Cells and methods for resisting transplant rejection - Patents.com

Abstract

The present invention relates to a cell that resists transplant immune rejection. The cell expresses a first protein that can recognize one or more immune effector cells of the host. Preferably, the cell has an inhibitory or killing function against the immune effector cells of the host. Also, the present invention relates to a method for preventing or modulating transplant immune rejection, and a method for preventing or modulating NK cell attack against exogenous cells.

Description

[Technical field]

[0001] The present invention relates to cells having the function of resisting transplant immune rejection, and to a method for resisting transplant immune rejection, in particular a method for resisting immune rejection of NK cells. [Background technology]

[0002] Due to the immunogenetic differences between donors and recipients, when a foreign-source donor is transplanted, as a foreign-source graft, the donor may be recognized and attacked by immune cells in the recipient's body, thereby inhibiting or eliminating the foreign-source graft, resulting in a host-versus-graft reaction (HVGR). By knocking out MHC molecules in transplanted cells, it is possible to effectively resist the host T-cell rejection of the graft, but it may cause the rejection of other immune cells in the host. For example, in allogeneic cell transplantation, the lack of MHC-I molecules in allogeneic cells will cause the rejection of NK cells in the host, promoting the elimination of allogeneic cells (Nat Biotechnol. 2017;35(8):765-772. doi:10.1038 / nbt.3860). Therefore, a method to effectively prevent the immune rejection of host NK cells is of great importance for the development of allogeneic cell transplantation therapy. DISCLOSURE OF THEINVENTION

[0003] It is an object of the present invention to provide cells that resist transplant immune rejection and methods for resisting inhibition and rejection.

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

[0005] In a first aspect of the present invention, a cell is provided which expresses a first protein capable of recognising one or more immune effector cells of a host, and preferably has an inhibitory or killing function on the host's immune effector cells.

[0006] In a preferred embodiment, said cells are immune effector cells or artificially modified cells having immune effector cell function.

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

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

[0009] In a preferred embodiment, the activation of said protein which recognises immune effector cells of the host is regulated by said second receptor.

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

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

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

[0013] In a preferred embodiment, said cells do not express HLA or the HLA gene endogenously expressed by said cells is silenced, preferably said HLA is the HLA-I gene.

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

[0015] In a preferred embodiment, the first protein comprises an antibody capable of recognizing a host's NK cell; Preferably, the antibody is capable of recognizing NKG2A, More preferably, the antibody comprises an HCDR1 as set forth in SEQ ID NO: 10, an HCDR2 as set forth in SEQ ID NO: 11, an HCDR3 as set forth in SEQ ID NO: 12, an LCDR1 as set forth in SEQ ID NO: 13, an LCDR2 as set forth in SEQ ID NO: 14, and an LCDR3 as set forth in SEQ ID NO: 15; Even more preferably, the antibody comprises a heavy chain variable region set forth in SEQ ID NO:1 or a light chain variable region set forth in SEQ ID NO:2.

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

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

[0018] In a preferred embodiment, the first protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain; Preferably, the cell transmits a signal via the intracellular signaling domain to mediate inhibition or killing of immune effector cells of the host.

[0019] In a preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain; Preferably, the cell transmits a signal via the intracellular signaling domain to mediate inhibition or killing of a tumor or pathogen.

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

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

[0022] In a preferred embodiment, gene editing techniques are used to silence the gene.

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

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

[0025] In a preferred embodiment, the first and second proteins may be in one chimeric receptor, i.e., preferably, said chimeric receptor comprises, in sequence, an antibody recognizing a host immune effector cell (first protein), an antibody recognizing a tumor or pathogen antigen (second protein), a transmembrane domain, and an intracellular domain, or The chimeric receptor comprises, in sequence, an antibody (second protein) that recognizes a tumor antigen or a pathogen antigen, an antibody (first protein) that recognizes a host immune effector cell, a transmembrane domain, and an intracellular domain; Preferably, the antibody that recognizes a host's immune effector cell (first protein) and the antibody that recognizes a tumor antigen or a pathogen antigen (second protein) are linked by a connecting peptide.

[0026] In a second aspect of the invention there is provided a cell for resisting transplant immune rejection, said cell being a T cell having a T cell receptor capable of recognising one or more immune effector cells of the host, preferably wherein said cell has an inhibitory or killing function against the host's immune effector cells.

[0027] In a preferred embodiment, said cells further express a second protein that recognizes a tumor or pathogen antigen, preferably said second protein is a chimeric receptor.

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

[0029] In a preferred embodiment, said cells do not express HLA or the HLA gene endogenously expressed by said cells is silenced, preferably said HLA is the HLA-I gene.

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

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

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

[0033] In a preferred embodiment, gene editing techniques are used to silence the gene.

[0034] Preferably, the gene editing technology is selected from CRISPR / Cas9 technology, Zinc Finger Nucleases (ZFN) technology, transcription activator-like effector (TALE) technology, or TALE-CRISPR / Cas9 technology; More preferably, the gene editing technology is CRISPR / Cas9 technology.

[0035] In a third aspect of the invention there is provided a method of preventing or modulating transplant immune rejection comprising administering a cell according to either the first or second aspect of the invention.

[0036] In a fourth aspect of the invention, there is provided a method of preventing or modulating NK cell attack against exogenous cells, comprising administering immune effector cells expressing a first protein that recognises NK cells, Optionally, the exogenous cells are T cells, NKT cells, stem cells, or engineered T cells, NKT cells, stem cells; Optionally, the immune effector cells are administered before, after, or simultaneously with administration of the exogenous cells.

[0037] In a preferred embodiment, the exogenous cell is an immune effector cell, and preferably, the exogenous cell expresses a second receptor.

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

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

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

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

[0042] In a preferred embodiment, cells according to either the first or second aspect of the invention are administered.

[0043] In a fifth aspect of the present invention, there is provided a method for preventing or modulating NK cell attack against exogenous cells, comprising administering immune effector cells expressing a first protein that recognizes NK cells, the method comprising: Optionally, the method is provided wherein the exogenous cells are T cells, NKT cells, stem cells, or engineered T cells, NKT cells, stem cells.

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

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

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

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

[0048] In a sixth aspect of the present invention, there is provided a method for preventing or modulating NK cell attack against exogenous immune effector cells expressing a first protein that recognizes NK cells, comprising the steps of: Preferably, the exogenous immune effector cells are cells that do not contain an HLA-I gene or cells in which the endogenous HLA-I gene has been silenced; More preferably, the method is provided wherein the exogenous immune effector cells are cells that do not contain the B2M gene or cells in which the B2M gene has been silenced.

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

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

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

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

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

[0054] In a preferred embodiment, the first protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain; Preferably, the cell transmits a signal via the intracellular signaling domain to mediate inhibition or killing of immune effector cells of the host.

[0055] In a preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain; Preferably, the cell transmits a signal via the intracellular signaling domain to mediate inhibition or killing of a tumor or pathogen.

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

[0057] [Figure 1] FIG. 1 shows the expression of NK cell surface markers. [Diagram 2] FIG. 2 shows the expression of NK cell surface markers in T cells. [Diagram 3] Figure 3 shows the proliferation characteristics of NKG2A CAR-T cells. A is the cell proliferation curve, B is the cell diameter, and C is the CAR positivity rate. [Figure 4] FIG. 4 shows the in vitro killing capacity of NKG2A CAR-T cells against NK cells after a total of 4 hours of incubation. [Diagram 5] FIG. 5 shows the in vitro killing capacity of NKG2A CAR-T cells against NK cells after a total of 18 hours of incubation. [Figure 6] FIG. 6 shows efficient knockout of TCR and B2M in CAR-T cells. [Figure 7] 7A, 7B, 7C, and 7D show detection of the resistance ability of NKG2A UCAR-T cells against NK cells by FACS. [Figure 8] FIG. 8 is a plasmid map of the NKG2A-targeting CAR. [Figure 9] FIG. 9 is a plasmid map of the BCMA-targeting CAR. [Figure 10] FIG. 10 shows detection of resistance ability of NKG2A UCAR-T cells against NK cells by FACS. [Figure 11] FIG. 11 shows the detection of UCAR-T cell survival in peripheral blood of mice by FACS. [Figure 12] FIG. 12 is a schematic diagram of the structure of BCMA-GS-NKG2A UCAR-T. [Figure 13] FIG. 13 shows the preparation of BCMA-GS-NKG2A UCAR-T cells. [Figure 14] FIG. 14 shows the in vitro antitumor effect of BCMA-GS-NKG2A UCAR-T cells. [Figure 15] FIG. 15 shows the resistant effect of BCMA-GS-NKG2A UCAR-T cells against NK cells. [Figure 16] FIG. 16 is a plasmid map of PRRL-BCMA-BBZ-F2A-EGFP. [Figure 17] FIG. 17 is a plasmid map of PRRL-NKG2A-28Z-F2A-EGFP. [Figure 18] FIG. 18 is a plasmid map of PRRL-BCMA-GS-NKG2A-BBZ. [Figure 19] FIG. 19 shows the in vivo resistance effect of BCMA-GS-NKG2A-UCAR-T cells against NK cells. Summary of the Invention

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art 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, among which suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, unless otherwise specified, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0059] Unless otherwise indicated, the practice of the present invention employs conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, all of which are within the skill of the art and are fully explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL,2000,Wiley and son Inc,Library of Congress,USA);Molecular Cloning: A Laboratory Manual,Third Edition,(Sambrooketal,2001,Cold Spring Harbor,NewYork: Cold Spring Harbor Laboratory Press);Oligonucleotide Synthesis (MJGaited., 1984);Mullis et al. US Pat. No. 4,683,195;Nucleic Acid Hybridization (BD Harries & SJ Higginseds. 1984);Transcription And Translation (BD Hames & SJ Higginseds. 1984);Culture Of Animal Cells (RI Freshney,Alan R. Liss,Inc.,1987);Immobilized Cells And Enzymes (IRL Press,1986);B. Perbal,A Practical Guide To Molecular Cloning (1984); the series,Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MP Caloseds.,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, Vol. I-IV (DM Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0060] In this disclosure, each aspect of the subject matter for which protection is sought is expressed in the form of a range. It should be understood that the description in the form of a range is merely for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the subject matter for which protection is sought. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual values ​​within that range. For example, when a range of values ​​is provided, all intermediate values ​​between the upper and lower limits of the range, and any other or intermediate values ​​within the range, are also included in the scope of the subject matter for which protection is sought, and the upper and lower limits of the range are also included in the scope of the subject matter for which protection is sought. The upper and lower limits of those smaller ranges may be independently included in the smaller ranges, and unless the upper and lower limits of the ranges are expressly excluded, they also belong to the scope of the subject matter for which protection is sought. If a stated range includes one or two limit values, the subject matter for which protection is sought also includes the ranges in which one or two of the limit values ​​are excluded. This applies regardless of the breadth of the range.

[0061] The term "about" as used herein refers to the normal error range of each value, which is readily known to those skilled in the art. Values ​​or parameters modified by "about" herein include (and describe) the embodiment of the value or parameter itself. For example, the description "about X" includes the description of "X". For example, "about" or "including" can mean within 1 or more than 1, according to the standard deviation in the art. Alternatively, "about" or "including" can mean a range of up to 10% (i.e., ±10%). For example, about 5 μM can include any number from 4.5 μM to 5.5 μM. When a specific value or composition is provided in the scope of the application and patent application, unless otherwise specified, "about" or "including" should be within the acceptable error range of the specific value or composition.

[0062] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include any integer number within that range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer), unless otherwise stated.

[0063] To better understand the present invention, relevant terms are defined as follows:

[0064] The term "transplant immune rejection" means that after a host receives a graft, such as an allogeneic tissue, organ, or cell, the foreign-source graft is recognized as a "foreign component" by the host's immune system and initiates an immunological response against the graft, such as attacking, destroying, and removing the graft.

[0065] The term "graft" refers to a biological material or preparation derived from an individual other than the host and used for transplantation into the 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 derived from the host, e.g., cells from the host are cultured or modified in vitro and then transplanted back into the host. In some embodiments, the graft may be derived from an allogeneic other individual, e.g., cells from another human are cultured or modified in vitro and then transplanted into the host. In some embodiments, the graft may be derived from a xenogeneic individual, such as transplanting organs from other species (such as mice, pigs, and monkeys) into a human.

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

[0067] The term "host" refers to the recipient of the graft transplant, which in some embodiments can be an individual, such as a human, receiving a foreign cell transplant.

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

[0069] The term "artificially modified cells with immune effector cell function" refers to cells or cell lines that have no immune effect and acquire immune effector cell function after being artificially modified or stimulated by a stimulant. For example, 293T cells are artificially modified to have immune effector cell function. For example, stem cells are induced in vitro to differentiate into immune effector cells.

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

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

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

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

[0074] The term "B2M" refers to beta-2 microglobulin, also called B2M, which is the light chain of the MHC class I molecule. In humans, B2M is encoded by the b2m gene located on chromosome 15, relative to other MHC genes that map as a gene cluster on chromosome 6. Hematopoietic grafts from mice lacking normal cell surface MHC I expression due to mutations in the B2M gene are rejected by normal mouse NK cells, indicating that defective expression of MHC I molecules renders cells more susceptible to rejection by the host immune system (Bix et al. 1991).

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

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

[0077] The term "T cell receptor (TCR)" includes classical and optimized TCR receptors, which mediate the recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens by T cells. Classical TCR receptors consist of two peptide chains, α and β, each of which is further divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. The antigen specificity resides in the V region. Each V region (Vα, Vβ) has three hypervariable regions, namely, CDR1, CDR2, and CDR3. In one aspect, T cells expressing classical TCR can induce the specificity of the T cell's TCR for a target antigen by using a method such as antigen stimulation of the T cell.

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

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

[0080] The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to regulate the activity of the cell through a defined signaling pathway, either by generating second messengers or by acting as an effector in response to such messengers. The intracellular signaling domain can include all the intracellular portion of the molecule, or all naturally occurring intracellular signaling domains, or functional fragments or derivatives thereof.

[0081] The term "costimulatory molecule" refers to a signal that binds to a cell stimulatory signal molecule, such as TCR / CD3, in combination resulting in T cell proliferation and / or up- or down-regulation of key molecules.

[0082] The terms "activate" and "activating" can refer to the process by which a cell changes from a resting state to an active state. This process can include a response to an antigen, transition, and / or a phenotypic or genetic change to a functionally active state. For example, the term "activation" can refer to the process of stepwise activation of a T cell. For example, a T cell may require at least one signal to become fully activated.

[0083] The term "gene editing" refers to the ability of humans to "edit" targeted genes to achieve, for example, the knockout or addition of specific DNA fragments.

[0084] The term "gene silencing" refers to the phenomenon of gene not being expressed or underexpressed for various reasons.Gene silencing may be gene silencing at the transcriptional level, which is caused by DNA methylation, heterochromatinization, and position effect, etc., or it may be post-transcriptional gene silencing, that is, at the level of gene transcription, inactivate gene by specific inhibition of target RNA, such as antisense RNA, co-inhibition, gene inhibition, RNA interference, and microRNA-mediated translation inhibition.

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

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

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

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

[0089] A "CRISPR / Cas9 system" refers collectively to the transcripts and other elements involved in the expression of or directing the activity of the Cas9 enzyme gene, including sequences encoding the Cas9 gene, tracr (transactivation CRISPR) sequences (such as tracrRNA or active partial tracrRNA), tracr pairing sequences (including "direct repeats" and partial direct repeats of tracrRNA processing in the context of endogenous CRISPR systems), guide sequences (also called "spacers" in the context of endogenous CRISPR systems, i.e., gRNA), or other sequences and transcripts from a CRISPR locus.

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

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

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

[0093] The term "Cas9 enzyme" may refer to a wild-type Cas9 or an artificially modified Cas9.

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

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

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

[0097] The Cas transgene can be delivered by vectors (e.g., AAV, adenovirus, lentivirus), and / or particles and / or nanoparticles, and / or electroporation.

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

[0099] By "inhibiting" or "suppressing" the expression of B2M or TCR, it is meant that the expression of B2M or TCR in a cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. More specifically, by "inhibiting" or "suppressing" the expression of B2M, it is meant that the content of B2M in a cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. Intracellular protein expression or content can be measured by any suitable method known in the art, such as ELISA, immunohistochemistry, Western blotting or flow cytometry, using B2M or TCR specific antibodies.

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

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

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

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

[0104] The term "peripheral blood mononuclear cells" (PBMCs) refers to mononuclear cells in peripheral blood, including lymphocytes, monocytes, etc.

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

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

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

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

[0109] The term "vector" as used herein refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside 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. The term can also include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc.

[0110] The term sequence "identity" as used herein refers to a percentage of identity determined by comparing two best-matched sequences over a comparison window (e.g., at least 20 positions), where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., indels), e.g., 20% or less indels (e.g., 5-15%, or 10-12%) for the two best-matched sequences compared to the reference sequence (not including additions or deletions). Typically, the percentage is calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences, thereby generating the number of correct matching positions, and dividing the number of correct matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to generate the percentage of sequence identity.

[0111] The term "exogenous" as used herein refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that has no endogenous expression in an organism or that has an expression level that is insufficient to achieve a function when overexpressed.

[0112] The term "endogenous" refers to a nucleic acid molecule or polypeptide, etc., that originates from an organism's own origin.

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

[0114] A chimeric antigen receptor typically comprises an extracellular antigen-binding region, which in some embodiments may be fully human, humanized, derived from a mouse, or chimeric, of said extracellular antigen-binding region, which is comprised of amino acid sequences from at least two different animals.

[0115] 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.

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

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

[0118] 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 to select the extracellular antigen binding region to have a higher affinity for its target. In some cases, the affinity of the extracellular antigen binding region for the target can be optimized to a target that can be expressed at a low level in normal tissues. This optimization can be performed to minimize potential toxicity. In other cases, clones of the extracellular antigen binding region that have a higher affinity for the membrane-bound form of the target may be superior to their soluble form counterparts. This modification can be performed because targets with different levels of soluble form can also be detected and those targets may cause undesirable toxicity.

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

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

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

[0122] The term "treatment" refers to intervention in the process of modifying a disease, and can be preventative as well as intervening in the clinical pathological process. Therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of a disease, prevention of metastasis, delay of disease progression, amelioration or alleviation of the condition, alleviation or improvement of prognosis, etc.

[0123] The term "prophylaxis" refers to an intervention that occurs before a disease (such as a rejection caused by a cell transplant) occurs.

[0124] The first protein of the present invention refers to the above protein that is capable of recognizing one or more immune effector cells of the host.

[0125] The second protein of the present invention refers to the above protein that recognizes a tumor antigen or a pathogen antigen.

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

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

[0128] When the "second receptor" of the present invention is expressed in parallel (tandem expression) with a "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 an antigen, such as a tumor antigen, recognized by the "second receptor."

[0129] "Tumor antigen" refers to an antigen that is newly emerged or overexpressed during the development and progression of a hyperproliferative disease. In a particular embodiment, the hyperproliferative disorder of the present invention refers to cancer.

[0130] The tumor antigen according to the present invention may be a solid tumor antigen or a hematoma antigen.

[0131] The tumor antigens of the present invention include thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor epithelial cell adhesion molecule 2 (EphA2);fucosyl GM1;sialyl Lewis adhesion molecule (sLe);ganglioside GM3;TGS5;high molecular weight melanin tumor associated antigen (HMWMAA);O-acetyl GD2 ganglioside (OAcGD2);folate receptor;tumor endothelial marker 1 (TEM1 / CD248);tumor endothelial marker 7 related (TEM7R);Claudin6, Claudin18.2, Claudin18.1;ASGPR1;CDH16;5T4;8H9;αvβ6 integrin;B cell maturation antigen (BCMA);CA9;κ light chain (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;tumor necrotic zone carcinoembryonic variant;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);breast differentiation antigen (NY-BR-1);uroplakin 2 (UPK2);hepatitis A virus cell receptor 1 (HAVCR1);adrenergic receptor beta 3 (ADRB3);pannexin 3 (PANX3);G protein-coupled receptor 20 (GPR20);lymphocyte antigen 6 complex locus K9 (LY6K);olfactory receptor 51E2 (OR51E2);TCR gamma alternate reading frame protein (TARP);Wilms tumor protein (WT1);ETS translocation variant gene 6 (ETV6-AML);sperm protein 17 (SP A17);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 transition breakpoint;melanoma inhibitor of apoptosis (ML-IAP);ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene);N-acetylglucosaminyltransferase V (NA17);pairing box protein Pax-3 (PAX3);androgen receptor;cyclin B1;V-myc avian myelosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); pairing box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79B; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF);The tumor antigens include, but are not limited to, C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing EGF-like module (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like peptide 1 (IGLL1). Preferably, the tumor antigen is BCMA or CD19.

[0132] The pathogen antigen is selected from a viral, bacterial, fungal, protozoan, or parasitic antigen. The viral antigen is selected from a cytomegalovirus antigen, an Epstein-Barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen.

[0133] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, the experimental methods that do not show specific conditions usually follow conventional conditions such as those described in J. Sambrook et al., eds. Molecular Cloning: A Laboratory Manual, Third Edition, Science Press, 2002, or follow the conditions recommended by the manufacturer. Example 1

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

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

[0136] Preparation of NKG2A UCAR-T cells 1. Knockout of TCR and B2M genes Conventional UTD cells, NKG2A CAR-T cells, and BCMA CAR-T cells (for control) were expanded in vitro for 48 h, and then the cell density was increased to 2 × 10 7 The RNP complex solution was adjusted to 1×10 / mL. Cas9 enzyme (purchased from NEB) and sgRNA were incubated at room temperature for 10 minutes in a ratio of 1:4 to obtain an RNP complex solution. The nucleic acid sequence of TRAC-sgRNA is shown in SEQ ID NO: 7, and the nucleic acid sequence of B2M-sgRNA is shown in SEQ ID NO: 8. 6 The cells were mixed with the RNP complex solution (final concentration of Cas9 enzyme was 3uM), and the RNP complex was introduced into the CAR-T cells individually using a maxcyte electrotransmitter. On the seventh day after electroporation, flow cytometry was used to detect the knockout of TCR and B2M genes. The experimental results show that the knockout efficiency of TRAC and B2M is more than 85% (see Figure 6). 2. TCR / B2M double negative cell screening B2M- and TCR-knockout CAR-T cells and UTD cells were expanded in vitro and the cell density was increased to 1 × 10 7The cells were adjusted to 100 μg / mL and labeled with anti-HLA-ABC and B2M antibodies, followed by a secondary antibody coupled to phycoerythrin (PE). After sorting the labeled cells with anti-PE magnetic beads on a sorting column, TCR and B2M double negative cells were collected (sorting kit purchased from Miltenyi Biotec), resulting in BCMA UCAR-T cells, NKG2A UCAR-T cells, and U-UTD cells lacking TCR and B2M. Example 4

[0137] Verification of the resistance function of NKG2A UCAR-T cells against NK cells 1. Detection of the rejection effect of UCAR-T cells against NK cells by LDH experiment UTD cells, BCMA-CAR T cells, NKG2A CAR-T cells, BCMA UCAR-T cells, NKG2A UCAR-T cells, and U-UTD cells were used as target cells, with a cell concentration of 5 × 10 5 The NK cells were adjusted to 100 μl / mL and inoculated into a 96-well plate, and the same amount (volume) and number of NK cells were inoculated according to the 1:1 ratio of expanded primary NK cells to target cells, and incubated in an incubator for 4 and 18 hours, respectively. 50 μl of the supernatant was taken to measure the content of lactate dehydrogenase (LDH) and calculate the lysis efficiency of CAR-T and UCAR-T cells. The detection results show that the LDH values ​​of the UTD and BCMA CAR-T groups are both very low, indicating that normal CAR-T cells do not trigger the attack of NK cells. In contrast, the U-UTD and BCMA UCAR-T groups both showed gradually increasing LDH values ​​at 4 and 18 hours, indicating that NK cells kill T cells lacking TCR and B2M. NKG2A UCAR-T cells showed lower levels of LDH, indicating that NKG2A UCAR-T cells have a resistant effect on NK cells. 2. To further demonstrate the resistance ability of NKG2A UCAR-T cells to NK cells, we selected BCMA UCAR-T cells as a control and set the cell concentration at 5 × 10 5The mixture was adjusted to 100 μl / mL and 100 μl was inoculated into a 96-well plate. The same volume and number of NK cells were inoculated at a 1:1 ratio of expanded primary NK cells to target cells and incubated in an incubator for 4, 18, 24, and 42 hours, respectively. Flow cytometry was used to label HLA-ABC positive NK cells and detect the percentage of UCAR-T cells incubated at various time points. The experimental results, as shown in Figures 7A-7D, show that BCMA UCAR-T was a low percentage of about 20% at 4 hours, and the percentage was always very low with the extension of the detection time, indicating that NK cells significantly inhibited the proliferation of BCMA UCAR-T cells. NKG2A UCAR-T cells were a low percentage of about 20% at 4 hours, but with the extension of the detection time, the percentage gradually increased, reaching nearly 60% at 42 hours. This indicates that the proliferation of NKG2A UCAR-T cells is inhibited by NK cells from the beginning, but the proliferation ability is gradually restored over time. The above results indicate that NKG2A UCAR-T can effectively resist the killing ability of NK cells. 3. To further demonstrate the resistance ability of NKG2A UCAR-T cells against 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. With reference to the operation of Example 2, a plasmid PRRL-BCMA-BBZ-F2A-EGFP expressing BCMA UCAR-T cells expressing GFP was constructed, and the plasmid map is shown in Figure 16. A plasmid PRRL-NKG2A-28Z-F2A-EGFP expressing NKG2A UCAR-T cells was constructed, and the plasmid map is shown in Figure 17. The constructed plasmid was packaged with lentivirus, transfected with T cells, and gene knockout and magnetic bead sorting were performed on CAR-T cells to obtain BCMA UCAR-T cells expressing GFP and NKG2A UCAR-T cells expressing GFP. The concentration of CAR-T cells was 5 × 10 5The cells were adjusted to 100 μl / mL and inoculated into 96-well plates with the same volume and number of NK cells according to a 1:1 ratio of expanded primary NK cells to target cells, and cultured in an incubator for 0, 4, 18, 24, and 48 h, respectively. Flow cytometry was used to detect the percentage of co-cultured GFP cells at different time points and was used to track the survival of UCAR-T cells. The experimental results, as shown in Figure 10, showed that the proportion of GFP-positive BCMA UCAR-T cells gradually decreased with time and were almost completely killed by NK cells after 48 hours, while the proportion of GFP-positive NKG2A UCAR-T cells slightly decreased at 4 hours and significantly increased after 18 hours, accounting for approximately 90% after 48 hours, indicating that NKG2A UCAR-T cells could significantly resist killing by NK cells. Example 5

[0138] Resistance of NKG2A UCAR-T cells to NK cells in vivo BCMA UCAR-T and NKG2A UCAR-T cells were cultured in vitro, and the CAR positivity rate was adjusted to 80%, with 8 × 10 6 The mice were injected into NPG immunodeficient mice via tail vein injection at a dose of 1000 cells / mouse, and the mice were divided into two groups: one group that received BCMA UCAR-T and NK cells (denoted as BCMA UCAR-T+NK) and one group that received NKG2A UCAR-T and NK cells (denoted as NKG2A-UCART+NK). The same amount of NK cells was injected 4 hours after UCAR T cell administration. The survival of human-derived CD4 and CD8 T cells in the peripheral blood of mice was detected by flow absolute technology (absolute cell counting by flow cytometry) on days 1, 3, and 6 after CAR T cell injection. The experimental results, as shown in FIG. 11, on the first day after injection, the number of UCAR-T cells (i.e., human-derived CD4 and CD8 T cells) in the BCMA UCAR-T+NK group and the NKG2A UCAR-T+NK group significantly decreased, indicating that UCAR-T cells were rejected by NK cells. On the third and sixth days after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group was always at a very low level, while the number of UCAR-T cells in the NKG2A UCAR-T+NK group increased significantly on the third and sixth days. The above results indicate that in an in vivo model, NK cells significantly inhibit the survival of BCMA UCAR-T cells, whereas NKG2A UCAR-T cells can effectively resist killing by NK cells and restore their proliferation ability. Example 6

[0139] Construction of BCMA- and NKG2A-targeting CAR T cells UCAR-T cells were prepared in which a BCMA-targeting scFv and a NKG2A-targeting scFv were linked in tandem (i.e., BCMA-GS-NKG2A UCAR-T) as shown in Figure 12. The amino acid sequence of BCMA-GS-NKG2A CAR is shown in SEQ ID NO:9. The plasmid PRRL-BCMA-GS-NKG2A-BBZ of BCMA-GS-NKG2A UCAR-T was constructed, and the plasmid map is shown in Figure 18. Referring to the operations in Examples 2 and 3, viral transfection was performed to obtain BCMA-GS-NKG2A UCAR-T cells, TRAC and B2M gene knockout was performed on BCMA-GS-NKG2A UCAR-T cells, and then more than 99% TCR and HLA-ABC negative BCMA-GS-NKG2A UCAR-T cells were obtained by magnetic bead selection. BCMA UCAR-T cells and NKG2A UCAR-T cells were prepared with reference to the procedures in Examples 2 and 3, respectively. The CAR expression of BCMA UCAR-T, NKG2A UCAR-T and BCMA-GS-NKG2A UCAR-T was detected respectively. The experimental results are shown in Figure 13, and the positive rate reached more than 60%, indicating that the preparation of BCMA-GS-NKG2A UCAR-T cells was successful. Example 7

[0140] Validation of in vitro functionality of BCMA-GS-NKG2A UCAR-T cells BCMA-positive multiple myeloma cell lines RPMI-8226 and NCI-H929 were cultured in vitro as target cells, and 1 × 10 4 of tumor cells were seeded into 96-well plates, and the corresponding number of UCAR-T cells were inoculated according to the T cell to tumor cell ratios of 3:1, 1:1, and 1:3, and after incubation for 18 h, 50 μl of the supernatant was aspirated for LDH content detection. The experimental results, as shown in FIG. 14, 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 of the BCMA-GS-NKG2A UCAR-T group was close to that of the BCMA UCAR-T group, indicating that BCMA-GS-NKG2A UCAR-T cells can effectively kill BCMA-positive tumor cells in vitro. Example 8

[0141] Verification of resistance function of BCMA-GS-NKG2A UCAR-T cells against NK cells As negative and positive controls, BCMA UCAR-T and NKG2A UCAR-T cells were selected, respectively, with a cell concentration of 5 × 10 5The BCMA UCAR-T cells were adjusted to 100 μl / mL, and 100 μl was inoculated into a 96-well plate. The same volume and number of NK cells were inoculated according to the NK cell to T cell ratio of 1:1, and cultured in an incubator for 0, 4, 18, 24, and 48 hours, respectively. Flow cytometry was used to label HLA-ABC positive NK cells and detect the proportion of co-cultured UCAR-T cells at different time points. The experimental results, as shown in Figure 15, show that the proportion of BCMA UCAR-T cells gradually decreased with the extension of incubation time and were mostly killed by NK cells after 48 hours, while BCMA-GS-NKG2A UCAR-T and NKG2A UCAR-T cells showed the same change trend, slightly decreased at 4 hours and then gradually increased, reaching about 70% or more after 48 hours, and BCMA-GS-NKG2A UCAR-T cells reached 90% after 48 hours, indicating that BCMA-GS-NKG2A UCAR-T cells can significantly resist killing by NK cells. Example 9

[0142] Resistance of BCMA-GS-NKG2A UCAR-T cells to NK cells in vivo BCMA UCAR-T and BCMA-GS-NKG2A UCAR-T cells were cultured in vitro, and the CAR positivity rate was adjusted to 60%, with 8 × 10 6 The CAR T cells were injected into NPG immunodeficient mice via tail vein injection at a dose of 10 ... The experimental results are shown in FIG. 19. Compared with the first day after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group did not significantly increase on the third and sixth days, indicating that the UCAR-T cells were rejected by the NK cells. The number of UCAR-T cells in the BCMA-GS-NKG2A UCAR-T+NK group significantly increased on both the third and sixth days, and the number of cells on the sixth day was more than 30 times higher than that on the first day. The above results indicate that in an in vivo model, NK cells significantly inhibit the survival of BCMA UCAR-T cells, whereas BCMA-GS-NKG2A UCAR-T cells can effectively resist killing by NK cells and restore their proliferation ability.

[0143] The sequences of the present application are shown in the following table:

[0144] [Table 1-1]

[0145] [Table 1-2]

[0146] [Table 1-3]

[0147] [Table 1-4]

[0148] [Table 1-5]

[0149] [Table 1-6]

Claims

1. A cell for resisting transplant immune rejection, the cell being an immune effector cell or an artificially modified cell having the function of an immune effector cell, expressing a first protein that is a chimeric receptor that recognizes a host's NK cells, the chimeric receptor comprising an extracellular domain including an antigen-binding region that recognizes the NK cells, a transmembrane domain, and an intracellular signaling domain, the cell having the function of inhibiting or killing the host's NK cells. A cell characterized by:

2. The immune effector cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, CIK cells, and immune effector cells derived from stem cells; The cell of claim 1.

3. The cell of claim 1 or 2, wherein the chimeric receptor is a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

4. The cell of claim 3 , wherein the immune effector cell is derived from the host's autologous cells or from another allogeneic source.

5. The antigen-binding region is selected from the group consisting of scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, and a single domain fragment. The cell according to any one of claims 1 to 4.

6. The chimeric receptor is the NKG2 receptor family, including NKG2A, NKG2D, and NKG2C; the killing immunoglobulin-like receptor (KIR) family, including KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2 / S3, KIR2DS4, KIR2DS5, and KIR3DS1; the natural cytotoxicity receptors (NCR), including NKP30, NKP44, NKP46, and NKp80; and other antigens specifically expressed by NK cells, including CD159a, CD159c, CD94, CD158, CD56, LIR / ILT2, CD244, CD226, CD2, CD16, and CD161; Specifically recognizes one or more NK cell antigens selected from the group consisting of: A cell according to any one of claims 1 to 5.

7. The cell of claim 6, wherein the chimeric receptor specifically recognizes one or more NK cell surface antigens selected from the group consisting of NKG2A, NKG2D, NKP30, NKP44, and NKP46.

8. The cell of claim 7 , wherein the chimeric receptor comprises an antigen-binding region that specifically recognizes NKG2A.

9. The cell of claim 8, wherein the antigen-binding region comprises an HCDR1 represented by SEQ ID NO: 10, an HCDR2 represented by SEQ ID NO: 11, an HCDR3 represented by SEQ ID NO: 12, an LCDR1 represented by SEQ ID NO: 13, an LCDR2 represented by SEQ ID NO: 14, and an LCDR3 represented by SEQ ID NO: 15; or wherein the antigen-binding region comprises a heavy chain variable region set forth in SEQ ID NO: 1 and a light chain variable region set forth in SEQ ID NO:

2.

10. the transmembrane domain is selected from CD28 and CD8α; and the intracellular signaling domain comprises a functional signaling domain of 4-1BB, CD27, CD28, CD3ζ, or a combination thereof; A cell according to any one of claims 1 to 9.

11. The cells do not contain MHC or HLA genes, or the MHC or HLA genes endogenously expressed in the cells are silenced or knocked out. The cell according to any one of claims 1 to 10.

12. The cell according to claim 11, wherein the MHC gene is a gene for an MHC class I molecule and the HLA is a gene for an HLA-I molecule.

13. The cell according to any one of claims 1 to 12, wherein the cell is a T cell in which an endogenous HLA-I gene and an endogenous TCR gene have been silenced or knocked out; or a T cell in which a B2M gene and a TCR gene have been silenced or knocked out.

14. The cell of claim 10 , wherein the first protein comprises an amino acid sequence represented by SEQ ID NO:

5.

15. The first protein further comprises an antigen-binding region that recognizes a tumor antigen or a pathogen antigen. The cell according to any one of claims 1 to 13.

16. The cell of claim 15, wherein the tumor or pathogen antigen comprises BCMA, GPC3, Claudin 18.2, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19, or a combination thereof.

17. The cell according to any one of claims 1 to 13, wherein the first protein further comprises an antigen-binding region that recognizes BCMA, and the first protein has an amino acid sequence represented by SEQ ID NO:

9.

18. The cells further express a second protein that includes an antigen-binding region that recognizes a tumor antigen or a pathogen antigen. The cell according to any one of claims 1 to 14.

19. The cell of claim 18, wherein the second protein is a CAR, a chimeric T cell receptor, or a TAC.

20. 20. The cell of claim 18 or 19, wherein the tumor or pathogen antigen comprises BCMA, GPC3, Claudin 18.2, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19 or a combination thereof.

21. The cell described in claim 18 or 19, wherein the antigen-binding region of the second protein specifically recognizes BCMA and includes an HCDR1 represented by SEQ ID NO: 16, an HCDR2 represented by SEQ ID NO: 17, an HCDR3 represented by SEQ ID NO: 18, an LCDR1 represented by SEQ ID NO: 19, an LCDR2 represented by SEQ ID NO: 20, and an LCDR3 represented by SEQ ID NO: 21; or the antigen-binding region includes a heavy chain variable region represented by SEQ ID NO: 22 and a light chain variable region represented by SEQ ID NO:

23.

22. The cell of claim 20 , wherein the tumor antigen is BCMA and the second protein has an amino acid sequence represented by SEQ ID NO:

6.

23. A composition for use in a method for preventing or modulating transplant immune rejection, the composition comprising a cell according to any one of claims 1 to 22.

24. A composition for use in a method for preventing or modulating NK cell attack against exogenous cells, the composition comprising a cell according to any one of claims 1 to 14.

25. The composition for use according to claim 24, wherein the exogenous cell is an exogenous immune effector cell selected from the group consisting of a T cell, a NK cell, a NKT cell, a macrophage, a CIK cell, and an immune effector cell derived from a stem cell.

26. The composition for use according to claim 25, wherein in the exogenous immune effector cells, the endogenous HLA-I gene is silenced or knocked out; or the B2M gene is silenced or knocked out.

27. The composition for use according to any one of claims 24 to 26, wherein the exogenous immune effector cells express a second protein comprising an antigen-binding region that recognises a tumor antigen or a pathogen antigen.

28. 28. The composition for use of claim 27, wherein the tumor or pathogen antigen comprises BCMA, GPC3, Claudin 18.2, CD20, CD22, CD38, GPRC5D, B7H3, Claudin 6, FAP, Mesothelin, CD19 or a combination thereof.

29. 29. The composition for use of claim 27 or 28, wherein the second protein is a chimeric receptor that is a CAR, a chimeric T cell receptor, or a TAC.

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