28Z(TM) lentivirus was added at an MOI of 10 to obtain CAR-T cells targeting NKG2A.

[0204] On day 6, CAR-T cells were taken for cell proliferation assay. The starting cell number was adjusted to 5*10^5. The cell number was measured at 24hr, 48hr, 72hr, and 96hr, and the cell diameter was recorded. Flow cytometry staining with anti-F(ab)'2 antibody was also performed to detect the expression of the CAR vector. The experimental results showed that NKG2A CAR-T cells and untransfected T cells (UTD) showed similar growth curves, and there was no significant difference in cell diameter. Approximately 80% of CAR-T cells expressed CAR molecules targeting NKG2A, indicating that the growth characteristics of NKG2A CAR-T cells were normal (see Figure 3 ).

[0205] 2. Preparation of BCMA-targeted CAR-T cells

[0206] Referring to the operation of 1, a plasmid of a chimeric antigen receptor targeting BCMA (amino acid sequence as shown in SEQ ID NO: 6) was constructed. Figure 9 As shown, lentivirus was packaged and transfected into T cells to obtain BCMA-CAR T cells targeting BCMA.

[0207] 3. In vitro killing function experiment.

[0208] Primary NK cells were expanded in vitro and used as target cells. The cell density was adjusted to 5*10^5 / mL, and 100μl was inoculated into a 96-well plate (three replicates were made in parallel). The corresponding CAR-T cells were inoculated at three ratios of effector T cells: target cells of 1:3, 1:1, and 3:1. MEM-α+5% FBS was used as the culture medium and incubated in a 37°C, 5% CO2 incubator for 4 hours and 18 hours, respectively. Using the cytotox-96 non-radioactive cytotoxicity assay kit (purchased from Thermo Fisher), 50μl of the supernatant was taken for the determination of lactate dehydrogenase (LDH) content, and the lysis efficiency of primary NK cells in the UTD and NKG2A CAR-T groups was calculated.

[0209] The test results showed that the LDH value in the NKG2A CAR-T group was significantly higher than that in the UTD group, indicating that NKG2A CAR-T can effectively kill primary NK cells (see Figure 4 and Figure 5 ).

[0210] Example 3. Preparation of NKG2A UCAR-T cells

[0211] 1. Knockout of TCR and B2M genes.

[0212] After 48 hours of in vitro expansion of conventional UTD cells, NKG2A CAR-T cells and BCMACAR-T cells (for control), the cell density was adjusted to 2*10^7 / mL. Cas 9 enzyme (purchased from NEB) and sgRNA were incubated at room temperature for 10 minutes at a ratio of 1:4 to obtain an RNP complex solution, wherein 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 Cas 9 enzyme was 3uM), and the RNP complex was introduced into CAR-T cells using a maxcyte electroporator. On the 7th day after electroporation, flow cytometry was used to detect the knockout of TCR and B2M genes. The experimental results showed that the knockout efficiency of TRAC and B2M was above 85% (see Figure 6 ).

[0213] 2.TCR / B2M double negative cell screening.

[0214] B2M and TCR knockout CAR-T cells and UTD cells were expanded in vitro. On day 8, 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 to phycoerythrin (PE). The labeled cells were sorted using anti-PE magnetic beads via a sorting column, and TCR and B2M double-negative cells were collected (sorting kit purchased from Miltenyi Biopharmaceuticals). Thus, TCR and B2M-deficient BCMA UCAR-T cells, NKG2A UCAR-T cells, and U-UTD cells were obtained.

[0215] Example 4: Verification of the Anti-NK Cell Function of NKG2A UCAR-T Cells

[0216] 1. LDH assay to detect the rejection of UCAR-T cells against NK cells

[0217] 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, and 100μl was inoculated into 96-well plates. Equal volumes and numbers of NK cells were inoculated at a 1:1 ratio of primary amplified NK cells to target cells. The plates were incubated in an incubator for 4 and 18 hours, respectively. 50μl of the supernatant was used to measure lactate dehydrogenase (LDH) content and calculate the lysis efficiency of CAR-T and UCAR-T cells. The test results showed that the LDH values ​​of the UTD and BCMA UCAR-T groups were very low, indicating that ordinary CAR-T cells would not cause attack by NK cells, while the U-UTD and BCMA UCAR-T groups showed gradually increasing LDH values ​​at 4hr and 18hr, indicating that NK cells would kill T cells lacking TCR and B2M. NKG2A UCAR-T cells showed even lower levels of LDH, indicating that NKG2A UCAR-T cells had resistance to NK cells.

[0218] 2. To further confirm the resistance of NKG2A UCAR-T cells to NK cells, BCMA UCAR-T cells were selected as a control, the cell concentration was adjusted to 5*10^5 / mL, 100μl was inoculated into a 96-well plate, and the ratio of primary amplified NK cells to target cells was 1:1. The same volume and number of NK cells were inoculated and incubated in the incubator for 4hr, 18hr, 24hr and 42hr respectively. HLA-ABC-positive NK cells were labeled by flow cytometry, and the proportion of UCAR-T cells at different time points of co-incubation was detected. The experimental results are shown in the figure below. Figures 7A-7DAs shown in the figure, BCMA UCAR-T cells were at a low ratio of around 20% at 4 hours and remained at a very low level as the detection time prolonged, indicating that NK cells significantly inhibited the growth of BCMA UCAR-T cells. In contrast, NKG2A UCAR-T cells, although at a low ratio of around 20% at 4 hours, gradually increased as the detection time prolonged, reaching nearly 60% at 42 hours, indicating that the growth of NKG2A UCAR-T cells was initially inhibited by NK cells, but gradually recovered their proliferation capacity over time. These results indicate that NKG2A UCAR-T cells can effectively resist the killing ability of NK cells.

[0219] 3. To further demonstrate the ability of NKG2A UCAR-T cells to resist primary NK cells, GFP-expressing BCMA UCAR-T cells and NKG2A UCAR-T cells 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.

[0220] Referring to the operation of Example 2, the plasmid PRRL-BCMA-BBZ-F2A-EGFP expressing BCMA UCAR-T cells was constructed. The plasmid map is shown in Figure 16 , construct the plasmid PRRL-NKG2A-28Z-F2A-EGFP expressing NKG2A UCAR-T cells, the plasmid map is shown in Figure 17 The constructed plasmid was packaged into lentivirus and transfected into T cells. The CAR-T cells were then gene-knocked out and magnetic bead-sorted to obtain GFP-expressing BCMA UCAR-T cells and GFP-expressing NKG2A UCAR-T cells.

[0221] The CAR-T cell concentration was adjusted to 5 x 10^5 / mL, and 100 μl was inoculated into a 96-well plate. Equal volumes and numbers of NK cells were inoculated at a 1:1 ratio of primary cultured NK cells to target cells. The plates were incubated in an incubator for 0, 4, 18, 24, and 48 hours. Flow cytometry was used to measure the proportion of GFP cells at different time points during incubation to track UCAR-T cell survival.

[0222] The experimental results are as follows Figure 10As shown in the figure, the proportion of GFP-positive BCMA UCAR-T cells gradually decreased with time, and was basically completely killed by NK cells after 48 hours. The proportion of GFP-positive NKG2A UCAR-T cells decreased slightly at 4 hours, increased significantly after 18 hours, and accounted for about 90% after 48 hours, indicating that NKG2A UCAR-T cells can significantly resist NK cell killing.

[0223] Example 5: NKG2A UCAR-T cells resist NK cells in vivo

[0224] BCMA UCAR-T and NKG2A UCAR-T cells were cultured in vitro, and the CAR positivity rate was adjusted to 80%. The cells were injected into NPG immunodeficient mice via the tail vein at a dose of 8*10^6 cells / mouse. The mice were divided into two groups: a BCMA UCAR-T and NK cell group (labeled as BCMA UCAR-T+NK) and an NKG2A UCAR-T and NK cell group (labeled as NKG2A-UCART+NK).

[0225] An equal amount of NK cells was injected 4 hours after UCAR T cell administration. On days 1, 3, and 6 after CAR T cell injection, the survival of human CD4 and CD8 T cells in the peripheral blood of mice was detected by flow cytometry.

[0226] The experimental results are as follows Figure 11 As shown in the figure, on the first day after injection, the number of UCAR-T cells (i.e., human CD4 and CD8 T cells) in the BCMA UCAR-T+NK group and the NKG2AUCAR-T+NK group decreased significantly, indicating that UCAR-T cells were rejected by NK cells. On the third and sixth days after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group remained very low, while the number of UCAR-T cells in the NKG2AUCAR-T+NK group increased significantly on the third and sixth days. The above results indicate that in the in vivo model, NK cells significantly inhibited the survival of BCMA UCAR-T cells, while NKG2AUCAR-T cells could effectively resist NK cell killing and restore proliferation capacity.

[0227] Example 6. Construction of CAR T cells targeting BCMA and NKG2A

[0228] like Figure 12As shown, UCAR-T cells containing scFv targeting BCMA and scFv targeting NKG2A in series (i.e., BCMA-GS-NKG2A UCAR-T) were prepared. The amino acid sequence of BCMA-GS-NKG2A CAR is shown in SEQ ID NO: 9.

[0229] The plasmid PRRL-BCMA-GS-NKG2A-BBZ for BCMA-GS-NKG2A UCAR-T was constructed. The plasmid map is shown in Figure 18 Referring to the procedures of Examples 2 and 3, viral transfection was performed to obtain BCMA-GS-NKG2AUCAR-T cells, TRAC and B2M genes were knocked out in BCMA-GS-NKG2A UCAR-T cells, and magnetic bead sorting was used to obtain BCMA-GS-NKG2AUCAR-T cells with more than 99% TCR and HLA-ABC negative results.

[0230] BCMA UCAR-T cells and NKG2AUCAR-T cells were prepared by referring to the procedures of Examples 2 and 3, respectively.

[0231] The CAR expression of BCMA UCAR-T, NKG2A UCAR-T and BCMA-GS-NKG2A UCAR-T was detected respectively. The experimental results are as follows Figure 13 As shown in the figure, the positive rates were all above 60%, indicating that BCMA-GS-NKG2A UCAR-T cells were successfully prepared.

[0232] Example 7. In vitro functional verification of BCMA-GS-NKG2A UCAR-T cells

[0233] BCMA-positive multiple myeloma cell lines RPMI-8226 and NCI-H929 were cultured in vitro as target cells. 1*10^4 tumor cells were inoculated in 96-well plates. The corresponding number of UCAR-T cells were inoculated at a T cell to tumor cell ratio of 3:1, 1:1, and 1:3. After incubation for 18 hours, 50 μl of supernatant was aspirated for LDH content detection.

[0234] The experimental results are as follows Figure 14 As shown in the data, in the UTD and NKG2A UCAR-T groups, the cell lysis rates of RPMI-8226 and NCI-H929 were very low; while the tumor cell lysis rate in the BCMA-GS-NKG2A UCAR-T group was comparable to that in the BCMA UCAR-T group, indicating that BCMA-GS-NKG2A UCAR-T cells can effectively kill BCMA-positive tumor cells in vitro.

[0235] Example 8: Verification of BCMA-GS-NKG2A UCAR-T Cells' Anti-NK Cell Function

[0236] BCMA UCAR-T and NKG2A UCAR-T cells were selected as negative and positive controls, respectively. The cell concentration was adjusted to 5*10^5 / mL, and 100μl was inoculated into a 96-well plate. The same volume and number of NK cells were inoculated at a 1:1 ratio of NK cells to T cells. The cells were co-incubated in the incubator for 0hr, 4hr, 18hr, 24hr and 48hr. HLA-ABC-positive NK cells were labeled by flow cytometry, and the proportion of UCAR-T cells at different time points of co-incubation was detected. The experimental results are shown in Figure 2. Figure 15 As shown, the proportion of BCMA UCAR-T cells gradually decreased with the extension of incubation time, and was basically killed by NK cells by 48 hours. However, BCMA-GS-NKG2A UCAR-T and NKG2A UCAR-T cells showed the same trend of change, with a slight decrease at 4 hours, then a gradual increase, reaching more than 70% by 48 hours, and BCMA-GS-NKG2A UCAR-T cells reaching a proportion of 90% at 48 hours. These results indicate that BCMA-GS-NKG2A UCAR-T cells can effectively resist NK cell killing.

[0237] Example 9: BCMA-GS-NKG2A UCAR-T cells resist NK cells in vivo

[0238] BCMA UCAR-T and BCMA-GS-NKG2A UCAR-T cells were cultured in vitro, and the CAR positivity was adjusted to 60%. They were then injected into NPG immunodeficient mice via the tail vein at a dose of 8*10^6 cells / mouse. The mice were then divided into two groups: a BCMA UCAR-T and NK cell group (labeled BCMA UCAR-T+NK) and a BCMA-GS-NKG2A UCAR-T and NK cell group (labeled BCMA-GS-NKG2A UCAR-T+NK). Four hours after UCAR-T cell administration, an equal number of NK cells were injected. On days 1, 3, and 6 after CAR T cell injection, the survival of human CD45-positive T cells in the mouse peripheral blood was assessed by flow cytometry.

[0239] The experimental results are as follows Figure 19As shown in the figure, compared with the first day after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group did not increase significantly on the third and sixth days, indicating that UCAR-T cells were rejected by NK cells. In contrast, the number of UCAR-T cells in the BCMA-GS-NKG2A UCAR-T+NK group increased significantly on the third and sixth days, and the number of cells on the sixth day increased by more than 30 times compared with the first day. The above results indicate that in the in vivo model, NK cells significantly inhibited the survival of BCMA UCAR-T cells, while BCMA-GS-NKG2A UCAR-T cells could effectively resist NK cell killing and restore proliferation capacity.

[0240] The sequences involved in this application are shown in the following table:

[0241]

[0242]

[0243]

Claims

1. A cell for resisting transplant rejection, characterized in that: The cells express chimeric receptors that recognize NK cells; preferably, the cells have an inhibitory or killing function on NK cells.

2. The cell according to claim 1, wherein The chimeric receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain; optionally, (1) The extracellular domain of the chimeric receptor recognizes one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCRs), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specific antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161; (2) The extracellular domain of the chimeric receptor recognizes one or more of the following: NKG2A, NKG2D, NKP30, NKP44, NKP46; preferably, the extracellular domain of the chimeric receptor recognizes NKG2A; more preferably, the extracellular domain of the chimeric receptor comprises an antibody that recognizes NKG2A; (3) The extracellular domain of the chimeric receptor comprises: scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, a single domain fragment, or a natural ligand and its derivatives; preferably, the chimeric receptor comprises: (i) 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, LCDR3 shown in SEQ ID NO: 15; (ii) the heavy chain variable region shown in SEQ ID NO: 1 or the light chain variable region shown in SEQ ID NO: 2; or (iii) the amino acid sequence shown in SEQ ID NO: 5 or 9; (4) The extracellular domain of the chimeric receptor further recognizes a tumor antigen or a pathogen antigen; preferably, the tumor antigen is a solid tumor antigen or a blood tumor antigen, and the pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; preferably, the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen; (5) The extracellular domain of the chimeric receptor further recognizes the following antigens, selected from: CD19, CD20, CD 22, CD 123, CD38, B7H3 (CD276), interleukin 13 receptor subunit α (IL-13Rα), mesothelin, epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), ephrin type A receptor 2 (EphA2), Claudin 6, Claudin18.2, B cell maturation antigen (BCMA), FAP, NKG2D ligand, G protein-coupled receptor class C 5 group-member D (GPRC5D), Wilms tumor protein (WT1), phosphatidylinositol proteoglycan-3 (GPC3) or Fc receptor-like 5 (FCRL5); preferably, the extracellular domain of the chimeric receptor further recognizes the following antigens, selected from: BCMA, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, Claudin18.2, GPC3, FCRL5 or a combination thereof; preferably, the chimeric receptor further comprises: (i) HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, HCDR3 set forth in SEQ ID NO: 18, and LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 20, and LCDR3 set forth in SEQ ID NO: 21; or (ii) a heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region set forth in SEQ ID NO: 23; (6) The intracellular signaling domain of the chimeric receptor includes a functional signaling domain of a stimulatory molecule and / or a costimulatory molecule; preferably, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, optionally, the intracellular signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 4; more preferably, the intracellular signaling domain further comprises the functional signaling domain of one or more costimulatory molecules, such as 4-1BB (i.e., CD137), CD27, CD28, or a combination thereof, optionally, the intracellular signaling domain of CD28 comprises the CD28 intracellular signaling domain shown in SEQ ID NO: 3; (7) The chimeric receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC); and / or (8) The chimeric receptor transmits signals through the intracellular signaling domain to mediate the inhibition or killing of the NK cells.

3. The cell according to claim 1 or 2, wherein Optionally, the cells (1) Immune effector cells or artificially modified cells with immune effector cell functions; (2) selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells; preferably, the cells are autologous T cells, xenogeneic T cells, or allogeneic T cells; (3) further expressing a second chimeric receptor; preferably, the second chimeric receptor contains an extracellular domain, a transmembrane domain and an intracellular signaling domain; preferably, the extracellular domain of the second chimeric receptor recognizes a tumor antigen or a pathogen antigen; preferably, the extracellular domain of the second chimeric receptor recognizes the following antigens, selected from the group consisting of: CD19, CD20, CD 22, CD 123, CD38, B7H3 (CD276), interleukin 13 receptor subunit α (IL-13Rα), mesothelin, epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), ephrin type A receptor 2 (EphA2), Claudin 6, Claudin18.2, B cell maturation antigen (BCMA), FAP, NKG2D ligand, G protein coupled receptor class C 5 group - member D (GPRC5D), Wilms tumor protein (WT1), Glypican-3 (GPC3) or Fc receptor-like 5 (FCRL5); preferably, the extracellular domain of the second chimeric receptor recognizes the following antigens selected from: BCMA, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, Claudin18.2, GPC3, FCRL5 or a combination thereof; preferably, the extracellular domain of the second chimeric receptor comprises: (i) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17, HCDR3 of SEQ ID NO: 18, and LCDR1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 20, and LCDR3 of SEQ ID NO: 21; (ii) The heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23, or (iii) the amino acid sequence shown in SEQ ID NO: 6; and / or (4) MHC or HLA is not expressed, or the endogenous MHC or HLA gene of the cell is silenced; preferably, the HLA gene is an HLA-1 class molecule; preferably, the HLA-1 class molecule is B2M; preferably, the endogenous HLA-1 gene and TCR gene of the cell are silenced; preferably, gene silencing is achieved by gene editing technology; preferably, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology, or TALE-CRISPR / Cas9 technology; preferably, the gene editing technology is CRISPR / Cas9 technology; preferably, gene editing is performed using CRISPR / Cas9 technology, and the gRNAs for targeting TCR and B2M genes include the sequences shown in SEQ ID NO: 7 and 8, respectively.

4. A nucleic acid encoding a chimeric receptor for anti-transplant rejection, characterized in that The chimeric receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain; the extracellular domain recognizes NK cell antigens; optionally, (1) The extracellular domain of the chimeric receptor recognizes one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCRs), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specific antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161; and / or (2) The extracellular domain of the chimeric receptor recognizes one or more of the following: NKG2A, NKG2D, NKP30, NKP44, NKP46; preferably, the extracellular domain of the chimeric receptor recognizes NKG2A; more preferably, the extracellular domain of the chimeric receptor comprises an antibody that recognizes NKG2A; and / or (3) The extracellular domain of the chimeric receptor comprises: scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, a single domain fragment, or a natural ligand and its derivatives; preferably, the chimeric receptor comprises: (i) 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, LCDR3 shown in SEQ ID NO: 15; (ii) the heavy chain variable region shown in SEQ ID NO: 1 or the light chain variable region shown in SEQ ID NO: 2; or (iii) the amino acid sequence shown in SEQ ID NO: 5 or 9; and / or (4) The extracellular antigen binding region of the chimeric receptor also recognizes a tumor antigen or a pathogen antigen; preferably, the tumor antigen is a solid tumor antigen or a blood tumor antigen, and the pathogen antigen is selected from: an antigen of a virus, a bacterium, a fungus, a protozoa, or a parasite; preferably, the viral antigen is selected from: a cytomegalovirus antigen, an Epstein-Barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen; and / or (5) The extracellular domain of the chimeric receptor further recognizes the following antigens, selected from: CD19, CD20, CD 22, CD 123, CD38, B7H3 (CD276), interleukin 13 receptor subunit α (IL-13Rα), mesothelin, epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), ephrin type A receptor 2 (EphA2), Claudin 6, Claudin18.2, B cell maturation antigen (BCMA), FAP, NKG2D ligand, G protein-coupled receptor class C 5 group-member D (GPRC5D), Wilms tumor protein (WT1), phosphatidylinositol proteoglycan-3 (GPC3) or Fc receptor-like 5 (FCRL5); preferably, the extracellular domain of the chimeric receptor further recognizes the following antigens, selected from: BCMA, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, Claudin18.2, GPC3, FCRL5 or a combination thereof; preferably, the chimeric receptor further comprises: (i) 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, LCDR3 shown in SEQ ID NO: 21; or (ii) the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23; and / or (6) The intracellular signaling domain of the chimeric receptor includes a functional signaling domain of a stimulatory molecule and / or a co-stimulatory molecule; preferably, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, optionally, the intracellular signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 4; more preferably, the intracellular signaling domain further comprises one or more functional signaling domains of co-stimulatory molecules, such as 4-1BB (i.e., CD137), CD27, CD28 or a combination thereof, optionally, the intracellular signaling domain of CD28 comprises the CD28 intracellular signaling domain shown in SEQ ID NO: 3; and / or (7) The chimeric receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC); and / or (8) The chimeric receptor transmits signals through the intracellular signaling domain to mediate the inhibition or killing of the NK cells; and / or (9) The nucleic acid is DNA or RNA; or the nucleic acid is single-stranded or double-stranded.

5. A chimeric receptor for anti-transplant immune rejection, characterized in that: The chimeric receptor comprises an extracellular domain, a transmembrane domain and an intracellular signaling domain; the extracellular domain recognizes NK cell antigens; optionally, (1) The extracellular domain of the chimeric receptor recognizes one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCRs), such as NKP30, NKP44, NKP46, NKp80, etc.; and other NK cell-specific antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, CD161; and / or (2) The extracellular domain of the chimeric receptor recognizes one or more of the following: NKG2A, NKG2D, NKP30, NKP44, NKP46; preferably, the extracellular domain of the chimeric receptor recognizes NKG2A; more preferably, the extracellular domain of the chimeric receptor comprises an antibody that recognizes NKG2A; and / or (3) The extracellular domain of the chimeric receptor comprises: scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, a single domain fragment, or a natural ligand and its derivatives; preferably, the chimeric receptor comprises: (i) 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, LCDR3 shown in SEQ ID NO: 15; (ii) the heavy chain variable region shown in SEQ ID NO: 1 or the light chain variable region shown in SEQ ID NO: 2; or (iii) the amino acid sequence shown in SEQ ID NO: 5 or 9; and / or (4) The extracellular domain of the chimeric receptor further recognizes a tumor antigen or a pathogen antigen; preferably, the tumor antigen is a solid tumor antigen or a blood tumor antigen, and the pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; preferably, the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen; and / or (5) The extracellular domain of the chimeric receptor further recognizes the following antigens, selected from: CD19, CD20, CD 22, CD 123, CD38, B7H3 (CD276), interleukin 13 receptor subunit α (IL-13Rα), mesothelin, epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), ephrin type A receptor 2 (EphA2), Claudin 6, Claudin18.2, B cell maturation antigen (BCMA), FAP, NKG2D ligand, G protein-coupled receptor class C 5 group-member D (GPRC5D), Wilms tumor protein (WT1), phosphatidylinositol proteoglycan-3 (GPC3) or Fc receptor-like 5 (FCRL5); preferably, the extracellular domain of the chimeric receptor further recognizes the following antigens, selected from: BCMA, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, Claudin18.2, GPC3, FCRL5 or a combination thereof; preferably, the extracellular domain of the chimeric receptor further comprises: (i) 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, LCDR3 shown in SEQ ID NO: 21; or (ii) the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23; and / or (6) The intracellular signaling domain of the chimeric receptor includes a functional signaling domain of a stimulatory molecule and / or a co-stimulatory molecule; preferably, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ, optionally, the intracellular signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 4; more preferably, the intracellular signaling domain further comprises one or more functional signaling domains of co-stimulatory molecules, such as 4-1BB (i.e., CD137), CD27, CD28 or a combination thereof, optionally, the intracellular signaling domain of CD28 comprises the CD28 intracellular signaling domain shown in SEQ ID NO: 3; and / or (7) The chimeric receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC); and / or (8) The chimeric receptor transmits signals through the intracellular signaling domain to mediate the inhibition or killing of the NK cells.

6. Use of the cell according to any one of claims 1 to 3, the nucleic acid according to claim 4, or the chimeric receptor according to claim 5 in the preparation of a medicament for preventing or regulating transplant rejection.

7. A method for preparing cells having an anti-transplant immune rejection effect, comprising transfecting the nucleic acid of claim 4 into the cells, or expressing the chimeric receptor of claim 5 in the cells; optionally, the cells: (1) Immune effector cells or artificially modified cells with immune effector cell functions; (2) selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells; preferably, the cells are autologous T cells, xenogeneic T cells, or allogeneic T cells; (3) further expressing a second chimeric receptor; preferably, the second chimeric receptor contains an extracellular domain, a transmembrane domain and an intracellular signaling domain; preferably, the extracellular domain of the second chimeric receptor recognizes a tumor antigen or a pathogen antigen; preferably, the extracellular domain of the second chimeric receptor recognizes the following antigens, selected from the group consisting of: CD19, CD20, CD 22, CD 123, CD38, B7H3 (CD276), interleukin 13 receptor subunit α (IL-13Rα), mesothelin, epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), ephrin type A receptor 2 (EphA2), Claudin 6, Claudin18.2, B cell maturation antigen (BCMA), FAP, NKG2D ligand, G protein-coupled receptor class C 5 group - member D (GPRC5D), Wilms tumor protein (WT1), Glypican-3 (GPC3) or Fc receptor-like 5 (FCRL5); preferably, the extracellular domain of the chimeric receptor further recognizes the following antigens selected from: BCMA, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, Claudin18.2, GPC3, FCRL5 or a combination thereof; preferably, the second chimeric receptor comprises: (i) HCDR1 set forth in SEQ ID NO: 16, HCDR2 set forth in SEQ ID NO: 17, HCDR3 set forth in SEQ ID NO: 18, and LCDR1 set forth in SEQ ID NO: 19, LCDR2 set forth in SEQ ID NO: 20, and LCDR3 set forth in SEQ ID NO: 21; or (ii) the heavy chain variable region set forth in SEQ ID NO: 22 and the light chain variable region set forth in SEQ ID NO: 23; and / or (4) MHC or HLA is not expressed, or the endogenous MHC or HLA gene of the cell is silenced; preferably, the HLA gene is an HLA-1 class molecule; preferably, the HLA-1 class molecule is B2M; preferably, the endogenous HLA-1 gene and TCR gene of the cell are silenced; preferably, gene silencing is achieved by gene editing technology; preferably, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology, or TALE-CRISPR / Cas9 technology; preferably, the gene editing technology is CRISPR / Cas9 technology; preferably, gene editing is performed using CRISPR / Cas9 technology, and the gRNAs for targeting TCR and B2M genes include the sequences shown in SEQ ID NO: 7 and 8, respectively.

8. Nucleic acid, including gRNA targeting TCR and B2M genes: the gRNA targeting the TCR gene includes the sequence shown in SEQ ID NO: 7, and the gRNA targeting the B2M gene includes the sequence shown in SEQ ID NO:

8.

9. Cells for resisting transplant rejection, characterized in that The endogenous TCR and B2M genes of the cell are silenced, and the gene silencing is performed by gene editing of the TCR and B2M genes using CRISPR / Cas9 technology, and the gRNAs for targeting the TCR and B2M genes include the sequences shown in SEQ ID NOs: 7 and 8, respectively; preferably, the cell comprises the nucleic acid of claim 4, or expresses the chimeric receptor of claim 5: Optionally, the cells: (1) further expressing a second chimeric receptor; preferably, the second chimeric receptor contains an extracellular antigen binding region, a transmembrane domain and an intracellular signaling domain; preferably, the extracellular domain of the second chimeric receptor recognizes a tumor antigen or a pathogen antigen; preferably, the extracellular antigen binding region of the second chimeric receptor recognizes the following antigens, selected from the group consisting of: CD19, CD20, CD 22, CD 123, CD38, B7H3 (CD276), interleukin 13 receptor subunit α (IL-13Rα), mesothelin, epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), ephrin type A receptor 2 (EphA2), Claudin 6, Claudin18.2, B cell maturation antigen (BCMA), FAP, NKG2D ligand, G protein coupled receptor class C 5 group - member D (GPRC5D), Wilms tumor protein (WT1), Glypican-3 (GPC3) or Fc receptor-like 5 (FCRL5) Preferably, the extracellular antigen binding region of the second chimeric receptor recognizes the following antigens selected from: BCMA, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, Claudin18.2, GPC3, FCRL5 or a combination thereof; Preferably, the second chimeric receptor comprises: (i) HCDR1 of SEQ ID NO: 16, HCDR2 of SEQ ID NO: 17, HCDR3 of SEQ ID NO: 18, and LCDR1 of SEQ ID NO: 19, LCDR2 of SEQ ID NO: 20, LCDR3 of SEQ ID NO: 21; (ii) the heavy chain variable region set forth in SEQ ID NO: 22 and the light chain variable region set forth in SEQ ID NO: 23, or (iii) the amino acid sequence set forth in SEQ ID NO: 6; (2) Immune effector cells or artificially modified cells with immune effector cell functions; (3) selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells; preferably, the cells are autologous T cells, xenogeneic T cells, or allogeneic T cells.

10. Cells for resisting transplant rejection, characterized in that: The cell expresses a chimeric receptor that recognizes one or more immune effector cells; preferably, the cell has an inhibitory or killing function on the one or more immune effector cells.

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