WO2020259707A1 - 抗移植反应的细胞和方法 - Google Patents

抗移植反应的细胞和方法 Download PDF

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WO2020259707A1
WO2020259707A1 PCT/CN2020/098930 CN2020098930W WO2020259707A1 WO 2020259707 A1 WO2020259707 A1 WO 2020259707A1 CN 2020098930 W CN2020098930 W CN 2020098930W WO 2020259707 A1 WO2020259707 A1 WO 2020259707A1
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cell
cells
receptor
protein
seq
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PCT/CN2020/098930
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English (en)
French (fr)
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李宗海
廖朝晖
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科济生物医药（上海）有限公司
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Priority to KR1020227003537A priority Critical patent/KR20220028090A/ko
Priority to JP2021577705A priority patent/JP2024500254A/ja
Priority to CN202080043759.1A priority patent/CN114729028A/zh
Priority to EP20830547.4A priority patent/EP3992204A4/en
Priority to US17/623,481 priority patent/US20220356447A1/en
Priority to MX2021015620A priority patent/MX2021015620A/es
Priority to AU2020304139A priority patent/AU2020304139A1/en
Priority to CA3144549A priority patent/CA3144549A1/en
Publication of WO2020259707A1 publication Critical patent/WO2020259707A1/zh
Priority to IL289353A priority patent/IL289353A/en

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    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Definitions

  • the present invention relates to a cell with the function of resisting transplant rejection, and also relates to a method of resisting transplantation immune rejection, in particular to a method of resisting NK cell immune rejection.
  • the donor may also be recognized and attacked by immune cells in the recipient, thereby inhibiting or eliminating the foreign source
  • the graft produces a host versus graft response (HVGR).
  • HVGR host versus graft response
  • By knocking out the MHC molecules in the transplant cells it can effectively resist the rejection of host T cells to the transplant, but it may cause rejection of other immune cells in the host.
  • allogeneic cell transplantation when the MHC-I molecules of allogeneic cells are missing, it will cause rejection of NK cells in the host and enhance the elimination of allogeneic cells (Nat Biotechnol. 2017; 35(8): 765-772. doi:10.1038/nbt.3860). Therefore, how to effectively prevent the immune rejection of host NK cells is crucial to the development of allogeneic cell transplantation therapy.
  • the purpose of the present invention is to provide a cell resistant to transplantation immune rejection and a method for preventing rejection.
  • a cell expressing a first protein capable of recognizing one or more immune effector cells of the host; preferably, the cell has inhibitory or inhibitory effects on the immune effector cells of the host Killing function.
  • the cells are immune effector cells or artificially modified cells with immune effector cell functions.
  • the cells are selected from T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells;
  • the cell is a T cell
  • the first protein is a chimeric receptor.
  • the cell also expresses a second protein that recognizes tumor antigens or pathogen antigens, preferably the second protein chimeric receptor or T cell receptor.
  • the activation of the protein recognizing host immune effector cells is regulated by the second receptor.
  • the activation of the second receptor is regulated by a protein that recognizes the host's immune effector cells.
  • the protein that recognizes the host's immune effector cell and the activation of the second receptor do not affect each other.
  • the cell does not express MHC, or the MHC gene endogenously expressed by the cell is silenced; preferably, the MHC gene is a MHC class I molecule gene.
  • the cell does not express HLA, or the HLA gene endogenously expressed by the cell is silenced; preferably, the HLA is an HLA class I gene.
  • the anti-transplant immune rejection is an attack against host NK cells, or the first protein can recognize host NK cells,
  • the first protein can specifically recognize one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1 , KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP46, NKp80, etc.; Sexually expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161,
  • the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP46.
  • the first protein contains an antibody capable of recognizing host NK cells
  • the antibody can recognize NKG2A;
  • the antibody contains HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, and LCDR1 shown in SEQ ID NO: 13, SEQ ID NO: LCDR2 shown in SEQ ID NO: LCDR3 shown in 15;
  • the antibody contains the heavy chain variable region described in SEQ ID NO:1 or the light chain variable region described in SEQ ID NO:2.
  • the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M; preferably, the HLA-I gene is B2M.
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the first protein includes an extracellular domain, a transmembrane domain, and an intracellular signal domain;
  • the cells transmit signals through the intracellular signal domain to mediate the inhibition or killing of the host's immune effector cells.
  • the second protein includes an extracellular domain, a transmembrane domain and an intracellular signal domain;
  • the cells transmit signals through the intracellular signal domain to mediate the inhibition or killing of tumors or pathogens.
  • the cell is a HLA-I gene and an endogenous TCR gene silenced T cell;
  • the cell is a T cell with B2M and TCR gene silenced.
  • the second protein can specifically recognize BCMA or CD19;
  • the second protein contains an antibody capable of specifically recognizing BCMA;
  • the antibody specifically recognizing BCMA contains HCDR1 shown in SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, and HCDR3 shown in SEQ ID NO: 19 LCDR1, LCDR2 shown in SEQ ID NO: 20, LCDR2 shown in SEQ ID NO: 21;
  • the antibody specifically recognizing BCMA contains the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23.
  • gene editing technology is used to silence genes.
  • the gene editing technology is selected from CRISPR/Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nuclease, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology, or TALE-CRISPR /Cas9 technology;
  • the gene editing technology is CRISPR/Cas9 technology.
  • the first protein includes an antibody that recognizes host immune effector cells, an antibody that recognizes tumor antigens or pathogen antigens, a transmembrane domain, and an intracellular domain;
  • the antibody recognizing host immune effector cells and the antibody recognizing tumor antigen or pathogen antigen are connected by a connecting peptide;
  • the first protein has a sequence shown in SEQ ID NO: 9.
  • the first protein and the second protein may be in a chimeric receptor, that is, preferably, the chimeric receptor contains an antibody (first protein) that recognizes host immune effector cells connected in sequence, Antibodies (second protein), transmembrane domains, and intracellular domains that recognize tumor antigens or pathogen antigens; or
  • the chimeric receptor contains an antibody (second protein) that recognizes tumor antigens or pathogen antigens, an antibody (first protein) that recognizes host immune effector cells, a transmembrane domain, and an intracellular domain that are sequentially connected;
  • the antibody (first protein) that recognizes host immune effector cells and the antibody (second protein) that recognizes tumor antigens or pathogen antigens are connected by a connecting peptide.
  • a cell resistant to transplantation immune rejection characterized in that the cell is a T cell, and the T cell has a T cell receptor capable of recognizing one or more immune effector cells of the host Preferably, the cells have inhibitory or killing functions on the immune effector cells of the host.
  • the cell also expresses a second protein that recognizes tumor antigens or pathogen antigens, preferably the second protein is a chimeric receptor.
  • the cell does not express MHC, or the MHC gene endogenously expressed by the cell is silenced; preferably, the MHC gene is a MHC class I molecule gene.
  • the cell does not express HLA, or the HLA gene endogenously expressed by the cell is silenced; preferably, the HLA is an HLA class I gene.
  • the T cell receptor can recognize the host's NK cells
  • the T cell receptor can specifically recognize one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like receptor (KIR) family, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP46, other NKp80, etc.; Specific expressed antigens, such as CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161,
  • the T cell receptor can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP46.
  • the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M; preferably, the HLA-I gene is B2M.
  • the second protein is a chimeric receptor
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric receptor comprising the second protein includes the second protein, the transmembrane domain, and the intracellular domain
  • the second protein can specifically recognize BCMA or CD19;
  • the second protein contains an antibody capable of specifically recognizing BCMA;
  • the antibody specifically recognizing BCMA contains HCDR1 shown in SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, and HCDR3 shown in SEQ ID NO: 19 LCDR1, LCDR2 shown in SEQ ID NO: 20, LCDR2 shown in SEQ ID NO: 21;
  • the antibody specifically recognizing BCMA contains the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23.
  • gene editing technology is used to silence genes.
  • the gene editing technology is selected from CRISPR/Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nuclease, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology, or TALE-CRISPR /Cas9 technology;
  • the gene editing technology is CRISPR/Cas9 technology.
  • the method for preventing or regulating transplantation immune rejection in the third aspect of the present invention includes administering the cells according to any one of the first or second aspects of the present invention.
  • a method for preventing or regulating foreign cells from being attacked by NK cells which is characterized by administering immune effector cells expressing the first protein that recognizes NK cells;
  • the exogenous cells are T cells, NK T cells, stem cells, or engineered T cells, NK T cells, or stem cells;
  • the immune effector cells are administered before, after, or simultaneously with the exogenous cells.
  • the exogenous cell is an immune effector cell.
  • the exogenous cell expresses a second receptor.
  • the second receptor is a chimeric receptor or a T cell receptor
  • the chimeric receptor is selected from: chimeric antigen receptor (CAR), chimeric T cell receptor, and T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like Receptor (KIR) family, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP44 , NKp80, etc.; and other antigens specifically expressed by NK cells, such as CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161,
  • the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP46.
  • the HLA-I gene is selected from one or more of HLA-A, HLA-B, HLA-C, and B2M; preferably, the HLA-I gene is B2M.
  • the second protein is a chimeric receptor
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the chimeric receptor comprising the second protein includes the second protein, the transmembrane domain, and the intracellular domain
  • the second protein can specifically recognize BCMA or CD19;
  • the second protein contains an antibody capable of specifically recognizing BCMA;
  • the antibody specifically recognizing BCMA contains HCDR1 shown in SEQ ID NO: 16, HCDR1 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, and HCDR3 shown in SEQ ID NO: 19 LCDR1, LCDR2 shown in SEQ ID NO: 20, LCDR2 shown in SEQ ID NO: 21;
  • the antibody specifically recognizing BCMA contains the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23.
  • the cell of any one of the first or second aspect of the present invention is administered.
  • a method for preventing or regulating foreign cells being attacked by NK cells is provided, and immune effector cells expressing the first protein that recognizes NK cells are administered;
  • the exogenous cells are T cells, NK T cells, stem cells, or engineered T cells, NK T cells, or stem cells.
  • the exogenous cell is an immune effector cell.
  • the exogenous cell expresses a second receptor.
  • the second receptor is a chimeric receptor or a T cell receptor
  • the chimeric receptor is selected from: chimeric antigen receptor (CAR), chimeric T cell receptor, and T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like Receptor (KIR) family, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxicity receptors (NCR), such as NKP30, NKP44, NKP44 , NKp80, etc.; and other antigens specifically expressed by NK cells, such as CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161,
  • the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP46.
  • the immune effector cells include T cells, NK cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells.
  • a method for preventing or regulating exogenous immune effector cells being attacked by NK cells characterized in that the exogenous immune effector cells express a first protein that recognizes NK cells;
  • the exogenous immune effector cells are cells that do not contain HLA-I genes or endogenously silenced HLA-I genes;
  • the exogenous immune effector cell is a cell that does not contain the B2M gene or the B2M gene is silenced.
  • the exogenous immune effector cells are T cells
  • the first protein that recognizes NK cells is a chimeric receptor or a T cell receptor.
  • the antigen recognized by the first protein that recognizes NK cells is one or more of the following antigens: NKG2 receptor family, such as NKG2A, NKG2D, NKG2C, etc.; killer immunoglobulin-like Receptor (KIR) family, such as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2/S3, KIR2DS4, KIR2DS5, KIR3DS1, etc.; natural cytotoxic receptor (NCR), such as NKP30, NKP44 , NKp80, etc.; and other antigens specifically expressed by NK cells, such as CD159a, CD159c, CD94, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161,
  • the first protein can specifically recognize one or more of the following NK cell surface antigens: NKG2A, NKG2D, NKP30, NKP44, NKP46.
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, and a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the exogenous immune effector cells also express a second protein that recognizes tumor antigens or pathogen antigens;
  • the second protein is a chimeric receptor
  • the chimeric receptor is selected from a chimeric antigen receptor (CAR), a chimeric T cell receptor, or a T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the first protein is a chimeric antigen receptor, a chimeric T cell receptor, or a T cell antigen coupler (TAC) containing antibodies that recognize NK cells and recognize tumor antigens or pathogen antigens. ).
  • TAC T cell antigen coupler
  • the first protein includes an extracellular domain, a transmembrane domain, and an intracellular signal domain;
  • the cells transmit signals through the intracellular signal domain to mediate the inhibition or killing of the host's immune effector cells.
  • the two protein includes an extracellular domain, a transmembrane domain and an intracellular signal domain;
  • the cells transmit signals through the intracellular signal domain to mediate the inhibition or killing of tumors or pathogens.
  • the first protein includes an antibody that recognizes host immune effector cells, an antibody that recognizes tumor antigens or pathogen antigens, a transmembrane domain, and an intracellular domain;
  • the antibody recognizing host immune effector cells and the antibody recognizing tumor antigen or pathogen antigen are connected by a connecting peptide;
  • the first protein has a sequence shown in SEQ ID NO: 9.
  • FIG. 1 Expression of NK cell surface markers in T cells. ;
  • Figure 3 Growth characteristics of NKG2A CAR-T cells.
  • A cell proliferation curve
  • B cell diameter
  • C CAR positive rate
  • Figure 4 In vitro killing ability of NKG2A CAR-T cells on NK cells after a total of 4 hours of incubation;
  • Figure 5 In vitro killing ability of NKG2A CAR-T cells on NK cells after a total of 18 hours of incubation;
  • FIG. 6 Efficient knockout of TCR and B2M in CAR-T cells
  • FIG. 7A, Figure 7B, Figure 7C and Figure 7D FACS detects the resistance of NKG2A UCAR-T cells to NK cells;
  • FIG. 8 Plasmid map of CAR targeting NKG2A
  • Figure 10 FACS detects the resistance of NKG2A UCAR-T cells to NK cells
  • Figure 11 FACS detection of UCAR-T cell survival in peripheral blood of mice
  • Figure 12 Schematic diagram of the structure of BCMA-GS-NKG2A UCAR-T;
  • Figure 15 BCMA-GS-NKG2A UCAR-T cell resistance to NK cells
  • Figure 16 Plasmid map of PRRL-BCMA-BBZ-F2A-EGFP
  • Figure 17 Plasmid map of PRRL-NKG2A-28Z-F2A-EGFP
  • Figure 18 Plasmid map of PRRL-BCMA-GS-NKG2A-BBZ;
  • Figure 19 shows the results of BCMA-GS-NKG2A UCAR-T cell resistance to NK cells in vivo.
  • the upper and lower limits of these smaller ranges can be independently included in the smaller range, and they also belong to the scope of the claimed subject matter, unless the upper and lower limits of the range are explicitly excluded.
  • the set range includes one or two limit values
  • the subject of protection also includes a range that excludes one or two of the limit values. This applies regardless of the width of the range.
  • any concentration range, percentage range, ratio range or integer range described herein should be understood to include any integer within the stated range, and where appropriate, a fraction thereof (such as one tenth of an integer and One percent).
  • transplant immune rejection refers to the host's transplantation of allogeneic tissues, organs, or cells.
  • the foreign graft is recognized by the host's immune system as a "foreign component” and initiated against the transplant. The immunological response of the attack, destruction and clearance.
  • graft refers to a biological material or preparation that is derived from an individual other than the host and is used to implant the host.
  • the graft can be from any animal source, such as a mammalian source, preferably from a human.
  • the graft may be derived from a host, for example, cells from the host are cultured in vitro or modified to be implanted into the host again.
  • the graft may be an allogeneic other individual, for example, cells from another person are cultured in vitro or transformed into a host.
  • the transplant may be a heterogeneous individual, such as transplanting organs from other species (such as mice, pigs, and monkeys) into humans.
  • cell and other grammatical forms can refer to cells of human or non-human animal origin.
  • the term "host” refers to a recipient who receives a graft transplant, and in some embodiments, it may be an individual, such as a human, who receives foreign cell transplantation.
  • immune effector cells refers to cells that participate in immune responses and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, and macrophages , Mast cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cell may be an autologous T cell, a heterologous T cell, or an allogeneic T cell.
  • the NK cells may be allogeneic NK cells.
  • artificially modified cell with immune effector cell function refers to a cell or cell line that does not have an immune effect after being artificially modified or stimulated by a stimulus, the cell acquires the immune effector cell function.
  • 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.
  • T cells may be pluripotent stem cells derived from bone marrow, which differentiate and mature into mature T cells with immunological activity in the thymus.
  • T cells may be cell populations with specific phenotypic characteristics, or mixed cell populations with different phenotypic characteristics, such as “T cells” may be cells containing at least one T cell subpopulation: memory Stem cell-like memory T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells ( Tem).
  • Tscm cells memory Stem cell-like memory T cells
  • Tcm central memory T cells
  • effector T cells Tef, Teff
  • Tregs regulatory T cells
  • Tem effector memory T cells
  • Tem effector memory T cells
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of techniques known to those skilled in the art, such as FicollTM isolation can be used to obtain T cells from blood collected from an individual.
  • cells from the circulating blood of the individual are obtained by a single blood collection.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets.
  • cells collected by apheresis collection can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from healthy donors, from patients diagnosed with cancer.
  • MHC is the histocompatibility complex, which is a collective term for all the gene groups encoding the antigens of the biocompatibility complex.
  • MHC antigens are expressed in the tissues of all higher vertebrates and are called HLA antigens in human cells.
  • the reaction plays an important role, and rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.
  • MHC protein plays a vital role in T cell stimulation.
  • Antigen presenting cells (usually dendritic cells) display peptides belonging to degradation products of foreign proteins on the cell surface on MHC, in the presence of co-stimulatory signals , T cells are activated and act on target cells that also display the same peptide/MHC complex.
  • stimulated T helper cells will target macrophages that display antigens bound to their MHC, or cytotoxic T cells (CTL) will act on virus-infected cells that display foreign viral peptides.
  • MHC antigens are divided into NHC class I antigens and MHC class II antigens.
  • HLA human leukocyte antigen
  • HLA Human leukocyte antigen
  • HLA human leukocyte antigen
  • HLA is the coding gene of the human major histocompatibility complex, 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 genes.
  • the antigens expressed by HLA class I and class II genes are located on the cell membrane, and are MHC-I (HLA-A, HLA-B, HLA-C site encoding) and MHC-II (HLA-D region encoding),
  • HLA I Class II is distributed on the surface of almost all cells of the body, and is a heterodimer composed of heavy chain ( ⁇ chain) and ⁇ 2 microglobulin (B2M).
  • Class II is mainly glycoprotein located on the surface of macrophages and B lymphocytes. .
  • B2M refers to ⁇ -2 microglobulin, also known as B2M, which is the light chain of MHC class I molecules.
  • B2M is encoded by the b2m gene located on chromosome 15, as opposed to other MHC genes located as gene clusters on chromosome 6.
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments or cDNAs corresponding to proteins from different sources using gene recombination technology, including extracellular domain, transmembrane domain and intracellular domain.
  • Chimeric receptors include but are not limited to: chimeric antigen receptor (CAR), chimeric T cell receptor (TCR), and T cell antigen coupler (TAC).
  • chimeric antigen receptor includes extracellular antigen binding domains, transmembrane domains, and intracellular signaling domains.
  • Intracellular signaling domains include functional signaling domains of stimulatory molecules and/or costimulatory molecules.
  • the stimulatory molecule is a delta chain that binds to the T cell receptor complex; in one aspect, cytoplasmic signaling
  • the conduction domain further includes one or more functional signal conduction domains of costimulatory molecules, such as 4-1BB (ie CD137), CD27 and/or CD28.
  • T cell receptor T cell receptor, TCR
  • MHC major histocompatibility complex
  • T cells including classic TCR receptors and optimized TCR receptors. body.
  • the classic TCR receptor is composed of two peptide chains, ⁇ and ⁇ . Each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region, and cytoplasmic region.
  • V region variable region
  • C region constant region
  • TCR ⁇ constant region
  • T cells expressing classic TCR can be stimulated by antigens to T cells. Induces the specificity of TCR of T cells to the target antigen.
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides that constitute TCR, which can bind to the surface antigens of target cells and interact with other polypeptides of the complete TCR complex, usually co-localized in T cell surface.
  • the chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain.
  • the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, and TCR cell
  • the stimulation domain of the intracellular signal domain of the intradomain; the TCR subunit and the antibody domain are effectively connected, wherein the extracellular, transmembrane, and intracellular signal domain of the TCR subunit is derived from CD3 ⁇ or CD3 ⁇ , and ,
  • the chimeric T cell receptor is integrated into the TCR expressed on the T cell.
  • T cell antigen coupler includes three functional domains: 1. The antigen binding domain, including single-chain antibodies, and designed ankyrin repeat protein (DARPin) Or other targeting groups; 2. The extracellular domain, a single-chain antibody that binds to CD3, so that the TAC receptor and TCR receptor are close; 3. The transmembrane region and the intracellular region of the CD4 co-receptor, where The intracellular domain is connected to the protein kinase LCK to catalyze the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • signal transduction domain refers to a functional part of a protein that functions by transmitting information in a cell, and is used to regulate the cell through a certain signal transduction pathway by generating a second messenger or acting as an effector in response to such a messenger. ⁇ activity.
  • the intracellular signaling domain may include all intracellular parts of the molecule, or all natural intracellular signaling domains, or functional fragments or derivatives thereof.
  • co-stimulatory molecule refers to a signal that binds to a cell stimulating signal molecule, such as TCR/CD3, and results in T cell proliferation and/or up-regulation or down-regulation of key molecules.
  • activation and “activation” are used interchangeably and can refer to the process by which a cell changes from a resting state to an active state.
  • the process can include a response to phenotypic or genetic changes in antigen, migration, and/or functional activity status.
  • activation can refer to the process of gradual activation of T cells.
  • T cells may require at least one signal to fully activate.
  • gene editing refers to the ability to allow humans to "edit” target genes to achieve the knockout and addition of specific DNA fragments.
  • Gene silencing refers to the phenomenon that genes are not expressed or underexpressed due to various reasons. Gene silencing can be gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization, and positional effects, or post-transcriptional gene silencing, that is, by specifically inhibiting target RNA at the post-transcriptional level It is gene inactivation, including antisense RNA, co-suppression, gene suppression, RNA interference, and microRNA-mediated translational inhibition.
  • TCR silencing means that the endogenous TCR is not expressed or is low expressed.
  • MHC silencing refers to the lack of expression or low expression of endogenous MHC.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • CRISPR-associated nuclease is a CRISPR-associated nuclease, which is an RNA-guided technology that uses Cas9 nuclease to edit targeted genes.
  • CRISPER/Cas9 system is collectively referred to as transcripts and other elements involved in the expression of Cas9 enzyme genes or directing its activity, including sequences encoding Cas9 genes, tracr (transactivation CRISPR) sequences (such as tracrRNA or active part of tracrRNA), tracr Matching sequence (covering "direct repeat” and partial direct repeat of tracrRNA processing in the context of endogenous CRISPR system), guide sequence (also called “spacer”, or gRNA in the context of endogenous CRISPR system ), or other sequences and transcripts from the CRISPR locus.
  • tracr transactivation CRISPR sequences
  • tracr Matching sequence covering "direct repeat” and partial direct repeat of tracrRNA processing in the context of endogenous CRISPR system
  • guide sequence also called “spacer”, or gRNA in the context of endogenous CRISPR system
  • gRNA guide sequence
  • target sequence refers to a sequence that has complementarity with a guide sequence, and complementary pairing between the target sequence and the guide sequence promotes the formation of a CRISPR complex.
  • a target sequence can contain any polynucleotide, such as DNA or RNA polynucleotide.
  • the target sequence is located in the nucleus or cytoplasm of the cell.
  • a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct the sequence-specific binding of the CRISPR complex to the target sequence.
  • the degree of complementarity between the guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80% , 85%, 90%, 95%, 97.5%, 99%, or more.
  • Any suitable algorithm for aligning sequences can be used to determine the best alignment, non-limiting examples of which include Smith-Waterman (Smith-Waterman) algorithm, Needleman-Wunsch (Needleman-Wunsch) algorithm, Algorithms based on Burrows-Wheeler Transform (such as Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technology Company), ELAND (Eland) Corporation (Illumina, San Diego, California), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Smith-Waterman Smith-Waterman
  • Needleman-Wunsch Needleman-Wunsch
  • Algorithms based on Burrows-Wheeler Transform such as Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • Novoalign Novocraft Technology Company
  • ELAND Eland
  • the CRISPR enzyme contains one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, excluding the CRISPR enzyme). 9, 10 or more domains) of the fusion protein.
  • the CRISPR enzyme fusion protein can include any other protein, and optionally a linking sequence between any two domains.
  • protein domains that can be fused to CRISPR enzymes 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 , Transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags .
  • Cas9 enzyme can be wild-type Cas9 or artificially modified Cas9.
  • sgRNA refers to short gRNA.
  • the given gRNA, tracr pairing sequence, and tracr sequence can be given separately, or a complete RNA sequence.
  • the combination of Cas9 protein and gRNA can cut DNA at specific sites.
  • the CRISPR/Cas system recognition sequence derived from Streptococcus pyogenes is 23 bp and can target 20 bp.
  • the last 3 NGG sequences of the recognition site are called PAM ( protospacer adjacent motif) sequence.
  • the Cas transgene can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electrotransduction.
  • vectors eg, AAV, adenovirus, lentivirus
  • particles and/or nanoparticles e.g., electrotransduction.
  • the exons of the corresponding coding genes in the constant regions of one or both of the ⁇ and ⁇ chains of the TCR are knocked out using CRISPER/Cas technology to make the endogenous TCR inactive,
  • the first exon of the constant region of the endogenous TCR ⁇ chain is targeted to be knocked out.
  • “Inhibiting” or “suppressing” the expression of B2M or TCR means that the expression of B2M or TCR in a cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. More specifically, “inhibiting” or “suppressing" the expression of B2M means that the content of B2M in a cell is reduced by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%.
  • the expression or content of protein in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western blotting, or flow cytometry, using B2M or TCR specific antibodies.
  • Modification used in the present invention refers to a change in the state or structure of the protein or polypeptide of the present invention. Modification methods can be chemical, structural, and functional.
  • transfection refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • nucleic acid molecule encoding refers to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. Therefore, the nucleic acid sequence encodes an amino acid sequence.
  • subject refers to any animal, such as a mammal or a marsupial.
  • Individuals of the present invention include, but are not limited to, humans, non-human primates (such as rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.
  • peripheral blood mononuclear cell refers to cells with mononuclear nuclei in peripheral blood, including lymphocytes and monocytes.
  • T cell activation or “T cell activation” and other grammatically other forms may refer to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • sequence When used in reference to a nucleotide sequence, the term "sequence" and other grammatical forms as used herein may include DNA or RNA, and may be single-stranded or double-stranded.
  • expression vector refers to a vector containing a recombinant polynucleotide, which contains 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 host cells or in vitro expression systems.
  • Expression vectors include all those known in the art, such as plasmids, viruses (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. It can also include non-plasmid and non-viral compounds that promote the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like.
  • sequence identity determines the percent identity by comparing two best-matched sequences over a comparison window (for example, at least 20 positions), where the portion of the polynucleotide or polypeptide sequence in the comparison window may include Additions or deletions (i.e. gaps), such as 20% or less gaps (e.g., 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the two sequences that best match %).
  • the percentage is usually calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of correct matching positions.
  • the number of correct matching positions is divided by the total number of positions in the reference sequence ( That is, the window size) and multiply the result by 100 to produce the percentage of sequence identity.
  • exogenous refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or the expression level is insufficient to achieve the function of overexpression.
  • endogenous means that a nucleic acid molecule or polypeptide is derived from the organism itself.
  • the chimeric receptor of the invention is a chimeric antigen receptor.
  • Chimeric antigen receptors usually contain an extracellular antigen binding region.
  • the extracellular antigen binding region can be fully human, humanized, murine, or the chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals.
  • extracellular antigen binding regions can be scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain fragments, or natural ligands that engage their cognate receptors, and their Any derivative of.
  • the extracellular antigen binding region may contain light chain CDRs specific for the antigen.
  • a light chain CDR may include two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, and the like.
  • the light chain CDR may comprise three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively.
  • a group of CDRs present on a common light chain can be collectively referred to as light chain CDRs.
  • the extracellular antigen binding region may contain a heavy chain CDR specific for the antigen.
  • the heavy chain CDR may be the heavy chain complementarity determining region of an antigen binding unit such as a scFv.
  • a heavy chain CDR may include two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, and the like.
  • the heavy chain CDR may comprise three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively.
  • a group of CDRs present on a common heavy chain can be collectively referred to as a heavy chain CDR.
  • the extracellular antigen-binding region can be modified in various ways.
  • the extracellular antigen binding region can be mutated so that the extracellular antigen binding region can be selected to have a higher affinity for its target.
  • the affinity of the extracellular antigen-binding region to its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity.
  • clones of extracellular antigen-binding regions that have higher affinity for the membrane-bound form of the target may be superior to their soluble counterparts. This modification can be made because different levels of targets in soluble form can also be detected, and their targeting can cause undesirable toxicity.
  • the extracellular antigen binding region also includes a hinge or spacer, and the terms hinge and spacer can be used interchangeably.
  • the hinge can be considered as part of the CAR used to provide flexibility to the extracellular antigen binding region.
  • the hinge may be the natural hinge region of the CD8 ⁇ molecule.
  • transmembrane domain can anchor the chimeric protein to the plasma membrane of the cell.
  • the transmembrane domain of CD28 and CD8 ⁇ can be used.
  • modulation refers to positive or negative changes. Examples of adjustments include 1%, 2%, 10%, 25%, 50%, 75%, or 100% changes. In a specific embodiment, it refers to a negative change.
  • treatment refers to interventions that are trying to change the course of the disease, either for prevention or intervention in the clinical pathological process.
  • the therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, reducing the symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progression of the disease, improving or relieving the condition, relieving or improving the prognosis, etc.
  • prevention refers to interventions that are attempted before disease (such as rejection caused by cell transplantation) occurs.
  • the first protein in the present invention refers to the aforementioned protein capable of recognizing one or more immune effector cells of the host.
  • the second protein in the present invention refers to the above-mentioned protein that recognizes tumor antigens or pathogen antigens.
  • the "second receptor” and "protein capable of recognizing one or more immune effector cells of the host” described in the present invention can be expressed in tandem or expressed separately.
  • the two have independent transmembrane domains and intracellular domains.
  • the expression method please refer to PCT/CN2015/095938 , Enhancing the specificity of T-cell cultures for adoptive immunotherapy of cancer, Duong CP et al., Immnuotherapy 3(1): 33-48, etc.
  • the protein that recognizes one or more immune effector cells of the host can also recognize the "second receptor".
  • the antigen recognized by the "second receptor" such as a tumor antigen.
  • Tumor antigen refers to an antigen that is newly emerged or overexpressed during the occurrence and development of hyperproliferative diseases.
  • the hyperproliferative disorder of the present invention refers to cancer.
  • the tumor antigens of the present invention can be solid tumor antigens or hematoma antigens.
  • the tumor antigens of the present invention include but are not limited to: Thyroid Stimulating Hormone Receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; Ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3(CD276), B7H6; KIT(CD117); Interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); Interleukin 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Re
  • the pathogen antigen is selected from: virus, bacteria, fungus, protozoa, or parasite antigen; virus antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.
  • NK cell separation kit purchased from Miltenyi
  • monocytes were separated from peripheral blood, and negative screening was performed with NK cell separation kit (purchased from Miltenyi) to remove T cells, B cells and monocytes After waiting for the cells, in vitro cell phenotype identification and expansion.
  • the results of flow cytometry showed that NKG2A, NKP30, NKP44, and NKP46 are expressed in about 80% of NK cells, while NKG2D is expressed in more than 90% of NK cells (see Figure 1).
  • T cells activated by CD3/CD28 magnetic beads purchased from Thermo Fisher
  • CD3/CD28 magnetic beads purchased from Thermo Fisher
  • plasmid targeting the chimeric antigen receptor of BCMA (the amino acid sequence is shown in SEQ ID NO: 6).
  • the plasmid map is shown in Figure 9.
  • the lentivirus is packaged and the T cell is transfected to obtain the target BCMA-CAR T cells of BCMA.
  • Amplify primary NK cells in vitro as target cells Adjust the cell density to 5*10 ⁇ 5/mL, inoculate 100 ⁇ l into a 96-well plate (make 3 multiple wells in parallel), according to the ratio of effector T cells: target cells of 1:3, 1:1 and 3:1 Inoculate the corresponding CAR-T cells.
  • MEM- ⁇ +5% FBS as the culture medium, incubate at 37°C and 5% CO 2 in an incubator for 4 hours and 18 hours, respectively.
  • cytotox-96non-radioactive cytotoxicity assay kit purchased from Thermo Fisher
  • LDH lactate dehydrogenase
  • NKG2A CAR-T cells and BCMA CAR-T cells (for control) cells in vitro for 48 hours, adjust the cell density to 2*10 ⁇ 7/mL.
  • TRAC-sgRNA 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 As shown in SEQ ID NO: 8, mix 1*10 ⁇ 6 cells with RNP complex solution (the final concentration of Cas 9 enzyme is 3uM), and use maxcyte electrotransmitter to separately introduce RNP complexes into CAR-T cells.
  • SEQ ID NO: 8 mix 1*10 ⁇ 6 cells with RNP complex solution (the final concentration of Cas 9 enzyme is 3uM), and use maxcyte electrotransmitter to separately introduce RNP complexes into CAR-T cells.
  • flow cytometry was used to detect the knockout of TCR and B2M genes.
  • the experimental results showed that the knockout efficiency of TRAC and B2M was above 85% (see Figure 6).
  • the test results show that the LDH values of the UTD and BCMA CAR-T groups are both very low, indicating that ordinary CAR-T cells will not cause NK cell attack, while the U-UTD and BCMA UCAR-T groups both exhibited at 4hr and 18hr.
  • the increasing LDH value indicates that NK cells will kill T cells lacking TCR and B2M.
  • NKG2A UCAR-T cells show lower levels of LDH, indicating that NKG2A UCAR-T cells are resistant to NK cells.
  • BCMA UCAR-T is at a low ratio of about 20% at 4 hours, and the ratio has been at a very low level with the extension of the detection time, indicating that NK cells significantly inhibited BCMA UCAR- The growth of T cells; while NKG2A UCAR-T cells are at a low proportion of about 20% at 4hr, but with the prolongation of the detection time, they show a gradually increasing proportion, reaching nearly 60% at 42hr, indicating that NKG2A The growth of UCAR-T cells was initially inhibited by NK cells, but gradually restored their proliferation ability over time. The above results indicate that NKG2A UCAR-T can effectively resist the killing ability of NK cells.
  • Example 2 Refer to the operation in Example 2 to construct the plasmid PRRL-BCMA-BBZ-F2A-EGFP expressing GFP-expressing BCMA UCAR-T cells, and the plasmid map is shown in Figure 16.
  • Construct the plasmid PRRL-NKG2A-28Z-F2A expressing NKG2A UCAR-T cells -EGFP the plasmid map is shown in Figure 17.
  • the constructed plasmid packs the lentivirus, transfects T cells, and performs 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.
  • CAR-T cells Adjust the concentration of CAR-T cells to 5*10 ⁇ 5/mL, inoculate 100 ⁇ l to 96-well plate, according to the ratio of primary expanded NK cells to target cells 1:1, inoculate the same volume and number of NK cells, in an incubator Incubate for 0hr, 4hr, 18hr, 24hr and 48hr. Flow cytometry was used to detect the proportion of GFP cells that were incubated at different time points, which was used to track the survival of UCAR-T cells.
  • BCMA UCAR-T and NKG2A UCAR-T cells in vitro, adjust the CAR positive rate to 80%, and inject 8*10 ⁇ 6 cells/mouse into NPG immunodeficient mice through the tail vein, and divide the mice into two Group: BCMA UCAR-T and NK cell group (labeled BCMA UCAR-T+NK), NKG2A UCAR-T group and NK cell group (labeled NKG2A-UCART+NK).
  • NK cells The same amount of NK cells was injected 4 hours after UCAR T cells were injected, and the survival of human-derived CD4 and CD8 T cells in the peripheral blood of mice were detected by flow absolute technology on the 1, 3, and 6 days after the CAR T cell injection. happening.
  • the experimental results are shown in Figure 11.
  • the number of UCAR-T cells ie, human-derived CD4 and CD8 T cells
  • the number of UCAR-T cells in the BCMA UCAR-T+NK group was always very low, while the number of UCAR-T cells in the NKG2A UCAR-T+NK group was on the 3rd day And there was a significant increase on the 6th day.
  • the above results indicate that in the in vivo model, NK cells significantly inhibited the survival of BCMA UCAR-T cells, and NKG2A UCAR-T cells can effectively resist the killing of NK cells and restore their proliferation ability.
  • UCAR-T cells ie, BCMA-GS-NKG2A UCAR-T
  • scFv targeting BCMA and scFv targeting NKG2A were prepared.
  • the amino acid sequence of BCMA-GS-NKG2A CAR is shown in SEQ ID NO: 9.
  • the plasmid PRRL-BCMA-GS-NKG2A-BBZ of BCMA-GS-NKG2A UCAR-T was constructed, and the plasmid map is shown in Figure 18.
  • the method obtained more than 99% TCR and HLA-ABC negative BCMA-GS-NKG2A UCAR-T cells.
  • BCMA UCAR-T cells and NKG2A UCAR-T cells were prepared respectively.
  • the 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 inoculated on 96-well plates, and the ratio of T cells to tumor cells was 3:1, 1:1 Inoculate the corresponding number of UCAR-T cells with 1:3, and after a total of 18 hours of incubation, draw 50 ⁇ l of supernatant for LDH content detection.
  • BCMA UCAR-T cells As the incubation time increases, the proportion of BCMA UCAR-T cells gradually decreases, and 48 hours have been basically killed by NK cells; while the performance of BCMA-GS-NKG2A UCAR-T and NKG2A UCAR-T cells Following the same trend, a slight decrease was found at 4hr, and then gradually increased, reaching more than 70% at 48hr, and the proportion of BCMA-GS-NKG2A UCAR-T cells reached 90% at 48hr. The above results indicate that BCMA-GS-NKG2A UCAR-T cells can effectively resist the killing of NK cells.
  • BCMA UCAR-T and BCMA-GS-NKG2A UCAR-T cells in vitro, adjust the CAR positive rate to 60%, and inject 8*10 ⁇ 6 cells/mouse into NPG immunodeficient mice through the tail vein.
  • the rats were divided into two groups: the BCMA UCAR-T and NK cell group (labeled BCMA UCAR-T+NK) and the BCMA-GS-NKG2A UCAR-T group and NK cell group (labeled BCMA-GS-NKG2A UCAR- T+NK).
  • BCMA-GS-NKG2A UCAR- T+NK The same amount of NK cells were injected 4 hours after the UCAR-T cell administration.
  • the flow cytometry was used to detect the human CD45-positive T in the peripheral blood of the mouse. The survival of the cells.

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Abstract

涉及一种抗移植免疫排斥的细胞，所述细胞表达有能够识别宿主一种或多种免疫效应细胞的第一蛋白；优选的，所述细胞对宿主的免疫效应细胞具有抑制或杀伤功能。还涉及一种预防或调控移植免疫排斥的方法和预防或调控外源细胞被NK细胞攻击的方法。

Description

抗移植反应的细胞和方法 技术领域
本发明涉及一种具有抗移植排斥功能的细胞，还涉及抗移植免疫排斥的方法，特别是涉及一种抗NK细胞免疫排斥的方法。
背景技术
由于供体和受体之间的免疫遗传学差异，在进行外源供体移植时，作为外源移植物，供体也可能受到受体体内的免疫细胞识别和攻击，进而抑制或者清除外源移植物，产生宿主抗移植物反应(HVGR)。通过敲除移植物细胞中的MHC分子，能够有效抵抗宿主T细胞对移植物的排斥反应，但是可能会引起宿主内其他免疫细胞的排斥反应。如在异体细胞移植中，当异体细胞的MHC-I类分子的缺失，会导致宿主体内NK细胞排斥反应，增强对异体细胞的清除作用(Nat Biotechnol.2017；35(8):765-772.doi:10.1038/nbt.3860)。因此，如何有效防止对宿主NK细胞的免疫排斥反应，对开发异体细胞移植治疗至关重要。
发明内容
本发明的目的在于提供一种抗移植免疫排斥的细胞及抗抑制排斥的方法。
本发明提供的技术方案包括：
在本发明的第一方面，提供了一种细胞，所述细胞表达有能够识别宿主一种或多种免疫效应细胞的第一蛋白；优选的，所述细胞对宿主的免疫效应细胞具有抑制或杀伤功能。
在一优选的实施例中，所述细胞为免疫效应细胞或经人工改造的具有免疫效应细胞功能的细胞。
在一优选的实施例中，所述细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞；
优选的，所述细胞是T细胞，
更优选的，所述第一蛋白是嵌合受体。
在一优选的实施例中，所述细胞还表达有识别肿瘤抗原或病原体抗原的第二蛋白，优选所述第二蛋白嵌合受体或T细胞受体。
在一优选的实施例中，所述识别宿主免疫效应细胞的蛋白的活化受所述第二受体的调控。
在一优选的实施例中，所述第二受体的活化受识别宿主的免疫效应细胞的蛋白的调控。
在一优选的实施例中，所述识别宿主的免疫效应细胞的蛋白和第二受体的活化不相互影响。
在一优选的实施例中，所述细胞不表达MHC，或所述细胞内源性表达的MHC基因被沉默；优选的，所述的MHC基因为MHC I类分子的基因。
在一优选的实施例中，所述的细胞不表达HLA，或所述细胞内源性表达的HLA基因被沉默；优选的，所述的HLA为HLA-I类基因。
在一优选的实施例中，所述的抗移植免疫排斥为抗宿主的NK细胞的攻击，或者所述第一蛋白能够识别宿主的NK细胞，
优选的，所述第一蛋白能够特异性识别以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
在一优选的实施例中，所述第一蛋白含有能够识别宿主NK细胞的抗体；
优选的，所述抗体能够识别NKG2A；
进一步优选的，所述抗体含有SEQ ID NO：10所示的HCDR1，SEQ ID NO：11所示的HCDR2，SEQ ID NO：12所示的HCDR3，SEQ ID NO：13所示的LCDR1，SEQ ID NO：14所示的LCDR2，SEQ ID NO：15所示的LCDR3；
更进一步优选的，所述抗体含有SEQ ID NO：1所述的重链可变区或SEQ ID NO：2所述的轻链可变区。
在一优选的实施例中，所述HLA-I基因选自HLA-A、HLA-B、HLA-C，B2M中的一种或两种以上；优选的，所述HLA-I基因为B2M。
在一优选的实施例中，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体或T细胞抗原耦合器(TAC)。
在一优选的实施例中，所述第一蛋白包含有胞外域、跨膜域和胞内信号域；
优选的，所述细胞通过胞内信号域传递信号介导对宿主的免疫效应细胞的抑制或 杀伤。
在一优选的实施例中，所述第二蛋白包含有胞外域、跨膜域和胞内信号域；
优选的，所述细胞通过胞内信号域传递信号介导对肿瘤或病原体的抑制或杀伤。
在一优选的实施例中，所述细胞为HLA-I基因和内源性的TCR基因沉默的T细胞；
优选的，所述细胞为B2M和TCR基因沉默的T细胞。
在一优选的实施例中，所述第二蛋白能够特异性识别BCMA或CD19；
优选的，所述第二蛋白含有能够特异性识别BCMA的抗体；
进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、以及SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；
更进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区。
在一优选的实施例中，采用基因编辑技术使基因沉默。
优选的，所述的基因编辑技术选自CRISPR/Cas9技术、人工锌指核酸酶(Zinc Finger Nucleases，ZFN)技术、转录激活样效应因子(transcription activator-like effector,TALE)技术、或TALE-CRISPR/Cas9技术；
更优选的，所述的基因编辑技术为CRISPR/Cas9技术。
在一优选的实施例中，所述第一蛋白包含识别宿主免疫效应细胞的抗体、识别肿瘤抗原或病原体抗原的抗体、跨膜域、及胞内域；
优选的，所述识别宿主免疫效应细胞的抗体和识别肿瘤抗原或病原体抗原的抗体通过连接肽相连；
更进一步优选的，所述第一蛋白具有SEQ ID NO：9所示的序列。
在一优选的实施例中，第一蛋白和第二蛋白可以在一个嵌合受体中，即优选，所述嵌合受体含有顺序连接的识别宿主免疫效应细胞的抗体(第一蛋白)、识别肿瘤抗原或病原体抗原的抗体(第二蛋白)、跨膜域、及胞内域；或者
所述嵌合受体含有顺序连接的识别肿瘤抗原或病原体抗原的抗体(第二蛋白)、识别宿主免疫效应细胞的抗体(第一蛋白)、跨膜域、及胞内域；
优选的，所述识别宿主免疫效应细胞的抗体(第一蛋白)和识别肿瘤抗原或病原体抗原的抗体(第二蛋白)之间通过连接肽相连。
在本发明的第二方面，提供了一种抗移植免疫排斥的细胞，其特征在于，所述细胞为T细胞，该T细胞具有能够识别宿主一种或多种免疫效应细胞的T细胞受体，优选的 所述细胞对宿主的免疫效应细胞具有抑制或杀伤功能。
在一优选的实施例中，所述细胞还表达有识别肿瘤抗原或病原体抗原的第二蛋白，优选所述第二蛋白是嵌合受体。
在一优选的实施例中，所述细胞不表达MHC，或所述细胞内源性表达的MHC基因被沉默；优选的，所述的MHC基因为MHC I类分子的基因。
在一优选的实施例中，所述的细胞不表达HLA，或所述细胞内源性表达的HLA基因被沉默；优选的，所述的HLA为HLA-I类基因。
在一优选的实施例中，所述T细胞受体能够识别宿主的NK细胞，
优选的，所述T细胞受体能够特异性识别以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
更优选的，所述T细胞受体能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
在一优选的实施例中，所述HLA-I基因选自HLA-A、HLA-B、HLA-C，B2M中的一种或两种以上；优选的，所述HLA-I基因为B2M。
在一优选的实施例中，所述第二蛋白是嵌合受体，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体或T细胞抗原耦合器(TAC)，包含第二蛋白的嵌合受体包括第二蛋白、跨膜域、及胞内域，
优选所述第二蛋白能够特异性识别BCMA或CD19；
优选的，所述第二蛋白含有能够特异性识别BCMA的抗体；
进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、以及SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；
更进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区。
在一优选的实施例中，采用基因编辑技术使基因沉默。
优选的，所述的基因编辑技术选自CRISPR/Cas9技术、人工锌指核酸酶(Zinc Finger Nucleases，ZFN)技术、转录激活样效应因子(transcription activator-like effector,TALE)技 术、或TALE-CRISPR/Cas9技术；
更优选的，所述的基因编辑技术为CRISPR/Cas9技术。
在本发明的第三方面预防或调控移植免疫排斥的方法，包括给予本发明第一方面或第二方面任一所述的细胞。
在本发明的第四方面，提供了预防或调控外源细胞被NK细胞攻击的方法，其特征在于，给予表达有识别NK细胞的第一蛋白的免疫效应细胞；
可选的，所述外源细胞是T细胞、NK T细胞、干细胞，或工程化的T细胞、NK T细胞、干细胞；
可选的，在给予所述外源细胞之前、之后、或与所述外源细胞同时给予所述免疫效应细胞。
在一优选的实施例中，所述外源细胞是免疫效应细胞，优选的，所述外源细胞表达有第二受体。
在一优选的实施例中，所述第二受体为嵌合受体或T细胞受体；
优选的，所述嵌合受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
在一优选的实施例中，所述识别NK细胞的第一蛋白识别的抗原为以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
在一优选的实施例中，所述HLA-I基因选自HLA-A、HLA-B、HLA-C，B2M中的一种或两种以上；优选的，所述HLA-I基因为B2M。
在一优选的实施例中，所述第二蛋白是嵌合受体，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体或T细胞抗原耦合器(TAC)，包含第二蛋白的嵌合受体包括第二蛋白、跨膜域、及胞内域，
优选所述第二蛋白能够特异性识别BCMA或CD19；
优选的，所述第二蛋白含有能够特异性识别BCMA的抗体；
进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、以及SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；
更进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区。
在一优选的实施例中，给予本发明第一方面或第二方面任一所述的细胞。
在本发明的第五方面，提供了预防或调控外源细胞被NK细胞攻击的方法，给予表达有识别NK细胞的第一蛋白的免疫效应细胞；
可选的，所述外源细胞是T细胞、NK T细胞、干细胞，或工程化的T细胞、NK T细胞、干细胞。
在一优选的实施例中，所述外源细胞是免疫效应细胞，优选的，所述外源细胞表达有第二受体。
在一优选的实施例中，所述第二受体为嵌合受体或T细胞受体；
优选的，所述嵌合受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
在一优选的实施例中，所述识别NK细胞的第一蛋白识别的抗原为以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
在一优选的实施例中，所述免疫效应细胞包括T细胞、NK细胞、NKT细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞。
在本发明的第六方面，提供了预防或调控外源免疫效应细胞被NK细胞攻击的方法，其特征在于，所述外源免疫效应细胞表达识别NK细胞的第一蛋白；
优选的，所述外源免疫效应细胞是不含有HLA-I基因或者内源性的HLA-I基因沉默的细胞；
更优选的，所述外源免疫效应细胞是不含有B2M基因或B2M基因被沉默的细胞。
在一优选的实施例中，所述外源免疫效应细胞是T细胞，
优选的，所述识别NK细胞的第一蛋白是嵌合受体或T细胞受体。
在一优选的实施例中，所述识别NK细胞的第一蛋白识别的抗原为以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
在一优选的实施例中，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
在一优选的实施例中，所述外源免疫效应细胞还表达识别肿瘤抗原或病原体抗原的第二蛋白；
优选的，所述第二蛋白是嵌合受体，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
在一优选的实施例中，所述的第一蛋白为含有识别NK细胞及识别肿瘤抗原或病原体抗原的抗体的嵌合抗原受体、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
在一优选的实施例中，所述第一蛋白包含有胞外域、跨膜域和胞内信号域；
优选的，所述细胞通过胞内信号域传递信号介导对宿主的免疫效应细胞的抑制或杀伤。
在一优选的实施例中，所述二蛋白包含有胞外域、跨膜域和胞内信号域；
优选的，所述细胞通过胞内信号域传递信号介导对肿瘤或病原体的抑制或杀伤。
在一优选的实施例中，所述第一蛋白包含识别宿主免疫效应细胞的抗体、识别肿瘤抗原或病原体抗原的抗体、跨膜域、及胞内域；
优选的，所述识别宿主免疫效应细胞的抗体和识别肿瘤抗原或病原体抗原的抗体通过连接肽相连；
更进一步优选的，所述第一蛋白具有SEQ ID NO：9所示的序列。
附图说明
图1：NK细胞表面标志物表达情况；
图2：T细胞中NK细胞表面标志物表达情况。；
图3：NKG2A CAR-T细胞的生长特性。A，细胞增殖曲线；B，细胞直径大小；C,CAR阳性率；
图4：共孵育4小时NKG2A CAR-T细胞对NK细胞的体外杀伤能力；
图5：共孵育18小时NKG2A CAR-T细胞对NK细胞的体外杀伤能力；
图6：高效敲除CAR-T细胞中的TCR和B2M；
图7A、图7B、图7C和图7D：FACS检测NKG2A UCAR-T细胞对NK细胞的抵抗能力；
图8：为靶向NKG2A的CAR的质粒图谱；
图9：为靶向BCMA的CAR的质粒图谱；
图10：FACS检测NKG2A UCAR-T细胞对NK细胞的抵抗能力；
图11：FACS检测小鼠外周血中UCAR-T细胞存活；
图12：BCMA-GS-NKG2A UCAR-T的结构示意图；
图13：BCMA-GS-NKG2A UCAR-T细胞的制备；
图14：BCMA-GS-NKG2A UCAR-T细胞的体外抗肿瘤作用；
图15：BCMA-GS-NKG2A UCAR-T细胞对NK细胞抵抗作用；
图16：为PRRL-BCMA-BBZ-F2A-EGFP的质粒图谱；
图17：为PRRL-NKG2A-28Z-F2A-EGFP的质粒图谱；
图18：为PRRL-BCMA-GS-NKG2A-BBZ的质粒图谱；
图19：显示了BCMA-GS-NKG2A UCAR-T细胞体内对NK细胞的抵抗结果。
具体实施方式
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis (M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries & S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames & S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
公开内容中，请求保护的主题的各个方面均以范围形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。这适用而无关范围的宽度。
本文使用的术语约是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”或“包含”应假定为在该特定值或组成的可接受误差范围内。
除非另外指出，本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值。
为便于更好地理解本发明，对相关术语定义如下：
术语“移植免疫排斥”是指宿主进行同种异体的组织、器官、或细胞等移植物移植 后，外源的移植物作为一种“异己成分”被宿主的免疫系统识别，并发起针对移植物的攻击、破坏和清除的免疫学反应。
术语“移植物”是指来源于宿主之外的个体，用于植入宿主的生物材料或制剂。移植物可来自任何动物来源，如哺乳动物来源，优选来自人类。在一些实施方式中，所述的移植物可以是来自宿主，如来自宿主的细胞经体外培养、或改造再次植入宿主。在一些实施方式中，所述的移植物可以是来自同种异体的其他个体，如来自其他人的的细胞经体外培养、或改造植入宿主。在一些实施方式中，所述的移植物可以是来自异种的个体，如来自其他种属(如鼠、猪、猴)的器官植入人。
术语“细胞”及其语法上的其他形式可以指人或非人动物来源的细胞。
术语“宿主”是指接受移植物移植的受体，在一些实施方式中，可以是接受外源细胞植入的个体，如人。
术语“免疫效应细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞等。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT细胞。
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。或者，可以从健康供体，来自诊断患有癌症的患者衍生细胞。
术语“MHC”为组织相容性复合物，是所有编码生物相容复合体抗原的基因群一种统称，MHC抗原表达于所有高等脊椎动物的组织，在人类细胞中称为HLA抗原，在移植反应中发挥重要作用，由对所植入的组织的表面上的组织相容性抗原产生反应的T细胞介导排异。MHC蛋白质在T细胞刺激中发挥至关重要的作用，抗原呈递细胞(通常是树突状细胞)展示属于MHC上的细胞表面上的外源蛋白的降解产物的肽，在共刺激信号的存在下，T细胞被活化并作用于也展示相同肽/MHC复合体的靶细胞。例如，刺激的T辅助细胞会靶向巨噬细胞，该巨噬细胞展示与其MHC结合的抗原，或细胞毒性T细胞(CTL)会作用于展示外源病毒肽的病毒感染的细胞。MHC抗原分为NHC I类抗原和MHC II类抗原。
术语“人类白细胞抗原”(Human leukocyte antigen，HLA)是人类的主要组织相容性复合体的编码基因，位于6号染色体上(6p21.31)，与人类的免疫系统功能密切相关。HLA包括有I类、II类和III类基因部分。HLA的I类和II类基因所表达的抗原位于细胞膜上，为MHC-I(HLA-A、HLA-B、HLA-C位点编码)和MHC-II(HLA-D区编码)，HLA I类几乎分布于身体全部细胞表面，是一个异二聚体，由重链(α链)与β2微球蛋白组成(B2M)，II类主要是定位于巨噬细胞和B淋巴细胞表面的糖蛋白。
术语“B2M”为β-2微球蛋白，也称为B2M，是MHC I类分子的轻链。在人类中，B2M由位于15号染色体上的b2m基因编码，与6号染色体上的作为基因簇定位的其他MHC基因相对。有研究表明，当B2M基因发生突变，来自缺乏正常细胞表面MHC I表达的小鼠的造血移植物被正常小鼠中的NK细胞排斥，说明MHC I分子的缺陷性表达使细胞易于被宿主免疫系统排斥(Bix et al.1991)。
术语“嵌合受体”，即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域，在一个方面，刺激性分子为与T细胞受体复合体结合的δ链；在一个方面，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。
术语“T细胞受体(T cell receptor，TCR)”介导T细胞对特异性主要组织相容性复合物(MHC)-限制性肽抗原进行识别，包括经典的TCR受体和优化的TCR受体。经典的TCR受体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等，其抗原特异性存在于V区，V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，在一个方面，表达经典的TCR的T细胞可以通过对T细胞采用如抗原刺激等方式，诱导T细胞的TCR对靶抗原的特异性。
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够 结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该嵌合T细胞受体整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。胞内信号传导结构域可以包括分子的全部细胞内部分、或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“共刺激分子”指与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号。
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指T细胞逐步活化的过程。例如，T细胞可能需要至少一个信号才能完全激活。
术语“基因编辑”，指能够让人类对目标基因进行“编辑”，实现对特定DNA片段的敲除、加入等。
术语“基因沉默”，是指由于各种原因，使基因不表达或低表达的现象。基因沉默可以是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默，也可以是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而是基因失活，包括反义RNA、共抑制、基因压抑、RNA干扰和微小RNA介导的翻译抑制等。
所述“TCR沉默”是指内源性的TCR不表达或低表达。
所述“MHC沉默”是指内源性的MHC不表达或低表达。
术语“CRISPR(Clustered regularly interspaced short palindromicrepeats)”是指规律成簇间隔短回文重复。
术语“Cas9(CRISPRassociated nuclease)”是CRISPR相关核酸酶，是一种由RNA指导的，利用Cas9核酸酶对靶向基因进行编辑的技术。
“CRISPER/Cas9系统”统称为转录物和涉及Cas9酶基因的表达或指导其活性的其他元件，包括编码Cas9基因的序列、tracr(反式激活CRISPR)序列(例如tracrRNA或活性部分tracrRNA)、tracr配对序列(涵盖“同向重复”和在内源CRISPR系统背景下的tracrRNA加工的部分同向重复)、指导序列(在内源CRISPR系统背景下也称为“间隔子(spacer)”，即gRNA)、或来自CRISPR座位的其他序列和转录物。
术语“靶序列”是指与指导序列具有互补性的序列，靶序列与指导序列之间互补配对促进CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。
一般而言，指导序列(gRNA)是与靶多核苷酸序列具有足够互补性以便与该靶序列杂交并且指导CRISPR复合物与该靶序列的序列特异性结合的任何多核苷酸序列。在一些实施例中，当使用适合的比对算法进行最佳比对时，在指导序列与其相应的靶序列之间的互补程度是约或多于约50％、60％、75％、80％、85％、90％、95％、97.5％、99％、或更多。可以使用用于比对序列的任何适合的算法来确定最佳比对，其非限制性实例包括史密斯-沃特曼(Smith-Waterman)算法、尼德曼-翁施(Needleman-Wunsch)算法、基于伯罗斯-惠勒变换(Burrows-Wheeler Transform)的算法(例如伯罗斯-惠勒比对工具(Burrows Wheeler Aligner))、ClustalW、Clustal X、BLAT、Novoalign(Novocraft技术公司)、ELAND(亿明达公司(Illumina)，圣地亚哥，加利福尼亚州)、SOAP(在soap.genomics.org.cn可获得)、以及Maq(在maq.sourceforge.net可获得)。
在一些实施例中，该CRISPR酶是包含一个或多个异源蛋白结构域(例如除了该CRISPR酶之外的约或多于约1、2、3、4、5、6、7、8、9、10个或更多个结构域)的融合蛋白的一部分。CRISPR酶融合蛋白可以包含任何其他蛋白质，以及任选地在任何两个结构域之间的连接序列。可以融合到CRISPR酶上的蛋白质结构域的实例包括但不限于，表位标签、报告基因序列、以及具有下列活性的一者或多者的蛋白质结构域：甲基酶活性、脱甲基酶活性、转录激活活性、转录阻抑活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。表位标签的非限制性实例包括组氨酸(His)标签、V5标签、FLAG标签、流感病毒血凝素(HA)标签、Myc标签、VSV-G标签、和硫氧还蛋白(Trx)标签。
术语“Cas9酶”可以是野生型Cas9或人工改造Cas9。
术语“sgRNA”指短小的gRNA。
在进行基因编辑时，給予的gRNA、tracr配对序列、及tracr序列可以单独给予，也可以一条完整的RNA序列。
Cas9蛋白与gRNA结合能够实现在特异位点处切割DNA，来源于Streptococcus pyogenes的CRISPR/Cas系统识别序列为23bp,并能靶向20bp,其识别位点最末3位NGG序列被称作PAM(protospacer adjacent motif)序列。
Cas转基因可以通过载体(例如AAV、腺病毒、慢病毒)、和/或粒子和/或纳米粒子、和/或电转来递送。
在一实施例中，对在TCR的α和β链中的一种或两种链的恒定区域的相应编码基因的外显子用CRISPER/Cas技术敲除，使内源性TCR不具有活性，优选为定点敲除内源性TCRα链恒定区的第一外显子。
“抑制”或“遏制”B2M或TCR的表达是指细胞中B2M或TCR的表达减少至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。更具体而言，“抑制”或“遏制”B2M的表达是指细胞中B2M的含量降低至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。可以通过本领域内已知的任何合适的方法，如ELISA、免疫组织化学、免疫印迹(Western Blotting)或流式细胞术使用B2M或TCR的特异性抗体测定细胞中蛋白的表达或含量。
本发明所使用的“修饰”一词是指本发明的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。
术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
术语“T细胞活化”或“T细胞激活”及其语法上的其他形式可以指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
当用于指核苷酸序列时，本文所用的术语“序列”及其语法上的其他形式可以包括DNA或RNA，并且可以是单链或双链。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表 达载体包括本领域所有已知的那些，如质粒、病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。
本文使用的术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
本文所用的术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。
术语“内源”是指一个核酸分子或多肽等来自生物体自身。
在一些实施方案中，本发明嵌合受体是嵌合抗原受体。
嵌合抗原受体通常包含胞外抗原结合区。在一些实施方案中，胞外抗原结合区可以是全人的，人源化，鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。
胞外抗原结合区的例子可以是scFv、Fv、Fab、Fab'、Fab'-SH、F(ab')2、单结构域片段、或与接合其同源受体的天然配体，以及它们的任何衍生物。
在一些方面，细胞外抗原结合区(例如scFv)，可以包含对抗原特异性的轻链CDR。在一些情况下，轻链CDR可以包含两个或更多个轻链CDR，其可以被称为轻链CDR-1，CDR-2等。在一些情况下，轻链CDR可以包含三个轻链CDR，其可分别称为轻链CDR-1，轻链CDR-2和轻链CDR-3。在一实施方式中，存在于普通轻链上的一组CDR可统称为轻链CDR。
在一些方面，细胞外抗原结合区(例如scFv)，可以包含对抗原特异的重链CDR。重链CDR可以是抗原结合单元例如scFv的重链互补决定区。在一些情况下，重链CDR可以包含两个或更多个重链CDR，其可以称为重链CDR-1，CDR-2等。在一些情况下，重链CDR可以包含三个重链CDR，其可分别称为重链CDR-1，重链CDR-2和重链CDR-3。在一实施方式中，存在于共同重链上的一组CDR可统称为重链CDR。
通过使用基因工程，可以以各种方式修饰细胞外抗原结合区。在一些情况下，可以突变细胞外抗原结合区域，从而可以选择细胞外抗原结合区域以对其靶标具有更 高的亲和力。在一些情况下，细胞外抗原结合区域对其靶标的亲和力可针对可在正常组织上以低水平表达的靶标进行优化。可以进行此优化，以尽量减少潜在的毒性。在其他情况下，对靶标的膜结合形式具有更高亲和力的细胞外抗原结合区域的克隆可以优于其可溶形式的对应物。可以进行这种修饰，因为也可以检测到不同水平的可溶形式的靶标，并且它们的靶向可引起不期望的毒性。
在一些情况下，细胞外抗原结合区域还包括铰链或间隔区，术语铰链和间隔区可以互换使用。铰链可以被认为是用于向细胞外抗原结合区提供柔性的CAR的一部分。例如，铰链可以是CD8α分子的天然铰链区。
术语“跨膜域”可以将嵌合蛋白锚定在细胞的质膜上。例如，可以采用CD28、CD8α的跨膜域。
术语“调控”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。在一具体实施方式中，是指负向改变。
术语“治疗”是指在试图改变疾病过程的干预措施，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
术语“预防”是指在试图在疾病(如细胞移植产生的排斥反应)产生前进行的干预措施。
本发明所述的第一蛋白是指上述能够识别宿主一种或多种免疫效应细胞的蛋白。
本发明所述的第二蛋白是指上述识别肿瘤抗原或病原体抗原的蛋白。
本发明所述的“第二受体”与“能够识别宿主一种或多种免疫效应细胞的蛋白”可以串联表达，也可以单独表达。
当“第二受体”与“能够识别宿主一种或多种免疫效应细胞的蛋白”单独表达时，二者具有独立的跨膜域和胞内域，其表达方法可以参考PCT/CN2015/095938、Enhancing the specificity of T-cell cultures for adoptive immunotherapy of cancer,Duong CP et al.,Immnuotherapy 3(1):33-48等。
当本发明所述的“第二受体”与“能够识别宿主一种或多种免疫效应细胞的蛋白”串联表达时，则识别宿主一种或多种免疫效应细胞的蛋白也能够识别“第二受体”识别的抗原，如肿瘤抗原。
“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指癌症。
本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)， B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2 (EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。优选的，所述肿瘤抗原为BCMA或者CD19。
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、NK细胞表面受体表达情况检测
利用Ficoll-Paque(GE bioscience)进行密度梯度离心，从外周血中分离出单核细胞，用NK细胞分离试剂盒(购自美天旎)进行阴性筛选，去除T细胞，B细胞以及单核细胞等细胞后，进行体外细胞表型鉴定和扩增。利用流式染色对分离的NK细胞表面的受体，如NKG2A,NKG2D,NKP30,NKP44,NKP46等标志物进行鉴定。流式细胞术检测结果显示，NKG2A,NKP30,NKP44,NKP46在80％左右的NK细胞表达，而NKG2D在90％以上的NK细胞中表达(见图1)。
进一步地，我们还检测了上述表面标志物在T细胞中的表达情况。取CD3/CD28磁珠(购自Thermo Fisher)活化的T细胞，培养至第8天进行流式染色。实验结果显示，NKG2A,NKG2D,NKP30,NKP44,NKP46在T细胞中几乎不表达，表明上述标志物可以作为靶向NK细胞的靶点(见图2)。
实施例2、CAR-T细胞的制备及功能验证
1.选取NKG2A为靶点作为代表，制备靶向NK细胞的CAR-T细胞。参照常规操作，设计并构建包含抗NKG2A的单链抗体(VH的氨基酸序列如SEQ ID NO：1所示，VL的氨基酸序列如SEQ ID NO：2所示)，CD28跨膜域和胞内域(氨基酸序列如SEQ ID NO：3所示)，T细胞激活因子CD3δ(氨基酸序列如SEQ ID NO：4所示)的嵌合抗原受体(氨基酸序列如SEQ ID NO：5所示)的载体，质粒图如图8所示，包装慢病毒，命名为VRRL-NKG2A-28Z(TM)。
T细胞活化与扩增48小时后，调整细胞密度至2*10^6/mL，按MOI＝10的比例加入VRRL-NKG2A-28Z(TM)慢病毒，得到靶向NKG2A的CAR-T细胞。
取第6天的CAR-T细胞进行细胞增殖检测，将起始细胞数调整至5*10^5，分别于24hr,48hr,72hr,96hr检测细胞数目，并记录细胞直径。同时用抗F(ab)’2抗体进行流式细胞术染色，检测CAR载体的表达情况。实验结果显示NKG2A CAR-T 细胞与未转染的T细胞(UTD)表现出类似的增长曲线，细胞直径大小也无显著差异，约80％的CAR-T细胞均表达靶向NKG2A的CAR分子，表明NKG2A CAR-T细胞的生长特性正常(见图3)。
2.制备靶向BCMA的CAR-T细胞
参照1的操作，构建靶向BCMA的嵌合抗原受体的质粒(氨基酸序列如SEQ ID NO：6所示)，质粒图如图9所示，包装慢病毒，转染T细胞，得到靶向BCMA的BCMA-CAR T细胞。
3.体外杀伤功能实验。
体外扩增原代NK细胞，作为靶细胞。调整细胞密度至5*10^5/mL，取100μl接种至96孔板(平行做3个复孔)，按效应T细胞：靶细胞为1：3，1：1和3：1三个比例接种相对应的CAR-T细胞。采用MEM-α+5％FBS作为培养基，于37℃、5％CO 2培养箱中分别共孵育4hr和18hr。利用cytotox-96non-radioactive cytotoxicity assay试剂盒(购自Thermo Fisher)，取上清50μl进行乳酸脱氢酶(LDH)含量测定，并计算UTD和NKG2A CAR-T两组中，原代NK细胞的裂解效率。
检测结果显示，NKG2A CAR-T组中的LDH值显著高于UTD组，表明NKG2A CAR-T能有效杀伤原代NK细胞(见图4和图5)。
实施例3、NKG2A UCAR-T细胞的制备
1.TCR和B2M基因的敲除。
体外扩增常规UTD细胞,NKG2A CAR-T细胞以及BCMA CAR-T细胞(用于对照)细胞48小时后，调整细胞密度至2*10^7/mL。Cas 9酶(购自NEB)和sgRNA按1：4比例进行室温孵育10分钟，得到RNP复合液，其中，TRAC-sgRNA的核酸序列如SEQ ID NO：7所示，B2M-sgRNA的核酸序列如SEQ ID NO：8所示，将1*10^6细胞与RNP复合液进行混合(Cas 9酶的终浓度为3uM)，利用maxcyte电转仪将RNP复合物分别导入到CAR-T细胞中。电转后第7天，利用流式细胞术检测TCR和B2M基因的敲除情况，实验结果显示TRAC和B2M敲除效率在85％以上(见图6)。
2.TCR/B2M双阴性细胞筛选。
体外扩增敲除B2M和TCR的CAR-T细胞和UTD细胞，于第8天调整细胞密度至1*10^7/mL，用抗HLA-ABC和B2M抗体对细胞进行标记，再用偶联藻红素(PE)的二抗进行标记，标记后的细胞用抗PE的磁珠经分选柱分选后，收集TCR和B2M双阴性的细胞(分选试剂盒购自美天旎)，即得到TCR和B2M缺失的BCMA UCAR-T细胞、NKG2A UCAR-T细胞、和U-UTD细胞。
实施例4、NKG2A UCAR-T细胞对NK细胞抵抗功能的验证
1.LDH实验检测UCAR-T细胞对NK细胞的排斥作用
将UTD细胞、BCMA-CAR T细胞、NKG2A CAR-T细胞、BCMA UCAR-T细胞、NKG2A UCAR-T细胞和U-UTD细胞作为靶细胞，调整细胞浓度至5*10^5/mL,接种100μl至96孔板，按原代扩增的NK细胞与靶细胞比例1：1，接种同等体积和数目的NK细胞，于培养箱中分别共孵育4hr和18hr。取上清50μl用于乳酸脱氢酶(LDH)含量测定，计算CAR-T和UCAR-T细胞的裂解效率。检测结果显示，UTD以及BCMA CAR-T组的LDH值均很低，表明普通的CAR-T细胞不会引起NK细胞的攻击，而U-UTD和BCMA UCAR-T组在4hr以及18hr均表现出逐渐升高的LDH值，说明NK细胞会对TCR和B2M缺失的T细胞进行杀伤，NKG2A UCAR-T细胞表现出更低水平的LDH，显示NKG2A UCAR-T细胞对NK细胞有抵抗作用。
2.为了进一步证实NKG2A UCAR-T细胞对NK细胞的抵抗能力，选取BCMA UCAR-T细胞进行对照，调整细胞浓度至5*10^5/mL，接种100μl至96孔板，按原代扩增的NK细胞与靶细胞比例1：1，接种同等体积和数目的NK细胞，于培养箱中分别共孵育4hr,18hr,24hr和42hr。利用流式细胞术标记HLA-ABC阳性的NK细胞，检测共孵育不同时间点UCAR-T细胞的所占比例。实验结果如图7A-7D所示，BCMA UCAR-T在4hr时就处于20％左右的低比例，且随着检测时间的延长一直在很低水平的比例，说明NK细胞显著抑制了BCMA UCAR-T细胞的生长；而NKG2A UCAR-T细胞虽然在4hr时处于20％左右的低比例，但随着检测时间的延长，表现出逐渐升高的比例，在42hr时达到了将近60％，说明NKG2A UCAR-T细胞的生长一开始被NK细胞所抑制，但随着时间的延长逐渐恢复了增殖能力。上述结果表明，NKG2A UCAR-T能够有效抵抗NK细胞的杀伤能力。
3.为了进一步地证明NKG2A UCAR-T细胞对原代NK细胞的抵抗能力，构建表达GFP的BCMA UCAR-T细胞和NKG2A UCAR-T细胞。BCMA-GFP的氨基酸序列如SEQ ID NO：24所示，NKG2A-GFP的氨基酸序列如SEQ ID NO：25所示。
参照实施例2的操作，构建表达GFP的BCMA UCAR-T细胞的质粒PRRL-BCMA-BBZ-F2A-EGFP，质粒图谱见图16，构建表达NKG2A UCAR-T细胞的质粒PRRL-NKG2A-28Z-F2A-EGFP，质粒图谱见图17。构建后的质粒包装慢病毒，转染T细胞，并对CAR-T细胞进行基因敲除和磁珠分选，得到表达GFP的BCMA UCAR-T细胞和表达GFP的NKG2A UCAR-T细胞。
调整CAR-T细胞浓度至5*10^5/mL，接种100μl至96孔板，按原代扩增的NK细胞与靶细胞比例1：1，接种同等体积和数目的NK细胞，于培养箱中分别共孵育0hr,4hr,18hr,24hr和48hr。利用流式细胞术检测共孵育不同时间点GFP细胞的比例，用于示踪UCAR-T细胞的存活情况。
实验结果如图10所示，GFP阳性的BCMA UCAR-T细胞随着时间的延长，比例逐渐下降，48hr后基本上完全被NK细胞所杀伤，而GFP阳性的NKG2A UCAR-T 细胞比例在4hr有轻微的下降，18hr后出现了明显的上升，并在48hr后占到90％左右，表明NKG2A UCAR-T细胞能显著抵抗NK细胞的杀伤。
实施例5、NKG2A UCAR-T细胞体内对NK细胞的抵抗
体外培养BCMA UCAR-T和NKG2A UCAR-T细胞，调整CAR阳性率至80％，按8*10^6细胞/只的剂量通过尾静脉注射到NPG免疫缺陷小鼠中，将小鼠分为两组：给予BCMA UCAR-T及NK细胞组(标记为BCMA UCAR-T+NK)、给予NKG2A UCAR-T组及NK细胞组(标记为NKG2A-UCART+NK)。
在UCAR T细胞给予4hr后注射等量的NK细胞，在注射CAR T细胞后的第1，3，6天，分别通过流式绝对技术检测小鼠外周血中人源的CD4和CD8T细胞的存活情况。
实验结果如图11所示，注射后第1天，BCMA UCAR-T+NK组和NKG2A UCAR-T+NK组的UCAR-T细胞数目(即人源的CD4和CD8T细胞)出现显著下降，表明UCAR-T细胞被NK细胞所排斥。在注射后的第3天和第6天，BCMA UCAR-T+NK组的UCAR-T细胞数目一直处于非常低的状态，而NKG2A UCAR-T+NK组的UCAR-T细胞数目在第3天和第6天出现了明显的上升。上述结果表明，在体内模型中，NK细胞显著抑制了BCMA UCAR-T细胞的存活，而NKG2A UCAR-T细胞能有效抵抗NK细胞的杀伤，恢复增殖能力。
实施例6、靶向BCMA和NKG2A的CAR T细胞的构建
如图12所示，制备靶向BCMA的scFv和靶向NKG2A的scFv串联的UCAR-T细胞(即：BCMA-GS-NKG2A UCAR-T)。BCMA-GS-NKG2A CAR的氨基酸序列如SEQ ID NO：9所示。
构建BCMA-GS-NKG2A UCAR-T的质粒PRRL-BCMA-GS-NKG2A-BBZ，质粒图谱如图18所示。参照实施例2和3的操作，进行病毒转染，得到BCMA-GS-NKG2A UCAR-T细胞，对BCMA-GS-NKG2A UCAR-T细胞进行TRAC和B2M基因的敲除，再用磁珠分选的方法得到99％以上TCR和HLA-ABC阴性的BCMA-GS-NKG2A UCAR-T细胞。
参照实施例2和3的操作，分别制备得到BCMA UCAR-T细胞和NKG2A UCAR-T细胞。
分别检测BCMA UCAR-T，NKG2A UCAR-T和BCMA-GS-NKG2A UCAR-T的CAR表达情况，实验结果如图13所示，阳性率均达到60％以上，表明BCMA-GS-NKG2A UCAR-T细胞制备成功。
实施例7、BCMA-GS-NKG2A UCAR-T细胞的体外功能验证
体外培养BCMA阳性的多发性骨髓瘤细胞系RPMI-8226和NCI-H929作为靶细胞，接种1*10^4个肿瘤细胞于96孔板，按T细胞比肿瘤细胞为3：1，1：1和1：3接种相对应数目的UCAR-T细胞，共孵育18小时后，吸取50μl上清进行LDH含量检测。
实验结果如图14所示，UTD和NKG2A UCAR-T组中，RPMI-8226和NCI-H929的细胞裂解率均很低；而BCMA-GS-NKG2A UCAR-T组的肿瘤细胞裂解率与BCMA UCAR-T组相当，表明BCMA-GS-NKG2A UCAR-T细胞在体外能够有效杀伤BCMA阳性的肿瘤细胞。
实施例8、BCMA-GS-NKG2A UCAR-T细胞对NK细胞抵抗功能的验证
选取BCMA UCAR-T和NKG2A UCAR-T细胞分别作为阴性和阳性对照，调整细胞浓度至5*10^5/mL，接种100μl至96孔板，按NK细胞与T细胞比例1：1，接种同等体积和数目的NK细胞，于培养箱中分别共孵育0hr,4hr,18hr,24hr和48hr。利用流式细胞术标记HLA-ABC阳性的NK细胞，检测共孵育不同时间点UCAR-T细胞的所占比例。实验结果如图15所示，随着孵育时间的延长，BCMA UCAR-T细胞的比例逐渐降低，48hr已经基本上被NK细胞杀伤；而BCMA-GS-NKG2A UCAR-T和NKG2A UCAR-T细胞表现出相同的变化趋势，在4hr发现轻微下降，然后逐渐升高，48hr到达70％以上，BCMA-GS-NKG2A UCAR-T细胞在48hr达到90％的比例。上述结果表明，BCMA-GS-NKG2A UCAR-T细胞能够有效抵抗NK细胞的杀伤。
实施例9、BCMA-GS-NKG2A UCAR-T细胞体内对NK细胞的抵抗
体外培养BCMA UCAR-T和BCMA-GS-NKG2A UCAR-T细胞，调整CAR阳性率至60％，按8*10^6细胞/只的剂量通过尾静脉注射到NPG免疫缺陷小鼠中，将小鼠分为两组：给予BCMA UCAR-T及NK细胞组(标记为BCMA UCAR-T+NK)和给予BCMA-GS-NKG2A UCAR-T组及NK细胞组(标记为BCMA-GS-NKG2A UCAR-T+NK)。在UCAR-T细胞给予4小时后注射等量的NK细胞，在注射CAR T细胞后的第1，3，6天，分别通过流式绝对技术，检测小鼠外周血中人源的CD45阳性T细胞的存活情况。
实验结果如图19所示，相比于注射后第1天，BCMA UCAR-T+NK组UCAR-T细胞数目在第3天和第6天均未出现明显的增加，表明UCAR-T细胞被NK细胞所排斥。而BCMA-GS-NKG2A UCAR-T+NK组的UCAR-T细胞数目，在第3天和第6天均出现了明显的上升，第6天时细胞数目较第1天时增加了30倍以上。上述结果表明，在体内模型中，NK细胞显著抑制了BCMA UCAR-T细胞的存活，而BCMA-GS-NKG2A UCAR-T细胞能有效抵抗NK细胞的杀伤，恢复增殖能力。
本申请涉及的序列如下表所示：
Figure PCTCN2020098930-appb-000001
Figure PCTCN2020098930-appb-000002
Figure PCTCN2020098930-appb-000003

Claims (38)

  1. 抗移植免疫排斥的细胞，其特征在于，所述细胞表达有能够识别宿主一种或多种免疫效应细胞的第一蛋白；优选的，所述细胞对宿主的免疫效应细胞具有抑制或杀伤功能。
  2. 如权利要求1所述的细胞，其特征在于，所述细胞为免疫效应细胞或经人工改造的具有免疫效应细胞功能的细胞。
  3. 如权利要求1或2所述的细胞，其特征在于，所述细胞选自T细胞、NK细胞、NKT细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞；
    优选的，所述细胞是T细胞，
    更优选的，所述第一蛋白是嵌合受体。
  4. 如权利要求1-3中任一所述的细胞，其特征在于，所述细胞还表达有识别肿瘤抗原或病原体抗原的第二蛋白，优选所述第二蛋白是嵌合受体或T细胞受体。
  5. 如权利要求1-4任一所述的细胞，其特征在于，所述细胞不表达MHC，或所述细胞内源性表达的MHC基因被沉默；优选的，所述的MHC基因为MHC I类分子的基因。
  6. 如权利要求5所述的细胞，其特征在于，所述的细胞不表达HLA，或所述细胞内源性表达的HLA基因被沉默；优选的，所述的HLA为HLA-I类基因。
  7. 如权利要求1-6任一所述的细胞，其特征在于，所述的抗移植免疫排斥为抗宿主的NK细胞的攻击，或者所述第一蛋白能够识别宿主的NK细胞，
    优选的，所述第一蛋白能够特异性识别以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
    更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
  8. 如权利要求7所述的细胞，其特征在于，所述第一蛋白含有能够识别宿主NK细胞的抗体；
    优选的，所述抗体能够识别NKG2A；
    进一步优选的，所述抗体含有SEQ ID NO：10所示的HCDR1，SEQ ID NO：11所示 的HCDR2，SEQ ID NO：12所示的HCDR3，SEQ ID NO：13所示的LCDR1，SEQ ID NO：14所示的LCDR2，SEQ ID NO：15所示的LCDR3；
    更进一步优选的，所述抗体含有SEQ ID NO：1所述的重链可变区或SEQ ID NO：2所述的轻链可变区。
  9. 如权利要求8所述的细胞，其特征在于，所述HLA-I基因选自HLA-A、HLA-B、HLA-C，B2M中的一种或两种以上；优选的，所述HLA-I基因为B2M。
  10. 如权利要求3或4所述的细胞，其特征在于，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体或T细胞抗原耦合器(TAC)。
  11. 如权利要求1所述的细胞，其特征在于，所述第一蛋白包含有胞外域、跨膜域和胞内信号域；
    优选的，所述细胞通过胞内信号域传递信号介导对宿主的免疫效应细胞的抑制或杀伤。
  12. 如权利要求4所述的细胞，其特征在于，所述二蛋白包含有胞外域、跨膜域和胞内信号域；
    优选的，所述细胞通过胞内信号域传递信号介导对肿瘤或病原体的抑制或杀伤。
  13. 如权利要求6所述的细胞，其特征在于，所述细胞为HLA-I基因和内源性的TCR基因沉默的T细胞；
    优选的，所述细胞为B2M和TCR基因沉默的T细胞。
  14. 如权利要求4所述的细胞，其特征在于，所述第二蛋白能够特异性识别BCMA或CD19；
    优选的，所述第二蛋白含有能够特异性识别BCMA的抗体；
    进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、以及SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；
    更进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区。
  15. 如权利要求5、6或13所述的细胞，其特征在于，采用基因编辑技术使基因沉默。
  16. 如权利要求10所述的细胞，其特征在于，所述第一蛋白包含识别宿主免疫效应细胞的抗体、识别肿瘤抗原或病原体抗原的抗体、跨膜域、及胞内域；
    优选的，所述识别宿主免疫效应细胞的抗体和识别肿瘤抗原或病原体抗原的抗体通过连接肽相连；
    更进一步优选的，所述第一蛋白具有SEQ ID NO：9所示的序列。
  17. 一种抗移植免疫排斥的细胞，其特征在于，所述细胞为T细胞，该T细胞具有能够识别宿主一种或多种免疫效应细胞的T细胞受体，优选的所述细胞对宿主的免疫效应细胞具有抑制或杀伤功能。
  18. 如权利要求17所述的细胞，其特征在于，所述细胞还表达有识别肿瘤抗原或病原体抗原的第二蛋白，优选所述第二蛋白是嵌合受体。
  19. 如权利要求17或18任一所述的细胞，其特征在于，所述细胞不表达MHC，或所述细胞内源性表达的MHC基因被沉默；优选的，所述的MHC基因为MHC I类分子的基因。
  20. 如权利要求19所述的细胞，其特征在于，所述的细胞不表达HLA，或所述细胞内源性表达的HLA基因被沉默；优选的，所述的HLA为HLA-I类基因。
  21. 如权利要求17-20任一所述的细胞，其特征在于，所述T细胞受体能够识别宿主的NK细胞，
    优选的，所述T细胞受体能够特异性识别以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
    更优选的，所述T细胞受体能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
  22. 如权利要求20所述的细胞，其特征在于，所述HLA-I基因选自HLA-A、HLA-B、HLA-C，B2M中的一种或两种以上；优选的，所述HLA-I基因为B2M。
  23. 如权利要求18所述的细胞，其特征在于，所述第二蛋白是嵌合受体，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体或T细胞抗原耦合器(TAC)，包含第二蛋白的嵌合受体包括第二蛋白、跨膜域、及胞内域，
    优选所述第二蛋白能够特异性识别BCMA或CD19；
    优选的，所述第二蛋白含有能够特异性识别BCMA的抗体；
    进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、以及SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；
    更进一步优选的，所述特异性识别BCMA的抗体含有SEQ ID NO：22所示的重链可变 区和SEQ ID NO：23所示的轻链可变区。
  24. 预防或调控移植免疫排斥的方法，其特征在于，给予权利要求1-23任一所述的细胞。
  25. 预防或调控外源细胞被NK细胞攻击的方法，其特征在于，给予表达有识别NK细胞的第一蛋白的免疫效应细胞；
    可选的，所述外源细胞是T细胞、NK T细胞、干细胞，或工程化的T细胞、NK T细胞、干细胞。
  26. 如权利要求25所述的方法，其特征在于，所述外源细胞是免疫效应细胞，优选的，所述外源细胞表达有第二受体。
  27. 如权利要求26所述的方法，其特征在于，所述第二受体为嵌合受体或T细胞受体；
    优选的，所述嵌合受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
  28. 如权利要求25所述的方法，其特征在于，所述识别NK细胞的第一蛋白识别的抗原为以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
    更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
  29. 如权利要求25所述的方法，其特征在于，所述免疫效应细胞包括T细胞、NK细胞、NKT细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞。
  30. 预防或调控外源免疫效应细胞被NK细胞攻击的方法，其特征在于，所述外源免疫效应细胞表达识别NK细胞的第一蛋白；
    优选的，所述外源免疫效应细胞是不含有HLA-I基因或者内源性的HLA-I基因沉默的细胞；
    更优选的，所述外源免疫效应细胞是不含有B2M基因或B2M基因被沉默的细胞。
  31. 如权利要求30所述的方法，其特征在于，所述外源免疫效应细胞是T细胞，
    优选的，所述识别NK细胞的第一蛋白是嵌合受体或T细胞受体。
  32. 如权利要求31所述的方法，其特征在于，所述识别NK细胞的第一蛋白识别的抗原为以下抗原中的一种或两种以上：NKG2受体家族，如NKG2A,NKG2D,NKG2C等；杀伤免疫球蛋白样受体(KIR)家族，如KIR2DL1,KIR2DL2/3,KIR2DL4,KIR2DL5,KIR3DL1,KIR3DL2,KIR2DS1,KIR2DS2/S3,KIR2DS4,KIR2DS5,KIR3DS1等；自然细胞毒性受体(NCR)，如NKP30,NKP44,NKP46，NKp80等；以及其他NK细胞特异性表达的抗原，如CD159a,CD159c,CD94,CD158,CD56,LIR/ILT2,CD244，CD226，CD2，CD16，CD161，
    更优选的，所述第一蛋白能够特异性识别以下NK细胞表面抗原中的一种或两种以上：NKG2A,NKG2D、NKP30,NKP44,NKP46。
  33. 如权利要求32所述的方法，其特征在于，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)。
  34. 如权利要求30-33任一权利要求所述的方法，其特征在于，所述外源免疫效应细胞还表达识别肿瘤抗原或病原体抗原的第二蛋白；
    优选的，所述第二蛋白是嵌合受体，所述嵌合受体选自嵌合抗原受体(CAR)、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
  35. 如权利要求34所述的方法，其特征在于，所述的第一蛋白为含有识别NK细胞及识别肿瘤抗原或病原体抗原的抗体的嵌合抗原受体、嵌合T细胞受体、或T细胞抗原耦合器(TAC)。
  36. 如权利要求30所述的细胞，其特征在于，所述第一蛋白包含有胞外域、跨膜域和胞内信号域；
    优选的，所述细胞通过胞内信号域传递信号介导对宿主的免疫效应细胞的抑制或杀伤。
  37. 如权利要求34所述的细胞，其特征在于，所述二蛋白包含有胞外域、跨膜域和胞内信号域；
    优选的，所述细胞通过胞内信号域传递信号介导对肿瘤或病原体的抑制或杀伤。
  38. 如权利要求30所述的细胞，其特征在于，所述第一蛋白包含识别宿主免疫效应细胞的抗体、识别肿瘤抗原或病原体抗原的抗体、跨膜域、及胞内域；
    优选的，所述识别宿主免疫效应细胞的抗体和识别肿瘤抗原或病原体抗原的抗体通过连接肽相连；
    更进一步优选的，所述第一蛋白具有SEQ ID NO：9所示的序列。
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Publication number Priority date Publication date Assignee Title
US11525006B2 (en) * 2017-01-23 2022-12-13 Crage Medical Co., Limited BCMA-targeting antibody and use thereof
EP3834849A4 (en) * 2018-07-24 2022-08-03 CRAGE medical Co., Limited METHOD OF TREATMENT OF A TUMOR USING AN IMMUNE EFFECTOR CELL
WO2023284874A1 (zh) * 2021-07-16 2023-01-19 克莱格医学有限公司 用于肿瘤免疫学的组合物和方法
WO2023284875A1 (zh) * 2021-07-16 2023-01-19 克莱格医学有限公司 嵌合抗原受体

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US20220356447A1 (en) 2022-11-10
BR112021026416A8 (pt) 2022-06-28
BR112021026416A2 (pt) 2022-02-08
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