JP2024500254A - Cells and methods for resisting transplant rejection - Google Patents

Abstract

Concerning cells that resist transplant immune rejection. The cell expresses a first protein capable of recognizing one or more immune effector cells of the host. Preferably, the cells have an inhibitory or killing function against immune effector cells of the host. It also relates to methods for preventing or regulating transplant immune rejection, and methods for preventing or regulating NK cell attack on exogenous cells. [Selection diagram] None

Description

The present invention relates to cells that have the function of resisting transplant immune rejection. The present invention also relates to methods of resisting transplant immune rejection, particularly methods of resisting immune rejection of NK cells.

Due to immunogenetic differences between donor and recipient, when a foreign source donor is transplanted, as a foreign source graft, the donor is recognized and attacked by immune cells in the recipient's body, thereby eliminating the foreign source graft. host-versus-graft reaction (HVGR) may occur. Knocking out MHC molecules within transplanted cells can effectively resist host T cell rejection of the transplant, but may cause rejection of other immune cells within the host. For example, in allogeneic cell transplantation, the absence of MHC-I molecules in allogeneic cells causes rejection of NK cells in the host, promoting elimination of allogeneic cells (Nat Biotechnol. 2017;35(8):765-772.doi:10.1038/nbt.3860). Therefore, methods to effectively prevent immune rejection of host NK cells are of great importance for the development of allogeneic cell transplantation therapy.

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

The technical solutions provided by the present invention include the following.

In a first aspect of the invention, a first protein is expressed that is capable of recognizing one or more immune effector cells of the host, and preferably has an inhibitory or killing function towards the immune effector cells of the host. Cells are provided.

In one preferred embodiment, the cell is an immune effector cell or an artificially modified cell with the function of an immune effector cell.

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

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

In a preferred embodiment, activation of said protein recognizing host immune effector cells is regulated by said second receptor.

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

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

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 of an MHC class I molecule.

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

In a preferred embodiment, the anti-transplant immune rejection is resisting attack from host NK cells, or the first protein is capable of recognizing host NK cells,
Preferably, said first protein is a member of the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C; Killing of IR3DS1 etc. Immunoglobulin-like receptor (KIR) family; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16 , can specifically recognize one or more of other antigens specifically expressed by NK cells, such as CD161;
More preferably, the first protein is capable of specifically recognizing one or more NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

In one preferred embodiment, the first protein comprises an antibody capable of recognizing host NK cells,
Preferably, the antibody is capable of recognizing NKG2A,
More preferably, the antibody comprises HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 14, 15,
Even more preferably, said antibody comprises a heavy chain variable region as set forth in SEQ ID NO:1 or a light chain variable region as set forth in SEQ ID NO:2.

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

In one 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).

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

In one preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain,
Preferably, said cells transmit signals through intracellular signaling domains to mediate inhibition or killing of tumors or pathogens.

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

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

In one preferred embodiment, genes are silenced using gene editing techniques.

Preferably, the gene editing technology is CRISPR/Cas9 technology, Zinc Finger Nucleases (ZFN) technology, transcription activator-like effector nuclease (TALE) technology, or T ALE-CRISPR/ Selected from Cas9 technology,
More preferably, said 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 that recognizes a host immune effector cell and the antibody that recognizes a tumor antigen or pathogen antigen are linked by a connecting peptide,
More preferably, the first protein has the sequence shown in SEQ ID NO:9.

In one preferred embodiment, the first protein and the second protein may be in one chimeric receptor. That is, preferably, the chimeric receptor comprises an antibody that recognizes a host immune effector cell (first protein), an antibody that recognizes a tumor antigen or a pathogen antigen (second protein), and a transmembrane receptor that are linked in order. domain, and an intracellular domain, or
The chimeric receptor comprises an antibody that recognizes a tumor or pathogen antigen (second protein), an antibody that recognizes a host immune effector cell (first protein), a transmembrane domain, and an intracellular domain linked in sequence. contains the domain,
Preferably, the antibody (first protein) that recognizes a host immune effector cell and the antibody (second protein) that recognizes a tumor antigen or pathogen antigen are linked by a connecting peptide.

In a second aspect of the invention, a cell that resists transplant immune rejection, said cell being a T cell having a T cell receptor capable of recognizing one or more immune effector cells of the host; Preferably, cells are provided that have the function of inhibiting or killing host immune effector cells.

In a preferred embodiment, said cell further expresses a second protein that recognizes a tumor or pathogen antigen, preferably said second protein is a chimeric receptor.

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 of an MHC class I molecule.

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

In one preferred embodiment, the T cell receptor is capable of recognizing host NK cells,
Preferably, said T cell receptor is of the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C; Killing of IR3DS1 etc. Immunoglobulin-like receptor (KIR) family; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16 , can specifically recognize one or more of other antigens specifically expressed by NK cells, such as CD161;
More preferably, said T cell receptor is capable of specifically recognizing one or more NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

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

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

In one preferred embodiment, genes are silenced using gene editing techniques.

Preferably, the gene editing technology is CRISPR/Cas9 technology, Zinc Finger Nucleases (ZFN) technology, transcription activator-like effector nuclease (TALE) technology, or T ALE-CRISPR/ Selected from Cas9 technology,
More preferably, said gene editing technology is CRISPR/Cas9 technology.

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.

A fourth aspect of the present invention provides a method for preventing or regulating NK cell attack on exogenous cells, the method comprising administering immune effector cells expressing a first protein that recognizes NK cells. is,
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 the administration of the exogenous cells.

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

In one 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).

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

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

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

In one preferred embodiment, cells according to either the first or second aspect of the invention are administered.

A fifth aspect of the invention provides a method for preventing or modulating NK cell attack on exogenous cells, comprising administering an immune effector cell 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.

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

In one 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).

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

In one preferred embodiment, the immune effector cells include immune effector cells derived from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cells.

A sixth aspect of the invention provides a method for preventing or modulating NK cell attack on exogenous immune effector cells expressing a first protein that recognizes NK cells, comprising:
Preferably, the exogenous immune effector cells are cells that do not contain the HLA-I gene or cells in which the endogenous HLA-I gene has been silenced,
More preferably, the method is provided, wherein the exogenous immune effector cell is a cell that does not contain the B2M gene or a cell in which the B2M gene has been silenced.

In one 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 that recognizes NK cells is a member of the NKG2 receptor family such as NKG2A, NKG2D, and NKG2C; KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, Killing immunoglobulin-like receptor (KIR) family such as KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56 , LIR/ILT2, CD244, CD226, CD2, CD16, CD161, etc., and
More preferably, the first protein is capable of specifically recognizing one or more NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46.

In one preferred embodiment, said chimeric receptor is selected from 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 or pathogen antigen;
Preferably, said second protein is a chimeric receptor, said 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) comprising an antibody that recognizes NK cells and an antibody that recognizes a tumor or pathogen antigen. It is.

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

In one preferred embodiment, the second protein comprises an extracellular domain, a transmembrane domain, and an extracellular signal domain,
Preferably, said cells transmit signals through intracellular signaling domains to mediate inhibition or killing of tumors or pathogens.

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 that recognizes a host immune effector cell and the antibody that recognizes a tumor antigen or pathogen antigen are linked by a connecting peptide,
More preferably, the first protein has the sequence shown in SEQ ID NO:9.

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

Unless otherwise defined, all technical and scientific terms used herein are as commonly understood by those skilled in the art of gene therapy, biochemistry, genetics, and molecular biology. has the same meaning as Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, including those described herein. There is. 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, unless otherwise stated, the materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, the practice of the invention will employ 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. Belongs to the technical scope. These techniques are well explained in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Clone ing: A Laboratory Manual, Third Edition, (Sambrooketal, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M.J. Gaited., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid H hybridization (B.D.Harries & S.J. Transcription And Translation (B. D. Hames & S. J. Higginseds. 1984); Culture Of Animal Cells (R. I. Freshne y, 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 (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, Vol. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spri ng Harbor, N.Y., 1986).

In this disclosure, each aspect of the subject matter for which protection is sought is presented in the form of a range. It is to be understood that descriptions in scope are merely for convenience and brevity and are not to be construed as inflexible limitations on the scope of the subject matter sought protection. Accordingly, range descriptions should be considered as specifically disclosing all possible subranges and individual values within that range. For example, if a range of values is provided, all intermediate values between the upper and lower limits of that range, and any other or intermediate values within that range, are within the scope of the subject matter for which protection is sought. and the upper and lower limits of that scope are also included within the scope of the subject matter for which protection is sought. The limits of those smaller ranges may be independently included in that smaller range and, unless the range limits are specifically excluded, are also within the scope of the subject matter for which protection is sought. If the set range includes one or two limit values, the subject matter for which protection is sought also includes the range in which one or two of the limit values are excluded. This applies regardless of the scope.

The term "about" as used herein refers to the normal margin of error for each value that is readily known to those skilled in the art. A value or parameter modified herein with "about" includes (and describes) embodiments that refer to the value or parameter itself. For example, the description "about X" includes a description regarding "X". For example, "about" or "comprising" can mean within 1 or more than 1, according to the standard deviation in practice in the art. Alternatively, "about" or "comprising" can mean a range of up to 10% (ie, ±10%). For example, about 5 μM can include any number from 4.5 μM to 5.5 μM. When specific values or compositions are provided in applications and patent applications, unless otherwise specified, "about" or "comprising" should be within the tolerance range of the specific value or composition.

Any concentration range, percentage range, ratio range, or integer range set forth herein refers to any whole number within the range, and where appropriate, any fraction thereof (e.g., one-tenth of an integer), unless otherwise specified. and 1/100).

In order to better understand the present invention, related terms are defined as follows.

The term "transplant immune rejection" refers to the term "transplant immune rejection" that occurs after a host has transplanted a graft, such as an allogeneic tissue, organ, or cell, in which the foreign source graft is recognized by the host's immune system as a "foreign component" and the graft is means to mount an immunological response such as attack, destruction, and elimination against.

The term "graft" refers to a biological material or preparation derived from an individual other than the host and used to transplant into the host. The graft may be from any animal source, including a mammalian source, preferably from a human. In some embodiments, the graft may be derived from a host, eg, cells from the host are cultured in vitro or modified and transplanted back into the host. In some embodiments, the graft may be derived from another individual allogeneic, e.g., cells from another human being cultured in vitro or modified and transplanted into the host. Ru. 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 humans.

The term "cell" and other grammatical forms can refer to cells of human or non-human animal origin.

The term "host" refers to a recipient receiving a graft transplant, which in some embodiments may be an individual, such as a human, receiving a foreign cell transplant.

The term "immune effector cell" refers to a cell that participates in the immune response and produces an immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, Examples include CIK cells, macrophages, and mast cells. In some embodiments, the immune effector cell is a T cell, NK cell, 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.

The term "artificially modified cells with immune effector cell function" refers to cells or cell lines that do not have immune effect after being artificially modified or stimulated by irritants to have immune effector cell function. refers to cells that have acquired For example, 293T cells are artificially modified to have the function of immune effector cells. For example, stem cells are induced in vitro to differentiate into immune effector cells.

In some cases, "T cells" are bone marrow-derived pluripotent stem cells that can differentiate and mature into immunologically active mature T cells in the thymus. In some cases, a "T cell" can be a population of cells with specific phenotypic characteristics, or a mixed population of cells with different phenotypic characteristics; for example, a "T cell" may be a memory stem cell-like memory T cell ( 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). The cells may include two T cell subpopulations. In some cases, a "T cell" can be a particular subtype of T cell, such as a γδ T cell.

T cells can be obtained from multiple sources including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural 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 in a suitable buffer or medium for subsequent processing steps. Alternatively, the cells can be derived from a healthy donor, a patient diagnosed with cancer.

The term "MHC" is a collective term for all gene groups that encode antigens of the biocompatible complex. MHC antigens are expressed in the tissues of all higher vertebrates and are called HLA antigens on human cells, and play an important role in the transplantation reaction, with rejection occurring due to histocompatibility antigens on the surface of the transplanted tissue. Mediated by responding T cells. MHC proteins play an important role in T cell stimulation, and 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 displaying the same peptide/MHC complex. For example, stimulated T helper cells target macrophages displaying MHC-bound antigens, or cytotoxic T cells (CTLs) act on virus-infected cells displaying foreign viral peptides. MHC antigens are divided into NHC class I antigens and MHC class II antigens.

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

The term "B2M" refers to β-2 microglobulin, also called B2M, which is the light chain of MHC class I molecules. In humans, B2M is encoded by the b2m gene located on chromosome 15, relative to other MHC genes located in a gene cluster on chromosome 6. Hematopoietic grafts from mice that lack normal cell surface MHC I expression due to mutations in the B2M gene are rejected by normal mouse NK cells, because defective expression of MHC I molecules causes the cells to be susceptible to the host's immune system. (Bix et al. 1991).

The term "chimeric receptor" refers to a fusion molecule formed by linking DNA fragments or protein-corresponding cDNAs from different sources by recombinant techniques, which contain an extracellular region, a transmembrane region, and an intracellular region. Contains areas. 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. Intracellular signaling domains include functional signaling domains of stimulatory and/or costimulatory molecules. In one aspect, the stimulatory molecule is a zeta chain associated with 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 (ie, CD137), CD27, and/or CD28.

The term "T cell receptor (TCR)" includes classic TCR receptors and optimized TCR receptors, and includes specific major histocompatibility complex (MHC)-restricted peptides by T cells. Mediates antigen recognition. A classic TCR receptor consists of two peptide chains, α and β, and each peptide chain is further divided into a variable region (V region), a constant region (C region), a transmembrane region, and a cytoplasmic region. Its antigen specificity resides in the V region. Each V region (Vα, Vβ) has three hypervariable regions: CDR1, CDR2, and CDR3. In one aspect, a T cell expressing a classical TCR can induce specificity of the T cell's TCR for a target antigen by using methods such as antigen stimulation of the T cell.

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

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

The term "signal transduction domain" refers to a functional part of a protein that functions by transmitting information within a cell, either by producing a second messenger or by acting as an effector in response to such a messenger. This is because it regulates cell activity through the signal transduction pathway. The intracellular signaling domain may include all intracellular portions of the molecule, or all naturally occurring intracellular signaling domains, or functional fragments or derivatives thereof.

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

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

The term "gene editing" refers to the ability of humans to "edit" targeted genes, such as knocking out or adding specific DNA fragments.

The term "gene silencing" refers to the phenomenon of not expressing or underexpressing genes for various reasons. Gene silencing may be gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization, position effects, etc., or post-transcriptional gene silencing, i.e. at the level of gene transcription, by antisense RNA. Genes may be inactivated by specific inhibition of target RNA, such as co-inhibition, gene inhibition, RNA interference, and microRNA-mediated translation inhibition.

"TCR silencing" refers to not expressing or underexpressing endogenous TCR.

"MHC silencing" refers to not expressing or underexpressing endogenous MHC.

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

The term "Cas9 (CRISPR associated nuclease)" is CRISPR-associated nuclease, an RNA-guided technology that uses Cas9 nuclease to edit target genes.

The "CRISPER/Cas9 system" refers collectively to transcripts and other elements involved in the expression of, or directing the activity of, the Cas9 enzyme gene, including the sequence encoding the Cas9 gene, tracr (transactivation CRISPR) tracrRNA sequences (such as tracrRNA or active part tracrRNA), tracr pairing sequences (including "direct repeats" and partial direct repeats of tracrRNA processing in the context of the endogenous CRISPR system), guide sequences (in the context of the endogenous CRISPR system) (also referred to as "spacers," i.e., gRNAs) or other sequences and transcripts from the CRISPR locus.

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

Generally speaking, a guide sequence (gRNA) has sufficient complementarity with a target polynucleotide sequence to hybridize to the target polynucleotide sequence and guide sequence-specific binding of the CRISPR complex to the target sequence. Any polynucleotide sequence that has In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50% or more, 60% or more, 75% or more, 80% when optimally aligned using a suitable alignment algorithm. 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, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, the Burrows-Wheeler Transform-based algorithm ( For example, Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies), ELAND ((Illumina, San Diego, CA), SOAP available at .genomics.org.cn) and Maq (available at maq.sourceforget (available at .net).

In some embodiments, the CRISPR enzyme comprises 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, other than the CRISPR enzyme), 8 or more domains, 9 or more domains, 10 or more domains). A CRISPR enzyme fusion protein can include any additional protein sequences and linker sequences between any two domains, which may be selected. Examples of protein domains that can be fused to CRISPR enzymes include epitope tags, reporter gene sequences, and methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and Included are, but are not limited to, protein domains having one or more activities selected from the group consisting of nucleic acid binding activities. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza virus hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. .

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

The term "sgRNA" refers to short gRNA.

When performing gene editing, specific gRNAs, tracr pairing sequences, and tracr sequences can be provided individually, or the complete RNA sequence can be provided.

Binding of Cas9 protein to gRNA allows it to cleave DNA at specific sites. The CRISPR/Cas system recognition sequence from Streptococcus pyogenes is 23 bp and can target 20 bp, and the last three NGG sequences of the recognition site are called PAM (protospacer adjacent motif) sequences.

Cas transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, 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 CRISPER/Cas technology to render the endogenous TCR inactive. . Preferably, the first exon of the constant region of the endogenous TCRα chain is targeted and knocked out.

"Inhibiting" or "suppressing" the expression of B2M or TCR means that the expression of B2M or TCR in the cell is 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, "inhibiting" or "suppressing" the expression of B2M means that the content of B2M in the cell is 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%. Protein expression or content within cells can be determined by any method known in the art such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies for B2M or TCR. It can be measured by any suitable method.

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.

The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfections include various techniques including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics. This can be accomplished by various means known in the art.

The terms "nucleic acid molecule encoding", "encoding DNA sequence" and "encoding DNA" refer to the order or order of deoxyribonucleotides along a deoxyribonucleic acid chain. 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 marsupial. Individuals of the invention include humans, non-human primates (such as rhesus macaques or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry; Not limited to these.

The term "peripheral blood mononuclear cell" (PBMC) refers to cells that have mononuclei in peripheral blood, including lymphocytes, monocytes, and the like.

The terms "T cell activates" or "T cell activates" and other grammatical forms are used to induce detectable cell proliferation, cytokine production, and/or detectable effector function. It can refer to the state of fully stimulated T cells.

When used to refer to a nucleotide sequence, the term "sequence" and other grammatical forms as used herein may include DNA or RNA, whether single-stranded or double-stranded. good.

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 containing a recombinant polynucleotide that includes an expression control sequence operatively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in vitro expression system. Expression vectors include all known in the art, such as plasmids and viruses such as lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses.

The term "vector" as used herein is a composition that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid inside 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 movement of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.

As used herein, the term sequence "identity" refers to determining percentage identity by comparing two best-matched sequences in a comparison window (e.g., at least 20 positions) and A portion of the nucleotide or polypeptide sequence may contain additions or deletions (i.e. indels), e.g. 20% for two optimally matched sequences compared to the reference sequence (not containing additions or deletions). May contain the following indels (eg, 5-15%, or 10-12%): Typically, a percentage is calculated by determining the number of positions where the same nucleobase or amino acid residue occurs in the two sequences, thereby generating the number of correct matching positions, and calculating the number of correct matching positions by determining the number of positions where the same nucleobase or amino acid residue occurs in the reference sequence. (i.e., the window size) and multiply the result by 100 to generate the percentage sequence identity.

As used herein, the term "exogenous" means that a nucleic acid molecule or polypeptide, cell, tissue, etc., has no endogenous expression or achieves a function when overexpressed in vivo. Indicates that the expression level is insufficient for

The term "endogenous" refers to a nucleic acid molecule, polypeptide, etc. derived from the organism itself.

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

Chimeric antigen receptors usually contain an extracellular antigen binding region. In some embodiments, the extracellular antigen binding region may be fully human, or may be humanized, derived from a mouse, or chimera of the extracellular antigen binding region from at least two different animals. It consists of the amino acid sequence of

Examples of extracellular antigen binding regions include scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain fragments, or natural ligands conjugated to their cognate receptors; It may be any derivative of.

In some aspects, the extracellular antigen binding region (eg, scFv) can include light chain CDRs specific for the antigen. In some cases, the light chain CDRs may include more than one light chain CDR, which can be referred to as light chain CDR-1, CDR-2, etc. In some cases, the light chain CDRs may 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.

In some embodiments, the extracellular antigen binding region (eg, scFv) may include heavy chain CDRs specific for the antigen. The heavy chain CDRs may be heavy chain complementarity determining regions of antigen binding units such as scFvs. In some cases, the heavy chain CDRs may include more than one heavy chain CDR, which can be referred to as heavy chain CDR-1, CDR-2, etc. In some cases, the heavy chain CDRs may include three heavy chain CDRs, which can be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In one embodiment, a set of CDRs present in a common heavy chain can be collectively referred to as a heavy chain CDR.

Using genetic engineering, extracellular antigen binding regions can be modified in a variety of ways. In some cases, the extracellular antigen binding region can be selected to have higher affinity for its target by mutating the extracellular antigen binding region. In some cases, the affinity of the extracellular antigen binding region for a target can be optimized to a target that can be expressed at low levels in normal tissues. This optimization can be done to minimize potential toxicity. In other cases, clones of extracellular antigen binding regions with higher affinity for the membrane-bound form of the target may be superior to their soluble form counterparts. This modification can be made because different levels of the target in soluble form can also be detected and may cause unwanted toxicity.

In some cases, the extracellular antigen binding region includes a hinge or spacer. The terms hinge and spacer can be used interchangeably. The hinge can be considered 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.

The term "transmembrane domain" is capable of anchoring the chimeric protein to the plasma membrane of a cell. For example, the transmembrane domains of CD28 and CD8α can be used.

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

The term "therapy" refers to interventional measures in the process of modifying the disease, which can also be preventive, as well as intervening in the clinicopathological process. Therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing direct or indirect pathological consequences of the disease, preventing metastasis, delaying disease progression, improving or alleviating the condition, and improving prognosis. This includes, but is not limited to, mitigation or improvement.

The term "prevention" refers to interventional measures taken before a disease (such as rejection caused by a cell transplant) occurs.

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

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

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. You can also do it.

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

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", it recognizes one or more immune effector cells of the host. The protein can also recognize an antigen, such as a tumor antigen, which is recognized by a "second receptor."

"Tumor antigen" refers to an antigen that is newly emerged or overexpressed during the development and progression of a hyperproliferative disease. In certain embodiments, the hyperproliferative disorder of the invention refers to cancer.

The tumor antigen according to the invention may be a solid tumor antigen or a hematoma antigen.

The tumor antigen of the present invention includes 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 α (IL-13Rα); Interleukin 11 receptor α (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 β (PDGFR-β); Stage-specific embryonic antigen- 4 (SSEA-4); Cell surface associated mucin 1 (MUC1), MUC6; Epidermal growth factor receptor family and its variants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); Neural cell adhesion molecule (NCAM); Carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanin tumor-associated antigen (HMWMAA); O-acetyl GD2 ganglioside (OAcGD2) ; Folate receptor; Tumor vascular endothelial marker 1 (TEM1/CD248); Tumor vascular 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; ); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); globoH hexose moiety of glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); ); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K) ; olfactory receptor 51E2 (OR51E2); TCRγ alternating reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); XAGE1); Angiopoietin-binding cell surface receptor 2 (Tie2); Melanoma cancer testis antigen-1 (MAD-CT-1); Melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; P53 variant ; human telomerase reverse transcriptase (hTERT); sarcoma 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 myelopathy virus 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 3 recognized by T cells (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); C-type lectin domain family 12 member A (CLEC12A) ; bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 (EMR2) containing an EGF-like module; lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5) ; including, but not limited to, immunoglobulin lambda-like peptide 1 (IGLL1). Preferably, said tumor antigen is BCMA or CD19.

Pathogen antigens are selected from viral, bacterial, fungal, protozoan, or parasitic antigens. The viral antigen is selected from cytomegalovirus antigen, Epstein Barr virus antigen, human immunodeficiency virus antigen or influenza virus antigen.

The invention is further described below in conjunction with specific examples. It is to be understood that these examples are used only to illustrate the invention and are not intended to limit the scope of the invention. In the examples below, experimental methods for which specific conditions are not indicated are generally described in J. Sambrook et al. , eds. Conventional conditions such as those described in Molecular Cloning: A Laboratory Manual, Third Edition, Kagaku Publishing, 2002 are followed or conditions recommended by the manufacturer are followed.

Detection of NK Cell Surface Receptor Expression Monocytes were isolated from peripheral blood using Ficoll-Paque (GE bioscience) for density gradient centrifugation and negative cells using an NK cell isolation kit (purchased from Miltenyi Biotec). Screening was performed to remove cells such as T cells, B cells, and monocytes, followed by in vitro cell phenotyping and expansion. Flow cytometry was used to identify receptors on the surface of isolated NK cells, including markers such as 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).
Furthermore, the expression of the above surface markers on T cells was also detected. T cells activated by CD3/CD28 magnetic beads (purchased from Thermo Fisher) were collected and cultured until day 8 and stained for flow cytometry. Experimental results show that NKG2A, NKG2D, NKP30, NKP44, and NKP46 are poorly expressed in T cells, indicating that the above markers can be used as targets for NK cells (see Figure 2).

Preparation of CAR-T cells and functional verification 1. NKG2A was selected as a target for preparing CAR-T cells targeting NK cells. With reference to conventional procedures, a single chain antibody comprising anti-NKG2A (the amino acid sequence of VH is shown in SEQ ID NO: 1 and the amino acid sequence of VL is shown in SEQ ID NO: 2), CD28 transmembrane domain and intracellular domain (the amino acid sequence is shown in SEQ ID No. 3), a chimeric antigen receptor (the amino acid sequence is shown in SEQ ID No. 5) of the T cell activating factor CD3ζ (the amino acid sequence is shown in SEQ ID No. 4), and constructed and the plasmid map is shown in Figure 8. The lentivirus was packaged and named VRRL-NKG2A-28Z(TM).
After activating and proliferating T cells for 48 hours, the cell density was adjusted to 2 × 10 ⁶ /mL, and VRRL-NKG2A-28Z (TM) lentivirus was added at a ratio of MOI = 10 to incubate NKG2A. Target CAR-T cells were obtained.
CAR-T cells were collected on day 6 for cell proliferation detection, the starting cell number was adjusted to 5 × 10 ⁵ , and the cell number was detected at 24 hours, 48 hours, 72 hours, and 96 hours. The diameter was recorded. At the same time, flow cytometry staining was performed using anti-F(ab)'2 antibody to detect the expression of the CAR vector. According to the experimental results, NKG2A CAR-T cells and untransfected T cells (UTD) showed similar growth curves, with no significant difference in cell diameter, and approximately 80% of CAR-T cells targeted NKG2A. expressed a CAR molecule. This indicates that the proliferation properties of NKG2A CAR-T cells are normal (see Figure 3).
2. Preparation of CAR-T cells targeting BCMA Referring to the procedure in 1, construct a plasmid (the amino acid sequence is shown in SEQ ID NO: 6) targeting the chimeric antigen receptor of BCMA, and the plasmid map is It is shown in FIG. Lentivirus was packaged and T cells were transfected to obtain BCMA-targeted BCMA-CAR T cells.
3. In vitro killing function experiment Primary NK cells were expanded in vitro as target cells. Cell density was adjusted to 5 x 10 ⁵ /mL, 100 μl was seeded into 96-well plates (three replicate wells were made in parallel), and effector T cell:target cell ratios of 1:3, 1:1, and The corresponding CAR-T cells were seeded according to three ratios of 3:1. MEM-α+5% FBS was used as the medium and incubated at 37° C. in a 5% CO ₂ incubator for 4 and 18 hours, respectively. Using the cytotox-96 non-radioactive cytotoxicity assay kit (purchased from Thermo Fisher), 50 μl of supernatant was taken and the content of lactate dehydrogenase (LDH) was determined and UTD The lysis efficiency of primary NK cells in the two groups of NKG2A and NKG2A CAR-T was calculated.
The detection results showed that the LDH value of NKG2A CAR-T group was significantly higher than that of UTD group, indicating that NKG2A CAR-T can effectively kill primary NK cells (Fig. 4 and see Figure 5).

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 grown in vitro for 48 hours, and then the cell density was reduced to 2. The concentration was adjusted to ×10 ⁷ /mL. Cas9 enzyme (purchased from NEB) and sgRNA were incubated at a ratio of 1:4 for 10 min at room temperature to obtain an RNP complex solution. The nucleic acid sequence of TRAC-sgRNA is shown in SEQ ID NO: 7, and the nucleic acid sequence of B2M-sgRNA is shown in SEQ ID NO: 8. 1×10 ⁶ cells were mixed with RNP complex solution (final concentration of Cas9 enzyme was 3 uM) and RNP complexes were introduced into CAR-T cells individually using maxcyte electrotransmitter. Seven days after electroporation, flow cytometry was used to detect the knockout of TCR and B2M genes. Experimental results show that the knockout efficiency of TRAC and B2M is over 85% (see Figure 6).
2. TCR/B2M double negative cell screening B2M and TCR-knockout CAR-T cells and UTD cells were grown in vitro, and on day 8, the cell density was adjusted to 1×10 ⁷ /mL, and the cells were treated with anti-HLA. - Labeled with ABC and B2M antibodies and then with a coupled phycoerythrin (PE) secondary antibody. After sorting the labeled cells with anti-PE magnetic beads on a sorting column, TCR and B2M double negative cells were collected (the sorting kit was purchased from Miltenyi Biotec), BCMA UCAR-T cells lacking TCR and B2M, NKG2A UCAR -T cells and U-UTD cells were obtained.

Verification of the resistance function of NKG2A UCAR-T cells against NK cells 1. Detection of the rejection effect of UCAR-T cells against NK cells by LDH experiment UTD cells, BCMA-CAR T cells, NKG2A CAR-T cells, BCMA UCAR-T cells , NKG2A UCAR-T cells, and U-UTD cells were used as target cells, the cell concentration was adjusted to 5 × 10 ⁵ /mL, 100 μl was inoculated into a 96-well plate, and the amplified primary NK cells and target cells were The same amount (volume) and number of NK cells were inoculated according to the ratio 1:1 and incubated in the incubator for 4 and 18 hours, respectively. 50 μl of the supernatant was taken, the content of lactate dehydrogenase (LDH) was measured, and the lysis efficiency of CAR-T and UCAR-T cells was calculated. The detection results show that the LDH values of both UTD and BCMA CAR-T groups are very low. This indicates that normal CAR-T cells do not cause NK cell attack. In contrast, both U-UTD and BCMA UCAR-T groups showed gradually increasing LDH values at 4 and 18 hours, indicating that NK cells kill T cells lacking TCR and B2M. ing. 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 prove the resistance ability of NKG2A UCAR-T cells against NK cells, we selected BCMA UCAR-T cells as a control, adjusted the cell concentration to 5×10 ⁵ /mL, and transferred 100 μl into a 96-well plate. Inoculated. The same volume and number of NK cells were inoculated at a ratio of 1:1 between expanded primary NK cells and target cells, and incubated in an incubator for 4 hours, 18 hours, 24 hours, and 42 hours, respectively. Flow cytometry was used to label HLA-ABC positive NK cells and detect the percentage of UCAR-T cells incubated at different time points. The experimental results show that, as shown in Figures 7A-7D, BCMA UCAR-T has a low rate of about 20% in 4 hours, and the rate is always very low as the detection time increases, which is due to the NK Figure 2 shows that the cells significantly inhibited the proliferation of BCMA UCAR-T cells. NKG2A UCAR-T cells had a low proportion of about 20% at 4 hours, but as the detection time increased, the proportion gradually increased and reached 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, we constructed GFP-expressing BCMA UCAR-T cells and NKG2A UCAR-T cells. The amino acid sequence of BCMA-GFP is shown in SEQ ID NO: 24, and the amino acid sequence of NKG2A-GFP is shown in SEQ ID NO: 25.
Referring to the procedure in Example 2, a plasmid PRRL-BCMA-BBZ-F2A-EGFP expressing GFP-expressing BCMA UCAR-T cells was constructed, and the plasmid map is shown in FIG. A plasmid PRRL-NKG2A-28Z-F2A-EGFP expressing NKG2A UCAR-T cells was constructed, and the plasmid map is shown in FIG. 17. Package lentivirus on the constructed plasmid, transfect T cells, perform gene knockout and magnetic bead selection on CAR-T cells to express GFP and BCMA UCAR-T cells expressing GFP. NKG2A UCAR-T cells were obtained.
Adjust the concentration of CAR-T cells to 5 × 10 ⁵ /mL, inoculate 100 μl into a 96-well plate, and inject the same volume and number of NK cells according to the 1:1 ratio of expanded primary NK cells to target cells. The cells were inoculated and cultured in an incubator for 0 hours, 4 hours, 18 hours, 24 hours, and 48 hours, respectively. Flow cytometry was used to detect the percentage of co-cultured GFP cells at various time points and was used to track UCAR-T cell survival.
The experimental results show that, as shown in Figure 10, the percentage of GFP-positive BCMA UCAR-T cells gradually decreased with time, and after 48 hours, they were almost completely killed by NK cells, but the percentage of GFP-positive NKG2A UCAR-T cells was almost completely killed by NK cells after 48 hours. The proportion of T cells decreased slightly at 4 h, increased significantly after 18 h, and accounted for approximately 90% after 48 h, indicating that NKG2A UCAR-T cells can significantly resist killing of NK cells. ing.

In vivo resistance of NKG2A UCAR-T cells to NK cells BCMA UCAR-T and NKG2A UCAR-T cells were cultured in vitro, the CAR positivity rate was adjusted to 80%, and the tail was incubated at a dose of 8 × ¹⁰ cells/mouse. NPG was injected into immunodeficient mice by intravenous injection, and the mice were divided into three groups: a group receiving BCMA UCAR-T and NK cells (designated as BCMA UCAR-T+NK), a group receiving NKG2A UCAR-T and a group receiving NK cells (NKG2A-UCART+NK). They were divided into two groups:
The same amount of NK cells were injected 4 hours after UCAR T cell administration. On days 1, 3, and 6 after CAR T cell injection, 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).
The experimental results show that, as shown in Figure 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 was , significantly decreased, indicating that UCAR-T cells are rejected by NK cells. On days 3 and 6 after injection, the number of UCAR-T cells in the BCMA UCAR-T+NK group was always at a very low level, and the number of UCAR-T cells in the NKG2A UCAR-T+NK group was at a very low level on days 3 and 6. There was a significant increase on day 1. The above results demonstrate 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 NK cell killing and recover their proliferative ability. It shows.

Construction of CAR T cells targeting BCMA and NKG2A As shown in FIG. -T) was prepared. The amino acid sequence of BCMA-GS-NKG2A CAR is shown in SEQ ID NO:9.
A plasmid PRRL-BCMA-GS-NKG2A-BBZ of BCMA-GS-NKG2A UCAR-T was constructed, and the plasmid map is shown in FIG. Referring to the procedures in Examples 2 and 3, viral transfection was performed to obtain BCMA-GS-NKG2A UCAR-T cells, and TRAC and B2M gene knockout was performed on BCMA-GS-NKG2A UCAR-T cells. After implementation, more than 99% of TCR and HLA-ABC negative BCMA-GS-NKG2A UCAR-T cells were obtained by magnetic bead sorting method.
Referring to the procedures in Examples 2 and 3, BCMA UCAR-T cells and NKG2A UCAR-T cells were prepared, respectively.
CAR expression of BCMA UCAR-T, NKG2A UCAR-T and BCMA-GS-NKG2A UCAR-T was detected, respectively. The experimental results, as shown in Figure 13, showed that the positive rate reached more than 60%, indicating that the BCMA-GS-NKG2A UCAR-T cells were successfully prepared.

Verification of in vitro function 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 tumor cells were placed in a 96-well plate. After inoculating and inoculating the corresponding number of UCAR-T cells according to the ratio of T cells and tumor cells 3:1, 1:1, and 1:3 and incubating for 18 hours, 50 μl for LDH content detection. The supernatant was aspirated.
The experimental results show that, as shown in Figure 14, the cell lysis rates of RPMI-8226 and NCI-H929 were very low in the UTD and NKG2A UCAR-T groups, and the tumor lysis rates in the BCMA-GS-NKG2A UCAR-T group were very low. The lysis rate of cells was close to that of BCMA UCAR-T group, indicating that BCMA-GS-NKG2A UCAR-T cells can effectively kill BCMA-positive tumor cells in vitro.

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

In vivo resistance of BCMA-GS-NKG2A UCAR-T cells to NK cells BCMA UCAR-T and BCMA-GS-NKG2A UCAR-T cells were cultured in vitro, the CAR positivity rate was adjusted to 60%, and 8 × 10 NPG was injected into immunodeficient mice by tail vein injection at a dose of ⁶ cells/mouse, and the mice were divided into groups that received BCMA UCAR-T and NK cells (designated as BCMA UCAR-T+NK), BCMA-GS-NKG2A UCAR-T group and a group to which NK cells were administered (denoted as BCMA-GS-NKG2A-UCAR-T+NK). The same amount of NK cells were injected 4 hours after UCAR-T cell administration. On the 1st, 3rd, and 6th day after CAR T cell injection, the survival of human-derived CD45-positive T cells in the peripheral blood of mice was detected by flow absolute technology.
The experimental results show that, as shown in Figure 19, the number of UCAR-T cells in the BCMA UCAR-T+NK group significantly increased on the 3rd and 6th day compared to the 1st day after injection. This indicates that UCAR-T cells are rejected by NK cells. The number of UCAR-T cells in the BCMA-GS-NKG2A UCAR-T+NK group significantly increased on both days 3 and 6, and the number of cells on day 6 was 30% lower than that on day 1. It has more than doubled. The above results demonstrate that in an in vivo model, NK cells significantly inhibit the survival of BCMA UCAR-T cells, whereas BCMA-GS-NKG2A UCAR-T cells effectively resist NK cell killing and proliferate. It shows that you can recover your abilities.

The sequences according to the present application are shown in the following table.

Claims (38)

for transplant immune rejection, characterized in that it expresses a first protein capable of recognizing one or more immune effector cells of the host, and preferably has an inhibitory or killing function against the immune effector cells of the host. cells that resist. The cell according to claim 1, wherein the cell is an immune effector cell or an artificially modified cell having the function of an immune effector cell. The cells are selected from immune effector cells derived from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cells,
Preferably, the cell is a T cell;
More preferably, the cell according to claim 1 or 2, characterized in that the first protein is a chimeric receptor. 1-1, characterized in that said cell further expresses 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. 3. The cell according to any one of 3. Claim characterized in that said cells do not express MHC or that the MHC genes endogenously expressed by said cells are silenced, preferably said MHC genes are genes of MHC class I molecules. 5. The cell according to any one of 1 to 4. According to claim 5, characterized in that 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. cells. Resisting transplant immune rejection means resisting attack from host NK cells, or the first protein being able to recognize host NK cells,
Preferably, said first protein is a member of the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C; Killing of IR3DS1 etc. Immunoglobulin-like receptor (KIR) family; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16 , can specifically recognize one or more of other antigens specifically expressed by NK cells, such as CD161;
More preferably, the first protein is capable of specifically recognizing one or more NK cell surface antigens such as NKG2A, NKG2D, NKP30, NKP44, and NKP46, any one of claims 1 to 6. Cell according to item 1. The first protein includes an antibody capable of recognizing host NK cells,
Preferably, the antibody is capable of recognizing NKG2A,
More preferably, the antibody comprises HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, LCDR2 shown in SEQ ID NO: 14, 15,
Even more preferably, the cell according to claim 7, characterized in that the antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 1 or a light chain variable region as set forth in SEQ ID NO: 2. The HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M, and preferably, the HLA-I gene is B2M. 9. The cell according to claim 8. Cell according to claim 3 or 4, characterized in that the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor or a T cell antigen coupler (TAC). The first protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain,
Cell according to claim 1, characterized in that the cell preferably transmits a signal via an intracellular signaling domain to mediate inhibition or killing of immune effector cells of the host. The second protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain,
Cell according to claim 4, characterized in that the cell preferably transmits a signal via an intracellular signaling domain to mediate inhibition or killing against a tumor or pathogen. The cell is a T cell silenced by the HLA-I gene and the endogenous TCR gene,
The cell according to claim 6, wherein the cell is preferably a T cell silenced by B2M and TCR genes. The second protein can specifically recognize BCMA or CD19,
Preferably, the second protein includes an antibody that can specifically recognize BCMA,
More preferably, the antibody that specifically recognizes BCMA includes HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 19, and HCDR1 shown in SEQ ID NO: 20. LCDR2 shown in, LCDR3 shown in SEQ ID NO: 21,
Even more preferably, the antibody that specifically recognizes BCMA comprises a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO: 23, according to claim 4. cells. Cell according to claim 5, 6 or 13, characterized in that the gene has been silenced by gene editing technology. The first protein includes 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 that recognizes a host immune effector cell and the antibody that recognizes a tumor antigen or pathogen antigen are linked by a connecting peptide,
More preferably, the cell according to claim 10, wherein the first protein has a sequence shown in SEQ ID NO:9. A cell that resists transplant immune rejection, said cell being a T cell having a T cell receptor capable of recognizing one or more immune effector cells of the host, preferably said cell being a T cell having a T cell receptor capable of recognizing one or more immune effector cells of the host; A cell characterized by having an inhibitory or killing function against effector cells. 18. Cell according to claim 17, characterized in that said cell further expresses a second protein recognizing a tumor or pathogen antigen, preferably said second protein is a chimeric receptor. Claim characterized in that said cells do not express MHC or that the MHC genes endogenously expressed by said cells are silenced, preferably said MHC genes are genes of MHC class I molecules. The cell according to item 17 or 18. 20. According to claim 19, characterized in that said cell does not express HLA or the HLA gene endogenously expressed by said cell is silenced, preferably said HLA is an HLA-I gene. cells. The T cell receptor is capable of recognizing host NK cells,
Preferably, said T cell receptor is of the NKG2 receptor family, such as NKG2A, NKG2D, NKG2C; Killing of IR3DS1 etc. Immunoglobulin-like receptor (KIR) family; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16 , can specifically recognize one or more of other antigens specifically expressed by NK cells, such as 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, and NKP46. Cell according to item 1. The HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M, and preferably, the HLA-I gene is B2M. 21. The cell according to claim 20. 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 comprises a second protein. The chimeric receptor comprises a second protein, a transmembrane domain, and an intracellular domain;
Preferably, the second protein can specifically recognize BCMA or CD19,
Preferably, the second protein includes an antibody that can specifically recognize BCMA,
More preferably, the antibody that specifically recognizes BCMA includes HCDR1 shown in SEQ ID NO: 16, HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, and LCDR1 shown in SEQ ID NO: 19, SEQ ID NO: 20, LCDR3 shown in SEQ ID NO: 21,
Even more preferably, the antibody that specifically recognizes BCMA comprises a heavy chain variable region shown in SEQ ID NO: 22 and a light chain variable region shown in SEQ ID NO: 23, according to claim 18. cells. A method for preventing or regulating transplant immune rejection, which comprises administering the cell according to any one of claims 1 to 23. A method of preventing or modulating NK cell attack on exogenous cells, the method comprising: administering an immune effector cell expressing a first protein that recognizes NK cells;
Optionally, said method, characterized in that said exogenous cells are T cells, NKT cells, stem cells, or engineered T cells, NKT cells, stem cells. 26. A method according to claim 25, characterized in that said exogenous cells are immune effector cells, preferably said exogenous cells express a second receptor. the second receptor is a chimeric receptor or a T cell receptor,
27. A method according to claim 26, characterized in that the chimeric receptor is preferably selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC). The antigen recognized by the first protein that recognizes NK cells includes the NKG2 receptor family such as NKG2A, NKG2D, and NKG2C; KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, Killing immunoglobulin-like receptor (KIR) family such as KIR2DS5, KIR3DS1; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244 , one or more of other antigens specifically expressed by NK cells, such as CD226, CD2, CD16, CD161;
More preferably, the method according to claim 25, characterized in that the first protein is capable of specifically recognizing one or more NK cell surface antigens, which are NKG2A, NKG2D, NKP30, NKP44, and NKP46. 26. The method of claim 25, wherein the immune effector cells include immune effector cells derived from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cells. 1. A method for preventing or modulating NK cell attack on exogenous immune effector cells, the exogenous immune effector cell expressing a first protein that recognizes NK cells;
Preferably, the exogenous immune effector cells are cells that do not contain the HLA-I gene or cells in which the endogenous HLA-I gene has been silenced,
More preferably, the method described above, wherein the exogenous immune effector cell is a cell that does not contain the B2M gene or a cell in which the B2M gene is silenced. The exogenous immune effector cell is a T cell,
31. The method according to claim 30, wherein the first protein that recognizes NK cells is preferably a chimeric receptor or a T cell receptor. The antigen recognized by the first protein that recognizes NK cells includes the NKG2 receptor family such as NKG2A, NKG2D, and NKG2C; KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, Killing immunoglobulin-like receptor (KIR) family such as KIR2DS5, KIR3DS1; natural cytotoxic receptors (NCR) such as NKP30, NKP44, NKP46, NKp80; and CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244 , one or more of other antigens specifically expressed by NK cells, such as CD226, CD2, CD16, CD161;
More preferably, the method according to claim 31, characterized in that the first protein is capable of specifically recognizing one or more NK cell surface antigens, which are NKG2A, NKG2D, NKP30, NKP44, and NKP46. 33. The method according to claim 32, characterized in that the chimeric receptor is selected from chimeric antigen receptors (CARs), chimeric T cell receptors, T cell antigen couplers (TACs). the exogenous immune effector cell further expresses a second protein that recognizes a tumor or pathogen antigen;
Preferably, said second protein is a chimeric receptor, said chimeric receptor selected from a chimeric antigen receptor (CAR), a chimeric T-cell receptor, or a T-cell antigen coupler (TAC). The method according to any one of claims 30 to 33, wherein: 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 an antibody that recognizes a tumor antigen or a pathogen antigen. 35. The method of claim 34. The first protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain,
31. A cell according to claim 30, characterized in that said cell preferably transmits a signal via an intracellular signaling domain to mediate inhibition or killing of immune effector cells of the host. The second protein includes an extracellular domain, a transmembrane domain, and an extracellular signal domain,
35. Cell according to claim 34, characterized in that the cell preferably transmits a signal via an intracellular signaling domain to mediate inhibition or killing against a tumor or pathogen. The first protein includes 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 that recognizes a host immune effector cell and the antibody that recognizes a tumor antigen or pathogen antigen are linked by a connecting peptide,
More preferably, the cell according to claim 30, wherein the first protein has a sequence shown in SEQ ID NO:9.