CN114729028A - Cells and methods for resisting transplant response - Google Patents

Abstract

Relates to a cell that is resistant to transplant immune rejection, said cell expressing a first protein capable of recognizing one or more immune effector cells of a host; preferably, the cell has an inhibitory or killing function on an immune effector cell of the host. Also relates to a method for preventing or regulating transplant immune rejection and a method for preventing or regulating attack of exogenous cells by NK cells.

Description

Cells and methods for resisting transplant response Technical Field

The invention relates to a cell with the function of resisting transplant rejection, and also relates to a method for resisting transplant rejection, in particular to a method for resisting NK cell rejection.

Background

Due to the immunogenetic differences between the donor and recipient, when an exogenous donor is transplanted, the donor may also be recognized and attacked by immune cells in the recipient as an exogenous graft, thereby inhibiting or eliminating the exogenous graft and generating a host-versus-graft response (HVGR). By knocking out MHC molecules in the transplanted cells, the rejection of host T cells to the transplanted cells can be effectively resisted, but the rejection of other immune cells in the host can be possibly caused. For example, in allogeneic cell transplantation, when the MHC-I molecules of allogeneic cells are deleted, NK cell rejection in the host body is caused, and the clearance effect on allogeneic cells is enhanced (Nat Biotechnol.2017; 35(8):765-772.doi: 10.1038/nbt.3860). Therefore, how to effectively prevent immune rejection to host NK cells is crucial for the development of allogeneic cell transplantation therapy.

Disclosure of Invention

The invention aims to provide a cell for resisting transplant immune rejection and a method for resisting rejection inhibition.

The technical scheme provided by the invention comprises the following steps:

in a first aspect of the invention, there is provided a cell expressing a first protein capable of recognising one or more immune effector cells of a host; preferably, the cell has an inhibitory or killing function on an immune effector cell of the host.

In a preferred embodiment, the cell is an immune effector cell or an artificially engineered cell with immune effector cell function.

In a preferred embodiment, the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a CIK cell, and a stem cell-derived immune effector cell;

preferably, the cell is a T cell,

more preferably, the first protein is a chimeric receptor.

In a preferred embodiment, the cell further expresses a second protein recognizing a tumor antigen or a pathogen antigen, preferably the second protein is a chimeric receptor or a T cell receptor.

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

In a preferred embodiment, activation of the second receptor is regulated by a protein that recognizes an immune effector cell of the host.

In a preferred embodiment, the protein that recognizes the immune effector cell of the host and the activation of the second receptor do not interact.

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

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

In a preferred embodiment, said anti-transplant immunity is repellent against attack by NK cells of the host, or said first protein is capable of recognizing NK cells of the host,

preferably, the first protein is capable of specifically recognizing one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.

In a preferred embodiment, the first protein comprises an antibody capable of recognizing a host NK cell;

preferably, the antibody is capable of recognizing NKG 2A;

further preferably, the antibody comprises SEQ ID NO: 10, HCDR1 shown in SEQ ID NO: HCDR2 as shown in SEQ ID NO: HCDR3 as shown in SEQ ID NO: LCDR1 shown in SEQ ID NO: LCDR2 shown in SEQ ID NO: LCDR3 shown at 15;

still further preferably, the antibody comprises SEQ ID NO: 1 or the heavy chain variable region of SEQ ID NO: 2, or a light chain variable region as set forth in claim 2.

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.

In a preferred embodiment, the chimeric receptor is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

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

preferably, the cell mediates inhibition or killing of an immune effector cell of the host by signaling through an intracellular signaling domain.

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

preferably, the cell mediates inhibition or killing of a tumor or pathogen by signaling through an intracellular signaling domain.

In a preferred embodiment, the cell is a T cell with HLA-I gene and endogenous TCR gene silencing;

preferably, the cell is a B2M and TCR gene silenced T cell.

In a preferred embodiment, the second protein is capable of specifically recognizing BCMA or CD 19;

preferably, the second protein comprises an antibody capable of specifically recognizing BCMA;

further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17 HCDR2, SEQ ID NO: 18, and the HCDR3 shown in SEQ ID NO: LCDR1 shown in SEQ ID NO: LCDR2 shown in fig. 20, SEQ ID NO: LCDR3 shown at 21;

still further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 22 and SEQ ID NO: 23, or a light chain variable region as shown.

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

Preferably, the gene editing technology is selected from CRISPR/Cas9 technology, artificial 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.

In a preferred embodiment, the first protein comprises an antibody that recognizes a host immune effector cell, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain;

preferably, the antibody recognizing the host immune effector cell and the antibody recognizing the tumor antigen or the pathogen antigen are linked by a linker peptide;

still further preferably, the first protein has the amino acid sequence of SEQ ID NO: 9, and (c) 9.

In a preferred embodiment, the first protein and the second protein may be in a chimeric receptor, i.e., preferably, the chimeric receptor comprises, in sequence, an antibody that recognizes a host immune effector cell (the first protein), an antibody that recognizes a tumor antigen or a pathogen antigen (the second protein), a transmembrane domain, and an intracellular domain; or

The chimeric receptor comprises an antibody (second protein) recognizing a tumor antigen or a pathogen antigen, an antibody (first protein) recognizing a host immune effector cell, a transmembrane domain, and an intracellular domain, which are connected in sequence;

preferably, the antibody (first protein) recognizing the host immune effector cell and the antibody (second protein) recognizing the tumor antigen or the pathogen antigen are linked via a linker peptide.

In a second aspect of the invention, there is provided a cell for use in anti-transplant immune rejection, wherein the cell is a T cell having a T cell receptor capable of recognising one or more immune effector cells of a host, preferably wherein the cell has an inhibitory or killing function against an immune effector cell of the host.

In a preferred embodiment, the cell further expresses a second protein recognizing a tumor antigen or a pathogen antigen, preferably the second protein is a chimeric receptor.

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

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

In a preferred embodiment, the T cell receptor is capable of recognizing NK cells of the host,

preferably, the T cell receptor is capable of specifically recognizing one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.

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.

In a preferred embodiment, the second protein is a chimeric receptor selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC), the chimeric receptor comprising the second protein comprises a second protein, a transmembrane domain, and an endodomain,

preferably the second protein is capable of specifically recognizing BCMA or CD 19;

preferably, the second protein comprises an antibody capable of specifically recognizing BCMA;

further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, and the HCDR3 shown in SEQ ID NO: LCDR1 as shown in SEQ ID NO: LCDR2 shown in fig. 20, SEQ ID NO: LCDR3 shown at 21;

still further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 22 and SEQ ID NO: 23, or a light chain variable region as shown.

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

Preferably, the gene editing technology is selected from CRISPR/Cas9 technology, artificial 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.

In a third aspect of the invention, a method of preventing or modulating transplant immune rejection comprising administering a cell according to any one of the first or second aspects of the invention.

In a fourth aspect of the present invention, there is provided a method for preventing or regulating the attack of foreign cells by NK cells, characterized by administering an immune effector cell expressing a first protein that recognizes NK cells;

optionally, the exogenous cell is a T cell, NK T cell, stem cell, or an engineered T cell, NK T cell, stem cell;

optionally, the immune effector cell is administered before, after, or concurrently with the exogenous cell.

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

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

preferably, the chimeric receptor is selected from: chimeric Antigen Receptors (CARs), chimeric T cell receptors, T cell antigen couplers (TAC).

In a preferred embodiment, the antigen recognized by the first protein recognizing NK cells is one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.

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.

In a preferred embodiment, the second protein is a chimeric receptor selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC), the chimeric receptor comprising the second protein comprises a second protein, a transmembrane domain, and an endodomain,

preferably the second protein is capable of specifically recognizing BCMA or CD 19;

preferably, the second protein comprises an antibody capable of specifically recognizing BCMA;

further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17, HCDR2 shown in SEQ ID NO: 18, and the HCDR3 shown in SEQ ID NO: LCDR1 as shown in SEQ ID NO: LCDR2 shown in fig. 20, SEQ ID NO: LCDR3 shown at 21;

still further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 22 and SEQ ID NO: 23, or a light chain variable region as shown.

In a preferred embodiment, the cell of any of the first or second aspects of the invention is administered.

In a fifth aspect of the present invention, there is provided a method for preventing or regulating the attack of foreign cells by NK cells, comprising administering to an immune effector cell expressing a first protein that recognizes NK cells;

optionally, the exogenous cell is a T cell, NK T cell, stem cell, or an engineered T cell, NK T cell, stem cell.

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

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

preferably, the chimeric receptor is selected from: chimeric Antigen Receptors (CARs), chimeric T cell receptors, T cell antigen couplers (TAC).

In a preferred embodiment, the antigen recognized by the first protein recognizing NK cells is one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCR), such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.

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.

In a sixth aspect of the present invention, there is provided a method of preventing or regulating the attack of an exogenous immune effector cell by an NK cell, characterized in that the exogenous immune effector cell expresses a first protein that recognizes the NK cell;

preferably, the exogenous immune effector cell is a cell that does not contain an HLA-I gene or that is endogenous to the cell that is silenced by an HLA-I gene;

more preferably, the exogenous immune effector cell is a cell that does not contain the B2M gene or the B2M gene is silenced.

In a preferred embodiment, the exogenous immune effector cell is a T cell,

preferably, the first protein that recognizes NK cells is a chimeric receptor or a T cell receptor.

In a preferred embodiment, the antigen recognized by the first protein recognizing NK cells is one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.

In a preferred embodiment, the chimeric receptor is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC).

In a preferred embodiment, the exogenous immune effector cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen;

preferably, the second protein is a chimeric receptor selected from a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).

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 an antibody that recognizes NK cells and recognizes a tumor antigen or a pathogen antigen.

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

preferably, the cell mediates inhibition or killing of an immune effector cell of the host by signaling through an intracellular signaling domain.

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

preferably, the cell mediates inhibition or killing of a tumor or pathogen by signaling through an intracellular signaling domain.

In a preferred embodiment, the first protein comprises an antibody that recognizes a host immune effector cell, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain;

preferably, the antibody recognizing the host immune effector cell and the antibody recognizing the tumor antigen or the pathogen antigen are linked by a linker peptide;

still further preferably, the first protein has the amino acid sequence of SEQ ID NO: 9, and (c) 9.

Drawings

FIG. 1: NK cell surface marker expression;

FIG. 2: NK cell surface marker expression in T cells. (ii) a

FIG. 3: growth characteristics of NKG2A CAR-T cells. A, cell proliferation curve; b, cell diameter size; c, CAR positive rate;

FIG. 4: the in vitro killing capacity of NKG2A CAR-T cells on NK cells after 4 hours of co-incubation;

FIG. 5: (ii) in vitro killing ability of NKG2A CAR-T cells against NK cells after 18 hours of co-incubation;

FIG. 6: high-efficiency knockout of TCR and B2M in CAR-T cells;

fig. 7A, 7B, 7C, and 7D: FACS detects the resistance of NKG2A UCAR-T cells to NK cells;

FIG. 8: is a plasmid map of NKG 2A-targeted CARs;

FIG. 9: a plasmid map of a CAR targeting BCMA;

FIG. 10: FACS detects the resistance of NKG2A UCAR-T cells to NK cells;

FIG. 11: FACS detects UCAR-T cell survival in peripheral blood of mice;

FIG. 12: a schematic structure diagram of BCMA-GS-NKG2A UCAR-T;

FIG. 13: preparing BCMA-GS-NKG2A UCAR-T cells;

FIG. 14: the in vitro anti-tumor effect of BCMA-GS-NKG2A UCAR-T cells;

FIG. 15: BCMA-GS-NKG2A UCAR-T cells resist NK cells;

FIG. 16: is a plasmid map of PRRL-BCMA-BBZ-F2A-EGFP;

FIG. 17: is a plasmid map of PRRL-NKG 2A-28Z-F2A-EGFP;

FIG. 18 is a schematic view of: is a plasmid map of PRRL-BCMA-GS-NKG 2A-BBZ;

FIG. 19: the results of resistance of BCMA-GS-NKG2A UCAR-T cells to NK cells in vivo are shown.

Detailed Description

Unless specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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, with suitable methods and materials being 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting unless otherwise specified.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wileyand sonInc, Library of Congress, USA); molecular Cloning A Laboratory Manual, Third Edition, (Sambrookettal, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); oligonucleotide Synthesis (m.j. gaited., 1984); mullis et al.U.S. Pat. No.4,683,195; nucleic Acid Hybridization (B.D. Harries & S.J. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, Alan r.loss, 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-coef, Academic Press, inc., New York), In particular vols.154 and 155(wuetal. eds.) and vol.185, "Gene Expression Technology" (d.goeddel., ed.); gene Transfer Vectors For mammlian Cells (J.H.Miller and M.P.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, volumes I-IV (d.m.weir and c.c.blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

In the disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated or intervening value, to the upper or lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the claimed subject matter, also falling within the scope of that claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where a range is stated to include one or both of the limits, the claimed subject matter also includes ranges excluding either or both of the limits. This applies regardless of the breadth of the range.

The term about as used herein refers to the usual error range for each value as would be readily understood by one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that point to that value or parameter itself. For example, a description of "about X" includes a description of "X". For example, "about" or "including" can mean within 1 or more than 1 according to the actual standard deviation in the field. Alternatively, "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 5.5 uM. When particular values or compositions are provided in the application and claims, unless otherwise indicated, "about" or "including" is to be assumed to be within an acceptable error range for the particular value or composition.

Unless otherwise indicated, any concentration range, percentage range, proportion range, or integer range recited herein is to be understood as including any integer within the stated range and, where appropriate, fractional (e.g., tenth to hundredth of an integer) values thereof.

To facilitate a better understanding of the invention, the relevant terms are defined as follows:

the term "transplant immune rejection" refers to the condition in which after a host has been transplanted with an allogeneic tissue, organ, or cell transplant, the foreign transplant is recognized by the host's immune system as a "xenogenic component" and initiates an immunological response to attack, destruction, and removal of the transplant.

The term "graft" refers to a biological material or formulation derived from an individual other than the host and intended for implantation into the host. The graft may be from any animal source, such as mammalian sources, preferably from humans. In some embodiments, the graft may be derived from a host, e.g., cells from a host are cultured in vitro, or engineered to be reimplanted into a host. In some embodiments, the graft may be cells from other individuals, such as others, from an allogeneic source, cultured in vitro, or engineered into the host. In some embodiments, the transplant may be an organ-implanted human from a xenogeneic individual, such as from another species (e.g., murine, porcine, monkey).

The term "cell" and grammatical variations thereof can refer to a cell of human or non-human animal origin.

The term "host" refers to a recipient that receives a graft, and in some embodiments, may be an individual, such as a human, who has received an implant of exogenous cells.

The term "immune effector cell" refers to a cell involved in an immune response, producing an immune effect, such as a T cell, B cell, Natural Killer (NK) cell, natural killer T (nkt) cell, dendritic cell, CIK cell, macrophage, mast cell, and the like. In some embodiments, the immune effector cell is a T cell, NK cell, NKT cell. In some embodiments, the T cell may be an autologous T cell, a xenogenic T cell, an allogeneic T cell. In some embodiments, the NK cell may be an allogeneic NK cell.

The term "engineered cell having immune effector cell function" refers to a cell or cell line that has no immune effect and which has acquired immune effector cell function after being engineered or stimulated by a stimulus. Such as 293T cells, which are artificially modified to have the function of immune effector cells; such as stem cells, are induced in vitro to differentiate into immune effector cells.

In some cases, a "T cell" may be a bone marrow-derived pluripotent stem cell that differentiates and matures within the thymus into an immunocompetent mature T cell. In some cases, a "T cell" may be a population of cells with a particular phenotypic characteristic, or a mixed population of cells with different phenotypic characteristics, e.g., a "T cell" may be a cell comprising at least one T cell subpopulation: memory stem-like 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, a "T cell" may be a particular subset of T cells, such as γ δ T cells.

T cells can be obtained from a number of sources, including PBMCs, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain instances, T cells may be obtained from blood collected from an individual using any number of techniques known to those skilled in the art, such as ficoll (tm) separation. In one embodiment, the cells from the circulating blood of the individual are obtained by a single blood draw. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis may be washed to remove plasma molecules and placed in a suitable buffer or culture medium for subsequent processing steps. Alternatively, the cells may be derived from a healthy donor, from a patient diagnosed with cancer.

The term "MHC" is a histocompatibility complex, a generic term for all populations of genes encoding antigens of biocompatible complexes, which are expressed in all tissues of higher vertebrates, called HLA antigens in human cells, and play an important role in transplantation reactions, mediated by T cells responding to histocompatibility antigens on the surface of the implanted tissue. MHC proteins play a crucial 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 the MHC, and in the presence of a costimulatory signal, the 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 displaying foreign viral peptides. MHC antigens are classified as NHC class I antigens and MHC class II antigens.

The term "Human leukocyte antigen" (HLA) is the coding gene of the major histocompatibility complex of humans, located on chromosome 6 (6p21.31), and is closely related to the function of the Human immune system. HLA includes class I, class II and class III gene portions. The antigens expressed by HLA class I and II genes are located on the cell membrane and are MHC-I (encoded by HLA-A, HLA-B, HLA-C locus) and MHC-II (encoded by HLA-D region), HLA class I is distributed on the whole cell surface of the body, is a heterodimer consisting of a heavy chain (alpha chain) and beta 2 microglobulin (B2M), and class II is mainly glycoprotein located on the surfaces of macrophages and B lymphocytes.

The term "B2M" is β -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, as opposed to other MHC genes located as gene clusters on chromosome 6. It has been shown that hematopoietic transplants from mice lacking normal cell surface MHC I expression are rejected by NK cells in normal mice when the B2M gene is mutated, suggesting that defective expression of MHC I molecules makes cells susceptible to rejection by the host immune system (Bix et al 1991).

The term "chimeric receptor" refers to a fusion molecule formed by connecting corresponding cDNAs of DNA fragments or proteins of different origins by using gene recombination technology, and comprises an extracellular domain, a transmembrane domain and an intracellular domain. Chimeric receptors include, but are not limited to: chimeric Antigen Receptors (CARs), chimeric T Cell Receptors (TCRs), T cell antigen couplers (TACs).

The term "chimeric antigen receptor" (CAR) includes 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 co-stimulatory molecule, in one aspect, the stimulatory molecule is a delta chain that binds to a T cell receptor complex; in one aspect, 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 CD 28.

The term "T Cell Receptor (TCR)" mediates the recognition by T cells of specific Major Histocompatibility Complex (MHC) -restricted peptide antigens, including classical TCR receptors and optimized TCR receptors. The classical TCR receptor is composed of two peptide chains of alpha and beta, each peptide chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region, a cytoplasmic region and the like, the antigen specificity of the peptide chain exists in the V region, and each V region (V alpha and V beta) is provided with three hypervariable regions of CDR1, CDR2 and CDR 3.

The term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides that make up a TCR, which are capable of binding to a surface antigen on a target cell, and interacting with other polypeptides of the intact TCR complex, typically being co-localized on the T cell surface. The chimeric T cell receptor consists of a TCR subunit and an antigen-binding domain consisting of a human or humanized antibody domain, wherein the TCR subunit comprises at least part of a TCR extracellular domain, a transmembrane domain, a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, intracellular signaling domain of the TCR subunit is derived from CD3 epsilon or CD3 gamma, and wherein the chimeric T cell receptor is integrated into a TCR expressed on a T cell.

The term "T cell antigen coupler (TAC)", includes three functional domains: 1. antigen binding domains, including single chain antibodies, designed ankyrin repeat proteins (darpins), or other targeting groups; 2. an extracellular domain, a single chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; 3. the transmembrane region and the intracellular region of the CD4 co-receptor, where the intracellular region is linked to the protein kinase LCK, catalyses phosphorylation of Immunoreceptor Tyrosine Activation Motifs (ITAMs) of the TCR complex as an initial step in T cell activation.

The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell to modulate the activity of the cell via a defined signaling pathway, either by generating second messengers or by acting as an effector in response to such messengers. The intracellular signaling domain may comprise the entire intracellular portion of the molecule, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

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

The terms "activation" and "activation" are used interchangeably and may refer to the process by which a cell transitions from a resting state to an active state. The process may include a response to a phenotypic or genetic change in antigen, migration and/or functional activity status. For example, the term "activation" may refer to the process of stepwise activation of T cells. For example, T cells may require at least one signal to become fully activated.

The term "gene editing" refers to enabling a human to "edit" a target gene, to achieve knockout, addition, etc. of a specific DNA fragment.

The term "gene silencing" refers to the phenomenon of non-expression or low expression of a gene for various reasons. Gene silencing may be at the transcriptional level due to DNA methylation, heteropigmentation, position effects, etc., or post-transcriptional gene silencing, i.e., inactivation of a gene at the post-transcriptional level by specific inhibition of a target RNA, including antisense RNA, co-suppression, gene suppression, RNA interference, microrna-mediated translation suppression, etc.

By "TCR silencing" is meant that endogenous TCRs are not expressed or are under expressed.

By "MHC silencing" is meant endogenous MHC non-expression or low expression.

The term "crispr (clustered regular interspersed short palindromic repeats") refers to regularly clustered interspaced short palindromic repeats.

The term "Cas 9(CRISPRassociated nuclease)" is a CRISPR-associated nuclease, an RNA-guided technique for editing a targeted gene using Cas9 nuclease.

The "CRISPER/Cas 9 system" collectively refers to the transcripts and other elements involved in expression of or directing the activity of the Cas9 enzyme gene, including sequences encoding the Cas9 gene, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portions of tracrRNA), tracr mate sequences (encompassing "direct repeats" and partial direct repeats of tracrRNA processing in the context of an endogenous CRISPR system), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system, i.e., grnas), or other sequences and transcripts from the CRISPR locus.

The term "target sequence" refers to a sequence having complementarity to a guide sequence, complementary pairings between which promote formation of a CRISPR complex. A target sequence may 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.

In general, a guide sequence (gRNA) is any polynucleotide sequence that is sufficiently complementary to a target polynucleotide sequence to hybridize to the target sequence and to 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 is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, when optimally aligned using a suitable alignment algorithm. The optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman (Smith-Waterman) algorithm, the nidman-Wunsch (Needleman-Wunsch) algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., Burrows-Wheeler alignment tool), ClustalW, Clustal X, BLAT, Novoalign (Novocraft technologies), ELAND (illumana, san diego, ca), net (available in SOAP).

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 other than the CRISPR enzyme). The CRISPR enzyme fusion protein can comprise any other protein, and optionally a linker sequence between any two domains. Examples of protein domains that can 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 a histidine (His) tag, a V5 tag, a FLAG tag, an influenza virus Hemagglutinin (HA) tag, a Myc tag, a VSV-G tag, and a thioredoxin (Trx) tag.

The term "Cas 9 enzyme" may be a wild-type Cas9 or an artificially engineered Cas 9.

The term "sgRNA" refers to short grnas.

In gene editing, the gRNA, tracr mate sequence, and tracr sequence to be administered may be administered alone or as a complete RNA sequence.

The combination of the Cas9 protein and gRNA can realize the DNA cutting at a specific site, the CRISPR/Cas system recognition sequence derived from Streptococcus pyogenes is 23bp and can target 20bp, and the last 3-bit NGG sequence of the recognition site is called PAM (protospacer adjacent motif) sequence.

The Cas transgene may be delivered by a vector (e.g., AAV, adenovirus, lentivirus), and/or a particle and/or nanoparticle, and/or electroporation.

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

By "inhibiting" or "suppressing" expression of B2M or a TCR is meant that expression of B2M or a TCR 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% in a cell. More specifically, "inhibiting" or "suppressing" expression of B2M 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%. Expression or amount of protein in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, immunoblotting (Western Blotting) or flow cytometry using antibodies specific for B2M or TCR.

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

The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection may be accomplished by a variety of 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 (biolistics).

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

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

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

The term "T cell activation" or "T cell activation" and grammatical variations thereof can refer to the state of a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.

As used herein, the term "sequence" and grammatical variations thereof, when used in reference to a nucleotide sequence, can include DNA or RNA, and can be single-stranded or double-stranded.

The term "effective amount" as used herein refers to an amount that provides a therapeutic or prophylactic benefit.

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. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or 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).

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 ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. Non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like, may also be included.

The term "sequence identity" as used herein is determined by comparing two best matched sequences over a comparison window (e.g., at least 20 positions) wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps), e.g., a gap of 20% or less (e.g., 5 to 15%, or 10 to 12%) for the best matched two sequences as compared to the reference sequence (which does not comprise additions or deletions). The percentage is typically calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield 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 yield the percentage of sequence identity.

The term "exogenous" as used herein refers to a nucleic acid molecule or polypeptide, cell, tissue, etc., that does not function when expressed endogenously in the organism itself, or at a level insufficient to achieve overexpression.

The term "endogenous" refers to a nucleic acid molecule or polypeptide or the like that is derived from the organism itself.

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

Chimeric antigen receptors typically comprise an extracellular antigen-binding region. In some embodiments, the extracellular antigen-binding region can be fully human, humanized, murine, or a chimera within the extracellular antigen-binding region consists of amino acid sequences from at least two different animals.

Examples of extracellular antigen-binding regions may be scFv, Fv, Fab '-SH, F (ab')2, single domain fragments, or natural ligands that bind their cognate receptor, and any derivatives thereof.

In some aspects, the extracellular antigen-binding region (e.g., scFv), can comprise light chain CDRs specific for the antigen. In some cases, the light chain CDRs may comprise two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, and the like. In some cases, the light chain CDR can comprise 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 may be collectively referred to as light chain CDRs.

In some aspects, the extracellular antigen-binding region (e.g., scFv), can comprise heavy chain CDRs specific for an antigen. The heavy chain CDRs may be antigen binding units such as heavy chain complementarity determining regions of scFv. In some cases, a heavy chain CDR can comprise two or more heavy chain CDRs, which can be referred to as heavy chain CDR-1, CDR-2, and the like. In some cases, a heavy chain CDR can comprise 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 set of CDRs present on a common heavy chain may be collectively referred to as a heavy chain CDR.

By using genetic engineering, the extracellular antigen-binding region can be modified in various ways. In some cases, the extracellular antigen-binding region may be mutated such that the extracellular antigen-binding region may be selected to have a higher affinity for its target. In some cases, the affinity of the extracellular antigen-binding region for its target may be optimized for targets that may be expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity. In other cases, cloning of extracellular antigen-binding regions with higher affinity for the membrane-bound form of the target may be preferred over their soluble form counterparts. Such modifications can be made because different levels of soluble forms of the target can also be detected and their targeting can cause undesirable toxicity.

In some cases, the extracellular antigen-binding region also includes a hinge or a spacer, and the terms hinge and spacer are used interchangeably. The hinge can be considered to be part of the CAR for providing flexibility to the extracellular antigen-binding region. For example, the hinge may be the native hinge region of the CD8 a molecule.

The term "transmembrane domain" may anchor the chimeric protein to the plasma membrane of a cell. For example, transmembrane domains of CD28, CD8 α may be employed.

The term "modulate" refers to a positive or negative change. Examples of adjustments include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes. In one embodiment, a negative change is meant.

The term "treatment" refers to an intervention in an attempt to alter the disease process, either prophylactically or during a clinical pathology. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating a disease, alleviating or improving prognosis, and the like.

The term "prevention" refers to intervention prior to the onset of an attempt to cause a disease, such as rejection by cell transplantation.

The first protein of the present invention refers to the above-mentioned protein capable of recognizing one or more immune effector cells of a host.

The second protein of the present invention refers to the above-mentioned protein recognizing a tumor antigen or a pathogen antigen.

The "second receptor" of the present invention and the "protein capable of recognizing one or more immune effector cells of the host" may be expressed in tandem or may be expressed individually.

When the "second receptor" is expressed separately from the "protein capable of recognising one or more immune effector cells of the host", they have separate transmembrane and intracellular domains, and their expression may be determined by reference to PCT/CN2015/095938, engineering the specificity of T-cell cultures for adaptive immunization of cancer, Duong CP et al, Immunothiapy 3(1):33-48, etc.

When a "second receptor" according to the present invention is expressed in tandem with a "protein capable of recognizing one or more immune effector cells of a host", the protein capable of recognizing one or more immune effector cells of the host is also capable of recognizing an antigen recognized by the "second receptor", such as a tumor antigen.

"tumor antigen" refers to an antigen that is newly present or overexpressed in the development, progression, or progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorders of the present invention refer to cancer.

The tumor antigen of the invention can be a solid tumor antigen or a blood tumor antigen.

Tumor antigens of the invention include, but are not limited to: thyroid Stimulating Hormone Receptor (TSHR); CD 171; CS-1; c-type lectin-like molecule-1; gangliosides GD 3; tn antigen; CD 19; CD 20; CD 22; CD 30; CD 70; CD 123; CD 138; CD 33; CD 44; CD44v 7/8; CD 38; CD44v 6; B7H3(CD276), B7H 6; KIT (CD 117); interleukin 13 receptor subunit alpha (IL-13 ra); interleukin 11 receptor alpha (IL-11R α); prostate Stem Cell Antigen (PSCA); prostate Specific Membrane Antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp 100; a tyrosinase enzyme; mesothelin; EpCAM; protease serine 21(PRSS 21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2(VEGFR 2); a lewis (Y) antigen; CD 24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); cell surface associated mucin 1(MUC1), MUC 6; epidermal growth factor receptor family and mutants thereof (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural Cell Adhesion Molecule (NCAM); carbonic anhydrase ix (caix); LMP 2; ephrin type a receptor 2(EphA 2); fucosyl GM 1; sialyl lewis adhesion molecule (sLe); ganglioside GM 3; TGS 5; high Molecular Weight Melanoma Associated Antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD 2); a folate receptor; tumor vascular endothelial marker 1(TEM1/CD 248); tumor vascular endothelial marker 7-associated (TEM 7R); claudin 6, Claudin18.2, Claudin 18.1; ASGPR 1; CDH 16; 5T 4; 8H 9; α v β 6 integrin; b Cell Maturation Antigen (BCMA); CA 9; kappa light chains (kappa light chain); CSPG 4; EGP2, EGP 40; FAP; FAR; FBP; embryonic type AchR; HLA-A1, HLA-A2; MAGEA1, MAGE 3; KDR; MCSP; NKG2D ligand; PSC 1; ROR 1; sp 17; SURVIVIN; TAG 72; a TEM 1; fibronectin; tenascin; carcinoembryonic variants of the necrotic area of the tumor; g protein-coupled receptor class C group 5-member D (GPRC 5D); x chromosome open reading frame 61(CXORF 61); CD 97; CD179 a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1(PLAC 1); hexose portion of globoH glycoceramide (globoH); mammary differentiation antigen (NY-BR-1); uroplakin 2(UPK 2); hepatitis a virus cell receptor 1(HAVCR 1); adrenergic receptor β 3(ADRB 3); pannexin 3(PANX 3); g protein-coupled receptor 20(GPR 20); lymphocyte antigen 6 complex locus K9(LY 6K); olfactory receptor 51E2(OR51E 2); TCR γ alternate reading frame protein (TARP); wilms tumor protein (WT 1); ETS translocation variant gene 6(ETV 6-AML); sperm protein 17(SPA 17); x antigen family member 1A (XAGE 1); angiogenin binds to cell surface receptor 2(Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-related antigen 1; a p53 mutant; human telomerase reverse transcriptase (hTERT); a sarcoma translocation breakpoint; melanoma inhibitors of apoptosis (ML-IAP); ERG (transmembrane protease serine 2(TMPRSS2) ETS fusion gene); n-acetylglucosaminyltransferase V (NA 17); paired box protein Pax-3(PAX 3); an androgen receptor; cyclin B1; a V-myc avian myelomatosis virus oncogene neuroblastoma-derived homolog (MYCN); ras homolog family member c (rhoc); cytochrome P4501B 1(CYP1B 1); CCCTC binding factor (zinc finger protein) like (BORIS); squamous cell carcinoma antigen 3 recognized by T cells (SART 3); paired box protein Pax-5(PAX 5); proacrosin binding protein sp32(OYTES 1); lymphocyte-specific protein tyrosine kinase (LCK); a kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2(SSX 2); CD79 a; CD79 b; CD 72; leukocyte-associated immunoglobulin-like receptor 1(LAIR 1); fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2(LILRA 2); CD300 molecular-like family member f (CD300 LF); c-type lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2(BST 2); comprising EGF-like module mucin-like hormone receptor-like 2 (EMR 2); lymphocyte antigen 75(LY 75); glypican-3 (GPC 3); fc receptor like 5(FCRL 5); immunoglobulin lambda-like polypeptide 1(IGLL 1). Preferably, the tumor antigen is BCMA or CD 19.

The pathogen antigen is selected from: an antigen of a virus, bacterium, fungus, protozoan, or parasite; the viral antigen is selected from: a cytomegalovirus antigen, an epstein-barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBrook et al, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 detection of NK cell surface receptor expression

Using Ficoll-Paque (GE bioscience) to carry out density gradient centrifugation, separating mononuclear cells from peripheral blood, carrying out negative screening by using an NK cell separation kit (purchased from Meitian and whirlwind), removing cells such as T cells, B cells and mononuclear cells, and then carrying out in vitro cell phenotype identification and amplification. Isolated NK cell surface receptors, such as NKG2A, NKG2D, NKP30, NKP44, NKP46, were identified by flow staining. Flow cytometry results showed that NKG2A, NKP30, NKP44, NKP46 were expressed in around 80% of NK cells, while NKG2D was expressed in more than 90% of NK cells (see fig. 1).

Furthermore, we also examined the expression of the above surface markers in T cells. CD3/CD28 magnetic beads (purchased from Thermo Fisher) activated T cells were cultured until day 8 for flow staining. The experimental results show that NKG2A, NKG2D, NKP30, NKP44 and NKP46 are hardly expressed in T cells, and the markers can be used as targets for targeting NK cells (see FIG. 2).

Example 2 preparation and functional validation of CAR-T cells

1. NKG2A is selected as a target point to be used as a representative, and the CAR-T cell targeting the NK cell is prepared. By referring to the conventional operation, a vector containing a single-chain antibody against NKG2A (the amino acid sequence of VH is shown in SEQ ID NO: 1, 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), a chimeric antigen receptor of T cell activator CD3 delta (the amino acid sequence is shown in SEQ ID NO: 4) (the amino acid sequence is shown in SEQ ID NO: 5) is designed and constructed, the plasmid map is shown in FIG. 8, and the packaged lentivirus is named as VRRL-NKG2A-28Z (TM).

After 48 hours of T cell activation and expansion, cell density was adjusted to 2 x 10^6/mL, and VRRL-NKG2A-28Z (TM) lentivirus was added at a MOI of 10 to obtain NKG 2A-targeted CAR-T cells.

Taking CAR-T cells on day 6 for cell proliferation detection, adjusting initial cell number to 5 x 10^5, detecting cell number at 24hr,48hr,72hr and 96hr respectively, and recording cell diameter. And meanwhile, carrying out flow cytometry staining by using an anti-F (ab)' 2 antibody, and detecting the expression condition of the CAR vector. The experimental results showed that NKG2A CAR-T cells showed similar growth curves to untransfected T cells (UTDs), with no significant difference in cell diameter size, and about 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).

2. Preparation of BCMA-targeted CAR-T cells

By referring to the operation of 1, a plasmid (the amino acid sequence is shown in SEQ ID NO: 6) targeting the chimeric antigen receptor of BCMA is constructed, the plasmid map is shown in FIG. 9, lentivirus is packaged, and T cells are transfected to obtain BCMA-CAR T cells targeting BCMA.

3. In vitro killing function experiment.

Amplifying primary NK cells in vitro to serve as target cells. Adjusting cell density to 5 x 10^5/mL, seeding 100 μ l into 96-well plates (3 duplicate wells in parallel), and following the ratio of effector T cells: the target cells are 1: 3,1: 1 and 3: 1 three ratios were inoculated with the corresponding CAR-T cells. MEM-alpha + 5% FBS was used as a medium at 37 ℃ in 5% CO₂Incubate in the incubator for 4hr and 18hr, respectively. Using cytox-96 non-radioactive cytoxicity assay kit (purchased from Thermo Fisher), 50. mu.l of supernatant was taken for Lactate Dehydrogenase (LDH) content determination, and the lysis efficiency of primary NK cells in both UTD and NKG2A CAR-T groups was calculated.

The detection results show that the LDH value in the NKG2A CAR-T group is significantly higher than that in the UTD group, indicating that NKG2A CAR-T can effectively kill primary NK cells (see FIG. 4 and FIG. 5).

Example 3 preparation of NKG2A UCAR-T cells

Knock-out of the TCR and B2M genes.

Cell density was adjusted to 2 x 10^7/mL 48 hours after expansion of conventional UTD cells, NKG2A CAR-T cells and BCMA CAR-T cells (for control). Cas9 enzyme (purchased from NEB) and sgRNA as 1: and 4, incubating at room temperature for 10 minutes to obtain RNP complex liquid, wherein the nucleic acid sequence of TRAC-sgRNA is shown as SEQ ID NO: 7, the nucleic acid sequence of B2M-sgRNA is shown in SEQ ID NO: 8, 1 x 10^6 cells were mixed with RNP complex solution (final concentration of Cas9 enzyme 3uM), and RNP complexes were introduced into CAR-T cells using a maxcyte electrotransfer. 7 days after electrotransformation, the knockout of TCR and B2M genes was detected by flow cytometry, and the experimental results showed that the knockout efficiency of TRAC and B2M was above 85% (see FIG. 6).

TCR/B2M double negative cell screen.

The method comprises the steps of carrying out in-vitro amplification on CAR-T cells and UTD cells for knocking out B2M and TCR, adjusting the cell density to 1 x 10^7/mL on the 8 th day, marking the cells by using anti-HLA-ABC and B2M antibodies, marking the cells by using secondary antibody coupled with Phycoerythrin (PE), sorting the marked cells by using anti-PE magnetic beads through a sorting column, and collecting TCR and B2M double-negative cells (a sorting kit is purchased from Meitian and Japan) to obtain TCR and B2M-deleted BCMA UCAR-T cells, NKG2A UCAR-T cells and U-UTD cells.

Example 4 verification of NK cell resistant function by NKG2A UCAR-T cells

LDH (layered double hydroxide) experiment for detecting rejection of UCAR-T cells to NK cells

Using UTD cells, BCMA-CAR T cells, NKG2A CAR-T cells, BCMA UCAR-T cells, NKG2A UCAR-T cells and U-UTD cells as target cells, adjusting the cell concentration to 5 x 10^5/mL, inoculating 100 μ l to a 96-well plate, and performing primary amplification on NK cells and the target cells according to the ratio of 1: 1, inoculating NK cells of the same volume and number, and incubating in an incubator for 4hr and 18hr, respectively. 50 μ l of supernatant was taken for Lactate Dehydrogenase (LDH) assay and the lysis efficiency of CAR-T and UCAR-T cells was calculated. The detection results show that the LDH values of the UTD and BCMA CAR-T groups are low, which indicates that the common CAR-T cells do not cause the attack of NK cells, while the U-UTD and BCMA UCAR-T groups show gradually increased LDH values at 4hr and 18hr, which indicates that the NK cells kill T cells with TCR and B2M deficiency, and NKG2A UCAR-T cells show lower levels of LDH, which indicates that the NKG2A UCAR-T cells have resistance effect on the NK cells.

2. To further confirm the resistance of NKG2A UCAR-T cells to NK cells, BCMA UCAR-T cells were selected for control, cell concentration was adjusted to 5 x 10^5/mL, 100. mu.l was inoculated into 96-well plates, and the ratio of primary expanded NK cells to target cells was 1: 1, inoculating NK cells of the same volume and number, and incubating in an incubator for 4hr,18hr,24hr and 42hr, respectively. And (3) marking the NK cells with positive HLA-ABC by using flow cytometry, and detecting the proportion of UCAR-T cells at different time points of co-incubation. The experimental results are shown in fig. 7A-7D, the BCMA UCAR-T is at a low rate of about 20% at 4hr, and is at a very low rate all the time as the detection time is prolonged, indicating that NK cells significantly inhibit the growth of BCMA UCAR-T cells; however, NKG2A UCAR-T cells were found to be low at about 20% at 4hr, but showed a gradually increasing proportion as the time of detection increased, reaching nearly 60% at 42hr, indicating that the NKG2A UCAR-T cells were initially inhibited by NK cells but gradually restored the proliferative capacity as the time increased. The results show that NKG2A UCAR-T can effectively resist the killing capacity of NK cells.

3. To further demonstrate the resistance of NKG2A UCAR-T cells to primary NK cells, BCMA UCAR-T cells expressing GFP and NKG2A UCAR-T cells were constructed. The amino acid sequence of BCMA-GFP is shown as SEQ ID NO: 24, the amino acid sequence of NKG2A-GFP is shown as SEQ ID NO: shown at 25.

With reference to the procedures of example 2, plasmid PRRL-BCMA-BBZ-F2A-EGFP of BCMA UCAR-T cells expressing GFP was constructed, the plasmid map is shown in FIG. 16, and plasmid PRRL-NKG2A-28Z-F2A-EGFP of NKG2A UCAR-T cells was constructed, the plasmid map is shown in FIG. 17. The constructed plasmid is used for packaging lentivirus, transfecting T cells, and performing gene knockout and magnetic bead sorting on CAR-T cells to obtain BCMA UCAR-T cells expressing GFP and NKG2A UCAR-T cells expressing GFP.

Adjusting CAR-T cell concentration to 5 x 10^5/mL, plating 100 μ l to 96-well plates, in a primary expanded NK cell to target cell ratio of 1: 1, inoculating NK cells of the same volume and number, and incubating in an incubator for 0hr,4hr,18hr,24hr and 48hr, respectively. The proportion of GFP cells at different time points of co-incubation was determined by flow cytometry and used to track the survival of UCAR-T cells.

The experimental results are shown in fig. 10, the proportion of GFP-positive BCMA UCAR-T cells is gradually decreased with the time being prolonged, and the GFP-positive NKG2A UCAR-T cells are basically completely killed by NK cells after 48hr, while the proportion of GFP-positive NKG2A UCAR-T cells is slightly decreased after 4hr, and is obviously increased after 18hr and accounts for about 90% after 48hr, which indicates that NKG2A UCAR-T cells can significantly resist the killing of NK cells.

Example 5 resistance of NKG2A UCAR-T cells to NK cells in vivo

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

Equal amounts of NK cells were injected 4hr after UCAR T cell administration, and survival of human CD4 and CD8T cells in mouse peripheral blood was examined by flow absolute technique on days 1, 3, and 6 after CAR T cell injection, respectively.

The results of the experiment are shown in FIG. 11, and on day 1 after injection, the numbers of UCAR-T cells (i.e., human-derived CD4 and CD8T cells) in BCMA UCAR-T + NK group and NKG2A UCAR-T + NK group were significantly decreased, indicating that UCAR-T cells were rejected by NK cells. On days 3 and 6 post-injection, the numbers of UCAR-T cells in the BCMA UCAR-T + NK group were always in a very low state, while the numbers of UCAR-T cells in the NKG2A UCAR-T + NK group appeared to rise significantly on days 3 and 6. The results show that in an in vivo model, the NK cells obviously inhibit the survival of BCMA UCAR-T cells, and the NKG2A UCAR-T cells can effectively resist the killing of the NK cells and restore the proliferation capacity.

Example 6 construction of BCMA and NKG 2A-Targeted CAR T cells

As shown in FIG. 12, UCAR-T cells (i.e., BCMA-GS-NKG2A UCAR-T) were generated in tandem with BCMA-targeting scFv and NKG 2A-targeting scFv. The amino acid sequence of BCMA-GS-NKG2A CAR is shown as SEQ ID NO: shown at 9.

The plasmid PRRL-BCMA-GS-NKG2A-BBZ of BCMA-GS-NKG2A UCAR-T was constructed, and the plasmid map is shown in FIG. 18. By referring to the procedures of examples 2 and 3, virus transfection was performed to obtain BCMA-GS-NKG2A UCAR-T cells, TRAC and B2M genes were knocked out from the BCMA-GS-NKG2A UCAR-T cells, and 99% or more of TCR-and HLA-ABC-negative BCMA-GS-NKG2A UCAR-T cells were obtained by magnetic bead sorting.

BCMA UCAR-T cells and NKG2A UCAR-T cells were prepared according to the procedures of examples 2 and 3, respectively.

The CAR expression conditions of BCMA UCAR-T, NKG2A UCAR-T and BCMA-GS-NKG2A UCAR-T are respectively detected, the experimental results are shown in figure 13, the positive rates are all over 60%, and the success of the preparation of BCMA-GS-NKG2A UCAR-T cells is shown.

Example 7 in vitro functional validation of BCMA-GS-NKG2A UCAR-T cells

In vitro culture of BCMA positive multiple myeloma cell lines RPMI-8226 and NCI-H929 as target cells, seeding 1 x 10^4 tumor cells in 96-well plates, 3: 1,1: 1 and 1: 3 inoculating a corresponding number of UCAR-T cells, incubating for 18 hours, and sucking 50 ul of supernatant for detecting the LDH content.

As shown in FIG. 14, both UTD and NKG2A UCAR-T groups showed low cell lysis rates for RPMI-8226 and NCI-H929; the tumor cell lysis rate of the BCMA-GS-NKG2A UCAR-T group is equivalent to that of the BCMA UCAR-T group, and the result shows that the BCMA-GS-NKG2A UCAR-T cells can effectively kill BCMA positive tumor cells in vitro.

Example 8 verification of NK cell resistant function by BCMA-GS-NKG2A UCAR-T cells

Selecting BCMA UCAR-T and NKG2A UCAR-T cells as negative and positive controls, adjusting the cell concentration to 5 x 10^5/mL, inoculating 100 mu l to a 96-pore plate, and mixing the positive control and the negative control with the cell concentration of NKG2A UCAR-T cells according to the ratio of NK cells to T cells of 1: 1, inoculating NK cells of the same volume and number, and incubating in an incubator for 0hr,4hr,18hr,24hr and 48hr, respectively. And (3) marking the NK cells with positive HLA-ABC by using flow cytometry, and detecting the proportion of UCAR-T cells at different time points of co-incubation. The experimental result is shown in fig. 15, the proportion of BCMA UCAR-T cells is gradually reduced along with the prolonging of the incubation time, and 48hr is basically killed by NK cells; the BCMA-GS-NKG2A UCAR-T cells and the NKG2A UCAR-T cells show the same change trend, slightly decrease in 4hr and then gradually increase, wherein 48hr reaches more than 70%, and the BCMA-GS-NKG2A UCAR-T cells reach 90% in 48 hr. The results show that the BCMA-GS-NKG2A UCAR-T cells can effectively resist the killing of NK cells.

Example 9 resistance of BCMA-GS-NKG2A UCAR-T cells to NK cells in vivo

In vitro culture of BCMA UCAR-T and BCMA-GS-NKG2A UCAR-T cells, adjusting CAR positivity to 60%, injection into NPG immunodeficient mice via tail vein at a dose of 8 x 10^6 cells/mouse, dividing the mice into two groups: the BCMA UCAR-T and NK cell groups (labeled BCMA UCAR-T + NK) and the BCMA-GS-NKG2A UCAR-T and NK cell groups (labeled BCMA-GS-NKG2A UCAR-T + NK) were administered. Equal amounts of NK cells were injected 4 hours after UCAR-T cell administration, and survival of human-derived CD45 positive T cells in peripheral blood of mice was examined by flow absolute technique on days 1, 3, and 6 after CAR T cell injection, respectively.

The experimental results are shown in FIG. 19, and compared to day 1 after injection, no significant increase in the number of UCAR-T cells in BCMA UCAR-T + NK group UCAR-T cells occurred on day 3 and day 6, indicating that UCAR-T cells were rejected by NK cells. The number of UCAR-T cells in the BCMA-GS-NKG2A UCAR-T + NK group is obviously increased on the 3 rd day and the 6 th day, and the number of the cells on the 6 th day is increased by more than 30 times compared with the number of the cells on the 1 st day. The results show that in an in vivo model, the NK cells obviously inhibit the survival of the BCMA UCAR-T cells, and the BCMA-GS-NKG2A UCAR-T cells can effectively resist the killing of the NK cells and restore the proliferation capacity.

The sequences involved in the present application are shown in the following table:

Claims (38)

1. a cell that is resistant to transplant immune rejection, wherein the cell expresses a first protein that recognizes one or more immune effector cells of the host; preferably, the cell has an inhibitory or killing function on an immune effector cell of the host.
2. The cell of claim 1, wherein the cell is an immune effector cell or an artificially engineered cell with immune effector cell function.
3. The cell of claim 1 or 2, wherein the cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a macrophage, a CIK cell, and a stem cell-derived immune effector cell;

   preferably, the cell is a T cell,

   more preferably, the first protein is a chimeric receptor.
4. The cell of any one of claims 1-3, wherein the cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen, preferably wherein the second protein is a chimeric receptor or a T cell receptor.
5. The cell of any one of claims 1-4, wherein the cell does not express MHC, or wherein an MHC gene endogenously expressed by the cell is silenced; preferably, the MHC gene is a gene of an MHC class I molecule.
6. The cell of claim 5, wherein said cell does not express HLA, or wherein an HLA gene endogenously expressed by said cell is silenced; preferably, the HLA is an HLA-class I gene.
7. The cell of any one of claims 1-6, wherein said anti-transplant immunity is directed against challenge by NK cells of the host, or wherein said first protein is capable of recognizing NK cells of the host,

   preferably, the first protein is capable of specifically recognizing one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.
8. The cell of claim 7, wherein the first protein comprises an antibody capable of recognizing a host NK cell;

   preferably, the antibody is capable of recognizing NKG 2A;

   further preferably, the antibody comprises SEQ ID NO: 10, HCDR1 shown in SEQ ID NO: HCDR2 as shown in SEQ ID NO: HCDR3 as shown in SEQ ID NO: LCDR1 shown in SEQ ID NO: LCDR2 shown in fig. 14, SEQ ID NO: LCDR3 shown at 15;

   still further preferably, the antibody comprises SEQ ID NO: 1 or the heavy chain variable region of SEQ ID NO: 2, or a light chain variable region as set forth in claim 2.
9. The cell of claim 8, wherein 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.
10. The cell of claim 3 or 4, wherein the chimeric receptor is selected from a Chimeric Antigen Receptor (CAR), a chimeric T-cell receptor, or a T-cell antigen coupler (TAC).
11. The cell of claim 1, wherein the first protein comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

    preferably, the cell mediates inhibition or killing of an immune effector cell of the host by signaling through an intracellular signaling domain.
12. The cell of claim 4, wherein the two proteins comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

    preferably, the cell mediates inhibition or killing of a tumor or pathogen by signaling through an intracellular signaling domain.
13. The cell of claim 6, wherein the cell is a T cell with HLA-I gene and endogenous TCR gene silencing;

    preferably, the cell is a B2M and TCR gene-silenced T cell.
14. The cell of claim 4, wherein the second protein is capable of specifically recognizing BCMA or CD 19;

    preferably, the second protein comprises an antibody capable of specifically recognizing BCMA;

    further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17 HCDR2, SEQ ID NO: 18, and the HCDR3 shown in SEQ ID NO: LCDR1 as shown in SEQ ID NO: LCDR2 shown in fig. 20, SEQ ID NO: LCDR3 shown at 21;

    still further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 22 and SEQ ID NO: 23, or a light chain variable region as shown.
15. The cell of claim 5, 6 or 13, wherein gene editing techniques are used to silence the gene.
16. The cell of claim 10, wherein the first protein comprises an antibody that recognizes a host immune effector cell, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain;

    preferably, the antibody recognizing the host immune effector cell and the antibody recognizing the tumor antigen or the pathogen antigen are linked by a linker peptide;

    still further preferably, the first protein has the amino acid sequence of SEQ ID NO: 9, and (c) 9.
17. A cell that is resistant to transplant immune rejection, wherein said cell is a T cell that has a T cell receptor capable of recognizing one or more immune effector cells of a host, preferably wherein said cell has an inhibitory or killing function against an immune effector cell of a host.
18. The cell of claim 17, wherein the cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen, preferably wherein the second protein is a chimeric receptor.
19. The cell of any one of claims 17 or 18, wherein the cell does not express MHC, or wherein an MHC gene endogenously expressed by the cell is silenced; preferably, the MHC gene is a gene of an MHC class I molecule.
20. The cell of claim 19, wherein the cell does not express HLA or is silenced for HLA genes endogenously expressed by the cell; preferably, the HLA is an HLA-class I gene.
21. The cell of any one of claims 17-20, wherein said T cell receptor is capable of recognizing a host's NK cell,

    preferably, the T cell receptor is capable of specifically recognizing one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.
22. The cell of claim 20, wherein 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.
23. The cell of claim 18, wherein the second protein is a chimeric receptor selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T-cell receptor, and a T-cell antigen coupler (TAC), wherein the chimeric receptor comprising the second protein comprises the second protein, a transmembrane domain, and an endodomain,

    preferably the second protein is capable of specifically recognizing BCMA or CD 19;

    preferably, the second protein comprises an antibody capable of specifically recognizing BCMA;

    further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17 HCDR2, SEQ ID NO: 18, and the HCDR3 shown in SEQ ID NO: LCDR1 as shown in SEQ ID NO: LCDR2 shown in fig. 20, SEQ ID NO: LCDR3 shown at 21;

    still further preferably, the antibody specifically recognizing BCMA comprises SEQ ID NO: 22 and SEQ ID NO: 23, or a light chain variable region as shown.
24. A method of preventing or modulating transplant immune rejection comprising administering the cell of any one of claims 1-23.
25. A method for preventing or regulating the attack of foreign cells by NK cells, characterized by administering immune effector cells expressing a first protein that recognizes NK cells;

    optionally, the exogenous cell is a T cell, NK T cell, stem cell, or an engineered T cell, NK T cell, stem cell.
26. The method of claim 25, wherein the exogenous cell is an immune effector cell, preferably wherein the exogenous cell expresses a second receptor.
27. The method of claim 26, wherein the second receptor is a chimeric receptor or a T cell receptor;

    preferably, the chimeric receptor is selected from: chimeric Antigen Receptors (CAR), chimeric T cell receptors, T cell antigen couplers (TAC).
28. The method of claim 25, wherein the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killing immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.
29. The method of claim 25, wherein the immune effector cells comprise T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells.
30. A method of preventing or regulating the attack of an exogenous immune effector cell by an NK cell, wherein the exogenous immune effector cell expresses a first protein that recognizes the NK cell;

    preferably, the exogenous immune effector cell is a cell that does not contain an HLA-I gene or that is endogenous to HLA-I gene silenced;

    more preferably, the exogenous immune effector cell is a cell that does not contain the B2M gene or the B2M gene is silenced.
31. The method of claim 30, wherein the exogenous immune effector cell is a T cell,

    preferably, the first protein that recognizes NK cells is a chimeric receptor or a T cell receptor.
32. The method of claim 31, wherein the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C and the like; killer immunoglobulin-like receptor (KIR) families, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, and the like; natural Cytotoxic Receptors (NCRs) such as NKP30, NKP44, NKP46, NKP80 and the like; and other NK cell-specific expressed antigens, 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 two or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP 46.
33. The method of claim 32, wherein the chimeric receptor is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC).
34. The method of any one of claims 30-33, wherein the exogenous immune effector cell further expresses a second protein that recognizes a tumor antigen or a pathogen antigen;

    preferably, the second protein is a chimeric receptor selected from a Chimeric Antigen Receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).
35. The method of claim 34, wherein the first protein is a chimeric antigen receptor, a chimeric T cell receptor, or a T cell antigen coupler (TAC) comprising an antibody that recognizes NK cells and recognizes a tumor antigen or a pathogen antigen.
36. The cell of claim 30, wherein the first protein comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

    preferably, the cell mediates inhibition or killing of an immune effector cell of the host by signaling through an intracellular signaling domain.
37. The cell of claim 34, wherein the two proteins comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain;

    preferably, the cell mediates inhibition or killing of a tumor or pathogen by signaling through an intracellular signaling domain.
38. The cell of claim 30, wherein the first protein comprises an antibody that recognizes a host immune effector cell, an antibody that recognizes a tumor antigen or a pathogen antigen, a transmembrane domain, and an intracellular domain;

    preferably, the antibody recognizing the host immune effector cell and the antibody recognizing the tumor antigen or the pathogen antigen are linked by a linker peptide;

    still further preferably, the first protein has the amino acid sequence of SEQ ID NO: 9, and (c) a sequence shown in figure 9.

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