WO2023284874A1 - Composition and method for tumor immunology - Google Patents

Composition and method for tumor immunology Download PDF

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Publication number
WO2023284874A1
WO2023284874A1 PCT/CN2022/106095 CN2022106095W WO2023284874A1 WO 2023284874 A1 WO2023284874 A1 WO 2023284874A1 CN 2022106095 W CN2022106095 W CN 2022106095W WO 2023284874 A1 WO2023284874 A1 WO 2023284874A1
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cells
seq
cell
antigen
nkg2a
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PCT/CN2022/106095
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French (fr)
Chinese (zh)
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李宗海
廖朝晖
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克莱格医学有限公司
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Priority to CN202280042414.3A priority Critical patent/CN117730094A/en
Publication of WO2023284874A1 publication Critical patent/WO2023284874A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present application provides the combined use of immune cells that recognize NKG2A and immune cells that recognize tumor and/or pathogen antigens.
  • the core problem facing allogeneic immune cell therapy is to avoid graft-versus-host (GVHD) reaction and host immune system rejection (HVGR). Therefore, how to increase the survival and expansion of allogeneic immune cells in the host and avoid host immune rejection is crucial to improving the efficacy of immune cell therapy.
  • GVHD graft-versus-host
  • HVGR host immune system rejection
  • the present application provides the combined use of a first CAR T cell targeting NKG2A and a second CAR T cell targeting a target antigen in disease treatment.
  • the endogenous NKG2A expression, activity and/or signal transduction of the first CAR T cell and/or the second CAR T cell is reduced.
  • the target antigen is a tumor antigen and the disease is a tumor.
  • the endogenous NKG2A expression, activity and/or signaling of the first CAR T cell and the second CAR T cell is reduced.
  • the tumor antigens are CD19 and BCMA.
  • the TCR molecule of the first CAR T cell and/or the second CAR T cell is silenced.
  • the TCR molecular silencing refers to the silencing of genes encoding one or both of the ⁇ and ⁇ chains of TCR;
  • the TCR molecular silencing refers to the silencing of the gene encoding the alpha chain of TCR (ie TRAC gene);
  • the TCR molecular silencing refers to the silencing of the gene encoding the ⁇ -chain constant region of TCR
  • the TCR molecular silencing refers to the silencing of the first exon of the gene encoding the ⁇ -chain constant region of TCR.
  • endogenous MHC molecules of the first CAR T cell and/or the second CAR T cell are silenced.
  • the MHC molecule refers to an HLA molecule
  • the HLA molecules are selected from HLA-I class and/or HLA-II molecules, including at least one of HLA-A, HLA-B, HLA-C, B2M and CIITA molecules;
  • the HLA molecules are class I HLA molecules.
  • the HLA molecule is a B2M molecule.
  • gene editing technology is used to silence endogenous TCR molecules, endogenous MHC molecules, or reduce endogenous NKG2A expression, activity and/or signal transduction.
  • the present application provides the combined use of a first CAR T cell targeting NKG2A and a second CAR T cell targeting a target antigen in disease treatment, characterized in that an inhibitor of NKG2A protein is administered at the same time.
  • the TCR molecule of the first CAR T cell and/or the second CAR T cell is silenced.
  • the TCR molecular silencing refers to the silencing of genes encoding one or both of the ⁇ and ⁇ chains of TCR;
  • the TCR molecular silencing refers to the silencing of the gene encoding the alpha chain of TCR (ie TRAC gene);
  • the TCR molecular silencing refers to the silencing of the gene encoding the ⁇ -chain constant region of TCR
  • the TCR molecular silencing refers to the silencing of the first exon of the gene encoding the ⁇ -chain constant region of TCR.
  • endogenous MHC molecules of the first CAR T cell and/or the second CAR T cell are silenced.
  • the MHC molecule refers to an HLA molecule
  • the HLA molecules are selected from HLA-I class and/or HLA-II molecules, including at least one of HLA-A, HLA-B, HLA-C, B2M and CIITA molecules;
  • the HLA molecules are class I HLA molecules.
  • the HLA molecule is a B2M molecule.
  • the endogenous NKG2A of the first CAR T cell and/or the second CAR T cell does not express.
  • gene editing technology is used to knock out the endogenous NKG2A of the first CAR T cell and/or the second CAR T cell;
  • the gene editing technology is selected from CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guided editing technology and/or homing endonuclease technology;
  • the gene editing technology is selected from CRISPR/Cas9 technology.
  • the gRNA used by the CRISPR/Cas9 technology comprises the sequence shown in SEQ ID NO: 10, 46, 47 or 48.
  • the endogenous NKG2A function of the first CAR T cell and/or the second CAR T cell is disrupted.
  • the T cells for preparing CAR T cells are natural T cells or T cells induced by pluripotent stem cells.
  • the antibody that recognizes NKG2A has: HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:5, LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8;
  • the antibody recognizing NKG2A contains the heavy chain variable region described in SEQ ID NO:1 and/or the light chain variable region described in SEQ ID NO:2.
  • the tumor antigen is WT1, HER2, GPC3, Claudin18.2, CD19 or EGFR.
  • the tumor antigen is BCMA
  • the antibody that recognizes BCMA contains: HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and SEQ ID NO HCDR3 shown in :15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18;
  • the antibody recognizing BCMA contains the heavy chain variable region shown in SEQ ID NO:19 and/or the light chain variable region shown in SEQ ID NO:20.
  • the application provides cell composition, it comprises:
  • the non-NKG2A antigens include tumor antigens and/or pathogen antigens.
  • the first immune cell includes a first exogenous receptor that recognizes NKG2A.
  • the first exogenous receptor is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor and T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the first exogenous receptor comprises a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor is a first CAR comprising an NKG2A antigen binding domain.
  • the NKG2A antigen binding domain can specifically recognize the NKG2A antigen, and the NKG2A antigen comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the NKG2A antigen binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determination as shown in any one of SEQ ID NOs: 6, 7, 8 region (LCDR) or a combination thereof.
  • VL light chain variable region
  • LCDR light chain complementarity determination
  • the light chain variable region comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3)
  • the LCDR1 comprises As shown in the amino acid sequence of SEQ ID NO:6, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:8.
  • the NKG2A antigen binding domain comprises a heavy chain variable region
  • the heavy chain variable region (VH) comprises a heavy chain complementarity determining region as shown in any one of SEQ ID NO3, 4, 5 ( HCDR) or a combination thereof.
  • the heavy chain variable region comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), heavy chain complementarity determining region 3 (HCDR3), and the HCDR1
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 heavy chain complementarity determining region 2
  • HCDR3 heavy chain complementarity determining region 3
  • the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:1
  • the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:2 .
  • the NKG2A antigen binding domain comprises a light chain comprising the light chain variable region (VL).
  • said NKG2A antigen binding domain comprises a heavy chain comprising said heavy chain variable region (VL).
  • the second immune cell includes a second exogenous receptor that recognizes a tumor antigen and/or a pathogen antigen.
  • the second exogenous receptor is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor and T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the second exogenous receptor comprises a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor is a second CAR comprising a tumor antigen binding domain.
  • the tumor antigen is selected from: WT1, HER2, EGFR, BCMA, CD19.
  • the tumor antigen comprises BCMA.
  • the BCMA antigen-binding domain can specifically recognize and/or bind to a BCMA antigen
  • the BCMA antigen comprises the amino acid sequence shown in SEQ ID NO:12.
  • the BCMA antigen binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determination as shown in any one of SEQ ID NOs: 16, 17, 18 region (LCDR) or a combination thereof.
  • VL light chain variable region
  • the light chain variable region comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3)
  • the LCDR1 comprises As shown in the amino acid sequence of SEQ ID NO: 16, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 18.
  • the BCMA antigen binding domain comprises a heavy chain variable region
  • the heavy chain variable region (VH) comprises a heavy chain complementarity determination as shown in any one of SEQ ID NO: 13, 14, 15 region (HCDR) or a combination thereof.
  • the heavy chain variable region comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), heavy chain complementarity determining region 3 (HCDR3), and the HCDR1
  • HCDR1 heavy chain complementarity determining region 1
  • HCDR2 heavy chain complementarity determining region 2
  • HCDR3 heavy chain complementarity determining region 3
  • the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 19
  • the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 20 .
  • the first CAR and the second CAR comprise a transmembrane domain.
  • the transmembrane domain of the first CAR and the transmembrane domain of the second CAR are each independently selected from the transmembrane domains of the following proteins: CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137).
  • the first CAR and the second CAR comprise an intracellular signaling domain.
  • the intracellular signaling domain of the first CAR and the intracellular signaling domain of the second CAR are each independently selected from the intracellular signaling domains of the following proteins: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10, DAP12.
  • the first CAR and the second CAR comprise a hinge domain.
  • the hinge domain of the first CAR and the hinge domain of the second CAR are independently selected from hinge domains of the following proteins: CD8 and CD28.
  • the first CAR and the second CAR comprise a co-stimulatory signaling domain.
  • the co-stimulatory signaling domain of the first CAR and the co-stimulatory signaling domain of the second CAR are each independently selected from the co-stimulatory signaling domains of the following proteins: CD27, CD28, 4-1BB( CD137), OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD1.
  • proteins CD27, CD28, 4-1BB( CD137), OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (
  • the first CAR further comprises the antigen-binding domain that recognizes tumors and/or pathogens.
  • the first CAR includes:
  • an antigen binding domain that recognizes NKG2A optionally an antigen binding domain that recognizes tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, a co-stimulatory signaling domain of CD28 and CD3 ⁇ ;
  • an antigen binding domain that recognizes NKG2A optionally an antigen binding domain that recognizes tumor and/or pathogen antigens, a transmembrane region of CD28 or CD8, a co-stimulatory signaling domain of CD137 and CD3 ⁇ ; and/or
  • an antigen-binding domain that recognizes a NKG2A polypeptide optionally an antigen-binding domain that recognizes tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and CD3 ⁇ ;
  • an antigen-binding domain that recognizes a NKG2A polypeptide optionally an antigen-binding domain that recognizes tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, and CD3 ⁇ ;
  • the second CAR includes:
  • an antigen-binding domain that recognizes tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, a co-stimulatory signaling domain of CD28 and CD3 ⁇ ;
  • the first immune cell and/or the second immune cell is selected from the group consisting of T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells and stem cell-derived immune cells.
  • the first immune cell and/or the second immune cell is selected from: autologous or allogeneic T cells, stem cell-derived T cells, primary T cells and autologous T cells derived from humans.
  • the endogenous HLA molecules of the first CAR T cell and/or the second CAR T cell have low expression or no expression.
  • the HLA molecules include HLA-class I and/or HLA-II molecules.
  • the first immune cell and/or the second immune cell include:
  • the low expression or no expression of the TCR molecule refers to the low expression or no expression of the gene encoding the ⁇ chain of TCR (ie TRAC gene);
  • the low expression or no expression of the TCR molecule refers to the low expression or no expression of the gene encoding the ⁇ chain constant region of TCR;
  • the low or no expression of the TCR molecule refers to the low or no expression of the first exon of the gene encoding the ⁇ -chain constant region of TCR.
  • the first immune cell and/or the second immune cell include:
  • the first immune cell and the second immune cell include:
  • CRISPR/Cas9 technology was used to knock out endogenous B2M/TCR/CIITA/NKG2A.
  • the antigen-binding domain that recognizes NKG2A includes:
  • the antigen-binding domain that recognizes tumor antigens includes:
  • the first CAR includes a sequence as shown in any one of SEQ ID NO: 9, 41, 42, 43, 44, 45, 53; and/or the second CAR includes: SEQ ID NO: The sequence shown in any one of ID NO: 26, 27 or 28, or the sequence formed by sequentially linking SEQ ID NO: 21, 22, 23, 24 or 25 with EQ ID NO: 49, 50, 51 respectively.
  • the gRNA used by the CRISPR/Cas9 technology includes a sequence as shown in any one of SEQ ID NO: 10, 46, 47, 48 or a combination thereof.
  • the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A are respectively present in different containers.
  • the cell composition comprises a cell mixture comprising the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A.
  • the present application also provides a pharmaceutical composition, which includes an effective amount of the cell composition described in the present application and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition comprises a first preparation and a second preparation
  • the first preparation comprises the first immune cells recognizing NKG2A and a pharmaceutically acceptable first adjuvant
  • the second preparation comprising said second immune cells recognizing antigens other than NKG2A and a pharmaceutically acceptable second adjuvant.
  • the pharmaceutical composition comprises a pharmaceutical mixture comprising the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A.
  • the present application also provides the use of the cell composition described in the present application in the preparation of medicaments for preventing and/or treating tumors.
  • the present application also provides a method for preventing, alleviating and/or treating tumors, which comprises administering the cell composition and the pharmaceutical composition described in the present application to a subject in need.
  • the present application also provides the cell composition described in the present application and the pharmaceutical composition described in the present application, which are used for preventing, alleviating and/or treating tumors.
  • the tumors include solid tumors and hematological tumors.
  • the hematological tumor comprises multiple myeloma.
  • the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A are configured to be administered to the subject at the same time.
  • the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A are configured to be administered to a subject separately.
  • the present application also provides a kit, which includes the cell composition or the pharmaceutical composition described in the present application.
  • the kit further includes written instructions for treating and/or preventing tumors, pathogenic infections, autoimmune diseases or allogeneic transplantation.
  • the present application also provides a method for increasing the survival time and/or expansion ability of the first immune cell that recognizes the NKG2A polypeptide and the second immune cell that recognizes the non-NKG2A antigen in the presence of host immune cells, including:
  • the second exogenous receptor targets tumor antigens and/or pathogen antigens.
  • the first exogenous receptor and/or the second exogenous receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC) or a combination thereof .
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • the first exogenous receptor is a first CAR
  • the second exogenous receptor is a second CAR
  • the first CAR includes:
  • an antibody that recognizes a NKG2A polypeptide optionally an antibody that recognizes a tumor and/or a pathogen antigen, CD28 or the transmembrane region of CD8, the co-stimulatory signal domain of CD28 and CD3 ⁇ ; and/or
  • antibodies that recognize NKG2A polypeptides optionally antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signal domain of CD28, the costimulatory signal domain of CD137 and CD3 ⁇ ;
  • antibodies that recognize NKG2A polypeptides optionally antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, and CD3 ⁇ ;
  • the second CAR includes:
  • the first immune cell and/or the second immune cell are selected from T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells or combination.
  • the first immune cell and/or the second immune cell are autologous or allogeneic T cells, stem cell-derived T cells, primary T cells or autologous T cells derived from humans.
  • the polypeptide in step b) is selected from HLA-I, TCR, HLA-II, NKG2A or a combination thereof.
  • said step b) includes:
  • CRISPR/Cas9 technology was used to knock out endogenous B2M/TCR/CIITA/NKG2A.
  • the tumor antigen includes WT1, HER2, EGFR, BCMA or a combination thereof.
  • the antibody recognizing NKG2A polypeptide includes:
  • the tumor-recognizing antibody includes:
  • HCDR1 shown in SEQ ID NO:13 HCDR2 shown in SEQ ID NO:14
  • HCDR3 shown in SEQ ID NO:15 LCDR1 shown in SEQ ID NO:16
  • LCDR2 shown in SEQ ID NO:17 HCDR3 shown in SEQ ID NO: 18; or
  • the scFv set forth in SEQ ID NO: 21, 22, 23, 24, 25, 36, 37, 38, 39 or 40.
  • the first CAR includes the sequence shown in SEQ ID NO: 9, 40, 41, 42, 43, 44, 45 or 53; and/or the second CAR includes SEQ ID NO: 26, The sequence shown in 27 or 28, or the sequence formed by sequentially linking SEQ ID NO: 21, 22, 23, 24 or 25 with EQ ID NO: 49, 50 or 51 respectively.
  • said step b) includes:
  • the gRNA used by the CRISPR/Cas9 technology includes sequences shown in SEQ ID NO: 10, 46, 47, 48 or combinations thereof.
  • the method is used to treat and/or prevent tumors.
  • Figure 1 shows that NKG2A-UCAR-T cells can promote the in vitro survival and/or expansion of UCAR-T cells in the composition.
  • Figure 2A shows that NKG2A-UCAR-T cells exert synergistic anti-tumor effect of BCMA-UCAR-T cells in vivo;
  • Figure 2B shows that UCAR-T cells that recognize NKG2A can significantly increase the number of BCMA UCAR-T cells in vivo;
  • Figure 2C shows that NKG2A -UCAR-T cells cooperate with BCMA-UCAR-T cells to specifically infiltrate into tumor tissue;
  • Figure 2D shows that the combination of NKG2A UCAR-T and BCMA UCAR-T does not produce obvious toxic side effects in mice;
  • Figure 2E shows that NKG2A - The combination of UCAR-T cells and BCMA-UCAR-T does not cause graft-versus-host reaction.
  • Figure 3 It shows that NKG2A-UCAR-T or BCMA-NKG2A UCAR-T can exert the anti-tumor effect of synergistic BCMA-UCAR-T cells in vivo.
  • Figure 4 Shows that NKG2A-UCAR-T cells knocked out of endogenous TCR/B2M/CIITA/NKG2A can not only promote the in vitro survival and/or expansion of UCAR-T cells in the composition, but also exert synergistic BCMA-UCAR - Antitumor effect of T cells.
  • Figure 5 Shows that endogenous TCR/B2M/CIITA/NKG2A knockout NKG2A-UCAR-T cells exert anti-tumor effects in vivo synergistically with BCMA-UCAR-T cells.
  • the invention relates to a cell composition, a preparation method thereof, and an application of the cell composition.
  • the cell composition of the present invention includes a first engineered cell that recognizes NKG2A and a second engineered cell (exemplary, CAR-T cells or UCAR-T cells) that recognizes tumors and/or pathogens, in the presence of host immune cells (exemplary, Primary NK cells) have a longer survival time and/or greater expansion capacity in the presence.
  • host immune cells exemplary, Primary NK cells
  • the cell composition of the present invention has stronger anti-tumor activity in vivo.
  • the first engineered cell that recognizes NKG2A in the composition is used as a general tool cell for resisting host immune rejection (such as NK cell attack), and can recognize one or more tumor antigens and/or pathogen antigens in the composition Engineered cells have wider applicability.
  • the term "about” refers to the usual error range for each value readily known to those skilled in the art. Reference herein to "about” a value or parameter includes embodiments referring to the value or parameter itself. For example, description of “about X” includes description of "X.” Herein, “about” may be an acceptable error range in the technical field; For example, “about” a value or parameter within ⁇ 10% of a value or parameter can be meant, eg, about 5 uM can include any number between 4.5 uM and 5.5 uM.
  • receptor is a kind of special protein or polypeptide that exists in the cell membrane or in the cell, can bind to the target molecule and activate a series of biochemical reactions in the cell, and make the cell respond to external stimuli.
  • Target molecules also referred to as biologically active substances
  • ligands or target antigens.
  • exogenous refers to a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or whose expression level is insufficient to achieve the function when overexpressed; includes any recombinant nucleic acid molecule or polypeptide expressed in the cell, such as exogenous , heterologous and overexpressed nucleic acid molecules and polypeptides.
  • exogenous receptor usually refers to a cell that does not express the receptor itself, but is connected and expressed by DNA fragments or cDNAs corresponding to proteins from different sources through genetic recombination technology. Fusion polypeptide molecules. Extracellular domains, transmembrane domains and intracellular domains may be included. Exogenous receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor (TCR), T Cell Antigen Coupler (TAC).
  • CAR Chimeric Antigen Receptor
  • TCR Chimeric T Cell Receptor
  • TAC T Cell Antigen Coupler
  • CAR chimeric antigen receptor
  • CAR refers to an engineered molecule that can be expressed by immune cells, including but not limited to T cells. CARs are expressed in T cells and can redirect T cells to induce killing of target cells with a specificity dictated by the chimeric receptor.
  • CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Intracellular signaling domains include primary signaling domains and/or co-stimulatory signaling domains.
  • the extracellular binding domains of CARs can be derived from murine, humanized or fully human monoclonal antibodies.
  • the term CAR is not specifically limited to CAR molecules, but also includes CAR variants.
  • CAR variants include split CARs in which the extracellular portion (eg, the ligand-binding portion) and the intracellular portion (eg, the intracellular signaling portion) of the CRA are present on two separate molecules.
  • CAR variants also include ON-switch CARs, which are conditionally activatable CARs, including, for example, split CARs in which conditional heterodimerization of the two parts of the split CAR is controlled by a drug .
  • CAR variants also include bispecific CARs that include a secondary CAR-binding domain that amplifies or inhibits the activity of the primary CAR.
  • CAR variants also include inhibitory chimeric antigen receptors (iCARs), which can, for example, be used as components of bispecific CAR systems, where binding of the secondary CAR binding domain results in inhibition of primary CAR activation.
  • iCARs inhibitory chimeric antigen receptors
  • T cell antigen coupler includes three functional domains: 1. Antigen binding domain, including single chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein) protein, DARPin) or other targeting groups; 2. The extracellular region domain, the single-chain antibody that binds to CD3, so that the TAC receptor and the TCR receptor are close; 3. The cell of the transmembrane region and the CD4 co-receptor The inner domain, where the intracellular domain is linked to the protein kinase LCK, catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides that make up the TCR, which are capable of binding to surface antigens on target cells, and interacting with other polypeptides of the complete TCR complex , usually co-localized on the surface of T cells.
  • a chimeric T cell receptor consists of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, TCR intracellular domain; the TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3 ⁇ , CD3 ⁇ , CD3z, the ⁇ chain of TCR, or the ⁇ chain of TCR , and, the chimeric T cell receptor is integrated into the TCR/CD3 complex expressed on T cells.
  • the term "independently” generally means that there is no relationship between the involved subjects, wherein the structure, type and/or quantity of one subject does not affect the structure, type and/or quantity of other subjects.
  • the "subject" can be the first exogenous receptor and the second exogenous receptor, which can be the same or different; for example, the "subject” can be the first CAR and the second CAR.
  • Each functional domain in CAR for example, hinge domain, transmembrane domain, intracellular signaling domain or co-stimulatory signal domain, wherein each functional domain may be the same or different.
  • Engineered cells can also refer to cells that contain added, deleted and/or altered genes.
  • cell or “engineered cell” may refer to a cell of human or non-human animal origin.
  • mice rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs , goat, cow, horse, ape, monkey.
  • the term "antigen-binding domain” refers to a molecule that specifically binds an antigenic determinant, including immunoglobulin molecules and immunologically active portions of immunological molecules, i.e., containing the antigen to which it specifically binds ("immunoreacts") Molecules at the binding site.
  • antibody includes not only intact antibody molecules but also fragments of antibody molecules that retain antigen-binding ability.
  • antibody is used interchangeably with the term “immunoglobulin” and "antigen binding domain” in this application.
  • an antibody comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • CH consists of three structural domains CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • CL consists of one domain.
  • VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains include binding domains that interact with antigen.
  • An antigen-binding domain "specifically binds" or is "immunogenic" to an antigen if the antigen-binding domain binds the antigen with greater affinity (or avidity) than other reference antigens (including polypeptides or other substances). Reactive".
  • activation of immune cells refers to changes in intracellular protein expression caused by signal transduction pathways, resulting in the initiation of an immune response.
  • the immune synapse formed after CAR binds to an antigen includes the aggregation of many molecules near the binding receptor (e.g., CD4 or CD8, CD3 ⁇ /CD ⁇ /CD ⁇ /CD ⁇ , etc.). This aggregation of membrane-bound signaling molecules phosphorylates the ITAM motif included in the CD3 molecule. This phosphorylation in turn initiates T cell activation pathways, ultimately activating transcription factors such as NF- ⁇ B and AP-1.
  • T cell activation or “T cell activation” refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • cell composition generally refers to a combined form comprising at least two types of cells, one of which recognizes NKG2A at least (e.g., only recognizes NKG2A; e.g., simultaneously recognizes NKG2A and a disease-associated antigen), and the other Classes recognize disease-associated antigens (eg, tumor antigens).
  • each type of cell can be present in a different container, and can also be formulated into a desired preparation with a suitable adjuvant when necessary; in some embodiments, each type of cell can be of different origin (e.g., prepared, produced, or sold by different manufacturers; e.g., naturally occurring T cells isolated from a donor and T cells derived from stem cells, respectively); in some embodiments, each type of cell can be prepared separately into separate formulations (solid, liquid, gel, etc.); in some embodiments, each type of cell may be present in admixed form.
  • each type of cell can be of different origin (e.g., prepared, produced, or sold by different manufacturers; e.g., naturally occurring T cells isolated from a donor and T cells derived from stem cells, respectively); in some embodiments, each type of cell can be prepared separately into separate formulations (solid, liquid, gel, etc.); in some embodiments, each type of cell may be present in admixed form.
  • the term “disease” refers to any condition that damages or interferes with the normal function of a cell, tissue or organ, such as a tumor (cancer) or a pathogenic infection.
  • the disease includes solid tumors, hematological tumors, autoimmune diseases, or combinations thereof.
  • NKG2A is a member of the NKG2 transcriptome, a heterodimeric inhibitory receptor CD94/NKG2A formed by NKG2A and CD94, expressed on subtypes of NK cells, ⁇ T cells, ⁇ T cells, and NKT cells. on the surface of the group.
  • “NKG2A” may be any variant, derivative or isoform of the NKG2A gene or encoded protein.
  • the NKG2A polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% of the amino acid sequence encoded by the transcript expressed by the gene of NCBI GenBank Gene ID: 3821 %, at least about 98%, at least about 99%, or at least about 100% homology or identity of amino acid sequences or fragments thereof, and/or may optionally include up to one or up to two or up to three conservative amino acid substitutions .
  • BCMA antigen or "BCMA” generally refers to B-cell maturation antigen, which belongs to the TNF receptor superfamily. After BCMA binds to its ligand, it can activate the proliferation and survival of B cells. BCMA is specifically highly expressed in plasma cells and multiple myeloma cells, but not expressed in hematopoietic stem cells and other normal tissue cells. "BCMA” may be any variant, derivative or isoform of the BCMA gene or encoded protein.
  • the term “recognize” refers to selective binding of a target antigen.
  • the engineered cells expressing exogenous receptors in the present invention can recognize cells expressing antigens specifically bound by the exogenous receptors.
  • binding partner e.g., tumor antigen
  • the term "immune cell” generally refers to a cell that participates in an immune response and performs effector functions.
  • the exercising effector functions may include clearing foreign antigens or promoting immune effector responses, etc.; for example, it may be cells of lymphoid lineage. Examples include T cells and NK cells.
  • tumor antigen refers to an antigen that appears newly or is overexpressed during the onset, progression of a hyperproliferative disease.
  • a hyperproliferative disorder refers to cancer/tumor.
  • it can be a solid tumor antigen, for example, it can also be a blood tumor antigen.
  • peptide As used interchangeably to refer to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, including any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof.
  • the nucleic acid molecule only needs to maintain basic identity with the endogenous nucleic acid sequence, and does not need to have 100% homology or identity with the endogenous nucleic acid sequence.
  • substantially identity or “substantial homology” refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence or nucleic acid sequence.
  • such a sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid or nucleic acid sequence used for comparison. origin or identity. Sequence identity can be measured by using sequence analysis software (eg, the BLAST, BESTFIT, GAP or PILEUP/PRETTYBOX programs).
  • the term "transmembrane domain” generally refers to a region of a protein sequence that spans a cell membrane.
  • the membrane-spanning region of the protein sequence is generally alpha-helical, comprising mostly hydrophobic amino acids.
  • the transmembrane domain may be obtained from a native protein (such as from CD8 or a functionally derived sequence thereof), or the transmembrane domain may be a synthetic non-naturally occurring protein segment , such as hydrophobic protein segments that are thermodynamically stable in cell membranes.
  • co-stimulatory domain generally refers to the intracellular domain of a co-stimulatory molecule that can provide an immune co-stimulatory signal, and the co-stimulatory molecule is a cell surface molecule required for an effective response of lymphocytes to an antigen .
  • the costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.
  • intracellular signaling domain also referred to as “primary signaling domain” generally refers to a signal transduction sequence containing a so-called immunoreceptor tyrosine-based activation motif or ITAM.
  • primary signaling domains derived from CD3 ⁇ , FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (Fc ⁇ R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10, and DAP12.
  • the intracellular signaling domain transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain.
  • a primary intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domain.
  • hinge domain generally refers to a stretch of amino acids between two domains of a protein that is capable of allowing flexibility of the protein and/or movement of one or more domains relative to each other.
  • hinge domains from IgG family such as IgG1 and IgG4
  • IgD IgD
  • other protein molecules such as hinge domains from CD8, CD28, HLA family.
  • HVGR host-versus-graft reaction
  • graft-versus-host disease generally refers to: due to the diversity of the TCR of the exogenously transplanted donor T lymphocytes and the incompatibility with the host HLA molecules, the donor T lymphocytes It will recognize the antigens on the normal tissues of the host, amplify and release a series of cytokines to attack the host cells.
  • endogenous means that the nucleic acid molecule or polypeptide comes from the organism itself.
  • HLA human leukocyte antigen
  • HLA human leukocyte antigen
  • HLA class I is a heterodimer consisting of a heavy chain ( ⁇ chain) and a light chain ⁇ 2 microglobulin (B2M).
  • HLA-II genes include the HLA-D family, mainly including HLA-DP, HLA-DQ and HLA-DR, etc., and are mainly distributed on the surface of professional antigen-presenting cells such as B lymphocytes, macrophages and dendritic cells.
  • TCR generally refers to the T cell receptor, which mediates the recognition of specific major histocompatibility complex (MHC)-peptide antigens by T cells.
  • MHC major histocompatibility complex
  • TCR is usually composed of two peptide chains ⁇ and ⁇ , and each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region, etc., and its antigen specificity exists in Zone V.
  • B2M refers to beta-2 microglobulin, also known as B2M, the light chain of an MHC class I molecule.
  • B2M is encoded by the b2m gene located on chromosome 15, opposite other MHC genes located as a gene cluster on chromosome 6.
  • Studies have shown that when the B2M gene is mutated, hematopoietic grafts from mice lacking normal cell surface MHC I expression are rejected by NK cells in normal mice, indicating that defective expression of MHC I molecules makes cells vulnerable to host immune system Repulsion (Bix et al. 1991).
  • CIITA generally refers to the transactivator of major histocompatibility complex class II (MHC II).
  • MHC II major histocompatibility complex class II
  • the transactivator may be a protein having an acidic transcription activation domain, 4 LRRs (leucine rich repeats) and a GTP binding domain.
  • the protein may be localized in the nucleus and acts as a positive regulator of transcription of major histocompatibility complex class II (MHC II) genes.
  • MHC II major histocompatibility complex class II
  • the protein is encoded by a gene located at 16p13.13 (information such as that shown in HGNC:7067), enabling the generation of several transcript variants encoding different isoforms.
  • preparation usually refers to a drug prepared according to a certain dosage form to meet the needs of treatment or prevention, and can be provided to the subject.
  • a formulation may contain active ingredients, adjuvants.
  • the active ingredient can be one or more immune cells with therapeutic effects.
  • the term "adjuvant” can generally refer to any substance other than the active ingredient in a pharmaceutical preparation.
  • a pharmaceutically acceptable compound, composition or vehicle involved in carrying, storing or transporting cells Such as buffers, stabilizers, preservatives, absorption enhancers for enhanced bioavailability, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials and/or other conventional protective or dispersing agents Wait.
  • Each adjuvant is "acceptable” in the sense of being compatible with the other ingredients of the formulation and not detrimental to the patient.
  • tumors generally refers to a neoplasm or solid lesion formed by abnormal cell growth/hyperproliferation.
  • tumors may be solid tumors or hematological tumors.
  • a tangible mass that can be palpable through clinical examinations such as X-rays, CT scans, B-ultrasound, or palpation can be called a solid tumor, and X-rays, CT scans, B-ultrasound and palpation cannot Tumors such as leukemia that are seen or felt are called hematomas.
  • the term "container” generally refers to any vessel or device suitable for containing a drug.
  • a drug for example, kits, vials, pouches, blisters, tubes, syringes, etc.
  • terapéuticaally effective amount refers to a compound effective to achieve a particular biological result as described herein, An amount of an agent, substance or composition, pharmaceutical composition, such as but not limited to an amount or dosage sufficient to promote a T cell response.
  • An effective amount of immune cells including but not limited to: the number of immune cells that can increase, enhance or prolong anti-tumor activity; increase the number of anti-tumor immune cells or the number of activated immune cells; promote IFN- ⁇ secretion, tumor regression, Tumor shrinkage, number of immune cells in tumor necrosis.
  • the present application provides a cell composition, which includes: a) a first engineered cell that recognizes NKG2A; and b) a second engineered cell that recognizes a non-NKG2A antigen.
  • the first engineered cell can express a first exogenous receptor that recognizes NKG2A; for example, the second engineered cell can express a second exogenous receptor that recognizes an antigen other than NKG2A.
  • the non-NKG2A antigens include tumor antigens and/or pathogen antigens.
  • the first exogenous receptor can also recognize tumor antigens and/or pathogen antigens.
  • the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may be the same.
  • the first exogenous receptor and the second exogenous receptor can be independently selected from: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), T cell fusion protein (TFP).
  • the first exogenous receptor and the second exogenous receptor can be the same; for example, the first exogenous receptor and the second exogenous receptor can both be chimeric antigen receptors (CAR) , a chimeric T cell receptor (TCR), a T cell antigen coupler (TAC), or a T cell fusion protein (TFP); for example, the first exogenous receptor and the second exogenous receptor can be different;
  • the first exogenous receptor can be a chimeric T cell receptor
  • the second exogenous receptor can be a CAR
  • the first exogenous receptor can be a CAR
  • the second exogenous receptor can be is a chimeric T cell receptor
  • the first exogenous receptor can be TAC
  • the first exogenous receptor provided in this application can recognize NKG2A.
  • the NKG2A can have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least Amino acid sequences or fragments thereof that are about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical, and/or may optionally include at most one or at most two or up to three conservative amino acid substitutions.
  • the NKG2A is human NKG2A, comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least Amino acid sequences or fragments thereof of about 97%, at least about 98%, at least about 99%, or at least about 100% homology or identity, and/or may optionally include up to one or up to two or up to three conserved Amino acid substitutions.
  • the first exogenous receptor provided by the present application recognizes NKG2A, and also recognizes tumor antigens and/or pathogen antigens.
  • the second exogenous receptor provided herein recognizes tumor antigens and/or pathogen antigens.
  • the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may be the same.
  • the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor may be different from the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor.
  • the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may target different tumor antigens in the same tumor.
  • the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may be different epitopes of the same tumor antigen.
  • tumor antigens are expressed as polypeptides or as intact proteins or parts thereof.
  • the tumor antigens of the present application 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); 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; Acidase; Mesothelin; EpCAM
  • the first exogenous receptor recognizes NKG2A and the second exogenous receptor recognizes BCMA.
  • the first exogenous receptor recognizes NKG2A and BCMA
  • the second exogenous receptor recognizes BCMA.
  • the BCMA can have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least Amino acid sequences or fragments thereof that are about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous or identical, and/or may optionally include at most one or at most two or at most Three conservative amino acid substitutions.
  • the human BCMA polypeptide can include the amino acid sequence shown in SEQ ID NO: 12.
  • the first exogenous receptor binds to the extracellular domain of NKG2A. In one example, the second exogenous receptor binds to the extracellular domain of BCMA. In one example, the first exogenous receptor binds to the extracellular domain of the NKG2A polypeptide and the BCMA polypeptide.
  • the exogenous receptor recognizes a pathogen antigen, eg, for the treatment and/or prevention of a pathogen infection or other infectious disease, eg, in an immunocompromised subject.
  • Pathogen antigens include, but are not limited to: antigens of viruses, bacteria, fungi, protozoa, or parasites; viral antigens include, but are not limited to: cytomegalovirus (CMV) antigens, Epstein-Barr virus (EBV) antigens, human immune Defective virus (HIV) antigen or influenza virus antigen.
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • HAV human immune Defective virus
  • the first exogenous receptor (for example, the first CAR) and the second exogenous receptor (for example, the second CAR) of the present application may include an antigen-binding domain.
  • the antigen binding domain comprises an antibody or fragment thereof.
  • the antigen binding domain comprises an antibody heavy chain variable region (VH) and/or light chain variable region (VL); or comprises a cross-linked Fab; or comprises F(ab) 2 .
  • the antigen binding domain comprises antibody VH and VL, forming a variable fragment (Fv).
  • the antigen binding domain comprises a scFv.
  • the first exogenous receptor (eg, first CAR) can comprise an NKG2A antigen binding domain.
  • the NKG2A antigen binding domain can be an antibody to which the NKG2A polypeptide specifically binds.
  • the first exogenous receptor includes an antibody that recognizes NKG2A and an antibody that recognizes a tumor antigen, and their connection is: (1) the light chain/heavy chain (or light chain variable region) of an antibody that recognizes NKG2A /heavy chain variable region)—heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes NKG2A—heavy chain/light chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen region/light chain variable region)—the light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes a tumor antigen; (2) the light chain (or light chain variable region) of an antibody that recognizes a tumor antigen Variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes NKG2A—the light chain (or light chain variable region) of an antibody that recognizes NKG2A—the heavy chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen variable
  • the first CAR includes a tandem antibody that specifically binds to the NKG2A polypeptide and the BCMA polypeptide; the antigen domain of the CAR includes Fv that specifically binds to the NKG2A polypeptide and the BCMA polypeptide, respectively.
  • the first CAR includes an antibody that specifically binds to the NKG2A polypeptide.
  • the NKG2A antigen binding domain comprises a light chain variable region (VL) and/or a heavy chain variable region (VH) of an NKG2A antibody.
  • VL light chain variable region
  • VH heavy chain variable region
  • the NKG2A antigen binding domain comprises the light chain and/or heavy chain of an NKG2A antibody.
  • the antibody heavy chain or VH of NKG2A comprises one or more heavy chain CDRs (HCDR): HCDR1 as shown in the sequence of SEQ ID NO:3, HCDR2 as shown in the sequence of SEQ ID NO:4, and HCDR2 as shown in SEQ ID NO:4 HCDR3 of the sequence shown in ID NO:5.
  • the heavy chain or VH of the NKG2A antibody comprises sequences as shown in SEQ ID NOs: 3-5.
  • the NKG2A antibody light chain or VL comprises one or more light chain CDRs (LCDRs): LCDR1 of the sequence shown in SEQ ID NO:6, LCDR2 of the sequence shown in SEQ ID NO:7, LCDR2 of the sequence shown in SEQ ID NO:7, LCDR3 of the sequence shown in NO:8.
  • the NKG2A antibody light chain or VL comprises sequences as shown in SEQ ID NOs: 6-8.
  • the NKG2A antibody heavy chain or VH comprises the sequence shown in SEQ ID NO: 1.
  • the NKG2A antibody light chain or VL comprises the sequence shown in SEQ ID NO:2.
  • the NKG2A antibody or the NKG2A antigen binding domain comprises the VH of the sequence shown in SEQ ID NO:1 and/or the VL of the sequence shown in SEQ ID NO:2. In one example, the NKG2A antibody or NKG2A antigen binding domain comprises the sequences shown in SEQ ID NO: 1 and 2. In one example, the NKG2A antibody or NKG2A antigen binding domain comprises a scFv sequence as shown in SEQ ID NO:52.
  • the BCMA antigen binding domain comprises a scFv of an antibody that recognizes BCMA (also referred to as a BCMA antibody).
  • the first exogenous receptor (for example, the first CAR) can include an NKG2A antigen-binding domain and a tumor and/or pathogen antigen-binding domain, and the antigen-binding domain can specifically bind NKG2A, tumor and/or Antibodies to pathogen antigens; in one example, the antigen-binding domain can be Fv that specifically binds NKG2A and pathogen antigens, respectively. In one example, the antigen-binding domain may be an Fv that specifically binds NKG2A and a tumor antigen, respectively.
  • the first exogenous receptor may include an NKG2A antigen-binding domain and a BCMA antigen-binding domain
  • the antigen-binding domain may be an antibody specifically binding to NKG2A and BCMA, respectively.
  • the antigen-binding domain may be an Fv that specifically binds NKG2A and BCMA, respectively.
  • the first exogenous receptor (for example, the first CAR) may comprise an NKG2A antigen binding domain and a tumor and/or pathogen antigen binding domain
  • the second exogenous receptor (for example, a second CAR) may comprise a tumor and/or a pathogen antigen binding domain. or pathogen antigen binding domain
  • the first exogenous receptor (for example, the first CAR) can include the NKG2A antigen-binding domain and the BCMA antigen-binding domain
  • the second exogenous receptor (for example, the second CAR) can include the BCMA antigen-binding domain.
  • the BCMA antigen binding domain comprises BCMA antibody VL and/or VH.
  • the BCMA antigen binding domain comprises a light chain and/or a heavy chain of an antibody to BCMA.
  • the heavy chain or VH of the BCMA antibody comprises one or more heavy chain CDRs (HCDR): HCDR1 of the sequence shown in SEQ ID NO: 13, HCDR2 of the sequence shown in SEQ ID NO: 14, HCDR3 of the sequence shown in NO:15.
  • the heavy chain or VH of the BCMA antibody comprises a sequence as shown in SEQ ID NOs: 13-15.
  • the BCMA antibody light chain or VL comprises one or more light chain CDRs (LCDR): LCDR1 of the sequence shown in SEQ ID NO: 16, LCDR2 of the sequence shown in SEQ ID NO: 17, LCDR2 of the sequence shown in SEQ ID NO: 17, LCDR3 of the sequence shown in NO:18.
  • the BCMA antibody light chain or VL comprises the sequences shown in SEQ ID NOs: 16-18.
  • the BCMA antibody heavy chain or VH comprises the sequence shown in SEQ ID NO: 19.
  • the BCMA antibody light chain or VL comprises the sequence shown in SEQ ID NO:20.
  • the BCMA antibody or BCMA antigen binding domain comprises the VH of the sequence shown in SEQ ID NO:19 and/or the VL of the sequence shown in SEQ ID NO:20. In one example, the BCMA antibody or BCMA antigen binding domain comprises the sequences shown in SEQ ID NOs: 19 and 20. In one example, the BCMA antigen binding domain comprises a scFv of an antibody that recognizes BCMA (also referred to as a BCMA antibody).
  • the first exogenous receptor (eg, the first CAR) comprises all CDR regions of the NKG2A antibody and the BCMA antibody. In one example, the first exogenous receptor (eg, the first CAR) comprises sequences shown in SEQ ID NOs: 3-8 and 13-18.
  • the application provides an anti-BCMA antibody comprising the scFv sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25; an anti-NKG2A antibody comprising the VH shown in SEQ ID NO: 1, SEQ ID NO: 2
  • the VL shown may include the scFv shown in SEQ ID NO: 52;
  • the BCMA-NKG2A tandem antibody includes the sequence shown in SEQ ID NO: 36, 37, 38, 39 or 40.
  • the present application contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, VH or VL) to produce a functionally equivalent molecule.
  • the VH or VL of the NKG2A antibody or BCMA antibody included in the CAR can be modified such that the NKG2A or BCMA antibody such as the VH or VL is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the exogenous receptor of the present application may be a chimeric antigen receptor (CAR), the first exogenous receptor is the first CAR, and the second exogenous receptor is the second CAR.
  • the various domains of the CAR polypeptide can be in the same polypeptide chain, eg, expressed as a single polypeptide chain.
  • the individual domains of the CAR polypeptide may not be contiguous to each other, e.g., be in different polypeptide chains.
  • antibodies or antibody fragments of the invention may be further modified such that they have changes in amino acid sequence (eg, relative to wild type) but no change in the desired activity.
  • additional nucleotide substitutions can be made to the protein, resulting in amino acid substitutions at "non-essential" amino acid residues.
  • a non-essential amino acid residue in a molecule can be replaced by another amino acid residue from the same side chain family.
  • amino acid stretches may be substituted with amino acid stretches that are structurally similar but differ in sequence and/or composition from members of the side chain family, for example, conservative substitutions may be made wherein amino acid residues are replaced by amino acids with similar side chains residue replaced.
  • the first CAR and/or the second CAR of the present application may further include a hinge domain.
  • the hinge domains of the first CAR and the second CAR can be independently selected from hinge domains of the following proteins: CD28, CD8, HLA, Fc, IgG, IgD, 4-1BB, CD4, CD27, CD7 and PD1.
  • the hinge domains of the first CAR and the second CAR can be the same or different; the first CAR or the second CAR can select any suitable hinge sequence known, and the hinge domain selected by the first CAR does not affect the second CAR. Selection of hinge domains in CAR.
  • the first CAR can contain the hinge domain of CD8, and the second CAR can contain the hinge domain of CD8 or any other suitable protein molecule; for example, the second CAR can contain the hinge domain of CD8, and the first CAR can contain the hinge domain of CD8.
  • the antigen binding domain is linked directly or via a hinge to the transmembrane domain.
  • each of the first CAR and the second CAR includes a CD8 hinge, for example, the CD8 hinge may include a sequence as shown in SEQ ID NO: 30 or a sequence having 95-99% identity with SEQ ID NO: 30.
  • the first CAR and/or the second CAR of the present application may further include a signal peptide.
  • the signal peptides of the first CAR and the second CAR may be the same or different.
  • the signal peptides of the first CAR and the second CAR may be the signal peptide of CD8.
  • the CD8 signal peptide comprises the sequence shown in SEQ ID NO:29.
  • the first CAR and/or the second CAR of the present application may also include a transmembrane domain.
  • the transmembrane domains of the first CAR and the second CAR can be independently selected from transmembrane domains of the following proteins: transmembrane domains of ⁇ , ⁇ , or ⁇ of T cell receptors, CD28, CD3 ⁇ , CD45, CD4, CD5 , CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D
  • the transmembrane domains of the first CAR and the second CAR can be the same or different; the first CAR or the second CAR can select any suitable transmembrane domain known, and the transmembrane domain selected by the first CAR is not Influences selection of transmembrane domains in the second CAR.
  • the first CAR may contain the transmembrane domain of CD8/CD28
  • the second CAR may contain the transmembrane domain of CD8/CD28 or any other suitable protein molecule
  • the second CAR may contain the transmembrane domain of CD8/CD28.
  • the first CAR may comprise the transmembrane domain of CD8/CD28 or any other suitable protein molecules; in one example, the first CAR and the second CAR each comprise the CD8 transmembrane domain; in one example wherein, the first CAR and the second CAR each comprise a CD28 transmembrane domain; in one example, the first CAR comprises a CD8 transmembrane domain, and the second CAR comprises a CD28 transmembrane domain; in one example, the first CAR comprises a CD28 transmembrane domain Membrane domain, the second CAR contains the CD8 transmembrane domain.
  • the CD8 transmembrane domain comprises at least one, two or three modifications of the sequence shown in SEQ ID NO:31, but no more than 20, 10 or 5 modified sequences, or the same sequence as SEQ ID NO: The amino acid sequence shown in 31 has a sequence of 95-99% identity.
  • the CD8 transmembrane domain comprises the sequence set forth in SEQ ID NO:31.
  • the CD28 transmembrane domain comprises at least one, two or three modifications of the sequence shown in SEQ ID NO:32, but no more than 20, 10 or 5 modified sequences, or the sequence with SEQ ID NO:32
  • the amino acid sequence of NO:32 has a sequence of 95-99% identity.
  • the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 32.
  • the first CAR and/or the second CAR of the present application may further include an intracellular signaling domain.
  • the intracellular signaling domains of the first CAR and the second CAR can be independently selected from the intracellular signaling domains of the following proteins: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, FcyRIIa, DAP10, and DAP12.
  • the intracellular signaling domains of the first CAR and the second CAR may be the same or different; the first CAR or the second CAR may select any suitable intracellular signaling domain known, and the first CAR selected
  • the intracellular signaling domain does not affect the choice of intracellular signaling domain in the second CAR.
  • the first CAR can comprise the intracellular signaling domain of CD3 ⁇
  • the second CAR can comprise the intracellular signaling domain of CD3 ⁇ or the intracellular signaling domain of any other suitable protein molecule; for example, the second CAR can comprise the intracellular signaling domain of CD3 ⁇ .
  • the intracellular signaling domain, the first CAR may comprise the intracellular signaling domain of CD3 ⁇ or the intracellular signaling domain of any other suitable protein molecule; in one example, the first CAR and the second CAR each comprise the intracellular signaling domain of CD3 ⁇ signaling domain.
  • the CD3 ⁇ intracellular signaling domain may comprise at least 1, 2, or 3 modified amino acid sequences but no more than 20, 10, or 5 modified amino acid sequences of the amino acid sequence shown in SEQ ID NO:35, Or a sequence with 95-99% identity to the amino acid sequence shown in SEQ ID NO:35.
  • the CD3 ⁇ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 35.
  • the first CAR and/or the second CAR of the present application may further include a co-stimulatory signaling domain.
  • the transmembrane domains of the first CAR and the second CAR can be independently selected from the co-stimulatory signal domains of the following proteins: CD27, CD28,
  • 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83 CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLA
  • the co-stimulatory signal domains of the first CAR and the second CAR may be the same or different; the first CAR or the second CAR may select any suitable co-stimulatory domains known, and the first CAR selected The co-stimulatory signaling domain does not affect the choice of the second CAR co-stimulatory signaling domain.
  • the first CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28
  • the second CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28
  • the first CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28 or any other suitable co-stimulatory signaling domain of protein molecules
  • the first CAR and the second CAR each comprise a co-stimulatory signaling domain of 4-1BB.
  • each of the first CAR and the second CAR comprises a co-stimulatory signaling domain of CD28.
  • the 4-1BB co-stimulatory signaling domain comprises an amino acid sequence of at least 1, 2, or 3 modifications but no more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO: 34, or A sequence with 95-99% identity to the amino acid sequence shown in SEQ ID NO:34.
  • the 4-1BB co-stimulatory signaling domain comprises the sequence of SEQ ID NO:34.
  • the CD28 co-stimulatory signaling domain comprises at least 1, 2, or 3 modified amino acid sequences but no more than 20, 10, or 5 modified amino acid sequences of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence with SEQ ID NO:33
  • the amino acid sequence shown in ID NO:33 has 95-99% identity sequence.
  • the CD28 co-stimulatory signaling domain comprises the sequence of SEQ ID NO:33.
  • the first CAR and the second CAR may contain the same number or different numbers of co-stimulatory signaling domains, and the number of co-stimulatory signaling domains of the first CAR is independent of the number of co-stimulatory signaling domains of the second CAR.
  • the first CAR contains 1 costimulatory signaling domain
  • the second CAR contains more than one (for example, 2) costimulatory signaling domains
  • the second CAR contains 1 costimulatory signaling domain
  • the first CAR contains one
  • the above (for example 2) co-stimulatory signal domains; for example, the first CAR and the second CAR both contain more than 1 (for example 2) costimulatory signal domains.
  • the multiple costimulatory signal domains in the CAR comprising more than one costimulatory signal domain may be the same or different, for example, contain 2 costimulatory signal domains both of 4-1BB/CD28; For example, a 4-1BB co-stimulatory signaling domain and a CD28 co-stimulatory signaling domain are included.
  • the first CAR provided by the present invention includes sequences shown in SEQ ID NO: 9, 41, 42, 43, 44, 45 and/or 53.
  • the first CAR includes a sequence formed by sequentially linking the sequence shown in SEQ ID NO: 36, 37, 38, 39, 40 or 52 with SEQ ID NO: 49, 50 or 51, respectively.
  • the first CAR of the present invention includes a sequence formed by sequentially connecting SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO: 49, or SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO : 50 sequentially concatenated sequence, or sequence concatenated by SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO: 51, SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 49 sequentially connected sequence, or SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 50 sequentially connected sequence, or SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 51 sequentially connected sequence, SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 49 sequentially connected sequence, or SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 50
  • the second CAR provided by the present invention includes the sequence shown in SEQ ID NO: 26, 27 or 28.
  • the second CAR includes a sequence in which the sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25 is sequentially connected to SEQ ID NO: 49, 50 or 51, respectively.
  • the second CAR includes the sequence of SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 49, or the sequence of SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 50 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 51, or a sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 49 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 50, or a sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 51 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 23, SEQ ID NO: 30 and SEQ ID NO: 49, or a sequence of SEQ ID NO: 23, SEQ ID NO: 30 and SEQ ID NO: 50 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 23, S
  • the first engineered cell expresses any one of the above-mentioned first CARs; the second engineered cell expresses any one of the above-mentioned second CARs.
  • the first engineered cell in the cell composition provided by the present invention is a CAR-T cell expressing any one of the above-mentioned first CARs; the second engineered cell is a CAR-T cell expressing any one of the above-mentioned second CARs.
  • the present application contemplates modification of the entire CAR molecule, eg, modification of one or more amino acid sequences of each domain of the CAR molecule, in order to generate a functionally equivalent molecule.
  • the modifiable CAR molecule retains at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% of the starting CAR molecule , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identity.
  • the exogenous receptor provided herein is capable of associating with a CD3 ⁇ polypeptide.
  • CD3 ⁇ polypeptide can be endogenous or exogenous.
  • binding of an antigen binding domain of an exogenous receptor to an antigen eg, NKG2A polypeptide, tumor antigen, pathogen antigen
  • binding of the antigen binding domain of the exogenous receptor to an antigen eg, NKG2A polypeptide, tumor antigen, pathogen antigen
  • binding of the antigen binding domain of the exogenous receptor to an antigen can activate the CD3 ⁇ signaling domain of the exogenous receptor.
  • the cell composition of the present application includes a first engineered cell that recognizes NKG2A and a second engineered cell that recognizes tumor and/or pathogen antigens.
  • the engineered cells are immune cells, neurons, epithelial cells, endothelial cells or stem cells.
  • Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSC) and human embryonic stem cells).
  • the engineered cells include immune cells.
  • the engineered cells are primary cells.
  • the immune cells are B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, regulatory T cells, helper Cytotoxic T cells, other T cells, or combinations thereof.
  • said first engineered cell comprises an amino acid sequence having at least 80% sequence homology to any one of SEQ ID NO: 9, 41, 42, 43, 44, 45, 53 .
  • the first engineering cell comprises the sequence shown in SEQ ID NO: 36, 37, 38, 39, 40 or 52 and the sequence of SEQ ID NO: 49, 50 or 51 respectively Amino acid sequences with at least 80% sequence homology to the linked sequences.
  • the second engineered cell comprises a nucleic acid sequence having at least 80% sequence homology to SEQ ID NO: 26, 27 or 28 or an amino acid sequence translated into it.
  • the second engineered cell (such as T, NKT cell) comprises sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25 connected with SEQ ID NO: 49, 50 or 51 sequence respectively Nucleic acid sequences or their translated amino acid sequences having at least 80% sequence homology.
  • the cell composition provided by the present invention includes any of the first engineered cells described herein and any of the second engineered cells described herein.
  • the cell composition provided by the present invention includes any one of the engineered cells for the first exogenous recipient described herein and any of the engineered cells for the second exogenous recipient described herein.
  • the cell composition provided by the present invention includes any one of the engineered cells of the first CAR described herein and any of the engineered cells of the second CAR described herein.
  • the cell composition provided by the present invention includes any of the first CAR-T cells described herein and any of the second CAR-T cells described herein.
  • the cell composition of the present application does not have to be a mixture in a mixed state, wherein the first engineered cells recognizing NKG2A and the second engineered cells recognizing non-NKG2A antigens may be separated from each other, for example, each exists in a different container.
  • the cell composition of the present application may be a mixture in a mixed state.
  • composition of the present application includes first engineered cells (such as T, NKT cells) that recognize NKG2A polypeptides and tumor antigens and second engineered cells (such as T, NKT cells) that recognize tumor and/or pathogen antigens.
  • first engineered cells such as T, NKT cells
  • second engineered cells such as T, NKT cells
  • the engineered cells in the composition of the present application have longer survival time and/or stronger expansion ability in the presence of host immune cells (eg, NK cells).
  • the second engineered cells in the composition of the present application have longer survival time and/or stronger expansion ability in the presence of host immune cells (eg, NK cells).
  • the composition comprising the first engineered cell and the second engineered cell exhibits a stronger cell killing effect in vivo and in vitro on the cell carrying the target antigen.
  • the cell composition of the present application specifically infiltrates the tissue expressing the target antigen.
  • the cell composition of the present application specifically infiltrates the tumor tissue expressing the target antigen. In one example, the cell composition of the present application does not cause a graft-versus-host reaction. In one example, the expression, activity and/or signal of at least one endogenous gene involved in responding to self and non-self antigen recognition polypeptides in the first engineered cell and/or the second engineered cell is reduced or inhibited; comprising said first The cell composition of the second engineered cell does not cause a graft-versus-host reaction.
  • Both the first engineered cell and the second engineered cell in the composition of the present application are immune cells.
  • the immune cells are derived from neurons, epithelial cells, endothelial cells or stem cells.
  • the immune cells can be cells of the lymphoid lineage.
  • the lymphoid lineage including B, T, and natural killer (NK) cells provide for antibody production, regulation of the cellular immune system, detection of exogenous agents in the blood, detection of foreign cells to the host, etc.
  • Non-limiting examples of immune cells of the lymphoid lineage include T cells, natural killer T (NKT) cells and precursors thereof, including embryonic stem cells and pluripotent stem cells (e.g., stem cells that differentiate into lymphoid cells or pluripotent stem cells).
  • T cells may be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. T cells can be of any type, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-like memory T cells (or stem-like memory T cells), and both effector Memory T cells: eg TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, ⁇ T cells or ⁇ T cells. Cytotoxic T cells (CTL or killer T cells) are T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • CTL or killer T cells are T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • the subject's own T cells can be engineered to express the exogenous receptors of the present application.
  • the immune cells in the composition are T cells.
  • the T cells can be CD4+ T cells and/or CD8+ T cells.
  • the immune cells in the composition are CD3+ T cells.
  • the engineered cells in the composition of the present application include cell populations collected from PBMC cells stimulated by CD3 magnetic beads.
  • the immune cells (eg, T cells) in the composition can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells. It can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMC peripheral blood mononuclear cells
  • T cells can be obtained from a blood sample collected from a subject using any number of techniques known to those of skill in the art, such as the Ficoll TM separation technique.
  • the cells from the circulating blood of the individual are obtained by apheresis.
  • 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 can be washed to remove the plasma fraction and placed in an appropriate buffer or culture medium for subsequent processing steps. Multiple rounds of selection can also be used in the context of the present application. In some aspects, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "Unselected" cells can also undergo additional rounds of selection.
  • composition of the present application can regulate the tumor microenvironment.
  • the source of unpurified CTLs can be any source known in the art, such as bone marrow, fetal, neonatal or adult or other source of hematopoietic cells, such as fetal liver, peripheral blood or umbilical cord blood.
  • Cells can be isolated using various techniques. For example, negative selection can initially remove non-CTLs.
  • mAbs are particularly useful for identifying markers associated with specific cell lineages and/or differentiation stages of positive and negative selection.
  • Most of the terminally differentiated cells can be removed initially by relatively rough dissection.
  • magnetic bead separation can be used initially to remove large numbers of irrelevant cells.
  • at least about 80%, usually at least about 70%, of the total hematopoietic cells will be removed prior to isolating the cells.
  • Separation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; agents, including but not limited to complement and cytotoxins; and panning with antibodies attached to a solid substrate (eg, plate, chip, elutriation) or any other convenient technique.
  • a solid substrate eg, plate, chip, elutriation
  • Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low- and obtuse-angle light-scattering detection channels, impedance channels.
  • Cells can be selected for dead cells by using dyes associated with dead cells, such as propidium iodide (PI).
  • PI propidium iodide
  • cells are harvested in medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, eg, sterile isotonic medium.
  • FCS fetal calf serum
  • BSA bovine serum albumin
  • the immune cells in the composition of the present application have a longer survival time and/or ability to expand.
  • the second immune cells in the composition of the present application have longer survival time and/or expansion ability in the presence of host immune cells (eg, NK cells).
  • the composition comprising the first immune cell and the second immune cell exhibits a stronger cell killing effect in vivo and in vitro on cells carrying a target tumor antigen .
  • the expression, activity and/or signaling of at least one endogenous gene involved in the response to self and non-self antigen recognition polypeptides in the first immune cell and/or the second immune cell is reduced or inhibited; comprising said first The cellular composition of the second immune cell does not cause a graft-versus-host reaction.
  • the types of the first immune cell and the second immune cell in this application may be the same or different.
  • the first immune cell or the second immune cell may be any known suitable immune cell, and the type of the first immune cell does not affect the type of the second immune cell.
  • the first immune cell can be T, NKT cell, and the second immune cell can be T, NKT cell or any other suitable immune cell;
  • the second immune cell can be T, NKT cell, and the second immune cell can be T, NKT cells or any other suitable immune cells; in one example, the first immune cells and the second immune cells include T, NKT cells.
  • the source/preparation method of the first immune cell and the second immune cell of the present application may be the same or different.
  • the first immune cell or the second immune cell may be prepared by any known method, and the preparation method of the first immune cell does not affect the preparation method of the second immune cell.
  • the first immune cell can be an innate immune cell isolated from a donor
  • the second immune cell can be an innate immune cell isolated from a donor or an immune cell prepared by any other known method (for example, derived from a neuron , epithelial cells, endothelial cells or stem cells of immune cells).
  • the second immune cell can be an innate immune cell isolated from a donor
  • the first immune cell can be an innate immune cell isolated from a donor or an immune cell prepared by any other known method (for example, derived from a neuron). , epithelial cells, endothelial cells or stem cells of immune cells).
  • the first immune cell or the second immune cell can be any known source of immune cells, and the source of the first immune cell does not affect the source of the second immune cell.
  • the first immune cell can be an autologous immune cell
  • the second immune cell can be an autologous cell or an immune cell of any other suitable source (e.g., allogeneic); for example, the second immune cell can be an autologous immune cell, and the second immune cell can be an autologous immune cell.
  • An immune cell can be an autologous cell or an immune cell from any other suitable source (eg, allogeneic); in one example, the first immune cell and the second immune cell include allogeneic immune cells.
  • the immune cells in the cell composition of the present application may also include genetic modification of endogenous genes related to graft-versus-host disease (GVHD) and/or host-versus-graft reaction (HVGR).
  • the genetic modification may include low or no expression of HLA-I molecules, TCR and/or HLA-II molecules; for example, the genetic modification may also include low or no expression of NKG2A molecules.
  • Low expression or no expression means that the expression of TCR, B2M, HLA-II or NKG2A in cells 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, low or no expression of TCR, B2M, HLA-II or NKG2A means that the content of TCR, B2M, HLA-II or NKG2A in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, respectively.
  • the amount 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 antibodies specific for TCR, B2M, HLA-II, or NKG2A. expression or content.
  • the genetic modification of the first immune cell and the second immune cell need not be the same, and they are independently endogenously modified, for example, the first immune cell contains the modification of TCR, and the second immune cell contains the modification of B2M, or both Modifications for both. Genetic modifications comprising any combination of TCR, B2M, HLA-II or NKG2A are within the scope of this application.
  • the donor due to the immunogenetic differences between the donor and the recipient (or host), when exogenous donor transplantation is performed, the donor as an exogenous graft will be recognized and recognized by immune cells (such as NK cells) in the host. Attack, and then inhibit or eliminate the donor (such as the first immune cell, the second immune cell), resulting in host-versus-graft response (HVGR).
  • HVGR host-versus-graft response
  • allogeneic cells such as the first immune cell and the second immune cell
  • the host CD8+-mediated cellular immune rejection can be reduced.
  • the present application provides a cell composition: comprising a first immune cell that recognizes NKG2A and has low or no endogenous B2M expression, and/or recognizes tumor and/or pathogen antigens and has low or no endogenous B2M expression Expressed secondary immune cells.
  • the present application provides a cell composition: including first immune cells that recognize NKG2A polypeptides and tumor antigens and have low or no expression of endogenous B2M, and/or recognize tumor antigens and have low or no expression of endogenous B2M Secondary immune cells that do not express.
  • GVHD graft-versus-host disease
  • CRISPR system uses the CRISPR system to knock out the gene TRAC of the ⁇ chain of the endogenous TCR to prepare cells with low or no expression of the endogenous TCR.
  • the present application provides a cell composition: including a first immune cell that recognizes NKG2A and has low or no expression of endogenous TCR, and/or recognizes tumor and/or pathogen antigens and has low expression of endogenous TCR or non-expressing second immune cells; optionally, the first and/or second immune cells have low or no expression of endogenous B2M.
  • the present application provides a cell composition: including first immune cells that recognize NKG2A and tumor antigens and have low or no endogenous TCR expression, and/or recognize tumor antigens and have low or no endogenous TCR expression A second immune cell that does not express; optionally, the first and/or second immune cell expresses low or no endogenous B2M.
  • the HLA-II gene encodes HLA-II antigens, presents extracellular antigens to CD4+ T cells, promotes the proliferation of CD4+ T cells, and then stimulates B cells to produce antigen-specific antibodies, mainly inducing humoral immunity for immune rejection.
  • MHC-II transactivator CIITA is a kind of positive regulator of HLA-II, which can coordinate the action of various transcription factors and HLA-II gene promoter to regulate the expression of HLA-II.
  • the present application provides a cell composition: including first immune cells that recognize NKG2A and have low or no expression of endogenous HLA-II, and/or recognize tumor and/or pathogen antigens, and endogenous HLA-II -Second immune cells with low or no expression of II; optionally, the first and/or second immune cells have low or no expression of endogenous B2M, low or no expression of endogenous TCR, or endogenous TCR Low expression or no expression of derived B2M/TCR.
  • the present application provides a cell composition: including first immune cells that recognize NKG2A and tumor antigens, and endogenous HLA-II is low or not expressed, and/or recognize tumor antigens, and endogenous HLA-II Second immune cells with low or no expression of II; optionally, the first and/or second immune cells have low or no expression of endogenous B2M, low or no expression of endogenous TCR, or endogenous Sexual B2M/TCR low expression or no expression.
  • NKG2A Under repeated stimulation of target cells (such as tumor cells expressing target antigens), the expression of endogenous NKG2A in donor immune cells of exogenous grafts is up-regulated, and will be killed by immune cells that recognize NKG2A in the cell composition of the present application. In addition, low expression or no expression of NKG2A may release the inhibitory effect of immune cells themselves, thus exerting stronger anti-tumor ability.
  • target cells such as tumor cells expressing target antigens
  • the present application provides a cell composition: comprising a first immune cell that recognizes NKG2A and has low or no expression of endogenous NKG2A, and/or recognizes tumor and/or pathogen antigens and has low expression of endogenous NKG2A or non-expressing second immune cells; optionally, the first and/or second immune cells have low or no expression of endogenous B2M, low or no expression of endogenous TCR, endogenous HLA-II Low or no expression, low or no expression of endogenous B2M/TCR, low or no expression of endogenous B2M/HLA-II, low or no expression of endogenous TCR/HLA-II, or endogenous Low expression or no expression of B2M/TCR/HLA-II.
  • the present application provides a cell composition: including first immune cells that recognize NKG2A and tumor antigens, and endogenous NKG2A low expression or no expression, and/or recognize tumor and/or pathogen antigens, and endogenous
  • the second immune cell with low or no expression of NKG2A optionally, the first and/or second immune cell has low or no expression of endogenous B2M, low or no expression of endogenous TCR, endogenous Low or no expression of HLA-II, low or no expression of endogenous B2M/TCR, low or no expression of endogenous B2M/HLA-II, low or no expression of endogenous TCR/HLA-II, or Low expression or no expression of endogenous B2M/TCR/HLA-II.
  • immune cells eg, T cells or NKT cells
  • genetic modification of immune cells can be accomplished by transducing a substantially homogeneous population of cells with a recombinant nucleic acid molecule.
  • retroviral vectors gamma-retroviruses or lentiviruses
  • a polynucleotide encoding a foreign receptor eg, CAR
  • Non-viral vectors can also be used.
  • Transduction can use any suitable viral vector or non-viral delivery system.
  • CARs can be constructed with accessory molecules (eg, cytokines) in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors.
  • elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF- ⁇ B IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, abaculovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers ( For example 2A peptides such as P2A, T2A, E2A and F2A peptides).
  • viral vectors that may be used include, for example, adenovirus, lentivirus and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus or herpes viruses such as Epstein-Barr virus.
  • Non-viral methods can also be used for the genetic modification of immune cells.
  • nucleic acid molecules can be introduced into immune cells by microinjection under lipofection, asialomucoid-polylysine coupling, or surgical conditions.
  • Other non-viral methods of gene transfer include in vitro transfection using liposomes, calcium phosphate, DEAE-dextran, electroporation and protoplast fusion. It is also possible to first transfer the nucleic acid molecule into a cell type that can be cultured in vitro (for example, an autologous or allogeneic primary cell or its progeny), and then inject the cell (or its progeny) modified by the nucleic acid molecule into Subject target tissue or systemic injection.
  • gene knockout technology and/or gene silencing technology to prepare engineered cells with low or no expression of endogenous TCR, B2M, HLA-II or NKG2A.
  • gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous NKG2A.
  • gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M.
  • gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M/HLA-II.
  • gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M/NKG2A. In one example, gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M/HLA-II/NKG2A.
  • Gene knockout technologies include Argonaute, CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guided editing technology and/or homing endonuclease technology.
  • Gene silencing techniques include, but are not limited to: antisense RNA, RNA interference, microRNA-mediated translational inhibition, etc.
  • the clustered regularly interspaced short palindromic repeat (CRISPR) system is used for genome editing.
  • the system consists of Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, comprising the RNA that Cas9 uses to guide it to the correct segment of host DNA, and a region (usually in the form of a hairpin) that binds to tracrRNA. loop form), which forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA, forms an active complex with Cas9), and an optional segment of the DNA repair template (which directs the cellular repair process to allow the insertion of specific DNA sequence of DNA).
  • Cas9 a protein capable of modifying DNA using crRNA as its guide
  • CRISPR RNA comprising the RNA that Cas9 uses to guide it to the correct segment of host DNA
  • a region usually in the form of a hairpin
  • tracrRNA which binds to crRNA, forms an active
  • CRISPR/Cas9 usually uses plasmids or electroporation to deliver nucleic acid fragments to target cells.
  • crRNA needs to be designed for each application because this is the sequence that Cas9 uses to recognize and directly bind to target DNA in cells.
  • Multiple crRNAs and tracrRNAs can be packaged together to form guide RNAs (gRNAs).
  • This gRNA can be ligated with the Cas9 gene and made into a plasmid to be transfected into cells.
  • the present invention relates to the sequence of gRNA, it may be a targeted DNA sequence, or it may be a complete Cas9 guide sequence formed by ribonucleotides corresponding to the DNA, crRNA and TracrRNA.
  • the administered gRNA, tracr paired sequence and tracr sequence can be administered alone, or a complete RNA sequence can be administered.
  • CRISPR/Cas9 transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
  • Zinc finger nuclease is an artificial restriction enzyme produced by combining a zinc finger DNA binding domain with a DNA cleavage domain. Zinc finger domains can be engineered to target specific DNA sequences that allow zinc finger nucleases to target sequences within the genome.
  • Transcription activator-like effector nucleases are restriction enzymes that can be engineered to cleave specific sequences of DNA.
  • the TALEN system works almost the same as the ZFN. They are produced by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain.
  • the present application also provides nucleic acid molecules encoding one or more exogenous receptors described herein (such as CAR), and nucleic acid molecules targeting endogenous TCR, B2M, CIITA or NKG2A nucleic acid inhibitory molecules or gRNA.
  • gRNAs targeting NKG2A, TRAC, B2M, and CIITA include sequences shown in SEQ ID NO: 10, 46, 47, and 48, respectively.
  • CARs encoding NKG2A and target antigens exemplary, BCMA
  • BCMA target antigens
  • targeting endogenous TCR, B2M, CIITA and/or NKG2A nucleic acid inhibitory molecules or gRNA nucleic acid molecules are introduced into engineered cells.
  • in vitro transcribed CAR nucleic acid molecules nucleic acid inhibitory molecules or gRNAs targeting endogenous TCR, B2M, CIITA, or NKG2A can be introduced into cells as a transient transfection.
  • An exemplary artificial DNA sequence is a sequence comprising portions of a gene joined together to form an open reading frame encoding a fusion protein. The DNA portions joined together can be from a single organism or from multiple organisms.
  • the application provides a pharmaceutical composition, which includes an effective amount of the cell composition provided by the application and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may be in the form of a mixture or in the form of separate preparation, storage and placement of different cell active components (eg first engineered cells, second engineered cells).
  • the pharmaceutical composition comprises a first preparation and a second preparation
  • the first preparation comprises the first engineered cells recognizing NKG2A and a pharmaceutically acceptable first adjuvant
  • the second preparation The second engineered cell comprising the antigen recognizing non-NKG2A and the second pharmaceutically acceptable adjuvant.
  • the first formulation and the second formulation may be in the same dosage form or in different dosage forms.
  • the first formulation and the second formulation may be placed in different containers, respectively.
  • the pharmaceutical composition comprises a pharmaceutical mixture, and the pharmaceutical mixture comprises the first engineered cells recognizing NKG2A and the second engineered cells recognizing antigens other than NKG2A.
  • compositions comprising the present application may conveniently be presented in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to the selected pH.
  • sterile liquid preparations such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to the selected pH.
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection.
  • viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue.
  • Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable suitable ones. mixture.
  • a carrier which can be a solvent or dispersion medium including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable suitable ones. mixture.
  • Sterile injectable solutions can be prepared by mixing the engineered cells in the pharmaceutical composition of the present application into a required amount of an appropriate solvent, and adding different amounts of other ingredients as required.
  • Such compositions can be mixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, glucose, dextrose and the like.
  • Compositions can also be lyophilized.
  • the composition may include auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-increasing agents, preservatives, flavoring agents, pigments, etc., This depends on the route of administration and formulation desired.
  • additives can be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical forms can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immune cells or progenitors thereof.
  • compositions may be isotonic, ie they may have the same osmotic pressure as blood and/or tear fluid.
  • the desired isotonicity of the compositions can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • Sodium chloride may be particularly useful for buffers containing sodium ions.
  • a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level.
  • methylcellulose is readily and economically available and easy to use.
  • suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.
  • concentration of the thickener can depend on the agent chosen. It is important to use the amount that will achieve the chosen viscosity.
  • suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or other liquid form, e.g. time-release or liquid-filled form).
  • the number of engineered cells in the composition to be administered will vary for the subject being treated. More effective engineered cells can be administered in smaller numbers.
  • the precise determination of an effective dose can be determined according to each subject's individual factors, including its size, age, sex, weight and the condition of the subject. Dosages can be readily determined by those skilled in the art from this application and knowledge in the art.
  • any additives are present in 0.001% to 50% by weight solution in phosphate-buffered saline, and the active ingredient is present in micrograms to The order of milligrams is present, for example from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.05 wt%, or from about 0.001 wt% to about 20 wt%, from about 0.01 wt% to about 10 wt% % or from about 0.05 wt% to about 5 wt%.
  • toxicity for example by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g. rodents such as mice; the dose of the composition, wherein The concentration of the components and the time of application of the composition elicit an appropriate response.
  • LD lethal dose
  • LD50 LD50
  • suitable animal model e.g. rodents such as mice
  • the application provides the use of the cell composition of the application, and the use of the cell composition in the preparation of medicines, and the cell composition or the medicine is used for the prevention and/or treatment of solid tumors, blood tumors, autoimmune disease or a combination thereof.
  • the cell composition provided by this application has been described above, and the use of the cell composition provided by this application in the preparation of medicine includes all technical solutions thereof.
  • the present application provides a method for preventing, alleviating and/or treating tumors, which includes administering the cell composition and the pharmaceutical composition to a subject in need.
  • the cell composition and pharmaceutical composition provided in this application have been described above, and the method for preventing, alleviating and/or treating tumors provided in this application includes all technical solutions thereof.
  • the present application provides a method for preventing, alleviating and/or treating tumors, which includes administering the first engineered cell and the second engineered cell described in the present application to a subject in need, and the first engineered cell and the second engineered cell can be administered sequentially or simultaneously.
  • the first engineered cells are administered first, and then the second engineered cells are administered, or, for example, the second engineered cells are administered first, and then the first engineered cells are administered.
  • the first engineered cell and the second engineered cell are administered to the subject at the same time.
  • the first engineered cell and the second engineered cell of this application have been described above, and the method for preventing, alleviating and/or treating tumors provided by this application includes all technical solutions thereof.
  • the methods for preventing, alleviating and/or treating tumors provided in the present application include methods for inducing and/or increasing immune response in a subject in need of the cell composition of the present application.
  • the composition of the present application can be used to treat and/or prevent tumors in a subject.
  • the cell compositions of the present application can be used to prolong the survival of a subject with a tumor.
  • the cell composition of the present application can also be used to treat and/or prevent pathogen infection or other infectious diseases such as immunocompromised subjects.
  • Such methods involve administering an effective amount of a cellular composition of the present application to achieve a desired effect, whether alleviating an existing condition or preventing relapse.
  • the amount administered is that effective to produce the desired effect.
  • An effective amount may be provided in one or more administrations. Effective amounts can be provided in boluses or by continuous infusion.
  • compositions comprising cells of the present application can be used to treat a subject having tumor cells with low expression of surface antigens, eg, due to relapse of the disease, where the subject has received treatment that resulted in residual tumor cells.
  • the tumor cell has a low density of the target molecule on the surface of the tumor cell.
  • a composition comprising cells of the present application can be used to treat a subject with relapsed disease, wherein the subject has received immune cells (eg, T cells) comprising administration of a CAR alone.
  • the tumor cells have a low density of tumor-specific antigens on the surface of the tumor cells.
  • the disease is a BCMA positive tumor.
  • the tumor cells have a low density of BCMA on the tumor cells.
  • Such methods include administering an effective amount of a cellular composition of the present application to achieve a desired effect, to alleviate an existing condition or to prevent relapse.
  • an “effective amount” is an amount sufficient to produce beneficial or desired clinical results following treatment.
  • An effective amount can be administered to a subject in one or more doses.
  • an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow the progression of the disease or otherwise reduce the pathological consequences of the disease.
  • Effective amounts are generally determined by a physician on a case-by-case basis and are within the capabilities of those skilled in the art. Several factors are generally considered when determining a suitable dosage to achieve an effective amount. These factors include the age, sex and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of the cellular composition of the application administered.
  • the first engineered cell recognizing NKG2A and the second engineered cell recognizing an antigen other than NKG2A are configured to be administered to the subject at the same time.
  • the simultaneous administration to the subject may include that the time interval between the administration of the first engineered cell and the second engineered cell to the subject is no more than 1 hour.
  • the time interval is 60 minutes, 45 minutes, 30 minutes, 15 minutes, 1 minute, or administered together in admixture.
  • the first engineered cell recognizing NKG2A and the second engineered cell recognizing an antigen other than NKG2A are configured to be administered to a subject separately.
  • the separate administration to the subject may include the time interval between administering the first engineered cell and the second engineered cell to the subject greater than 1 hour.
  • the time interval is 5 hours, 10 hours, 15 hours, 24 hours, 3 days, 5 days, 7 days, or longer.
  • compositions of the present application can be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.
  • the present application provides methods for treating and/or preventing tumors in a subject.
  • the method may comprise administering an effective amount of a cell composition of the present application to a subject having a tumor.
  • the tumors include solid tumors and hematological tumors.
  • tumors include hematological tumors (such as leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer , gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcomas, and various carcinomas (including prostate cancer and small cell lung cancer).
  • hematological tumors such as leukemia, lymphoma, and myeloma
  • ovarian cancer such as leukemia, lymphoma, and myeloma
  • breast cancer breast cancer
  • bladder cancer brain cancer
  • colon cancer intestinal cancer
  • liver cancer liver cancer
  • lung cancer pancreatic cancer
  • prostate cancer skin cancer
  • gastric cancer glioblastoma
  • Non-limiting examples of tumors include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), Chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphatic Sarcoma, lymphangioendothelial sarcoma, liver cancer, cholangiocarcinoma, synovial tumor, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell
  • the tumor is selected from hematological cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer , prostate, skin, stomach, glioblastoma, and throat cancers.
  • the composition of the present application can be used for the treatment and/or prevention of unsuitable or relapsed refractory solid tumors, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer.
  • the tumor is a hematoma.
  • the hematological tumor comprises multiple myeloma.
  • the therapeutic goals of the composition of the present application may include alleviating or reversing disease progression and/or alleviating side effects, or the therapeutic goals may include reducing or delaying the risk of relapse.
  • the present application provides methods for treating and/or preventing a pathogenic infection (eg, viral, bacterial, fungal, parasitic, or protozoan infection) in, eg, an immunocompromised subject.
  • the method may comprise administering an effective amount of a composition of the present application to a subject suffering from a pathogenic infection.
  • a pathogenic infection eg, viral, bacterial, fungal, parasitic, or protozoan infection
  • a pathogenic infection eg, viral, bacterial, fungal, parasitic, or protozoan infection
  • the method may comprise administering an effective amount of a composition of the present application to a subject suffering from a pathogenic infection.
  • Exemplary viral infections that are amenable to treatment include, but are not limited to, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus infections.
  • enhancing refers to allowing a subject or a tumor cell to improve its ability to respond to the treatments disclosed herein.
  • enhanced response can include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% in responsiveness %, 75%, 80%, 85%, 90%, 95%, or 98% or more increase.
  • enhancing can also refer to increasing the number of subjects who respond to treatment, eg, immune cell therapy.
  • an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% more.
  • the cell composition is used to treat BCMA-positive tumors. In one example, the composition is used to treat multiple myeloma.
  • composition comprising the present application can be provided systemically or directly to a subject to induce and/or enhance an immune response to an antigen and/or treat and/or prevent tumors, pathogenic infections or infectious diseases.
  • a composition of the present application is injected directly into an organ of interest (eg, an organ affected by a tumor).
  • the compositions of the present application are provided to the organ of interest indirectly, eg, by administration to the circulatory system (eg, vein, tumor vasculature).
  • Expansion and differentiation agents can be provided before, simultaneously with or after administration of the composition to increase the production of T cells, NKT cells or CTL cells in vitro or in vivo.
  • Engineered cells in the compositions of the present application may include purified cell populations.
  • One skilled in the art can readily determine the percentage of engineered cells of the present application in a population using various well-known methods, such as fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • Suitable ranges for purity in a population comprising engineered cells of the present application are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%.
  • the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%.
  • the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art (eg, decreased purity may require increased dosages).
  • Cells can be introduced by injection, catheter, and the like.
  • the composition of the present application may be a pharmaceutical composition comprising the cells of the present application or their progenitor cells and a pharmaceutically acceptable carrier.
  • Administration can be autologous or allogeneic.
  • immune cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject.
  • Peripheral blood-derived immune cells or their progeny eg, in vivo, ex vivo, or in vitro sources
  • they may be formulated in unit dose injectable forms (solutions, suspensions, emulsions, etc.).
  • kits comprising the cell composition of the present application or the pharmaceutical composition of the present application.
  • the kit is a kit for inducing and/or enhancing immune response and/or treating and/or preventing tumor or pathogen infection in a subject.
  • the kit includes an effective amount of the cell composition or pharmaceutical composition of the present application or the first engineered cell and the second engineered cell of the present application.
  • kits include sterile containers; such containers can be in the form of boxes, ampoules, bottles, vials, tubes, bags, sachets, blister packs, or other suitable container forms known in the art.
  • Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug.
  • instructions for administering the cell composition of the present application to a subject suffering from or developing a tumor or pathogenic or immune disease are provided together.
  • the instructions generally include information about the use of the composition in the treatment and/or prophylaxis of tumors or pathogenic infections.
  • the instructions include at least one of the following: a description of the therapeutic agent; a dosage form and administration for the treatment or prevention of tumors, pathogenic infections, or immune diseases or symptoms thereof; precautions; warnings; indications; incompatibility ; Medication Information; Adverse Reactions; Animal Pharmacology; Clinical Studies; and/or References.
  • These instructions may be printed directly on the container, or as a label affixed to the container, or provided within or with the container as separate sheets, booklets, cards or file folders.
  • NK cells Primary PBMCs isolated from healthy donors monocytes Primary PBMCs isolated from healthy donors Cell line RPMI-8226 Purchased from ATCC MM.1S tumor cells Purchased from ATCC NPG immunodeficient mice Purchased from Beijing Weitongda Biotechnology Co., Ltd.
  • BCMA CAR-T cells, NKG2A CAR-T cells, and BCMA-NKG2A CAR-T cells expressing BCMA-CAR, NKG2A-CAR, and BCMA-NKG2A-CAR were respectively constructed using conventional molecular biology methods in the field.
  • BCMA-CAR includes the single-chain antibody of BCMA in turn (the amino acid sequence of VH is shown in SEQ ID NO: 19, and the amino acid sequence of VL is shown in SEQ ID NO: 20), CD8hinge, CD8 transmembrane domain, 4-1BB co- Stimulatory factors and CD3 ⁇ .
  • NKG2A-CAR includes NKG2A single-chain antibody in turn (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), CD8hinge, CD28 transmembrane domain, and CD28 co-stimulatory factor and CD3 ⁇ .
  • BCMA-NKG2A-CAR sequentially includes the tandem single-chain antibody of BCMA and NKG2A (the amino acid sequence is shown in SEQ ID NO: 36), CD8hinge, CD8 transmembrane domain, 4-1BB co-stimulatory factor and CD3 ⁇ .
  • the CD8hinge sequence in the above CAR is shown in SEQ ID NO: 30
  • the CD8 transmembrane domain sequence is shown in SEQ ID NO: 31
  • the CD28 transmembrane domain sequence is shown in SEQ ID NO: 32
  • 4-1BB co-stimulatory factor The sequence is shown in SEQ ID NO: 34
  • the CD28 costimulator sequence is shown in SEQ ID NO: 33
  • the CD3 ⁇ sequence is shown in SEQ ID NO: 35.
  • Embodiment 2 the preparation of UCAR-T cell
  • the gRNA sequences targeting TRAC, B2M and NKG2A were synthesized in vitro according to the reagent instructions (GeneArt TM Precision gRNA Synthesis Kit, Thermo Tisher).
  • the triple knockout of TCR/B2M/NKG2A was performed on the BCMA CAR-T cells, NKG2A CAR-T and BCMA-NKG2A CAR-T cells obtained in Example 1, respectively.
  • Cas 9 enzyme and gRNA were incubated at room temperature at a ratio of 1:4, cells were mixed with Cas9 enzyme and gRNA complex (RNP) (the final concentration of Cas 9 enzyme was 3uM), and the RNP complex was electrotransferred into BCMA CAR- From T cells, NKG2A CAR-T, and BCMA-NKG2A CAR-T cells, TCR/B2M/NKG2A knockout CAR-T cells were obtained, and by removing TCR/B2M positive cells, they were obtained and named BCMA UCAR-T- NKG2A KO (or BCMA UCAR-T-TKO), NKG2A UCAR-T-NKG2A KO (or NKG2A UCAR-T-TKO) and BCMA-NKG2A UCAR-T-NKG2A KO (or BCMA-NKG2A UCAR -T-TKO).
  • UTD-TKO cells that also knocked out TCR/B2M/
  • UCAR-T cells that recognize NKG2A can promote the survival and/or expansion of UCAR-T cells in vitro
  • NK cells were isolated from peripheral blood mononuclear cells using an NK cell isolation kit (purchased from Miltenyi), and cultured in vitro for 14 days using NK cell medium containing IL-2.
  • Target cells multiple myeloma RPMI-8226 cells;
  • Effector cell 1 primary cultured NK cells
  • Effector cell 2 NKG2A UCAR-T-TKO, BCMA
  • the tumor cells (RPMI-8226 cells) were identified by flow staining, NK cells and UCAR-T cells, CFSE+HLA-ABC+ represents tumor cells, CFSE-HLA-ABC+ represents NK cells, CFSE-HLA-ABC- represents UCAR-T cells in the detection system, and the absolute count is used to compare UCAR-T Quantitative analysis of cells was performed to detect the survival and/or expansion of UCAR-T cells in vitro, and the statistics of the number are shown in Figure 1.
  • Example 4 In vivo synergistic anti-tumor effect of UCAR-T cells recognizing NKG2A
  • mice 5 ⁇ 10 6 RPMI-8226 cells were inoculated subcutaneously in NPG immunodeficient mice. The average tumor volume was about 200 mm 3 10 days after inoculation. The mice were divided into 4 groups (UTD-TKO, NKG2A UCAR-T-TKO, BCMA UCAR-T-TKO+UTD-TKO, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO), 5 rats in each group.
  • the peripheral blood of the mice was collected, and flow staining and absolute quantification were performed with anti-human-CD45 antibody and anti-human-BCMA antibody to detect the number of BCMA UCAR-T cells in the peripheral blood of the mice.
  • test results are shown in Figure 2A.
  • UCAR-T cells that recognize NKG2A exert the anti-tumor effect of synergistic BCMA-UCAR-T cells in vivo.
  • UCAR-T cells recognizing NKG2A significantly increased the number of BCMA UCAR-T cells in vivo compared with UTD-TKO.
  • Example 5 UCAR-T cells that recognize NKG2A cooperate with UCAR-T cells to specifically infiltrate tumor tissue
  • mice in the three groups of UTD-TKO, BCMA UCAR-T-TKO+UTD-TKO, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO were collected. normal kidney tissue. After being fixed with paraformaldehyde, the sections and anti-human-CD45 immunohistochemical staining were performed to detect the infiltration of T cells in tumor cells and normal tissues.
  • Example 6 The combination of UCAR-T cells recognizing NKG2A and UCAR-T recognizing tumor antigens does not cause graft-versus-host reaction
  • BCMA UCAR-T-TKO and NKG2A UCAR-T-TKO cells were expanded and cultured in vitro, and untransfected T cells (UTD) and PBS were used as controls. Inject 1 ⁇ 10 7 UTD, 1 ⁇ 10 7 BCMA UCAR-T-TKO, 5 ⁇ 10 6 BCMA UCAR-T-TKO+5 ⁇ 10 6 NKG2A UCAR-T-TKO into NPG mice , body weight was weighed twice a week, and the hair and basic conditions of the mice were observed to determine whether there was a graft-versus-host reaction (GVHD).
  • GVHD graft-versus-host reaction
  • mice in the UTD group experienced obvious slowing or decreasing of body weight growth from D51, and showed obvious GVHD symptoms such as hair loss and decreased activity.
  • the BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO cell group was similar to PBS, and the mice all grew normally and gradually gained weight. The above results indicated that co-administration of NKG2A-recognizing UCAR-T cells and BCMA UCAR-T cells did not cause GVHD.
  • Example 7 In vivo synergistic anti-tumor effect of UCAR-T cells that recognize NKG2A and tumor antigens
  • mice 5 ⁇ 10 6 RPMI-8226 cells were inoculated subcutaneously in NPG mice, and the average tumor volume was about 250mm 3 10 days after inoculation.
  • the mice were divided into 4 groups (UTD-TKO, BCMA UCAR-T-TKO+UTD-TKO, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO, BCMA UCAR-T-TKO+BCMA-NKG2A UCAR-T-TKO), 5 rats in each group.
  • 0.4 ⁇ 10 6 UTD-TKO, 0.4 ⁇ 10 6 BCMA UCAR-T-TKO and 0.4 ⁇ 10 6 UTD-TKO, 0.4 ⁇ 10 6 BCMA were injected three times into the tail vein according to the above groups UCAR-T-TKO and 0.4 ⁇ 10 6 NKG2A UCAR-T-TKO, 0.4 ⁇ 10 6 BCMA UCAR-T-TKO and 0.4 ⁇ 10 6 BCMA-NKG2A UCAR-T-TKO cells.
  • the tumor growth curve was drawn according to the method described in Example 4.
  • Example 8 UCAR-T cells that recognize NKG2A promote the survival and/or expansion of UCAR-T cells in vitro
  • UTD cells that also knocked out TCR/B2M/NKG2A/CIITA but were not transfected with virus were used as negative controls.
  • Target cells multiple myeloma MM.1S-GFP cells
  • Effector cell 1 primary cultured NK cells
  • Effector cell 2 BCMA UCAR-T-FKO, NKG2A UCAR-T-FKO cells.
  • Example 9 UCAR-T cells that recognize NKG2A exert synergistic anti-tumor ability in vivo
  • mice 5 ⁇ 10 6 RPMI-8226 cells were inoculated subcutaneously in NPG mice. The average tumor volume was about 250mm 3 13 days after inoculation. The mice were divided into 4 groups (UTD-FKO, BCMA UCAR-T-FKO, BCMA UCAR-T -FKO+UTD+NK, BCMA UCAR-T-FKO+NKG2A UCAR-T-FKO+NK), 5 rats in each group.

Abstract

The present invention relates to a composition comprising a first immune cell identifying an NKG2A polypeptide and a second immune cell identifying a tumor and/or pathogen antigen, as well as a use thereof in disease treatment.

Description

用于肿瘤免疫学的组合物和方法Compositions and methods for tumor immunology
优先权信息priority information
本申请要求：于2021年7月16日提交的中国专利申请CN202110807344.3、于2021年9月8日提交的中国专利申请CN202111052328.4，于2022年4月27日提交的中国专利申请CN202210456417.3，它们的全部内容通过整体引用并入本文。This application requires: Chinese patent application CN202110807344.3 submitted on July 16, 2021, Chinese patent application CN202111052328.4 submitted on September 8, 2021, and Chinese patent application CN202210456417 submitted on April 27, 2022. 3, their entire contents are incorporated herein by reference in their entirety.
同时提交的序列表文件Sequence listing files submitted at the same time
下列XML文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(名称：FF00650PCT-sequence listing.xml，日期：20220715，大小：58KB)。The entire content of the following XML file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (Name: FF00650PCT-sequence listing.xml, Date: 20220715, Size: 58KB).
技术领域technical field
本申请提供识别NKG2A的免疫细胞和识别肿瘤和/或病原体抗原的免疫细胞的联合使用的用途。The present application provides the combined use of immune cells that recognize NKG2A and immune cells that recognize tumor and/or pathogen antigens.
背景技术Background technique
同种异体免疫细胞治疗面临的核心问题是避免移植物抗宿主(GVHD)反应和宿主免疫***的排斥反应(HVGR)。因此，如何增加异体免疫细胞在宿主体内存活与扩增，避免宿主免疫排斥，这些问题的解决对于提高免疫细胞治疗疗效至关重要。The core problem facing allogeneic immune cell therapy is to avoid graft-versus-host (GVHD) reaction and host immune system rejection (HVGR). Therefore, how to increase the survival and expansion of allogeneic immune cells in the host and avoid host immune rejection is crucial to improving the efficacy of immune cell therapy.
发明内容Contents of the invention
一方面，本申请提供靶向NKG2A的第一CAR T细胞与靶向靶抗原的第二CAR T细胞的联合使用在疾病治疗中的用途。In one aspect, the present application provides the combined use of a first CAR T cell targeting NKG2A and a second CAR T cell targeting a target antigen in disease treatment.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的内源性的NKG2A表达、活性和/或信号传导减少。In one example, the endogenous NKG2A expression, activity and/or signal transduction of the first CAR T cell and/or the second CAR T cell is reduced.
在一实例中，所述靶抗原是肿瘤抗原，所述疾病是肿瘤。In one example, the target antigen is a tumor antigen and the disease is a tumor.
在一实例中，所述第一CAR T细胞和第二CAR T细胞的内源性的NKG2A表达、活性和/或信号传导减少。In one example, the endogenous NKG2A expression, activity and/or signaling of the first CAR T cell and the second CAR T cell is reduced.
在一实例中，所述肿瘤抗原是CD19和BCMA。In one example, the tumor antigens are CD19 and BCMA.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的TCR分子沉默。In one example, the TCR molecule of the first CAR T cell and/or the second CAR T cell is silenced.
在一实例中，所述TCR分子沉默是指编码TCR的α和β链中的一种或两种链的基因发生沉默；In one example, the TCR molecular silencing refers to the silencing of genes encoding one or both of the α and β chains of TCR;
优选地，所述TCR分子沉默是指编码TCR的α链的基因(即TRAC基因)发生沉默；Preferably, the TCR molecular silencing refers to the silencing of the gene encoding the alpha chain of TCR (ie TRAC gene);
更优选地，所述TCR分子沉默是指编码TCR的α链恒定区的基因发生沉默；More preferably, the TCR molecular silencing refers to the silencing of the gene encoding the α-chain constant region of TCR;
进一步优选地，所述TCR分子沉默是指编码TCR的α链恒定区的基因的第一个外显子发生沉默。Further preferably, the TCR molecular silencing refers to the silencing of the first exon of the gene encoding the α-chain constant region of TCR.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的内源性MHC分子沉默。In one example, endogenous MHC molecules of the first CAR T cell and/or the second CAR T cell are silenced.
在一实例中，所述MHC分子是指HLA分子；In one example, the MHC molecule refers to an HLA molecule;
优选的，所述HLA分子选自HLA-I类和/或HLA-II分子，包含HLA-A、HLA-B、HLA-C，B2M和CIITA分子中的至少一种；Preferably, the HLA molecules are selected from HLA-I class and/or HLA-II molecules, including at least one of HLA-A, HLA-B, HLA-C, B2M and CIITA molecules;
更优选的，所述HLA分子为I类的HLA分子。More preferably, the HLA molecules are class I HLA molecules.
在一实例中，所述HLA分子为B2M分子。In one example, the HLA molecule is a B2M molecule.
在一实例中，采用基因编辑技术使内源性TCR分子沉默、内源性MHC分子沉默、或内源性的NKG2A表达、活性和/或信号传导减少。In one example, gene editing technology is used to silence endogenous TCR molecules, endogenous MHC molecules, or reduce endogenous NKG2A expression, activity and/or signal transduction.
一方面，本申请提供靶向NKG2A的第一CAR T细胞与靶向靶抗原的第二CAR T细胞的联合使用在疾病治疗中的用途，其特征在于，同时给予NKG2A蛋白的抑制剂。In one aspect, the present application provides the combined use of a first CAR T cell targeting NKG2A and a second CAR T cell targeting a target antigen in disease treatment, characterized in that an inhibitor of NKG2A protein is administered at the same time.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的TCR分子沉默。In one example, the TCR molecule of the first CAR T cell and/or the second CAR T cell is silenced.
在一实例中，所述TCR分子沉默是指编码TCR的α和β链中的一种或两种链的基因发生沉默；In one example, the TCR molecular silencing refers to the silencing of genes encoding one or both of the α and β chains of TCR;
优选地，所述TCR分子沉默是指编码TCR的α链的基因(即TRAC基因)发生沉默；Preferably, the TCR molecular silencing refers to the silencing of the gene encoding the alpha chain of TCR (ie TRAC gene);
更优选地，所述TCR分子沉默是指编码TCR的α链恒定区的基因发生沉默；More preferably, the TCR molecular silencing refers to the silencing of the gene encoding the α-chain constant region of TCR;
进一步优选地，所述TCR分子沉默是指编码TCR的α链恒定区的基因的第一个外显子发生沉默。Further preferably, the TCR molecular silencing refers to the silencing of the first exon of the gene encoding the α-chain constant region of TCR.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的内源性MHC分子沉默。In one example, endogenous MHC molecules of the first CAR T cell and/or the second CAR T cell are silenced.
在一实例中，所述MHC分子是指HLA分子；In one example, the MHC molecule refers to an HLA molecule;
优选的，所述HLA分子选自HLA-I类和/或HLA-II分子，包含HLA-A、HLA-B、HLA-C，B2M和CIITA分子中的至少一种；Preferably, the HLA molecules are selected from HLA-I class and/or HLA-II molecules, including at least one of HLA-A, HLA-B, HLA-C, B2M and CIITA molecules;
更优选的，所述HLA分子为I类的HLA分子。More preferably, the HLA molecules are class I HLA molecules.
在一实例中，所述HLA分子为B2M分子。In one example, the HLA molecule is a B2M molecule.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的内源性的NKG2A不表达。In one example, the endogenous NKG2A of the first CAR T cell and/or the second CAR T cell does not express.
在一实例中，采用基因编辑技术敲除所述第一CAR T细胞和/或第二CAR T细胞的内源性的NKG2A；In one example, gene editing technology is used to knock out the endogenous NKG2A of the first CAR T cell and/or the second CAR T cell;
优选的，所述基因编辑技术选自CRISPR/Cas9技术、ZFN技术、TALE技术、TALE-CRISPR/Cas9技术、Base Editor技术、引导编辑技术和/或归巢核酸内切酶技术；Preferably, the gene editing technology is selected from CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guided editing technology and/or homing endonuclease technology;
更优选的，所述基因编辑技术选自CRISPR/Cas9技术。More preferably, the gene editing technology is selected from CRISPR/Cas9 technology.
在一实例中，所述CRISPR/Cas9技术使用的gRNA包含SEQ ID NO：10、46、47或48所示的序列。In one example, the gRNA used by the CRISPR/Cas9 technology comprises the sequence shown in SEQ ID NO: 10, 46, 47 or 48.
在一实例中，第一CAR T细胞和/或第二CAR T细胞的内源性的NKG2A的功能被破坏。In one example, the endogenous NKG2A function of the first CAR T cell and/or the second CAR T cell is disrupted.
在一实例中，制备CAR T细胞的T细胞为天然的T细胞或经多能干细胞诱导产生的T细 胞。In one example, the T cells for preparing CAR T cells are natural T cells or T cells induced by pluripotent stem cells.
在一实例中，所述第一CAR T细胞中，识别NKG2A的抗体具有：SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2、SEQ ID NO:5所示的HCDR3、SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、和SEQ ID NO:8所示的LCDR3；In one example, in the first CAR T cell, the antibody that recognizes NKG2A has: HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:5, LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8;
优选的，所述识别NKG2A的抗体含有SEQ ID NO:1所述的重链可变区和/或SEQ ID NO:2所述的轻链可变区。Preferably, the antibody recognizing NKG2A contains the heavy chain variable region described in SEQ ID NO:1 and/or the light chain variable region described in SEQ ID NO:2.
在一实例中，所述肿瘤抗原是WT1、HER2、GPC3、Claudin18.2、CD19或EGFR。In one example, the tumor antigen is WT1, HER2, GPC3, Claudin18.2, CD19 or EGFR.
在一实例中，所述肿瘤抗原是BCMA，所述第二CAR T细胞中，识别BCMA的抗体含有：SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2、和SEQ ID NO:18所示的LCDR3；In one example, the tumor antigen is BCMA, and in the second CAR T cell, the antibody that recognizes BCMA contains: HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and SEQ ID NO HCDR3 shown in :15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18;
优选的，所述识别BCMA的抗体含有含有SEQ ID NO:19所示的重链可变区和/或SEQ ID NO:20所示的轻链可变区。Preferably, the antibody recognizing BCMA contains the heavy chain variable region shown in SEQ ID NO:19 and/or the light chain variable region shown in SEQ ID NO:20.
一方面，本申请提供了细胞组合物，其包括：On the one hand, the application provides cell composition, it comprises:
a)识别NKG2A的第一免疫细胞；以及a) a first immune cell that recognizes NKG2A; and
b)识别非NKG2A抗原的第二免疫细胞。b) Second immune cells that recognize non-NKG2A antigens.
在一实例中，所述非NKG2A抗原包括肿瘤抗原和/或病原体抗原。In one example, the non-NKG2A antigens include tumor antigens and/or pathogen antigens.
在一实例中，所述第一免疫细胞包括识别NKG2A的第一外源受体。In one example, the first immune cell includes a first exogenous receptor that recognizes NKG2A.
在一实例中，所述第一外源受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体和T细胞抗原耦合器(TAC)。In one example, the first exogenous receptor is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor and T cell antigen coupler (TAC).
在一实例中，所述第一外源受体包含嵌合抗原受体(CAR)。In one example, the first exogenous receptor comprises a chimeric antigen receptor (CAR).
在一实例中，所述嵌合抗原受体(CAR)为第一CAR，所述第一CAR包含NKG2A抗原结合域。In one example, the chimeric antigen receptor (CAR) is a first CAR comprising an NKG2A antigen binding domain.
在一实例中，所述NKG2A抗原结合域能够特异性识别NKG2A抗原，所述NKG2A抗原包含如SEQ ID NO:11所示的氨基酸序列。In one example, the NKG2A antigen binding domain can specifically recognize the NKG2A antigen, and the NKG2A antigen comprises the amino acid sequence shown in SEQ ID NO: 11.
在一实例中，所述NKG2A抗原结合域包含轻链可变区，所述轻链可变区(VL)包含如SEQ ID NO:6、7、8中任一项所示的轻链互补决定区(LCDR)或其组合。In one example, the NKG2A antigen binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determination as shown in any one of SEQ ID NOs: 6, 7, 8 region (LCDR) or a combination thereof.
在一实例中，所述轻链可变区(VL)包含轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)、轻链互补决定区3(LCDR3)，所述LCDR1包含如SEQ ID NO:6所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:8所示的氨基酸序列。In one example, the light chain variable region (VL) comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3), and the LCDR1 comprises As shown in the amino acid sequence of SEQ ID NO:6, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:7, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:8.
在一实例中，所述NKG2A抗原结合域包含重链可变区，所述重链可变区(VH)包含 如SEQ ID NO3、4、5中任一项所示的重链互补决定区(HCDR)或其组合。In one example, the NKG2A antigen binding domain comprises a heavy chain variable region, and the heavy chain variable region (VH) comprises a heavy chain complementarity determining region as shown in any one of SEQ ID NO3, 4, 5 ( HCDR) or a combination thereof.
在一实例中，所述重链可变区(VH)包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)、重链互补决定区3(HCDR3)，所述HCDR1的氨基酸序列如SEQ ID NO:3所示，所述HCDR2的氨基酸序列如SEQ ID NO:4所示，所述HCDR3的氨基酸序列如SEQ ID NO:5所示。In one example, the heavy chain variable region (VH) comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), heavy chain complementarity determining region 3 (HCDR3), and the HCDR1 The amino acid sequence is shown in SEQ ID NO:3, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:4, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:5.
在一实例中，所述重链可变区(VH)包含如SEQ ID NO:1所示的氨基酸序列，所述轻链可变区(VL)包含如SEQ ID NO:2所示的氨基酸序列。In one example, the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO:2 .
在一实例中，所述NKG2A抗原结合域包含轻链，所述轻链包含所述轻链可变区(VL)。In one example, the NKG2A antigen binding domain comprises a light chain comprising the light chain variable region (VL).
在一实例中，所述NKG2A抗原结合域包含重链，所述重链包含所述重链可变区(VL)。In one example, said NKG2A antigen binding domain comprises a heavy chain comprising said heavy chain variable region (VL).
在一实例中，所述第二免疫细胞包括识别肿瘤抗原和/或病原体抗原的第二外源受体。In one example, the second immune cell includes a second exogenous receptor that recognizes a tumor antigen and/or a pathogen antigen.
在一实例中，第二外源受体选自：嵌合抗原受体(CAR)、嵌合T细胞受体和T细胞抗原耦合器(TAC)。In one example, the second exogenous receptor is selected from the group consisting of chimeric antigen receptor (CAR), chimeric T cell receptor and T cell antigen coupler (TAC).
在一实例中，所述第二外源受体包含嵌合抗原受体(CAR)。In one example, the second exogenous receptor comprises a chimeric antigen receptor (CAR).
在一实例中，所述嵌合抗原受体(CAR)为第二CAR，所述第二CAR包含肿瘤抗原结合域。In one example, the chimeric antigen receptor (CAR) is a second CAR comprising a tumor antigen binding domain.
在一实例中，所述肿瘤抗原选自：WT1、HER2、EGFR、BCMA、CD19。In one example, the tumor antigen is selected from: WT1, HER2, EGFR, BCMA, CD19.
在一实例中，所述肿瘤抗原包含BCMA。In one example, the tumor antigen comprises BCMA.
在一实例中，所述BCMA抗原结合域能够特异性识别和/或结合BCMA抗原，所述BCMA抗原包含如SEQ ID NO:12所示的氨基酸序列。In one example, the BCMA antigen-binding domain can specifically recognize and/or bind to a BCMA antigen, and the BCMA antigen comprises the amino acid sequence shown in SEQ ID NO:12.
在一实例中，所述BCMA抗原结合域包含轻链可变区，所述轻链可变区(VL)包含如SEQ ID NO:16、17、18中任一项所示的轻链互补决定区(LCDR)或其组合。In one example, the BCMA antigen binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determination as shown in any one of SEQ ID NOs: 16, 17, 18 region (LCDR) or a combination thereof.
在一实例中，所述轻链可变区(VL)包含轻链互补决定区1(LCDR1)、轻链互补决定区2(LCDR2)、轻链互补决定区3(LCDR3)，所述LCDR1包含如SEQ ID NO:16所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:17所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:18所示的氨基酸序列。In one example, the light chain variable region (VL) comprises light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3), and the LCDR1 comprises As shown in the amino acid sequence of SEQ ID NO: 16, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 17, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 18.
在一实例中，所述BCMA抗原结合域包含重链可变区，所述重链可变区(VH)包含如SEQ ID NO:13、14、15中任一项所示的重链互补决定区(HCDR)或其组合。In one example, the BCMA antigen binding domain comprises a heavy chain variable region, and the heavy chain variable region (VH) comprises a heavy chain complementarity determination as shown in any one of SEQ ID NO: 13, 14, 15 region (HCDR) or a combination thereof.
在一实例中，所述重链可变区(VH)包含重链互补决定区1(HCDR1)、重链互补决定区2(HCDR2)、重链互补决定区3(HCDR3)，所述HCDR1的氨基酸序列如SEQ ID NO:13所示，所述HCDR2的氨基酸序列如SEQ ID NO:14所示，所述HCDR3的氨基酸序列如SEQ ID NO:15所示。In one example, the heavy chain variable region (VH) comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), heavy chain complementarity determining region 3 (HCDR3), and the HCDR1 The amino acid sequence is shown in SEQ ID NO:13, the amino acid sequence of the HCDR2 is shown in SEQ ID NO:14, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:15.
在一实例中，所述重链可变区(VH)包含如SEQ ID NO:19所示的氨基酸序列，所述 轻链可变区(VL)包含如SEQ ID NO:20所示的氨基酸序列。In one example, the heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 19, and the light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: 20 .
在一实例中，所述第一CAR、所述第二CAR包含跨膜域。In one example, the first CAR and the second CAR comprise a transmembrane domain.
在一实例中，所述第一CAR的跨膜域、所述第二CAR的跨膜域各自独立地选自如下蛋白的跨膜域：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)。In one example, the transmembrane domain of the first CAR and the transmembrane domain of the second CAR are each independently selected from the transmembrane domains of the following proteins: CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137).
在一实例中，所述第一CAR、所述第二CAR包含胞内信号传导域。In one example, the first CAR and the second CAR comprise an intracellular signaling domain.
在一实例中，所述第一CAR的胞内信号传导域、所述第二CAR的胞内信号传导域各自独立地选自如下蛋白的胞内信号传导域：CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10、DAP12。In one example, the intracellular signaling domain of the first CAR and the intracellular signaling domain of the second CAR are each independently selected from the intracellular signaling domains of the following proteins: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, DAP12.
在一实例中，所述第一CAR、所述第二CAR包含铰链域。In one example, the first CAR and the second CAR comprise a hinge domain.
在一实例中，所述第一CAR的铰链域、所述第二CAR的铰链域各自独立地选自如下蛋白的铰链域：CD8、CD28。In one example, the hinge domain of the first CAR and the hinge domain of the second CAR are independently selected from hinge domains of the following proteins: CD8 and CD28.
在一实例中，所述第一CAR、所述第二CAR包含共刺激信号结构域。In one example, the first CAR and the second CAR comprise a co-stimulatory signaling domain.
在一实例中，所述第一CAR的共刺激信号结构域、所述第二CAR的共刺激信号结构域各自独立地选自如下蛋白的共刺激信号结构域：CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD1。In one example, the co-stimulatory signaling domain of the first CAR and the co-stimulatory signaling domain of the second CAR are each independently selected from the co-stimulatory signaling domains of the following proteins: CD27, CD28, 4-1BB( CD137), OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD1.
在一实例中，所述第一CAR还包含所述识别肿瘤和/或病原体的抗原结合域。In one example, the first CAR further comprises the antigen-binding domain that recognizes tumors and/or pathogens.
在一实例中，所述第一CAR包括：In an example, the first CAR includes:
a)识别NKG2A的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD28的共刺激信号结构域和CD3ζ；和/或a) an antigen binding domain that recognizes NKG2A, optionally an antigen binding domain that recognizes tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, a co-stimulatory signaling domain of CD28 and CD3ζ; and/or
b)识别NKG2A的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD137的共刺激信号结构域和CD3ζ；和/或b) an antigen binding domain that recognizes NKG2A, optionally an antigen binding domain that recognizes tumor and/or pathogen antigens, a transmembrane region of CD28 or CD8, a co-stimulatory signaling domain of CD137 and CD3ζ; and/or
c)识别NKG2A多肽的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD28的共刺激信号结构域，CD137的共刺激信号结构域和CD3ζ；c) an antigen-binding domain that recognizes a NKG2A polypeptide, optionally an antigen-binding domain that recognizes tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and CD3ζ ;
d)识别NKG2A多肽的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，和CD3ζ；d) an antigen-binding domain that recognizes a NKG2A polypeptide, optionally an antigen-binding domain that recognizes tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, and CD3ζ;
所述第二CAR包括：The second CAR includes:
a)识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD28的共刺激信号结构域和CD3ζ；和/或a) an antigen-binding domain that recognizes tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, a co-stimulatory signaling domain of CD28 and CD3ζ; and/or
b)识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD137的共刺激信号结构域和CD3ζ；和/或b) antigen-binding domains that recognize tumor and/or pathogen antigens, transmembrane regions of CD28 or CD8, co-stimulatory signaling domains of CD137 and CD3ζ; and/or
c)识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD28的共刺激信号结构域，CD137的共刺激信号结构域和CD3ζ；c) antigen-binding domains that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, costimulatory signal domains of CD28, costimulatory signal domains of CD137 and CD3ζ;
d)识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，和CD3ζ。d) Antigen-binding domains that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, and CD3ζ.
在一实例中，所述第一免疫细胞和/或第二免疫细胞选自：T细胞、NK细胞、细胞毒性T细胞、NKT细胞、巨噬细胞、CIK细胞和以及干细胞衍生的免疫细胞。In one example, the first immune cell and/or the second immune cell is selected from the group consisting of T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells and stem cell-derived immune cells.
在一实例中，所述第一免疫细胞和/或第二免疫细胞选自：自体或同种异体T细胞、干细胞衍生的T细胞、原代T细胞和来源于人的自体T细胞。In one example, the first immune cell and/or the second immune cell is selected from: autologous or allogeneic T cells, stem cell-derived T cells, primary T cells and autologous T cells derived from humans.
在一实例中，所述第一CAR T细胞和/或第二CAR T细胞的内源性HLA分子低表达或不表达。In one example, the endogenous HLA molecules of the first CAR T cell and/or the second CAR T cell have low expression or no expression.
在一实例中，所述HLA分子包括HLA-I类和/或HLA-II分子。In one example, the HLA molecules include HLA-class I and/or HLA-II molecules.
在一实例中，所述第一免疫细胞和/或第二免疫细胞包括：In an example, the first immune cell and/or the second immune cell include:
a)内源性HLA-I分子低表达或不表达；a) Low expression or no expression of endogenous HLA-I molecules;
b)内源性TCR分子低表达或不表达；b) low or no expression of endogenous TCR molecules;
c)内源性HLA-II分子低表达或不表达；和/或c) low or no expression of endogenous HLA-II molecules; and/or
d)内源性NKG2A分子低表达或不表达。d) Low expression or no expression of endogenous NKG2A molecules.
在一实例中，所述TCR分子低表达或不表达是指编码TCR的α链的基因(即TRAC基因)低表达或不表达；In one example, the low expression or no expression of the TCR molecule refers to the low expression or no expression of the gene encoding the α chain of TCR (ie TRAC gene);
更优选地，所述TCR分子低表达或不表达是指编码TCR的α链恒定区的基因低表达或不表达；More preferably, the low expression or no expression of the TCR molecule refers to the low expression or no expression of the gene encoding the α chain constant region of TCR;
进一步优选地，所述TCR分子低表达或不表达是指编码TCR的α链恒定区的基因的第一个外显子低表达或不表达。Further preferably, the low or no expression of the TCR molecule refers to the low or no expression of the first exon of the gene encoding the α-chain constant region of TCR.
在一实例中，所述第一免疫细胞和/或第二免疫细胞包括：In an example, the first immune cell and/or the second immune cell include:
a)内源性HLA-I分子低表达或不表达包括编码HLA-I蛋白的基因的敲除；a) Low or no expression of endogenous HLA-I molecules including knockout of genes encoding HLA-I proteins;
b)内源性TCR分子低表达或不表达包括编码TCR蛋白的基因的敲除；b) Low or no expression of endogenous TCR molecules including knockout of genes encoding TCR proteins;
c)内源性HLA-II分子低表达或不表达包括编码HLA-II蛋白的基因的敲除；和/或c) low or no expression of endogenous HLA-II molecules including knockout of genes encoding HLA-II proteins; and/or
d)内源性NKG2A分子低表达或不表达包括编码NKG2A蛋白的基因的敲除。d) Low or no expression of endogenous NKG2A molecules including knockout of the gene encoding the NKG2A protein.
在一实例中，所述第一免疫细胞和第二免疫细胞包括：In an example, the first immune cell and the second immune cell include:
a)采用CRISPR/Cas9技术敲除内源性B2M；a) Using CRISPR/Cas9 technology to knock out endogenous B2M;
b)采用CRISPR/Cas9技术敲除内源性B2M/TCR；b) Using CRISPR/Cas9 technology to knock out endogenous B2M/TCR;
c)采用CRISPR/Cas9技术敲除内源性B2M/TCR/CIITA；c) Using CRISPR/Cas9 technology to knock out endogenous B2M/TCR/CIITA;
d)采用CRISPR/Cas9技术敲除内源性B2M/TCR/NKG2A；或d) Knocking out endogenous B2M/TCR/NKG2A using CRISPR/Cas9 technology; or
e)采用CRISPR/Cas9技术敲除内源性B2M/TCR/CIITA/NKG2A。e) CRISPR/Cas9 technology was used to knock out endogenous B2M/TCR/CIITA/NKG2A.
在一实例中，所述识别NKG2A的抗原结合域包括：In one example, the antigen-binding domain that recognizes NKG2A includes:
如SEQ ID NO：36、37、38、39、40、52中任一项所示的氨基酸序列。Amino acid sequence as shown in any one of SEQ ID NO:36,37,38,39,40,52.
在一实例中，所述识别肿瘤抗原的抗原结合域包括：In one example, the antigen-binding domain that recognizes tumor antigens includes:
SEQ ID NO：21、22、23、24、25所示的scFv。scFv shown in SEQ ID NO: 21, 22, 23, 24, 25.
在一实例中，所述第一CAR包括如SEQ ID NO:9、41、42、43、44、45、53中任一项所示的序列；和/或所述第二CAR包括：如SEQ ID NO:26、27或28中任一项所示的序列、或SEQ ID NO：21、22、23、24或25分别与EQ ID NO：49、50、51顺序连接而成的序列。In an example, the first CAR includes a sequence as shown in any one of SEQ ID NO: 9, 41, 42, 43, 44, 45, 53; and/or the second CAR includes: SEQ ID NO: The sequence shown in any one of ID NO: 26, 27 or 28, or the sequence formed by sequentially linking SEQ ID NO: 21, 22, 23, 24 or 25 with EQ ID NO: 49, 50, 51 respectively.
在一实例中，所述CRISPR/Cas9技术使用的gRNA包括如SEQ ID NO：10、46、47、48中任一项所示的序列或其组合。In an example, the gRNA used by the CRISPR/Cas9 technology includes a sequence as shown in any one of SEQ ID NO: 10, 46, 47, 48 or a combination thereof.
在一实例中，所述识别NKG2A的第一免疫细胞与所述识别非NKG2A的抗原的第二免疫细胞各自存在于不同的容器中。In one example, the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A are respectively present in different containers.
在一实例中，所述细胞组合物包含细胞混合物，所述细胞混合物包含所述识别NKG2A的第一免疫细胞与所述识别非NKG2A的抗原的第二免疫细胞。In one example, the cell composition comprises a cell mixture comprising the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A.
另一方面，本申请还提供了一种药物组合物，其包括有效量的本申请所述的细胞组合物和药学上可接受的佐剂。On the other hand, the present application also provides a pharmaceutical composition, which includes an effective amount of the cell composition described in the present application and a pharmaceutically acceptable adjuvant.
在一实例中，所述药物组合物包含第一制剂和第二制剂，所述第一制剂包含所述识别NKG2A的第一免疫细胞和药学上可接受的第一佐剂，所述第二制剂包含所述识别非NKG2A的抗原的第二免疫细胞和药学上可接受的第二佐剂。In one example, the pharmaceutical composition comprises a first preparation and a second preparation, the first preparation comprises the first immune cells recognizing NKG2A and a pharmaceutically acceptable first adjuvant, the second preparation comprising said second immune cells recognizing antigens other than NKG2A and a pharmaceutically acceptable second adjuvant.
在一实例中，所述药物组合物包含药物混合物，所述药物混合物包含所述识别NKG2A的第一免疫细胞与所述识别非NKG2A的抗原的第二免疫细胞。In one example, the pharmaceutical composition comprises a pharmaceutical mixture comprising the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A.
另一方面，本申请还提供了本申请所述的细胞组合物在制备药物中的用途，所述药物用于预防和/或***。On the other hand, the present application also provides the use of the cell composition described in the present application in the preparation of medicaments for preventing and/or treating tumors.
另一方面，本申请还提供了预防、缓解和/或***的方法，其包括向有需要的受试者施用本申请所述的细胞组合物、本申请所述的药物组合物。On the other hand, the present application also provides a method for preventing, alleviating and/or treating tumors, which comprises administering the cell composition and the pharmaceutical composition described in the present application to a subject in need.
另一方面，本申请还提供了本申请所述的细胞组合物、本申请所述的药物组合物，其用于预防、缓解和/或***。On the other hand, the present application also provides the cell composition described in the present application and the pharmaceutical composition described in the present application, which are used for preventing, alleviating and/or treating tumors.
在一实例中，所述肿瘤包括实体瘤和血液瘤。In one example, the tumors include solid tumors and hematological tumors.
在一实例中，所述血液瘤包括多发性骨髓瘤。In one example, the hematological tumor comprises multiple myeloma.
在一实例中，所述识别NKG2A的第一免疫细胞与所述识别非NKG2A的抗原的第二免疫细胞被配置为同时向受试者施用。In one example, the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A are configured to be administered to the subject at the same time.
在一实例中，所述识别NKG2A的第一免疫细胞与所述识别非NKG2A的抗原的第二免疫细胞被配置为分别向受试者施用。In one example, the first immune cell recognizing NKG2A and the second immune cell recognizing an antigen other than NKG2A are configured to be administered to a subject separately.
另一方面，本申请还提供了试剂盒，其包括本申请所述的细胞组合物或者本申请所述的药物组合物。On the other hand, the present application also provides a kit, which includes the cell composition or the pharmaceutical composition described in the present application.
在一实例中，所述试剂盒还包括用于治疗和/或预防肿瘤、病原体感染、自身免疫性疾病或同种异体移植的书面说明书。In one example, the kit further includes written instructions for treating and/or preventing tumors, pathogenic infections, autoimmune diseases or allogeneic transplantation.
另一方面，本申请还提供了增加识别NKG2A多肽的第一免疫细胞和识别非NKG2A的抗原的第二免疫细胞在有宿主免疫细胞存在时的存活时间和/或扩增能力的方法，包括：In another aspect, the present application also provides a method for increasing the survival time and/or expansion ability of the first immune cell that recognizes the NKG2A polypeptide and the second immune cell that recognizes the non-NKG2A antigen in the presence of host immune cells, including:
a)提供第一和第二免疫细胞；a) providing first and second immune cells;
b)任选地，通过编码参与响应自体和非自体抗原识别多肽的至少一种内源基因表达、活性和/或信号被降低或抑制来修饰所述第一和/或第二免疫细胞；b) optionally modifying said first and/or second immune cells by reducing or inhibiting the expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in the response to self and non-self antigen recognition;
c)编码识别NKG2A多肽的第一外源受体的多核苷酸来修饰所述第一免疫细胞；c) modifying said first immune cell with a polynucleotide encoding a first exogenous receptor that recognizes an NKG2A polypeptide;
d)编码识别非NKG2A的抗原的第二外源受体的多核苷酸来修饰所述第二免疫细胞。d) modifying said second immune cell with a polynucleotide encoding a second exogenous receptor that recognizes an antigen other than NKG2A.
在一实例中，所述第二外源受体靶向肿瘤抗原和/或病原体抗原。In one example, the second exogenous receptor targets tumor antigens and/or pathogen antigens.
在一实例中，所述第一外源受体和/或第二外源受体包括嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)或其组合。In one example, the first exogenous receptor and/or the second exogenous receptor comprises a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC) or a combination thereof .
在一实例中，所述第一外源受体是第一CAR、和/或所述第二外源受体是第二CAR；In one example, the first exogenous receptor is a first CAR, and/or the second exogenous receptor is a second CAR;
所述第一CAR包括：The first CAR includes:
a)识别NKG2A多肽的抗体、任选地识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，CD28的共刺激信号结构域和CD3ζ；和/或a) an antibody that recognizes a NKG2A polypeptide, optionally an antibody that recognizes a tumor and/or a pathogen antigen, CD28 or the transmembrane region of CD8, the co-stimulatory signal domain of CD28 and CD3ζ; and/or
b)识别NKG2A多肽的抗体、任选地识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，CD137的共刺激信号结构域和CD3ζ；和/或b) antibodies that recognize NKG2A polypeptides, optionally antibodies that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signal domain of CD137 and CD3ζ; and/or
c)识别NKG2A多肽的抗体、任选地识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，CD28的共刺激信号结构域，CD137的共刺激信号结构域和CD3ζ；c) antibodies that recognize NKG2A polypeptides, optionally antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signal domain of CD28, the costimulatory signal domain of CD137 and CD3ζ;
d)识别NKG2A多肽的抗体、任选地识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，和CD3ζ；d) antibodies that recognize NKG2A polypeptides, optionally antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, and CD3ζ;
所述第二CAR包括：The second CAR includes:
a)识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，CD28的共刺激信号结构域和CD3ζ；和/或a) antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD28 and CD3ζ; and/or
b)识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，CD137的共刺激信号结构域和CD3ζ；和/或b) antibodies that recognize tumor and/or pathogen antigens, the transmembrane region of CD28 or CD8, the co-stimulatory signaling domain of CD137 and CD3ζ; and/or
c)识别别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，CD28的共刺激信号结 构域，CD137的共刺激信号结构域和CD3ζ；c) antibodies that recognize other tumors and/or pathogen antigens, CD28 or the transmembrane region of CD8, the costimulatory signal domain of CD28, the costimulatory signal domain of CD137 and CD3ζ;
d)识别肿瘤和/或病原体抗原的抗体，CD28或CD8的跨膜区，和CD3ζ。d) Antibodies that recognize tumor and/or pathogen antigens, CD28 or the transmembrane region of CD8, and CD3ζ.
在一实例中，所述第一免疫细胞和/或第二免疫细胞，选自T细胞、NK细胞、细胞毒性T细胞、NKT细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫细胞或其组合。In one example, the first immune cell and/or the second immune cell are selected from T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune cells or combination.
在一实例中，所述第一免疫细胞和/或第二免疫细胞，为自体或同种异体T细胞、干细胞衍生的T细胞、原代T细胞或来源于人的自体T细胞。In one example, the first immune cell and/or the second immune cell are autologous or allogeneic T cells, stem cell-derived T cells, primary T cells or autologous T cells derived from humans.
在一实例中，所述步骤b)中的所述多肽选自HLA-I、TCR、HLA-II、NKG2A或其组合。In one example, the polypeptide in step b) is selected from HLA-I, TCR, HLA-II, NKG2A or a combination thereof.
在一实例中，所述步骤b)包括：In an example, said step b) includes:
a)采用CRISPR/Cas9技术敲除内源性B2M；a) Using CRISPR/Cas9 technology to knock out endogenous B2M;
b)采用CRISPR/Cas9技术敲除内源性B2M/TCR；b) Using CRISPR/Cas9 technology to knock out endogenous B2M/TCR;
c)采用CRISPR/Cas9技术敲除内源性B2M/TCR/CIITA；c) Using CRISPR/Cas9 technology to knock out endogenous B2M/TCR/CIITA;
d)采用CRISPR/Cas9技术敲除内源性B2M/TCR/NKG2A；或d) Knocking out endogenous B2M/TCR/NKG2A using CRISPR/Cas9 technology; or
e)采用CRISPR/Cas9技术敲除内源性B2M/TCR/CIITA/NKG2A。e) CRISPR/Cas9 technology was used to knock out endogenous B2M/TCR/CIITA/NKG2A.
在一实例中，所述肿瘤抗原包括WT1、HER2、EGFR、BCMA或其组合。In one example, the tumor antigen includes WT1, HER2, EGFR, BCMA or a combination thereof.
在一实例中，所述识别NKG2A多肽的抗体包括：In one example, the antibody recognizing NKG2A polypeptide includes:
SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2、SEQ ID NO:5所示的HCDR3、SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、和SEQ ID NO:8所示的LCDR3；或SEQ ID NO:1所述的重链可变区和/或SEQ ID NO:2所述的轻链可变区；或SEQ ID NO：52所示的scFv序列；或SEQ ID NO：36、37、38、39或40所示的串联抗体序列。HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, HCDR3 shown in SEQ ID NO:5, LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and the LCDR3 shown in SEQ ID NO:8; or the heavy chain variable region described in SEQ ID NO:1 and/or the light chain variable region described in SEQ ID NO:2; or shown in SEQ ID NO:52 or the tandem antibody sequence shown in SEQ ID NO: 36, 37, 38, 39 or 40.
在一实例中，所述识别肿瘤抗体包括：In an example, the tumor-recognizing antibody includes:
SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2、和SEQ ID NO:18所示的LCDR3；或HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO: 18; or
SEQ ID NO:19所示的重链可变区和/或SEQ ID NO:20所示的轻链可变区；或The heavy chain variable region set forth in SEQ ID NO:19 and/or the light chain variable region set forth in SEQ ID NO:20; or
SEQ ID NO：21、22、23、24、25、36、37、38、39或40所示的scFv。The scFv set forth in SEQ ID NO: 21, 22, 23, 24, 25, 36, 37, 38, 39 or 40.
在一实例中，所述第一CAR包括SEQ ID NO:9、40、41、42、43、44、45或53所示的序列；和/或所述第二CAR包括SEQ ID NO:26、27或28所示的序列、或SEQ ID NO：21、22、23、24或25分别与EQ ID NO：49、50或51顺序连接而成的序列。In one example, the first CAR includes the sequence shown in SEQ ID NO: 9, 40, 41, 42, 43, 44, 45 or 53; and/or the second CAR includes SEQ ID NO: 26, The sequence shown in 27 or 28, or the sequence formed by sequentially linking SEQ ID NO: 21, 22, 23, 24 or 25 with EQ ID NO: 49, 50 or 51 respectively.
在一实例中，所述步骤b)包括：In an example, said step b) includes:
所述CRISPR/Cas9技术使用的gRNA包括SEQ ID NO：10、46、47、48所示的序列或其组合。The gRNA used by the CRISPR/Cas9 technology includes sequences shown in SEQ ID NO: 10, 46, 47, 48 or combinations thereof.
在一实例中，所述方法用于治疗和/或预防肿瘤。In one example, the method is used to treat and/or prevent tumors.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1:显示了NKG2A-UCAR-T细胞能促进组合物中UCAR-T细胞的体外存活和/或扩增。Figure 1: shows that NKG2A-UCAR-T cells can promote the in vitro survival and/or expansion of UCAR-T cells in the composition.
图2A显示了NKG2A-UCAR-T细胞发挥体内协同BCMA-UCAR-T细胞抗肿瘤作用；图2B显示了识别NKG2A的UCAR-T细胞能显著增加体内BCMA UCAR-T细胞数量；图2C显示了NKG2A-UCAR-T细胞协同BCMA-UCAR-T细胞特异性向肿瘤组织浸润；图2D显示了NKG2A UCAR-T与BCMA UCAR-T联用在小鼠体内不会产生明显的毒副作用；图2E显示了NKG2A-UCAR-T细胞与BCMA-UCAR-T联用不引起移植物抗宿主反应。Figure 2A shows that NKG2A-UCAR-T cells exert synergistic anti-tumor effect of BCMA-UCAR-T cells in vivo; Figure 2B shows that UCAR-T cells that recognize NKG2A can significantly increase the number of BCMA UCAR-T cells in vivo; Figure 2C shows that NKG2A -UCAR-T cells cooperate with BCMA-UCAR-T cells to specifically infiltrate into tumor tissue; Figure 2D shows that the combination of NKG2A UCAR-T and BCMA UCAR-T does not produce obvious toxic side effects in mice; Figure 2E shows that NKG2A - The combination of UCAR-T cells and BCMA-UCAR-T does not cause graft-versus-host reaction.
图3.显示了NKG2A-UCAR-T、或BCMA-NKG2A UCAR-T均能发挥体内协同BCMA-UCAR-T细胞的抗肿瘤作用。Figure 3. It shows that NKG2A-UCAR-T or BCMA-NKG2A UCAR-T can exert the anti-tumor effect of synergistic BCMA-UCAR-T cells in vivo.
图4.显示了内源性TCR/B2M/CIITA/NKG2A敲除的NKG2A-UCAR-T细胞既能促进组合物中UCAR-T细胞的体外存活和/或扩增、又能发挥协同BCMA-UCAR-T细胞的抗肿瘤作用。Figure 4. Shows that NKG2A-UCAR-T cells knocked out of endogenous TCR/B2M/CIITA/NKG2A can not only promote the in vitro survival and/or expansion of UCAR-T cells in the composition, but also exert synergistic BCMA-UCAR - Antitumor effect of T cells.
图5.显示了内源性TCR/B2M/CIITA/NKG2A敲除的NKG2A-UCAR-T细胞发挥体内协同BCMA-UCAR-T细胞的抗肿瘤作用。Figure 5. Shows that endogenous TCR/B2M/CIITA/NKG2A knockout NKG2A-UCAR-T cells exert anti-tumor effects in vivo synergistically with BCMA-UCAR-T cells.
具体实施方式detailed description
本发明是关于一种细胞组合物和其制备方法，及所述细胞组合物的用途。本发明的细胞组合物包括识别NKG2A的第一工程细胞和识别肿瘤和/或病原体的第二工程细胞(示例性，CAR-T细胞或UCAR-T细胞)，在有宿主免疫细胞(示例性，原代NK细胞)存在时具有更长存活时间和/或更强的扩增能力。与第一工程细胞或第二工程细胞相比，本发明的细胞组合物在体内抗肿瘤活性更强。组合物中识别NKG2A的第一工程细胞作为抵抗宿主免疫排斥(如NK细胞攻击)的通用工具细胞，可以和组合物中识别一种或多种肿瘤抗原和/或病原体抗原的一种或多种工程细胞进行联用适用性更广。The invention relates to a cell composition, a preparation method thereof, and an application of the cell composition. The cell composition of the present invention includes a first engineered cell that recognizes NKG2A and a second engineered cell (exemplary, CAR-T cells or UCAR-T cells) that recognizes tumors and/or pathogens, in the presence of host immune cells (exemplary, Primary NK cells) have a longer survival time and/or greater expansion capacity in the presence. Compared with the first engineered cells or the second engineered cells, the cell composition of the present invention has stronger anti-tumor activity in vivo. The first engineered cell that recognizes NKG2A in the composition is used as a general tool cell for resisting host immune rejection (such as NK cell attack), and can recognize one or more tumor antigens and/or pathogen antigens in the composition Engineered cells have wider applicability.
除非专门定义，否则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那些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通过 引用并入本文。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，否则本发明的材料、方法和实施例仅是说明性的，而并非旨在进行限制。根据本发明内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等价的方法和组分在本发明的范围内。本发明包括对本发明的主题进行变型和修改来用于各种用途和条件。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, in which case suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples of the present invention are illustrative only and not intended to be limiting unless otherwise specified. Those of skill in the art should, in light of the teachings of the present invention, appreciate that many changes and modifications can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The invention is not to be limited in scope by the specific embodiments described herein, which are intended only as illustrations of various aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. The invention includes variations and modifications of the inventive subject matter for various usages and conditions.
除非另有说明，否则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)。The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrooke et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis(M.J.Gaited., 1984); Mullis et al.U.S.Pat.No.4,683,195; Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984);Transcription And Translation(B.D.Hames&Sed.J 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 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 Expert Immental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986) and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
在本申请中，范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本申请的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。In this application, the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the application. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range.
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。在本文中，“约”可以是在所述技术领域内可以接受的误差范围；。例如，可以是指“约”值或参数的±10％范围内的值或参数，例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。As used herein, the term "about" refers to the usual error range for each value readily known to those skilled in the art. Reference herein to "about" a value or parameter includes embodiments referring to the value or parameter itself. For example, description of "about X" includes description of "X." Herein, "about" may be an acceptable error range in the technical field; For example, "about" a value or parameter within ±10% of a value or parameter can be meant, eg, about 5 uM can include any number between 4.5 uM and 5.5 uM.
术语the term
术语“受体”是一类存在于胞膜或胞内的，能与目标分子结合进而激活细胞内一系列生物化学反应，使细胞对外界刺激产生相应的效应的特殊蛋白质或多肽。与受体结合的目标分子(也称为生物活性物质)统称为配体(ligand)，或称为靶抗原。The term "receptor" is a kind of special protein or polypeptide that exists in the cell membrane or in the cell, can bind to the target molecule and activate a series of biochemical reactions in the cell, and make the cell respond to external stimuli. Target molecules (also referred to as biologically active substances) that bind to receptors are collectively referred to as ligands, or target antigens.
术语“外源”，是指核酸分子或多肽不是内源性存在细胞中的，或表达水平不足以实现过表达时具有的功能；包括在细胞中表达的任何重组核酸分子或多肽，例如外源、异源和过表达的核酸分子和多肽。The term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or whose expression level is insufficient to achieve the function when overexpressed; includes any recombinant nucleic acid molecule or polypeptide expressed in the cell, such as exogenous , heterologous and overexpressed nucleic acid molecules and polypeptides.
在本申请中，术语“外源受体”，通常是指非表达该受体的细胞自身所天然具有的、而是通过基因重组技术将不同来源的DNA片段或蛋白质对应的cDNA连接、表达的融合多肽分子。可以包括胞外域、跨膜域和胞内域。外源受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)。In this application, the term "exogenous receptor" usually refers to a cell that does not express the receptor itself, but is connected and expressed by DNA fragments or cDNAs corresponding to proteins from different sources through genetic recombination technology. Fusion polypeptide molecules. Extracellular domains, transmembrane domains and intracellular domains may be included. Exogenous receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor (TCR), T Cell Antigen Coupler (TAC).
术语“嵌合抗原受体”或“CAR”是指可以由包括但不限于T细胞的免疫细胞表达的工程化分子。CAR在T细胞中表达并且可以重定向T细胞以诱导以由嵌合受体决定的特异性杀死靶细胞。CAR包括抗原结合域、跨膜结构域和胞内信号结构域。胞内信号结构域包括一级信号结构域和/或共刺激信号结构域。CAR的细胞外结合结构域可以衍生自鼠、人源化或完全人单克隆抗体。术语CAR不具体地限于CAR分子，而且还包括CAR变体。CAR变体包括拆分CAR，其中CRA的细胞外部分(例如，配体结合部分)和细胞内部分(例如，细胞内信号传导部分)存在于两个独立的分子上。CAR变体还包括启动开关CAR(ON-switch CAR)，其为可条件性激活的CAR，例如，包括拆分CAR，其中通过药物控制拆分CAR的两个部分的条件性异源二聚化。CAR变体还包括双特异性CAR，其包括可放大或抑制主要CAR的活性的次要CAR结合结构域。CAR变体还包括抑制性嵌合抗原受体(iCAR)，其可以例如用作双特异性CAR***的组分，其中次要CAR结合结构域的结合导致主要CAR激活的抑制。The term "chimeric antigen receptor" or "CAR" refers to an engineered molecule that can be expressed by immune cells, including but not limited to T cells. CARs are expressed in T cells and can redirect T cells to induce killing of target cells with a specificity dictated by the chimeric receptor. CAR includes an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Intracellular signaling domains include primary signaling domains and/or co-stimulatory signaling domains. The extracellular binding domains of CARs can be derived from murine, humanized or fully human monoclonal antibodies. The term CAR is not specifically limited to CAR molecules, but also includes CAR variants. CAR variants include split CARs in which the extracellular portion (eg, the ligand-binding portion) and the intracellular portion (eg, the intracellular signaling portion) of the CRA are present on two separate molecules. CAR variants also include ON-switch CARs, which are conditionally activatable CARs, including, for example, split CARs in which conditional heterodimerization of the two parts of the split CAR is controlled by a drug . CAR variants also include bispecific CARs that include a secondary CAR-binding domain that amplifies or inhibits the activity of the primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs), which can, for example, be used as components of bispecific CAR systems, where binding of the secondary CAR binding domain results in inhibition of primary CAR activation.
在本申请中，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。In this application, the term "T cell antigen coupler (TAC)" includes three functional domains: 1. Antigen binding domain, including single chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein) protein, DARPin) or other targeting groups; 2. The extracellular region domain, the single-chain antibody that binds to CD3, so that the TAC receptor and the TCR receptor are close; 3. The cell of the transmembrane region and the CD4 co-receptor The inner domain, where the intracellular domain is linked to the protein kinase LCK, catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
在本申请中，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，通常共定位在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；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、CD3γ、CD3z、TCR的α链、或TCR的β链，并且，该嵌合T细胞受体整合进T细胞上表达的TCR/CD3复合物。In this application, the term "chimeric T cell receptor" includes recombinant polypeptides derived from various polypeptides that make up the TCR, which are capable of binding to surface antigens on target cells, and interacting with other polypeptides of the complete TCR complex , usually co-localized on the surface of T cells. A chimeric T cell receptor consists of a TCR subunit and an antigen-binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, TCR intracellular domain; the TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε, CD3γ, CD3z, the α chain of TCR, or the β chain of TCR , and, the chimeric T cell receptor is integrated into the TCR/CD3 complex expressed on T cells.
在本申请中，术语“各自独立地”通常是指涉及的主体之间没有关联性，其中一个主体的结构、类型和/或数量不影响其他主体的结构、类型和/或数量。例如在本申请中，“主体”可以是第一外源受体和第二外源受体，其可以是相同的，也可以是不同的；例如，“主体”可以是第一CAR和第二CAR中的各功能域(例如，铰链域，跨膜域，胞内信号传导域或共刺激信号结构域)，其中的各功能域可以是相同的也可以是不同的。In this application, the term "independently" generally means that there is no relationship between the involved subjects, wherein the structure, type and/or quantity of one subject does not affect the structure, type and/or quantity of other subjects. For example, in this application, the "subject" can be the first exogenous receptor and the second exogenous receptor, which can be the same or different; for example, the "subject" can be the first CAR and the second CAR. Each functional domain in CAR (for example, hinge domain, transmembrane domain, intracellular signaling domain or co-stimulatory signal domain), wherein each functional domain may be the same or different.
术语“工程化”是指应用细胞生物学和分子生物学的原理和方法，通过某种工程学手段，在细胞整体水平或细胞器水平上，改变细胞内的遗传物质或获得细胞产品。工程细胞还可以指含有加入、缺失和/或改变的基因的细胞。The term "engineering" refers to applying the principles and methods of cell biology and molecular biology to change the genetic material in the cell or obtain cell products through some engineering means at the level of the whole cell or the organelle level. Engineered cells can also refer to cells that contain added, deleted and/or altered genes.
术语“细胞”或“工程细胞”可以指人或非人动物来源的细胞。The term "cell" or "engineered cell" may refer to a cell of human or non-human animal origin.
术语“个体”和“受试者”可互换，包括人或来自其他种属的动物，其包括但不限于人、小鼠、大鼠、仓鼠和豚鼠、兔子、狗、猫、绵羊、猪、山羊、牛、马、猿、猴子。The terms "individual" and "subject" are interchangeable and include humans or animals from other species including, but not limited to, humans, mice, rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs , goat, cow, horse, ape, monkey.
在本申请中，术语“抗原结合域”是指特异性结合抗原决定簇的分子，包括免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗体”不仅包括完整的抗体分子，也包括保留抗原结合能力的抗体分子的片段。本申请中术语“抗体”与术语“免疫球蛋白”“抗原结合域”可互换使用。抗体，包括但不限于单克隆抗体、多克隆抗体、天然抗体、双特异性抗体、嵌合抗体、Fv、Fab、Fab’、Fab’-SH、F(ab’)2、线性抗体、单链抗体(例如scFv)、单域抗体。在一实例中，抗体包括通过二硫键连接的至少两个重(H)链和两个轻(L)链。每条重链由重链可变区(VH)和重链恒定区(CH)组成。CH有三个结构域CH1、CH2、CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区(CL)。CL由一个结构域组成。VH和VL可进一步细分为高变区，称为互补决定区(CDR)，其间散布有更保守的区域，称为框架区(FR)。每个VH和VL均由三个CDR和四个FR组成，从氨基端到羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区包括与抗原相互作用的结合结构域。如果抗原结合域以与其它参考抗原(包括多肽或其他物质)结合相比更大亲和力(或称亲合力)结合抗原，则所述抗原结合域与抗原“特异性结合”或与抗原是“免疫反应性的”。In this application, the term "antigen-binding domain" refers to a molecule that specifically binds an antigenic determinant, including immunoglobulin molecules and immunologically active portions of immunological molecules, i.e., containing the antigen to which it specifically binds ("immunoreacts") Molecules at the binding site. The term "antibody" includes not only intact antibody molecules but also fragments of antibody molecules that retain antigen-binding ability. The term "antibody" is used interchangeably with the term "immunoglobulin" and "antigen binding domain" in this application. Antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, natural antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single chain Antibodies (eg scFv), single domain antibodies. In one example, an antibody comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH). CH consists of three structural domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). CL consists of one domain. VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains include binding domains that interact with antigen. An antigen-binding domain "specifically binds" or is "immunogenic" to an antigen if the antigen-binding domain binds the antigen with greater affinity (or avidity) than other reference antigens (including polypeptides or other substances). Reactive".
术语“激活免疫细胞”，是指信号转导通路引起的细胞内蛋白质表达的变化，导致免疫应答的启动。在一实例中，CAR与抗原结合后形成的免疫突触，包括在结合受体 (例如，CD4或CD8，CD3γ/CDδ/CDε/CDζ等)附近的许多分子的聚集。膜结合信号分子的这种聚集使CD3分子中包括的ITAM基序磷酸化。该磷酸化进而启动T细胞激活通路，最终激活转录因子，例如NF-κB和AP-1。这些转录因子诱导T细胞的整体基因表达，包括上调IL-2生成，促进T细胞增殖，进而启动T细胞介导的免疫应答。“T细胞活化”或“T细胞激活”指被刺激后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。The term "activation of immune cells" refers to changes in intracellular protein expression caused by signal transduction pathways, resulting in the initiation of an immune response. In one example, the immune synapse formed after CAR binds to an antigen includes the aggregation of many molecules near the binding receptor (e.g., CD4 or CD8, CD3γ/CDδ/CDε/CDζ, etc.). This aggregation of membrane-bound signaling molecules phosphorylates the ITAM motif included in the CD3 molecule. This phosphorylation in turn initiates T cell activation pathways, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce the overall gene expression of T cells, including the upregulation of IL-2 production, the promotion of T cell proliferation, and the initiation of T cell-mediated immune responses. "T cell activation" or "T cell activation" refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
在本申请中，术语“细胞组合物”通常是指至少包含两类细胞的组合形式，其中一类至少能够识别NKG2A(例如，仅识别NKG2A；例如，同时识别NKG2A和疾病相关抗原)，另一类识别疾病相关抗原(例如，肿瘤抗原)。在一些实施方式中，每一类细胞可以存在于不同的容器中，还可以在需要的时候同时或分别与合适的佐剂配制成期望的制剂；在一些实施方式中，每一类细胞可以是不同来源的(例如不同的厂商制备、生产或销售的；例如分别是分离自供体的天然存在的T细胞和衍生自干细胞的T细胞)；在一些实施方式中，每一类细胞可以各自制备成独立的制剂(固体、液体、凝胶体等)；在一些实施方式中，每一类细胞可以以混合的形式存在。In this application, the term "cell composition" generally refers to a combined form comprising at least two types of cells, one of which recognizes NKG2A at least (e.g., only recognizes NKG2A; e.g., simultaneously recognizes NKG2A and a disease-associated antigen), and the other Classes recognize disease-associated antigens (eg, tumor antigens). In some embodiments, each type of cell can be present in a different container, and can also be formulated into a desired preparation with a suitable adjuvant when necessary; in some embodiments, each type of cell can be of different origin (e.g., prepared, produced, or sold by different manufacturers; e.g., naturally occurring T cells isolated from a donor and T cells derived from stem cells, respectively); in some embodiments, each type of cell can be prepared separately into separate formulations (solid, liquid, gel, etc.); in some embodiments, each type of cell may be present in admixed form.
在本申请中，术语“疾病”是指损害或干扰细胞、组织或器官的正常功能的任何病症，例如肿瘤(癌症)或病原体感染。在一实例中，所述疾病包括实体肿瘤、血液肿瘤、自身免疫性疾病或其组合。In this application, the term "disease" refers to any condition that damages or interferes with the normal function of a cell, tissue or organ, such as a tumor (cancer) or a pathogenic infection. In one example, the disease includes solid tumors, hematological tumors, autoimmune diseases, or combinations thereof.
在本申请中，术语“NKG2A”是NKG2转录物组的成员，NKG2A与CD94形成的异源二聚体抑制性受体CD94/NKG2A，表达于NK细胞、αβT细胞、γδT细胞和NKT细胞的亚群的表面上。“NKG2A”可以是NKG2A基因或编码的蛋白的任何变体、衍生物或同种型。NKG2A多肽具有与由NCBI GenBank Gene ID：3821的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。In this application, the term "NKG2A" is a member of the NKG2 transcriptome, a heterodimeric inhibitory receptor CD94/NKG2A formed by NKG2A and CD94, expressed on subtypes of NK cells, αβT cells, γδT cells, and NKT cells. on the surface of the group. "NKG2A" may be any variant, derivative or isoform of the NKG2A gene or encoded protein. The NKG2A polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97% of the amino acid sequence encoded by the transcript expressed by the gene of NCBI GenBank Gene ID: 3821 %, at least about 98%, at least about 99%, or at least about 100% homology or identity of amino acid sequences or fragments thereof, and/or may optionally include up to one or up to two or up to three conservative amino acid substitutions .
在本申请中，术语“BCMA抗原”或“BCMA”通常是指B细胞成熟抗原(B-cell maturation antigen)，属TNF受体超家族。BCMA与其配体结合后，可激活B细胞的增殖和存活。BCMA特异地高表达于浆细胞和多发性骨髓瘤细胞，而在造血干细胞和其他正常组织细胞中均不表达。“BCMA”可以是BCMA基因或编码的蛋白的任何变体、衍生物或同种型。In this application, the term "BCMA antigen" or "BCMA" generally refers to B-cell maturation antigen, which belongs to the TNF receptor superfamily. After BCMA binds to its ligand, it can activate the proliferation and survival of B cells. BCMA is specifically highly expressed in plasma cells and multiple myeloma cells, but not expressed in hematopoietic stem cells and other normal tissue cells. "BCMA" may be any variant, derivative or isoform of the BCMA gene or encoded protein.
在本申请中，术语“识别”是指选择性结合靶抗原。本发明中表达外源受体的工程细胞能识别表达所述外源受体特异性结合的抗原的细胞。In this application, the term "recognize" refers to selective binding of a target antigen. The engineered cells expressing exogenous receptors in the present invention can recognize cells expressing antigens specifically bound by the exogenous receptors.
术语“特异性结合”是指识别并且结合存在于样品中的结合配偶体(例如肿瘤抗原)蛋 白质的抗体或配体，但是该抗体或配体基本上不会识别或结合样品中的其它分子。The term "specifically binds" refers to an antibody or ligand that recognizes and binds a binding partner (e.g., tumor antigen) protein present in a sample, but that does not substantially recognize or bind other molecules in the sample.
在本申请中，术语“免疫细胞”通常是指参与免疫应答、行使效应功能的细胞。例如所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等；例如可以是淋巴谱系的细胞。例如可以是T细胞、NK细胞。In this application, the term "immune cell" generally refers to a cell that participates in an immune response and performs effector functions. For example, the exercising effector functions may include clearing foreign antigens or promoting immune effector responses, etc.; for example, it may be cells of lymphoid lineage. Examples include T cells and NK cells.
在本申请中，术语“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。例如，过度增生性病症是指癌症/肿瘤。例如，可以是实体瘤抗原，例如，也可以是血液瘤抗原。In the present application, the term "tumor antigen" refers to an antigen that appears newly or is overexpressed during the onset, progression of a hyperproliferative disease. For example, a hyperproliferative disorder refers to cancer/tumor. For example, it can be a solid tumor antigen, for example, it can also be a blood tumor antigen.
在本申请中，术语“肽”、“多肽”和“蛋白质”可互换使用，是指由通过肽键共价连接的氨基酸残基组成的化合物。In this application, the terms "peptide", "polypeptide" and "protein" are used interchangeably to refer to a compound consisting of amino acid residues covalently linked by peptide bonds.
术语“核酸”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物，包括编码目的多肽或其片段的任何核酸分子。所述核酸分子只需要与内源性核酸序列保持基本同一性即可，不需要与内源性核酸序列100％同源性或同一性。术语“基本同一性”或“基本同源性”，是指与参考氨基酸序列或核酸序列表现出至少约50％同源性或同一性的多肽或核酸分子。在一实例中，这样的序列与用于比较的氨基酸或核酸序列为至少约60％、65％、70％、75％、80％、85％、90％、95％、99％或100％同源性或同一性。序列同一性可以通过使用序列分析软件(例如，BLAST、BESTFIT、GAP或PILEUP/PRETTYBOX程序)进行测量。The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, including any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof. The nucleic acid molecule only needs to maintain basic identity with the endogenous nucleic acid sequence, and does not need to have 100% homology or identity with the endogenous nucleic acid sequence. The term "substantial identity" or "substantial homology" refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence or nucleic acid sequence. In one example, such a sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid or nucleic acid sequence used for comparison. origin or identity. Sequence identity can be measured by using sequence analysis software (eg, the BLAST, BESTFIT, GAP or PILEUP/PRETTYBOX programs).
在本申请中，术语“跨膜域”通常是指蛋白质序列中跨越细胞膜的区域。在某些实施方式中，蛋白质序列中跨越细胞膜的区域通常为α-螺旋结构，包含大部分为疏水性的氨基酸。在某些实施方式中，所述跨膜域可获取自天然蛋白质(例如来自于CD8的跨膜域或其功能性衍生序列)，或者，跨膜域可以是合成的非天然存在的蛋白质区段，例如在细胞膜中热力学稳定的疏水性蛋白质区段。In this application, the term "transmembrane domain" generally refers to a region of a protein sequence that spans a cell membrane. In certain embodiments, the membrane-spanning region of the protein sequence is generally alpha-helical, comprising mostly hydrophobic amino acids. In certain embodiments, the transmembrane domain may be obtained from a native protein (such as from CD8 or a functionally derived sequence thereof), or the transmembrane domain may be a synthetic non-naturally occurring protein segment , such as hydrophobic protein segments that are thermodynamically stable in cell membranes.
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激信号的共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。In this application, the term "co-stimulatory domain" generally refers to the intracellular domain of a co-stimulatory molecule that can provide an immune co-stimulatory signal, and the co-stimulatory molecule is a cell surface molecule required for an effective response of lymphocytes to an antigen . The costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.
在本申请中，术语“胞内信号传导域”也称作“初级信号传导结构域”，通常是指含有称作基于免疫受体酪氨酸的活化基序或ITAM的信号转导序列。例如，衍生自CD3ζ、FcRγ(FCER1G)、FcγRIIa、FcRβ(FcεR1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12的初级信号传导结构域。在某些实施方式中，胞内信号传导域转导效应功能信号并指导细胞进行特化功能。虽然可以使用整个胞内信号传导域，但在许多情况下，不必使用整个链。就使用胞内信号传导域的截短部分而言，此类截短部分可 用于代替完整链，只要其能够转导效应子功能信号即可。因此，初胞内信号传导域意在包括足以转导效应子功能信号的胞内信号传导结构域的任一截短部分。胞内信号传导域。In the present application, the term "intracellular signaling domain", also referred to as "primary signaling domain", generally refers to a signal transduction sequence containing a so-called immunoreceptor tyrosine-based activation motif or ITAM. For example, primary signaling domains derived from CD3ζ, FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In certain embodiments, the intracellular signaling domain transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signaling domain can be used, in many cases it is not necessary to use the entire chain. To the extent that truncated portions of intracellular signaling domains are used, such truncated portions can be used in place of the intact chain, as long as they are capable of transducing effector function signals. Thus, a primary intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domain.
在本申请中，术语“铰链域”通常是指在蛋白质的两个域之间的氨基酸区段，其能够允许蛋白质的柔性和/或一个或两个以上的域相对于彼此的运动。例如来源于IgG家族(例如IgG1和IgG4)、IgD以及其他蛋白分子的铰链域，例如来自于CD8、CD28、HLA家族的铰链域。In this application, the term "hinge domain" generally refers to a stretch of amino acids between two domains of a protein that is capable of allowing flexibility of the protein and/or movement of one or more domains relative to each other. For example, hinge domains from IgG family (such as IgG1 and IgG4), IgD and other protein molecules, such as hinge domains from CD8, CD28, HLA family.
在本申请中，术语“宿主抗移植物反应(HVGR)”通常是指：由于供体和受体(或称为宿主)之间的免疫遗传学差异，在进行外源供体移植时，作为外源移植物的供体会受到宿主体内的免疫细胞(例如NK细胞)识别和攻击，进而抑制或者清除供体。In this application, the term "host-versus-graft reaction (HVGR)" generally refers to: due to immunogenetic differences between the donor and the recipient (or called the host), during exogenous donor transplantation, as The donor of the exogenous graft will be recognized and attacked by immune cells (such as NK cells) in the host, thereby inhibiting or eliminating the donor.
在本申请中，术语“移植物抗宿主病(GVHD)”通常是指：由于外源移植供体T淋巴细胞的TCR的多样性，以及与宿主HLA分子的不兼容性，供体T淋巴细胞会识别宿主正常组织上的抗原，经扩增并释放一系列细胞因子，攻击宿主细胞。In this application, the term "graft-versus-host disease (GVHD)" generally refers to: due to the diversity of the TCR of the exogenously transplanted donor T lymphocytes and the incompatibility with the host HLA molecules, the donor T lymphocytes It will recognize the antigens on the normal tissues of the host, amplify and release a series of cytokines to attack the host cells.
在本申请中，术语“内源”，是指核酸分子或多肽等来自生物体自身。In this application, the term "endogenous" means that the nucleic acid molecule or polypeptide comes from the organism itself.
在本申请中，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，与人类的免疫***功能密切相关。HLA包括有I类、II类和III类基因部分。HLA I类是一个异二聚体，由重链(α链)与轻链β2微球蛋白(B2M)组成。HLA-II类基因包括HLA-D家族，主要有HLA-DP、HLA-DQ和HLA-DR等，主要分布于B淋巴细胞、巨噬细胞和树突状细胞等专职抗原提呈细胞表面。In this application, the term "human leukocyte antigen" (Human leukocyte antigen, HLA) is the gene encoding human major histocompatibility complex, which is closely related to the function of the human immune system. HLA includes class I, class II and class III gene portions. HLA class I is a heterodimer consisting of a heavy chain (α chain) and a light chain β2 microglobulin (B2M). HLA-II genes include the HLA-D family, mainly including HLA-DP, HLA-DQ and HLA-DR, etc., and are mainly distributed on the surface of professional antigen-presenting cells such as B lymphocytes, macrophages and dendritic cells.
在本申请中，术语“TCR”通常是指T细胞受体(T cell receptor)，其介导T细胞对特异性主要组织相容性复合物(MHC)-肽抗原进行识别。TCR通常由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。In this application, the term "TCR" generally refers to the T cell receptor, which mediates the recognition of specific major histocompatibility complex (MHC)-peptide antigens by T cells. TCR is usually composed of two peptide chains α and β, and each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region, etc., and its antigen specificity exists in Zone V.
在本申请中，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***排斥(Bix et al.1991)。In this application, the term "B2M" refers to beta-2 microglobulin, also known as B2M, the light chain of an MHC class I molecule. In humans, B2M is encoded by the b2m gene located on chromosome 15, opposite other MHC genes located as a gene cluster on chromosome 6. Studies have shown that when the B2M gene is mutated, hematopoietic grafts from mice lacking normal cell surface MHC I expression are rejected by NK cells in normal mice, indicating that defective expression of MHC I molecules makes cells vulnerable to host immune system Repulsion (Bix et al. 1991).
在本申请中，术语“CIITA”通常是指Ⅱ类主要组织相容性复合体(MHCⅡ)的反式激活因子。所述反式激活因子可以是具有酸性转录激活结构域、4个LRR(富含亮氨酸的重复序列)和GTP结合结构域的蛋白质。所述蛋白质可位于细胞核中，作为II类主要组织相容性复合体(MHCⅡ)基因转录的正向调节剂。在人类中，所述蛋白质由位于 16p13.13的基因(例如HGNC:7067所示的信息)编码，能够产生几种编码不同同工型的转录物变体。In this application, the term "CIITA" generally refers to the transactivator of major histocompatibility complex class II (MHC II). The transactivator may be a protein having an acidic transcription activation domain, 4 LRRs (leucine rich repeats) and a GTP binding domain. The protein may be localized in the nucleus and acts as a positive regulator of transcription of major histocompatibility complex class II (MHC II) genes. In humans, the protein is encoded by a gene located at 16p13.13 (information such as that shown in HGNC:7067), enabling the generation of several transcript variants encoding different isoforms.
在本申请中，术语“制剂”，也可称作药物制剂，通常是指为适应治疗或预防的需要，按照一定的剂型要求所制成的可以提供给受试者使用的药物。例如，制剂可以包含活性成分、佐剂。例如活性成分可以是一种或以上具有治疗作用的免疫细胞。In this application, the term "preparation", which can also be referred to as pharmaceutical preparation, usually refers to a drug prepared according to a certain dosage form to meet the needs of treatment or prevention, and can be provided to the subject. For example, a formulation may contain active ingredients, adjuvants. For example, the active ingredient can be one or more immune cells with therapeutic effects.
在本申请中，术语“佐剂”通常可以是一种药物制剂中活性成分之外的其他任何物质。例如药学上可接受的涉及携带、储存或转运细胞的化合物、组合物或媒介物。例如缓冲液、稳定剂、防腐剂、用于增强生物利用度的吸收促进剂、液体或固体填充剂、稀释剂、赋形剂、溶剂、包囊材料和/或其他常规的保护剂或分散剂等。各佐剂在与该制剂的其它成分在可相容的意义上是“可接受的”且不会对患者产生损害。In this application, the term "adjuvant" can generally refer to any substance other than the active ingredient in a pharmaceutical preparation. For example a pharmaceutically acceptable compound, composition or vehicle involved in carrying, storing or transporting cells. Such as buffers, stabilizers, preservatives, absorption enhancers for enhanced bioavailability, liquid or solid fillers, diluents, excipients, solvents, encapsulating materials and/or other conventional protective or dispersing agents Wait. Each adjuvant is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not detrimental to the patient.
在本申请中，术语“肿瘤”或“癌症“通常指由异常细胞生长/过度增生形成的赘生物或实体病变。在本申请中，肿瘤可以是实体瘤或血液瘤。在某些实施方式中，可通过临床检查如x线摄片、CT扫描，B超、或触诊扪及到的有形肿块可称为实体瘤，X线、CT扫描，B超及触诊无法看到或扪及到的肿瘤例如白血病可称为血液瘤。In this application, the term "tumor" or "cancer" generally refers to a neoplasm or solid lesion formed by abnormal cell growth/hyperproliferation. In this application, tumors may be solid tumors or hematological tumors. In some embodiments, a tangible mass that can be palpable through clinical examinations such as X-rays, CT scans, B-ultrasound, or palpation can be called a solid tumor, and X-rays, CT scans, B-ultrasound and palpation cannot Tumors such as leukemia that are seen or felt are called hematomas.
在本申请中，术语“容器”通常是指任何适用于盛装药物的器皿或装置。例如，药盒、药瓶、药袋、泡罩、管、注射器等。In this application, the term "container" generally refers to any vessel or device suitable for containing a drug. For example, kits, vials, pouches, blisters, tubes, syringes, etc.
术语“治疗有效量”、“治疗有效的”、“有效量”或“以有效的量”在本文中可互换地使用，是指如本文中所述有效地实现特定生物学结果的化合物、制剂、物质或组合物、药物组合物的量，例如但不限于足以促进T细胞应答的量或剂量。有效量的免疫细胞，包括指但不限于：能使抗肿瘤活性增加、增强或延长的免疫细胞的数量；抗肿瘤免疫细胞数目或活化免疫细胞数目的增加；促进IFN-γ分泌、肿瘤消退、肿瘤缩小、肿瘤坏死的免疫细胞的数量。The terms "therapeutically effective amount", "therapeutically effective", "effective amount" or "in an effective amount" are used interchangeably herein to refer to a compound effective to achieve a particular biological result as described herein, An amount of an agent, substance or composition, pharmaceutical composition, such as but not limited to an amount or dosage sufficient to promote a T cell response. An effective amount of immune cells, including but not limited to: the number of immune cells that can increase, enhance or prolong anti-tumor activity; increase the number of anti-tumor immune cells or the number of activated immune cells; promote IFN-γ secretion, tumor regression, Tumor shrinkage, number of immune cells in tumor necrosis.
发明详述Detailed description of the invention
外源受体、嵌合抗原受体(CAR)Foreign receptors, chimeric antigen receptors (CAR)
本申请提供了细胞组合物，其包括：a)识别NKG2A的第一工程细胞；以及b)识别非NKG2A抗原的第二工程细胞。例如，所述第一工程细胞可以表达识别NKG2A的第一外源受体；例如，所述第二工程细胞可以表达识别非NKG2A抗原的第二外源受体。例如，所述非NKG2A抗原包括肿瘤抗原和/或病原体抗原。例如，所述第一外源受体还可以识别肿瘤抗原和/或病原体抗原。例如，所述第一外源受体识别的肿瘤抗原和/或病原体抗原和第二外源受体识别的肿瘤抗原和/或病原体抗原可以是相同的。The present application provides a cell composition, which includes: a) a first engineered cell that recognizes NKG2A; and b) a second engineered cell that recognizes a non-NKG2A antigen. For example, the first engineered cell can express a first exogenous receptor that recognizes NKG2A; for example, the second engineered cell can express a second exogenous receptor that recognizes an antigen other than NKG2A. For example, the non-NKG2A antigens include tumor antigens and/or pathogen antigens. For example, the first exogenous receptor can also recognize tumor antigens and/or pathogen antigens. For example, the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may be the same.
例如，第一外源受体、第二外源受体可以各自独立地选自：嵌合抗原受体(CAR)、 嵌合T细胞受体、T细胞抗原耦合器(TAC)、T细胞融合蛋白(TFP)。例如，所述第一外源受体和第二外源受体可以是相同的；例如，所述第一外源受体和第二外源受体可以均为嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)或T细胞融合蛋白(TFP)；例如，所述第一外源受体和第二外源受体可以是不同的；例如，所述第一外源受体可以是嵌合T细胞受体，第二外源受体可以是CAR；例如，所述第一外源受体可以是CAR，第二外源受体可以是嵌合T细胞受体；例如，所述第一外源受体可以是TAC，所述第二外源受体可以是CAR；例如，所述第一外源受体可以是TFP，所述第二外源受体可以是CAR；例如，所述第一外源受体可以是TFP，所述第二外源受体可以是嵌合T细胞受体；例如，所述第一外源受体可以是嵌合T细胞受体，所述第二外源受体可以是TAC。例如，所述第一外源受体和所述第二外源受体可以是CAR。For example, the first exogenous receptor and the second exogenous receptor can be independently selected from: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), T cell fusion protein (TFP). For example, the first exogenous receptor and the second exogenous receptor can be the same; for example, the first exogenous receptor and the second exogenous receptor can both be chimeric antigen receptors (CAR) , a chimeric T cell receptor (TCR), a T cell antigen coupler (TAC), or a T cell fusion protein (TFP); for example, the first exogenous receptor and the second exogenous receptor can be different; For example, the first exogenous receptor can be a chimeric T cell receptor, and the second exogenous receptor can be a CAR; for example, the first exogenous receptor can be a CAR, and the second exogenous receptor can be is a chimeric T cell receptor; for example, the first exogenous receptor can be TAC, and the second exogenous receptor can be CAR; for example, the first exogenous receptor can be TFP, the The second exogenous receptor can be CAR; for example, the first exogenous receptor can be TFP, and the second exogenous receptor can be a chimeric T cell receptor; for example, the first exogenous receptor The body can be a chimeric T cell receptor, and the second exogenous receptor can be TAC. For example, the first exogenous receptor and the second exogenous receptor can be CARs.
抗原antigen
本申请提供的第一外源受体可以识别NKG2A。在一实例中，所述NKG2A可以具有与由NCBI GenBank Gene ID：3821的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。在一实例中，所述NKG2A为人NKG2A,包括与SEQ ID No:11所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或至少约100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。The first exogenous receptor provided in this application can recognize NKG2A. In one example, the NKG2A can have an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least Amino acid sequences or fragments thereof that are about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical, and/or may optionally include at most one or at most two or up to three conservative amino acid substitutions. In one example, the NKG2A is human NKG2A, comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least Amino acid sequences or fragments thereof of about 97%, at least about 98%, at least about 99%, or at least about 100% homology or identity, and/or may optionally include up to one or up to two or up to three conserved Amino acid substitutions.
在一实例中，本申请提供的第一外源受体识别NKG2A,还识别肿瘤抗原和/或病原体抗原。In one example, the first exogenous receptor provided by the present application recognizes NKG2A, and also recognizes tumor antigens and/or pathogen antigens.
在一实例中，本申请提供的第二外源受体识别肿瘤抗原和/或病原体抗原。In one example, the second exogenous receptor provided herein recognizes tumor antigens and/or pathogen antigens.
在一实例中，所述第一外源受体识别的肿瘤抗原和/或病原体抗原和第二外源受体识别肿瘤抗原和/或病原体抗原可以是相同的。在一实例中，所述第一外源受体识别的肿瘤抗原和/或病原体抗原和第二外源受体识别的肿瘤抗原和/或病原体抗原可以是不相同的。例如，所述第一外源受体识别的肿瘤抗原和/或病原体抗原和第二外源受体识别的肿瘤抗原和/或病原体抗原可以是靶向同种肿瘤中的不同肿瘤抗原。例如，所述第一外源受体识别的肿瘤抗原和/或病原体抗原和第二外源受体识别的肿瘤抗原和/或病原体抗原可以是相同肿瘤抗原的不同表位。In one example, the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may be the same. In one example, the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor may be different from the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor. For example, the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may target different tumor antigens in the same tumor. For example, the tumor antigen and/or pathogen antigen recognized by the first exogenous receptor and the tumor antigen and/or pathogen antigen recognized by the second exogenous receptor may be different epitopes of the same tumor antigen.
任何肿瘤抗原均可用于本申请所述的肿瘤相关的实施例(本文中“实例”与“实施例”可互换使用)中。抗原表达为多肽或完整蛋白或其部分。本申请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)；***干细胞抗原(PSCA)；***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(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；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)；***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)；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)。Any tumor antigen can be used in the tumor-related embodiments described herein ("example" and "embodiment" are used interchangeably herein). Antigens are expressed as polypeptides or as intact proteins or parts thereof. The tumor antigens of the present application 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); 11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Acidase; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2); Lewis (Y) Antigen; CD24; Platelet-Derived Growth Factor Receptor Beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4 , EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe ); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside ( OAcGD2); Folate receptor; Tumor vascular endothelial marker 1 (TEM1/CD248); Tumor vascular endothelial marker 7-related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integration B cell maturation antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR ; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; Anaplastic lymphoma kinase (ALK); placenta-specific 1 (PLAC1); hexose moiety of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); Pannexin receptor β3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); Wilms tumor protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); Sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); 2 (Tie2); Melanoma Cancer Testis Antigen-1 (MAD-CT-1); Melanoma Cancer Testis Antigen-2 (MAD-CT-2); Fos-associated Antigen 1; p53 Mutant; Human Telomerase Reverse Transcriptase (hTERT); Sarcoma Translocation Breakpoint; Melanoma Inhibitor of Apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl Transferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; ; Ras homologue family member C (RhoC); cytochrome P450 1B1 (CYP1B1); BORIS; squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin binding protein sp32(OYTES1); Lymphocyte-specific protein tyrosine kinase (LCK); A kinase-anchored protein 4 (AKAP-4); Synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; Globulin-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 structure Domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); 3 (GPC3); Fc receptor-like 5 (FCRL5); Immunoglobulin lambda-like polypeptide 1 (IGLL1).
在一实例中，第一外源受体识别NKG2A，第二外源受体识别BCMA。在一实例中，第一外源受体识别NKG2A和BCMA，第二外源受体识别BCMA。在一实例中，BCMA可以具有与由NCBI GenBank Gene ID：NP_001183.2的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。在一实例中，人BCMA多肽可以包括SEQ ID NO:12所示的氨基酸序列。在一实例中，第一外源受体结合至NKG2A的胞外结构域。在一实例中，第二外源受体结合至BCMA的胞外结构域。在一实例中，第一外源受体结合至NKG2A多肽和BCMA多肽的胞外结构域。In one example, the first exogenous receptor recognizes NKG2A and the second exogenous receptor recognizes BCMA. In one example, the first exogenous receptor recognizes NKG2A and BCMA, and the second exogenous receptor recognizes BCMA. In one example, the BCMA can have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least Amino acid sequences or fragments thereof that are about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homologous or identical, and/or may optionally include at most one or at most two or at most Three conservative amino acid substitutions. In one example, the human BCMA polypeptide can include the amino acid sequence shown in SEQ ID NO: 12. In one example, the first exogenous receptor binds to the extracellular domain of NKG2A. In one example, the second exogenous receptor binds to the extracellular domain of BCMA. In one example, the first exogenous receptor binds to the extracellular domain of the NKG2A polypeptide and the BCMA polypeptide.
在一实例中，外源受体识别病原体抗原，例如用于治疗和/或预防病原体感染或其他感染性疾病，例如在免疫受损的受试者中。病原体抗原包括但不限于：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原包括但不限于：巨细胞病毒(CMV)抗原、爱泼斯坦-巴尔病毒(EBV)抗原、人类免疫缺陷病毒(HIV)抗原或流感病毒抗原。In one example, the exogenous receptor recognizes a pathogen antigen, eg, for the treatment and/or prevention of a pathogen infection or other infectious disease, eg, in an immunocompromised subject. Pathogen antigens include, but are not limited to: antigens of viruses, bacteria, fungi, protozoa, or parasites; viral antigens include, but are not limited to: cytomegalovirus (CMV) antigens, Epstein-Barr virus (EBV) antigens, human immune Defective virus (HIV) antigen or influenza virus antigen.
抗原结合域antigen binding domain
本申请的第一外源受体(例如第一CAR)、第二外源受体(例如第二CAR)可以包括抗原结合域。在一实例中，抗原结合域包括抗体或其片段。在某些实例中，抗原结合域包括抗体重链可变区(VH)和/轻链可变区(VL)；或者包括交联的Fab；或者包括F(ab) 2。在一实例中，抗原结合域包括抗体VH和VL，形成可变片段(Fv)。在一实例中，抗原结合域包括scFv。在一实例中，第一外源受体(例如第一CAR)可以包含NKG2A抗原结合域。在一实例中，NKG2A抗原结合域可以是NKG2A多肽特异性结合的抗体。 The first exogenous receptor (for example, the first CAR) and the second exogenous receptor (for example, the second CAR) of the present application may include an antigen-binding domain. In one example, the antigen binding domain comprises an antibody or fragment thereof. In certain examples, the antigen binding domain comprises an antibody heavy chain variable region (VH) and/or light chain variable region (VL); or comprises a cross-linked Fab; or comprises F(ab) 2 . In one example, the antigen binding domain comprises antibody VH and VL, forming a variable fragment (Fv). In one example, the antigen binding domain comprises a scFv. In one example, the first exogenous receptor (eg, first CAR) can comprise an NKG2A antigen binding domain. In one example, the NKG2A antigen binding domain can be an antibody to which the NKG2A polypeptide specifically binds.
在一实例中，第一外源受体包含识别NKG2A的抗体和包含识别肿瘤抗原的抗体，它们的连接方式是：(1)识别NKG2A的抗体的轻链/重链(或轻链可变区/重链可变区)—识别NKG2A的抗体的重链/轻链(或重链可变区/轻链可变区)—识别肿瘤抗原的抗体的重链/轻链(或重链可变区/轻链可变区)—识别肿瘤抗原的抗体的轻链/重链(或轻链可变区/重链可变区)；(2)识别肿瘤抗原的抗体的轻链(或轻链可变区)—识别NKG2A的抗体的重链(或重链可变区)—识别NKG2A的抗体的轻链(或轻链可变区)—识别肿瘤 抗原的抗体的重链(或重链可变区)；和/或(3)识别NKG2A的抗体的轻链(或轻链可变区)—识别肿瘤抗原的抗体的重链(或重链可变区)—识别肿瘤抗原的抗体的轻链(或轻链可变区)—识别NKG2A的抗体的重链(或重链可变区)。In one example, the first exogenous receptor includes an antibody that recognizes NKG2A and an antibody that recognizes a tumor antigen, and their connection is: (1) the light chain/heavy chain (or light chain variable region) of an antibody that recognizes NKG2A /heavy chain variable region)—heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes NKG2A—heavy chain/light chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen region/light chain variable region)—the light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes a tumor antigen; (2) the light chain (or light chain variable region) of an antibody that recognizes a tumor antigen Variable region)—the heavy chain (or heavy chain variable region) of an antibody that recognizes NKG2A—the light chain (or light chain variable region) of an antibody that recognizes NKG2A—the heavy chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen variable region); and/or (3) the light chain (or light chain variable region) of an antibody that recognizes NKG2A—the heavy chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen—the light chain (or variable region of a heavy chain) of an antibody that recognizes a tumor antigen Chain (or light chain variable region) - The heavy chain (or heavy chain variable region) of an antibody that recognizes NKG2A.
在一实例中，第一CAR包括与NKG2A多肽和BCMA多肽特异性结合的串联抗体；所述CAR的抗原结构域包括分别特异性结合NKG2A多肽、BCMA多肽的Fv。在一实例中，第一CAR包括与NKG2A多肽特异性结合的抗体。在一实例中，NKG2A抗原结合域包含NKG2A抗体的轻链可变区(VL)和/或重链可变区(VH)。在一实例中，NKG2A抗原结合域包含NKG2A抗体的轻链和/或重链。在一实例中，NKG2A的抗体重链或VH包含一个或多个重链CDR(HCDR)：如SEQ ID NO:3序列所示的HCDR1、如SEQ ID NO:4所示序列的HCDR2、如SEQ ID NO:5所示序列的HCDR3。在一实例中，NKG2A抗体重链或VH包含如SEQ ID NOs:3-5所示序列。在一实例中，NKG2A抗体轻链或VL包含一个或多个轻链CDR(LCDR)：如SEQ ID NO:6所示序列的LCDR1、如SEQ ID NO:7所示序列的LCDR2、如SEQ ID NO:8所示序列的LCDR3。在一实例中，NKG2A抗体轻链或VL包含如SEQ ID NOs:6-8所示序列。在一实例中，NKG2A抗体重链或VH包含如SEQ ID NO:1所示的序列。在一实例中，NKG2A抗体轻链或VL包含如SEQ ID NO:2所示的序列。在一实例中，NKG2A抗体或NKG2A抗原结合域包含如SEQ ID NO:1所示序列的VH和/或如SEQ ID NO:2所示序列的VL。在一实例中，NKG2A抗体或NKG2A抗原结合域包含如SEQ ID NO:1和2所示序列。在一实例中，NKG2A抗体或NKG2A抗原结合域包含如SEQ ID NO:52所示的scFv序列。In one example, the first CAR includes a tandem antibody that specifically binds to the NKG2A polypeptide and the BCMA polypeptide; the antigen domain of the CAR includes Fv that specifically binds to the NKG2A polypeptide and the BCMA polypeptide, respectively. In one example, the first CAR includes an antibody that specifically binds to the NKG2A polypeptide. In one example, the NKG2A antigen binding domain comprises a light chain variable region (VL) and/or a heavy chain variable region (VH) of an NKG2A antibody. In one example, the NKG2A antigen binding domain comprises the light chain and/or heavy chain of an NKG2A antibody. In one example, the antibody heavy chain or VH of NKG2A comprises one or more heavy chain CDRs (HCDR): HCDR1 as shown in the sequence of SEQ ID NO:3, HCDR2 as shown in the sequence of SEQ ID NO:4, and HCDR2 as shown in SEQ ID NO:4 HCDR3 of the sequence shown in ID NO:5. In one example, the heavy chain or VH of the NKG2A antibody comprises sequences as shown in SEQ ID NOs: 3-5. In one example, the NKG2A antibody light chain or VL comprises one or more light chain CDRs (LCDRs): LCDR1 of the sequence shown in SEQ ID NO:6, LCDR2 of the sequence shown in SEQ ID NO:7, LCDR2 of the sequence shown in SEQ ID NO:7, LCDR3 of the sequence shown in NO:8. In one example, the NKG2A antibody light chain or VL comprises sequences as shown in SEQ ID NOs: 6-8. In one example, the NKG2A antibody heavy chain or VH comprises the sequence shown in SEQ ID NO: 1. In one example, the NKG2A antibody light chain or VL comprises the sequence shown in SEQ ID NO:2. In one example, the NKG2A antibody or the NKG2A antigen binding domain comprises the VH of the sequence shown in SEQ ID NO:1 and/or the VL of the sequence shown in SEQ ID NO:2. In one example, the NKG2A antibody or NKG2A antigen binding domain comprises the sequences shown in SEQ ID NO: 1 and 2. In one example, the NKG2A antibody or NKG2A antigen binding domain comprises a scFv sequence as shown in SEQ ID NO:52.
在一实例中，BCMA抗原结合域包含识别BCMA的抗体(也称BCMA抗体)的scFv。In one example, the BCMA antigen binding domain comprises a scFv of an antibody that recognizes BCMA (also referred to as a BCMA antibody).
在一实例中，第一外源受体(例如第一CAR)可以包括NKG2A抗原结合域和肿瘤和/或病原体抗原结合域，所述抗原结合域可以是分别特异性结合NKG2A、肿瘤和/或病原体抗原的抗体；在一实例中，所述抗原结合域可以是分别特异性结合NKG2A、病原体抗原的Fv。在一实例中，所述抗原结合域可以是分别特异性结合NKG2A、肿瘤抗原的Fv。In an example, the first exogenous receptor (for example, the first CAR) can include an NKG2A antigen-binding domain and a tumor and/or pathogen antigen-binding domain, and the antigen-binding domain can specifically bind NKG2A, tumor and/or Antibodies to pathogen antigens; in one example, the antigen-binding domain can be Fv that specifically binds NKG2A and pathogen antigens, respectively. In one example, the antigen-binding domain may be an Fv that specifically binds NKG2A and a tumor antigen, respectively.
在一实例中，第一外源受体(例如第一CAR)可以包括NKG2A抗原结合域和BCMA抗原结合域，所述抗原结合域可以是分别特异性结合NKG2A、BCMA的抗体。在一实例中，所述抗原结合域可以是分别特异性结合NKG2A、BCMA的Fv。In an example, the first exogenous receptor (eg, the first CAR) may include an NKG2A antigen-binding domain and a BCMA antigen-binding domain, and the antigen-binding domain may be an antibody specifically binding to NKG2A and BCMA, respectively. In one example, the antigen-binding domain may be an Fv that specifically binds NKG2A and BCMA, respectively.
在一实例中，第一外源受体(例如第一CAR)可以包括NKG2A抗原结合域和肿瘤和/或病原体抗原结合域,第二外源受体(例如第二CAR)可以包含肿瘤和/或病原体抗原结合域。在一实例中，第一外源受体(例如第一CAR)可以包括NKG2A抗原结合域和BCMA抗原结合域,第二外源受体(例如第二CAR)可以包含BCMA抗原结合域。In one example, the first exogenous receptor (for example, the first CAR) may comprise an NKG2A antigen binding domain and a tumor and/or pathogen antigen binding domain, and the second exogenous receptor (for example, a second CAR) may comprise a tumor and/or a pathogen antigen binding domain. or pathogen antigen binding domain. In one example, the first exogenous receptor (for example, the first CAR) can include the NKG2A antigen-binding domain and the BCMA antigen-binding domain, and the second exogenous receptor (for example, the second CAR) can include the BCMA antigen-binding domain.
在一实例中，BCMA抗原结合域包含BCMA抗体VL和/或VH。在一实例中，BCMA 抗原结合域包含BCMA抗体的轻链和/或重链。在一实例中，BCMA抗体重链或VH包含一个或多个重链CDR(HCDR)：如SEQ ID NO:13所示序列的HCDR1、如SEQ ID NO:14所示序列的HCDR2、如SEQ ID NO:15所示序列的HCDR3。在一实例中，BCMA抗体重链或VH包含如SEQ ID NOs:13-15所示序列。在一实例中，BCMA抗体轻链或VL包含一个或多个轻链CDR(LCDR)：如SEQ ID NO:16所示序列的LCDR1、如SEQ ID NO:17所示序列的LCDR2、如SEQ ID NO:18所示序列的LCDR3。在一实例中，BCMA抗体轻链或VL包含如SEQ ID NOs:16-18所示序列。在一实例中，BCMA抗体重链或VH包含如SEQ ID NO:19所示的序列。在一实例中，BCMA抗体轻链或VL包含如SEQ ID NO:20所示的序列。在一实例中，BCMA抗体或BCMA抗原结合域包含如SEQ ID NO:19所示序列的VH和/或如SEQ ID NO:20所示序列的VL。在一实例中，BCMA抗体或BCMA抗原结合域包含如SEQ ID NOs:19和20所示序列。在一实例中，BCMA抗原结合域包含识别BCMA的抗体(也称BCMA抗体)的scFv。In one example, the BCMA antigen binding domain comprises BCMA antibody VL and/or VH. In one example, the BCMA antigen binding domain comprises a light chain and/or a heavy chain of an antibody to BCMA. In one example, the heavy chain or VH of the BCMA antibody comprises one or more heavy chain CDRs (HCDR): HCDR1 of the sequence shown in SEQ ID NO: 13, HCDR2 of the sequence shown in SEQ ID NO: 14, HCDR3 of the sequence shown in NO:15. In one example, the heavy chain or VH of the BCMA antibody comprises a sequence as shown in SEQ ID NOs: 13-15. In one example, the BCMA antibody light chain or VL comprises one or more light chain CDRs (LCDR): LCDR1 of the sequence shown in SEQ ID NO: 16, LCDR2 of the sequence shown in SEQ ID NO: 17, LCDR2 of the sequence shown in SEQ ID NO: 17, LCDR3 of the sequence shown in NO:18. In one example, the BCMA antibody light chain or VL comprises the sequences shown in SEQ ID NOs: 16-18. In one example, the BCMA antibody heavy chain or VH comprises the sequence shown in SEQ ID NO: 19. In one example, the BCMA antibody light chain or VL comprises the sequence shown in SEQ ID NO:20. In one example, the BCMA antibody or BCMA antigen binding domain comprises the VH of the sequence shown in SEQ ID NO:19 and/or the VL of the sequence shown in SEQ ID NO:20. In one example, the BCMA antibody or BCMA antigen binding domain comprises the sequences shown in SEQ ID NOs: 19 and 20. In one example, the BCMA antigen binding domain comprises a scFv of an antibody that recognizes BCMA (also referred to as a BCMA antibody).
在一实例中，第一外源受体(例如第一CAR)包含NKG2A抗体和BCMA抗体全部CDR区域。在一实例中，第一外源受体(例如第一CAR)包括如SEQ ID NOs:3-8和13-18所示序列。In one example, the first exogenous receptor (eg, the first CAR) comprises all CDR regions of the NKG2A antibody and the BCMA antibody. In one example, the first exogenous receptor (eg, the first CAR) comprises sequences shown in SEQ ID NOs: 3-8 and 13-18.
示例性，本申请提供抗BCMA的抗体包括SEQ ID NO：21、22、23、24或25所示的scFv序列；抗NKG2A的抗体包括SEQ ID NO：1所示的VH、SEQ ID NO：2所示的VL，或包括SEQ ID NO：52所示的scFv；BCMA-NKG2A串联抗体包括SEQ ID NO：36、37、38、39或40所示的序列。Exemplarily, the application provides an anti-BCMA antibody comprising the scFv sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25; an anti-NKG2A antibody comprising the VH shown in SEQ ID NO: 1, SEQ ID NO: 2 The VL shown may include the scFv shown in SEQ ID NO: 52; the BCMA-NKG2A tandem antibody includes the sequence shown in SEQ ID NO: 36, 37, 38, 39 or 40.
在一个方面，本申请考虑到产生功能上等同的分子的起始抗体或片段(例如，VH或VL)氨基酸序列的修饰。例如，可修饰CAR中包括的NKG2A抗体或BCMA抗体的VH或VL，保留NKG2A或BCMA抗体如VH或VL至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。In one aspect, the present application contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, VH or VL) to produce a functionally equivalent molecule. For example, the VH or VL of the NKG2A antibody or BCMA antibody included in the CAR can be modified such that the NKG2A or BCMA antibody such as the VH or VL is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% identity.
CARCAR
在一实例中，本申请的外源受体可以是嵌合抗原受体(CAR)，第一外源受体为第一CAR，第二外源受体为第二CAR。在一实例中，CAR多肽的各个结构域可以处于相同的多肽链中，例如，作为单个多肽链表达。在一些实例中，CAR多肽的各个结构域可以彼此不邻接，例如，处于不同的多肽链中。In one example, the exogenous receptor of the present application may be a chimeric antigen receptor (CAR), the first exogenous receptor is the first CAR, and the second exogenous receptor is the second CAR. In one example, the various domains of the CAR polypeptide can be in the same polypeptide chain, eg, expressed as a single polypeptide chain. In some examples, the individual domains of the CAR polypeptide may not be contiguous to each other, e.g., be in different polypeptide chains.
本领域普通技术人员将会理解，可进一步修饰本发明的抗体或抗体片段，使得它们在氨基酸序列上(例如，相对于野生型)有所变化，但在所需活性上没有变化。例如，可对蛋白质进行另外的核苷酸置换，导致“非必需”氨基酸残基处的氨基酸置换。例如，分子中的 非必需氨基酸残基可被来自相同侧链家族的另一个氨基酸残基取代。在另一个实施方案中，氨基酸片段可被结构相似但在顺序和/或组成上与侧链家族成员不同的氨基酸片段取代，例如，可进行保守置换，其中氨基酸残基被具有相似侧链的氨基酸残基所取代。Those of ordinary skill in the art will appreciate that antibodies or antibody fragments of the invention may be further modified such that they have changes in amino acid sequence (eg, relative to wild type) but no change in the desired activity. For example, additional nucleotide substitutions can be made to the protein, resulting in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule can be replaced by another amino acid residue from the same side chain family. In another embodiment, amino acid stretches may be substituted with amino acid stretches that are structurally similar but differ in sequence and/or composition from members of the side chain family, for example, conservative substitutions may be made wherein amino acid residues are replaced by amino acids with similar side chains residue replaced.
信号肽和铰链域Signal peptide and hinge domain
本申请的第一CAR和/或第二CAR还可以包括铰链域。第一CAR、第二CAR的铰链域可以各自独立地选自如下蛋白的铰链域：CD28、CD8、HLA、Fc、IgG、IgD、4-1BB、CD4、CD27、CD7和PD1。第一CAR和第二CAR的铰链域可以是相同的，也可以是不同的；第一CAR或第二CAR可以选择已知的任何合适的铰链序列，第一CAR选择的铰链域不影响第二CAR中铰链域的选择。例如，第一CAR可以包含CD8的铰链域，第二CAR可以包含CD8的铰链域或者其他任何合适的蛋白分子的铰链域；例如，第二CAR可以包含CD8的铰链域，第一CAR可以包含CD8的铰链域或者其他任何合适的蛋白分子的铰链域。在一个实例中，抗原结合域与跨膜结构域直接连接或通过铰链连接。在一实例中，第一CAR、第二CAR各自包括CD8铰链，例如，CD8铰链可以包含如SEQ ID NO:30所示的序列或与SEQ ID NO:30具有95-99％同一性的序列。The first CAR and/or the second CAR of the present application may further include a hinge domain. The hinge domains of the first CAR and the second CAR can be independently selected from hinge domains of the following proteins: CD28, CD8, HLA, Fc, IgG, IgD, 4-1BB, CD4, CD27, CD7 and PD1. The hinge domains of the first CAR and the second CAR can be the same or different; the first CAR or the second CAR can select any suitable hinge sequence known, and the hinge domain selected by the first CAR does not affect the second CAR. Selection of hinge domains in CAR. For example, the first CAR can contain the hinge domain of CD8, and the second CAR can contain the hinge domain of CD8 or any other suitable protein molecule; for example, the second CAR can contain the hinge domain of CD8, and the first CAR can contain the hinge domain of CD8. The hinge domain of or any other suitable protein molecule. In one example, the antigen binding domain is linked directly or via a hinge to the transmembrane domain. In one example, each of the first CAR and the second CAR includes a CD8 hinge, for example, the CD8 hinge may include a sequence as shown in SEQ ID NO: 30 or a sequence having 95-99% identity with SEQ ID NO: 30.
本申请的第一CAR和/或第二CAR还可以包括信号肽。第一CAR和第二CAR的信号肽可以是相同的，也可以是不同的。在一实例中，第一CAR和第二CAR的信号肽可以是CD8的信号肽。例如所述CD8信号肽包含如SEQ ID NO:29所示序列。The first CAR and/or the second CAR of the present application may further include a signal peptide. The signal peptides of the first CAR and the second CAR may be the same or different. In one example, the signal peptides of the first CAR and the second CAR may be the signal peptide of CD8. For example, the CD8 signal peptide comprises the sequence shown in SEQ ID NO:29.
跨膜域transmembrane domain
本申请的第一CAR和/或第二CAR还可以包括跨膜域。第一CAR、第二CAR的跨膜域可以各自独立地选自如下蛋白的跨膜域:T细胞受体的α、β、或ζ的跨膜结构域、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D和/或NKG2C的跨膜结构域。第一CAR和第二CAR的跨膜域可以是相同的，也可以是不同的；第一CAR或第二CAR可以选择已知的任何合适的跨膜域，第一CAR选择的跨膜域不影响第二CAR 中跨膜域的选择。例如，第一CAR可以包含CD8/CD28的跨膜域，第二CAR可以包含CD8/CD28的跨膜域或者其他任何合适的蛋白分子的跨膜域；例如，第二CAR可以包含CD8/CD28的跨膜域，第一CAR可以包含CD8/CD28的跨膜域或者其他任何合适的蛋白分子的跨膜域；在一实例中，第一CAR、第二CAR各自包含CD8跨膜域；在一实例中，第一CAR、第二CAR各自包含CD28跨膜域；在一实例中，第一CAR包含CD8跨膜域，第二CAR包含CD28跨膜域；在一实例中，第一CAR包含CD28跨膜域，第二CAR包含CD8跨膜域。在一实例中，所述CD8跨膜域包含SEQ ID NO:31所示序列的至少一个、两个或三个修饰，但不超过20，10或5个修饰的序列,或与SEQ ID NO:31所示的氨基酸序列具有95-99％同一性的序列。在一个实例中，CD8跨膜域包括SEQ ID NO:31所示的序列。在一实例中，所述CD28跨膜域，包含如SEQ ID NO:32所示序列的至少一个、两个或三个修饰，但不超过20，10或5个修饰的序列,或与SEQ ID NO:32的氨基酸序列具有95-99％同一性的序列。在一个实例中，CD28跨膜域包括SEQ ID NO:32的序列。The first CAR and/or the second CAR of the present application may also include a transmembrane domain. The transmembrane domains of the first CAR and the second CAR can be independently selected from transmembrane domains of the following proteins: transmembrane domains of α, β, or ζ of T cell receptors, CD28, CD3ε, CD45, CD4, CD5 , CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244 , 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3 ), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D and/or the transmembrane domain of NKG2C. The transmembrane domains of the first CAR and the second CAR can be the same or different; the first CAR or the second CAR can select any suitable transmembrane domain known, and the transmembrane domain selected by the first CAR is not Influences selection of transmembrane domains in the second CAR. For example, the first CAR may contain the transmembrane domain of CD8/CD28, and the second CAR may contain the transmembrane domain of CD8/CD28 or any other suitable protein molecule; for example, the second CAR may contain the transmembrane domain of CD8/CD28. Transmembrane domain, the first CAR may comprise the transmembrane domain of CD8/CD28 or any other suitable protein molecules; in one example, the first CAR and the second CAR each comprise the CD8 transmembrane domain; in one example wherein, the first CAR and the second CAR each comprise a CD28 transmembrane domain; in one example, the first CAR comprises a CD8 transmembrane domain, and the second CAR comprises a CD28 transmembrane domain; in one example, the first CAR comprises a CD28 transmembrane domain Membrane domain, the second CAR contains the CD8 transmembrane domain. In one example, the CD8 transmembrane domain comprises at least one, two or three modifications of the sequence shown in SEQ ID NO:31, but no more than 20, 10 or 5 modified sequences, or the same sequence as SEQ ID NO: The amino acid sequence shown in 31 has a sequence of 95-99% identity. In one example, the CD8 transmembrane domain comprises the sequence set forth in SEQ ID NO:31. In one example, the CD28 transmembrane domain comprises at least one, two or three modifications of the sequence shown in SEQ ID NO:32, but no more than 20, 10 or 5 modified sequences, or the sequence with SEQ ID NO:32 The amino acid sequence of NO:32 has a sequence of 95-99% identity. In one example, the CD28 transmembrane domain comprises the sequence of SEQ ID NO: 32.
胞内信号传导域intracellular signaling domain
本申请的第一CAR和/或第二CAR还可以包括胞内信号传导域。第一CAR、第二CAR的胞内信号传导域可以各自独立地选自如下蛋白的胞内信号传导域：CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10、和DAP12。第一CAR和第二CAR的胞内信号传导域可以是相同的，也可以是不同的；第一CAR或第二CAR可以选择已知的任何合适的胞内信号传导域，第一CAR选择的胞内信号传导域不影响第二CAR中胞内信号传导域的选择。例如，第一CAR可以包含CD3ζ的胞内信号传导域，第二CAR可以包含CD3ζ的胞内信号传导域或者其他任何合适的蛋白分子的胞内信号传导域；例如，第二CAR可以包含CD3ζ的胞内信号传导域，第一CAR可以包含CD3ζ的胞内信号传导域或者其他任何合适的蛋白分子的胞内信号传导域；在一实例中，第一CAR、第二CAR各自包含CD3ζ的胞内信号传导域。在一实例中，所述CD3ζ胞内信号传导域可包括如SEQ ID NO:35所示的氨基酸序列的至少1、2、或3个修饰但不超过20、10或5个修饰的氨基酸序列，或与SEQ ID NO:35所示的氨基酸序列有95-99％同一性的序列。在一实例中，CD3ζ胞内信号传导域包括SEQ ID NO:35的氨基酸序列。The first CAR and/or the second CAR of the present application may further include an intracellular signaling domain. The intracellular signaling domains of the first CAR and the second CAR can be independently selected from the intracellular signaling domains of the following proteins: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcyRIIa, DAP10, and DAP12. The intracellular signaling domains of the first CAR and the second CAR may be the same or different; the first CAR or the second CAR may select any suitable intracellular signaling domain known, and the first CAR selected The intracellular signaling domain does not affect the choice of intracellular signaling domain in the second CAR. For example, the first CAR can comprise the intracellular signaling domain of CD3ζ, the second CAR can comprise the intracellular signaling domain of CD3ζ or the intracellular signaling domain of any other suitable protein molecule; for example, the second CAR can comprise the intracellular signaling domain of CD3ζ. The intracellular signaling domain, the first CAR may comprise the intracellular signaling domain of CD3ζ or the intracellular signaling domain of any other suitable protein molecule; in one example, the first CAR and the second CAR each comprise the intracellular signaling domain of CD3ζ signaling domain. In one example, the CD3ζ intracellular signaling domain may comprise at least 1, 2, or 3 modified amino acid sequences but no more than 20, 10, or 5 modified amino acid sequences of the amino acid sequence shown in SEQ ID NO:35, Or a sequence with 95-99% identity to the amino acid sequence shown in SEQ ID NO:35. In one example, the CD3ζ intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 35.
共刺激信号结构域co-stimulatory signaling domain
本申请的第一CAR和/或第二CAR还可以包括共刺激信号结构域。第一CAR、第二CAR的跨膜域可以各自独立地选自如下蛋白的共刺激信号结构域：:CD27、CD28、The first CAR and/or the second CAR of the present application may further include a co-stimulatory signaling domain. The transmembrane domains of the first CAR and the second CAR can be independently selected from the co-stimulatory signal domains of the following proteins: CD27, CD28,
4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、 CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244,2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、或NKG2D。第一CAR和第二CAR的共刺激信号结构域可以是相同的，也可以是不同的；第一CAR或第二CAR可以选择已知的任何合适的共刺激信号结构域，第一CAR选择的共刺激信号结构域不影响第二CAR中共刺激信号结构域的选择。例如，第一CAR可以包含4-1BB/CD28的共刺激信号结构域，第二CAR可以包含4-1BB/CD28的共刺激信号结构域或者其他任何合适的蛋白分子的共刺激信号结构域；例如，第二CAR可以包含4-1BB/CD28的共刺激信号结构域，第一CAR可以包含4-1BB/CD28的共刺激信号结构域或者其他任何合适的蛋白分子的共刺激信号结构域；在一实例中，第一CAR、第二CAR各自包含4-1BB的共刺激信号结构域。在一实例中，第一CAR、第二CAR各自包含CD28的共刺激信号结构域。4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83 CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4(CD244,2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB- A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, or NKG2D. The co-stimulatory signal domains of the first CAR and the second CAR may be the same or different; the first CAR or the second CAR may select any suitable co-stimulatory domains known, and the first CAR selected The co-stimulatory signaling domain does not affect the choice of the second CAR co-stimulatory signaling domain. For example, the first CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28, and the second CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28 or any other suitable co-stimulatory signaling domain of protein molecules; for example , the second CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28, the first CAR may comprise a co-stimulatory signaling domain of 4-1BB/CD28 or any other suitable co-stimulatory signaling domain of protein molecules; in a In an example, the first CAR and the second CAR each comprise a co-stimulatory signaling domain of 4-1BB. In one example, each of the first CAR and the second CAR comprises a co-stimulatory signaling domain of CD28.
在一实例中，所述4-1BB共刺激信号结构域包括如SEQ ID NO:34的氨基酸序列的至少1、2、或3个修饰但不超过20、10或5个修饰的氨基酸序列，或与SEQ ID NO:34所示的氨基酸序列有95-99％同一性的序列。在一实例中，4-1BB共刺激信号结构域包括SEQ ID NO:34的序列。In one example, the 4-1BB co-stimulatory signaling domain comprises an amino acid sequence of at least 1, 2, or 3 modifications but no more than 20, 10, or 5 modifications of the amino acid sequence of SEQ ID NO: 34, or A sequence with 95-99% identity to the amino acid sequence shown in SEQ ID NO:34. In one example, the 4-1BB co-stimulatory signaling domain comprises the sequence of SEQ ID NO:34.
在一实例中，所述CD28共刺激信号结构域包括如SEQ ID NO:33的氨基酸序列的至少1、2、或3个修饰但不超过20、10或5个修饰的氨基酸序列，或与SEQ ID NO:33所示的氨基酸序列有95-99％同一性的序列。在一实例中，CD28共刺激信号结构域包括SEQ ID NO:33的序列。In one example, the CD28 co-stimulatory signaling domain comprises at least 1, 2, or 3 modified amino acid sequences but no more than 20, 10, or 5 modified amino acid sequences of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence with SEQ ID NO:33 The amino acid sequence shown in ID NO:33 has 95-99% identity sequence. In one example, the CD28 co-stimulatory signaling domain comprises the sequence of SEQ ID NO:33.
第一CAR、第二CAR可以包含相同数目或不同数目的共刺激信号结构域，第一CAR共刺激信号结构域的数目与第二CAR的共刺激信号结构域的数目互不关联。例如，第一CAR包含1个共刺激信号结构域，第二CAR包含一个以上(例如2个)共刺激信号结构域；例如，第二CAR包含1个共刺激信号结构域，第一CAR包含一个以上(例如2个)共刺激信号结构域；例如第一CAR、第二CAR均包含1个以上(例如2个)共刺激信号结构域。The first CAR and the second CAR may contain the same number or different numbers of co-stimulatory signaling domains, and the number of co-stimulatory signaling domains of the first CAR is independent of the number of co-stimulatory signaling domains of the second CAR. For example, the first CAR contains 1 costimulatory signaling domain, and the second CAR contains more than one (for example, 2) costimulatory signaling domains; for example, the second CAR contains 1 costimulatory signaling domain, and the first CAR contains one The above (for example 2) co-stimulatory signal domains; for example, the first CAR and the second CAR both contain more than 1 (for example 2) costimulatory signal domains.
包含1个以上的共刺激信号结构域的CAR中的多个共刺激信号结构域可以是相同的也可以是不同的，例如，包含均为4-1BB/CD28的2个共刺激信号结构域；例如，包含一个4-1BB共刺激信号结构域和一个CD28共刺激信号结构域。The multiple costimulatory signal domains in the CAR comprising more than one costimulatory signal domain may be the same or different, for example, contain 2 costimulatory signal domains both of 4-1BB/CD28; For example, a 4-1BB co-stimulatory signaling domain and a CD28 co-stimulatory signaling domain are included.
示例性，本发明提供的第一CAR包括SEQ ID NO：9、41、42、43、44、45和/或53所示序列。示例性，第一CAR包括SEQ ID NO：36、37、38、39、40或52所示序列分别与SEQ ID NO：49、50或51顺序连接而成的序列。示例性，本发明的第一CAR包括SEQ ID NO：36、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：36、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：36、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、SEQ ID NO：37、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：37、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：37、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、SEQ ID NO：38、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：38、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：38、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、SEQ ID NO：39、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：39、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：39、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、SEQ ID NO：40、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：40、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：40、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、SEQ ID NO：52、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：52、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：52、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列。Exemplarily, the first CAR provided by the present invention includes sequences shown in SEQ ID NO: 9, 41, 42, 43, 44, 45 and/or 53. Exemplarily, the first CAR includes a sequence formed by sequentially linking the sequence shown in SEQ ID NO: 36, 37, 38, 39, 40 or 52 with SEQ ID NO: 49, 50 or 51, respectively. Exemplarily, the first CAR of the present invention includes a sequence formed by sequentially connecting SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO: 49, or SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO : 50 sequentially concatenated sequence, or sequence concatenated by SEQ ID NO: 36, SEQ ID NO: 30 and SEQ ID NO: 51, SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 49 sequentially connected sequence, or SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 50 sequentially connected sequence, or SEQ ID NO: 37, SEQ ID NO: 30 and SEQ ID NO: 51 sequentially connected sequence, SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 49 sequentially connected sequence, or SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 50 The sequence formed by sequential connection, or the sequence formed by sequential connection of SEQ ID NO: 38, SEQ ID NO: 30 and SEQ ID NO: 51, the sequence of SEQ ID NO: 39, SEQ ID NO: 30 and SEQ ID NO: 49 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 39, SEQ ID NO: 30 and SEQ ID NO: 50, or a sequence of SEQ ID NO: 39, SEQ ID NO: 30 and SEQ ID NO: 51 A sequence formed by concatenation, a sequence formed by sequential concatenation of SEQ ID NO: 40, SEQ ID NO: 30 and SEQ ID NO: 49, or a sequential concatenation of SEQ ID NO: 40, SEQ ID NO: 30 and SEQ ID NO: 50 The sequence formed, or the sequence formed by sequential connection of SEQ ID NO: 40, SEQ ID NO: 30 and SEQ ID NO: 51, the sequential connection of SEQ ID NO: 52, SEQ ID NO: 30 and SEQ ID NO: 49 The sequence formed, or the sequence formed by sequential connection of SEQ ID NO: 52, SEQ ID NO: 30 and SEQ ID NO: 50, or the sequential connection of SEQ ID NO: 52, SEQ ID NO: 30 and SEQ ID NO: 51 into a sequence.
示例性，本发明提供的第二CAR包括SEQ ID NO：26、27或28所示序列。示例性，第二CAR包括SEQ ID NO：21、22、23、24或25所示的序列分别与SEQ ID NO：49、50或51顺序连接的序列。示例性，第二CAR包括SEQ ID NO：21、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：21、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：21、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、或SEQ ID NO：22、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：22、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：22、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、或SEQ ID NO：23、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：23、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：23、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、或SEQ ID NO：24、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：24、SEQ ID NO：30 与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：24、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列、或SEQ ID NO：25、SEQ ID NO：30与SEQ ID NO：49顺序连接而成的序列、或SEQ ID NO：25、SEQ ID NO：30与SEQ ID NO：50顺序连接而成的序列、或SEQ ID NO：25、SEQ ID NO：30与SEQ ID NO：51顺序连接而成的序列。Exemplarily, the second CAR provided by the present invention includes the sequence shown in SEQ ID NO: 26, 27 or 28. Exemplarily, the second CAR includes a sequence in which the sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25 is sequentially connected to SEQ ID NO: 49, 50 or 51, respectively. Exemplarily, the second CAR includes the sequence of SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 49, or the sequence of SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 50 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 21, SEQ ID NO: 30 and SEQ ID NO: 51, or a sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 49 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 50, or a sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 51 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 23, SEQ ID NO: 30 and SEQ ID NO: 49, or a sequence of SEQ ID NO: 23, SEQ ID NO: 30 and SEQ ID NO: 50 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 23, SEQ ID NO: 30 and SEQ ID NO: 51, or a sequence of SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 49 A concatenated sequence, or a concatenated sequence of SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 50, or a sequence of SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 51 The sequence formed by concatenation, or the sequence concatenated by SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 49, or the sequence of SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 50 The sequence formed by connection, or the sequence formed by sequential connection of SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 51.
示例性的，本发明提供的细胞组合物中第一工程细胞表达上述任一第一CAR；第二工程细胞表达上述任一第二CAR。示例性的，本发明提供的细胞组合物中第一工程细胞为表达上述任一第一CAR的CAR-T细胞；第二工程细胞为表达上述任一第二CAR的CAR-T细胞。Exemplarily, in the cell composition provided by the present invention, the first engineered cell expresses any one of the above-mentioned first CARs; the second engineered cell expresses any one of the above-mentioned second CARs. Exemplarily, the first engineered cell in the cell composition provided by the present invention is a CAR-T cell expressing any one of the above-mentioned first CARs; the second engineered cell is a CAR-T cell expressing any one of the above-mentioned second CARs.
本申请考虑到整个CAR分子的修饰，例如，CAR分子的各个结构域的一个或多个氨基酸序列的修饰，以便产生功能上等同的分子。可修饰CAR分子保留起始CAR分子的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。The present application contemplates modification of the entire CAR molecule, eg, modification of one or more amino acid sequences of each domain of the CAR molecule, in order to generate a functionally equivalent molecule. The modifiable CAR molecule retains at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% of the starting CAR molecule , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identity.
CD3复合物CD3 complex
在一实例中，本申请提供的外源受体能够与CD3ζ多肽缔合。CD3ζ多肽可以是内源性，也可以是外源性的。在一实例中，外源受体的抗原结合域与抗原(例如NKG2A多肽、肿瘤抗原、病原体抗原)的结合能够激活与外源受体缔合的CD3ζ多肽。在一实例中，外源受体的抗原结合域与抗原(例如NKG2A多肽、肿瘤抗原、病原体抗原)的结合能够激活与外源受体的CD3ζ信号结构域。In one example, the exogenous receptor provided herein is capable of associating with a CD3ζ polypeptide. CD3ζ polypeptide can be endogenous or exogenous. In one example, binding of an antigen binding domain of an exogenous receptor to an antigen (eg, NKG2A polypeptide, tumor antigen, pathogen antigen) activates a CD3ζ polypeptide associated with the exogenous receptor. In one example, binding of the antigen binding domain of the exogenous receptor to an antigen (eg, NKG2A polypeptide, tumor antigen, pathogen antigen) can activate the CD3ζ signaling domain of the exogenous receptor.
细胞组合物cell composition
在一实例中，本申请的细胞组合物包括识别NKG2A的第一工程细胞和识别肿瘤和/或病原体抗原的第二工程细胞。在一实例中，工程细胞是免疫细胞、神经元、上皮细胞、内皮细胞或干细胞。干细胞包括人多能干细胞(包括人诱导多能干细胞(iPSC)和人胚胎干细胞)。在一实例中，所述工程细胞包括免疫细胞。在一实例中，所述工程细胞为原代细胞。在一实例中，免疫细胞是B细胞、单核细胞、自然杀伤细胞、嗜碱性粒细胞、嗜酸性粒细胞、中性粒细胞、树突状细胞、巨噬细胞、调节性T细胞、辅助性T细胞、细胞毒性T细胞、其他T细胞或其组合。In one example, the cell composition of the present application includes a first engineered cell that recognizes NKG2A and a second engineered cell that recognizes tumor and/or pathogen antigens. In one example, the engineered cells are immune cells, neurons, epithelial cells, endothelial cells or stem cells. Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSC) and human embryonic stem cells). In one example, the engineered cells include immune cells. In one example, the engineered cells are primary cells. In one example, the immune cells are B cells, monocytes, natural killer cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, regulatory T cells, helper Cytotoxic T cells, other T cells, or combinations thereof.
在一实例中，所述第一工程细胞(例如T、NKT细胞)包括与SEQ ID NO：9、41、42、43、44、45、53中任何一个具有至少80％序列同源性氨基酸序列。在一实例中，所述第一工程细胞(例如T、NKT细胞)包括与SEQ ID NO：36、37、38、39、40或52所示序列分别与SEQ ID NO：49、50或51顺序连接而成的序列具有至少80％序列同源 性的氨基酸序列。在一实例中，所述第二工程细胞(例如T、NKT细胞)包括与SEQ ID NO：26、27或28具有至少80％序列同源性的核酸序列或其翻译成的氨基酸序列。在一实例中，所述第二工程细胞(例如T、NKT细胞)包括与SEQ ID NO：21、22、23、24或25所示序列分别与SEQ ID NO：49、50或51顺序连接而成的序列具有至少80％序列同源性的核酸序列或其翻译成的氨基酸序列。In one example, said first engineered cell (e.g. T, NKT cell) comprises an amino acid sequence having at least 80% sequence homology to any one of SEQ ID NO: 9, 41, 42, 43, 44, 45, 53 . In one example, the first engineering cell (such as T, NKT cell) comprises the sequence shown in SEQ ID NO: 36, 37, 38, 39, 40 or 52 and the sequence of SEQ ID NO: 49, 50 or 51 respectively Amino acid sequences with at least 80% sequence homology to the linked sequences. In one example, the second engineered cell (such as T, NKT cell) comprises a nucleic acid sequence having at least 80% sequence homology to SEQ ID NO: 26, 27 or 28 or an amino acid sequence translated into it. In one example, the second engineered cell (such as T, NKT cell) comprises sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25 connected with SEQ ID NO: 49, 50 or 51 sequence respectively Nucleic acid sequences or their translated amino acid sequences having at least 80% sequence homology.
示例性的，本发明提供的细胞组合物包括本文任一所述的第一工程细胞和本文任一所述的第二工程细胞。示例性的，本发明提供的细胞组合物包括本文任一所述的第一外源受体的工程细胞和本文任一所述的第二外源受体的工程细胞。示例性的，本发明提供的细胞组合物包括本文任一所述的第一CAR的工程细胞和本文任一所述的第二CAR的工程细胞。示例性的，本发明提供的细胞组合物包括本文任一所述的第一CAR-T细胞和本文任一所述的第二CAR-T细胞。Exemplarily, the cell composition provided by the present invention includes any of the first engineered cells described herein and any of the second engineered cells described herein. Exemplarily, the cell composition provided by the present invention includes any one of the engineered cells for the first exogenous recipient described herein and any of the engineered cells for the second exogenous recipient described herein. Exemplarily, the cell composition provided by the present invention includes any one of the engineered cells of the first CAR described herein and any of the engineered cells of the second CAR described herein. Exemplarily, the cell composition provided by the present invention includes any of the first CAR-T cells described herein and any of the second CAR-T cells described herein.
本申请的细胞组合物不必是混合状态的混合物，其中识别NKG2A的第一工程细胞与所述识别非NKG2A的抗原的第二工程细胞可以是互相分离的，例如各自存在于不同的容器中。The cell composition of the present application does not have to be a mixture in a mixed state, wherein the first engineered cells recognizing NKG2A and the second engineered cells recognizing non-NKG2A antigens may be separated from each other, for example, each exists in a different container.
在一实例中，本申请的细胞组合物可以是混合状态的混合物。In one example, the cell composition of the present application may be a mixture in a mixed state.
在一实例中，本申请的组合物包括识别NKG2A多肽和肿瘤抗原的第一工程细胞(例如T、NKT细胞)和识别肿瘤和/或病原体抗原的第二工程细胞(例如T、NKT细胞)。In one example, the composition of the present application includes first engineered cells (such as T, NKT cells) that recognize NKG2A polypeptides and tumor antigens and second engineered cells (such as T, NKT cells) that recognize tumor and/or pathogen antigens.
在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，本申请的组合物中的工程细胞具有更长存活时间和/或更强的扩增能力。在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，本申请的组合物中的第二工程细胞具有更长存活时间和/或更强的扩增能力。在一实例中，与第一工程细胞或第二工程细胞相比，包括第一工程细胞和第二工程细胞的的组合物对携带靶抗原的细胞表现出更强的体内外的细胞杀伤作用。在一实例中，本申请的细胞组合物特异性向表达靶抗原的组织浸润。在一实例中，本申请的细胞组合物特异性向表达靶抗原的肿瘤组织浸润。在一实例中，本申请的细胞组合物不引起移植物抗宿主反应。在一实例中，第一工程细胞和/或第二工程细胞中参与响应自体和非自体抗原识别多肽的至少一种内源基因表达、活性和/或信号被降低或抑制；包含所述第一和第二工程细胞的细胞组合物不引起移植物抗宿主反应。In one example, the engineered cells in the composition of the present application have longer survival time and/or stronger expansion ability in the presence of host immune cells (eg, NK cells). In one example, the second engineered cells in the composition of the present application have longer survival time and/or stronger expansion ability in the presence of host immune cells (eg, NK cells). In one example, compared with the first engineered cell or the second engineered cell, the composition comprising the first engineered cell and the second engineered cell exhibits a stronger cell killing effect in vivo and in vitro on the cell carrying the target antigen. In one example, the cell composition of the present application specifically infiltrates the tissue expressing the target antigen. In one example, the cell composition of the present application specifically infiltrates the tumor tissue expressing the target antigen. In one example, the cell composition of the present application does not cause a graft-versus-host reaction. In one example, the expression, activity and/or signal of at least one endogenous gene involved in responding to self and non-self antigen recognition polypeptides in the first engineered cell and/or the second engineered cell is reduced or inhibited; comprising said first The cell composition of the second engineered cell does not cause a graft-versus-host reaction.
本申请组合物中的第一工程细胞、第二工程细胞均为免疫细胞。在一实例中，所述免疫细胞来源于神经元、上皮细胞、内皮细胞或干细胞。在一实例中，所述免疫细胞可以是淋巴谱系的细胞。包括B、T和自然杀伤(NK)细胞的淋巴谱系提供抗体的产生、细胞免疫***的调节、血液中外源试剂的检测、宿主外源细胞的检测等。淋巴谱系的免疫细胞的非限制性实例包括T细胞、自然杀伤T(NKT)细胞及其前体，包括胚胎干细胞和多能干细胞(例 如，分化成淋巴样细胞的干细胞或多能干细胞)。T细胞可以是在胸腺中成熟的淋巴细胞，主要负责细胞介导的免疫。T细胞参与适应性免疫***。T细胞可以是任何类型的T细胞，包括但不限于辅助T细胞、细胞毒性T细胞、记忆T细胞(包括中央记忆T细胞、干细胞样记忆T细胞(或干样记忆T细胞)和两种效应记忆T细胞：例如TEM细胞和TEMRA细胞)、调节性T细胞(也称为抑制性T细胞)、自然杀伤T细胞、粘膜相关性不变T细胞、γδT细胞或αβT细胞。细胞毒性T细胞(CTL或杀伤性T细胞)是能够诱导被感染的体细胞或肿瘤细胞死亡的T淋巴细胞。受试者自身的T细胞可以被工程化改造以表达本申请外源受体。在一实例中，组合物中免疫细胞是T细胞。在一实例中，T细胞可以是CD4+T细胞和/或CD8+T细胞。在一实例中，组合物中免疫细胞是CD3+T细胞。在一实例中，本申请组合物中的工程细胞包括由PBMC细胞经CD3磁珠刺激后收集的细胞群。Both the first engineered cell and the second engineered cell in the composition of the present application are immune cells. In one example, the immune cells are derived from neurons, epithelial cells, endothelial cells or stem cells. In one example, the immune cells can be cells of the lymphoid lineage. The lymphoid lineage including B, T, and natural killer (NK) cells provide for antibody production, regulation of the cellular immune system, detection of exogenous agents in the blood, detection of foreign cells to the host, etc. Non-limiting examples of immune cells of the lymphoid lineage include T cells, natural killer T (NKT) cells and precursors thereof, including embryonic stem cells and pluripotent stem cells (e.g., stem cells that differentiate into lymphoid cells or pluripotent stem cells). T cells may be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. T cells can be of any type, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-like memory T cells (or stem-like memory T cells), and both effector Memory T cells: eg TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, γδ T cells or αβ T cells. Cytotoxic T cells (CTL or killer T cells) are T lymphocytes capable of inducing the death of infected somatic or tumor cells. The subject's own T cells can be engineered to express the exogenous receptors of the present application. In one example, the immune cells in the composition are T cells. In one example, the T cells can be CD4+ T cells and/or CD8+ T cells. In one example, the immune cells in the composition are CD3+ T cells. In one example, the engineered cells in the composition of the present application include cell populations collected from PBMC cells stimulated by CD3 magnetic beads.
组合物中免疫细胞(例如，T细胞)可以是自体的、非自体的(例如，同种异体的)、或者是体外从工程化的祖细胞或干细胞衍生而来。可从许多来源获得，包括外周血单个核细胞(PBMC)、骨髓、***组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。The immune cells (eg, T cells) in the composition can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells. It can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
在本申请的某些方面，可使用本领域技术人员已知的任意数量的技术如Ficoll TM分离技术从收集自受试者的血液样品中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体的循环血液的细胞。单采血液成分术产物通常含有淋巴细胞，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。在一个方面，可洗涤通过单采血液成分术收集的细胞以去除血浆部分并将细胞置于适当的缓冲液或培养基中以供后续处理步骤。在本申请的背景下还可使用多轮选择。在某些方面，可能需要进行选择程序并在激活和扩充过程中使用“未选择的”细胞。“未选择的”细胞也可以经受其他轮选择。 In certain aspects of the present application, T cells can be obtained from a blood sample collected from a subject using any number of techniques known to those of skill in the art, such as the Ficoll ™ separation technique. In a preferred aspect, the cells from the circulating blood of the individual are obtained by apheresis. Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or culture medium for subsequent processing steps. Multiple rounds of selection can also be used in the context of the present application. In some aspects, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "Unselected" cells can also undergo additional rounds of selection.
本申请的组合物能够调节肿瘤微环境。The composition of the present application can regulate the tumor microenvironment.
未纯化的CTL来源可以是本领域已知的任何来源，例如骨髓、胎儿、新生儿或成年或其它造血细胞来源，例如胎儿肝、外周血或脐带血。可以采用各种技术来分离细胞。例如，阴性选择法可以最初去除非CTL。mAb对于鉴定与特定细胞谱系和/或阳性和阴性选择的分化阶段相关的标志物特别有用。The source of unpurified CTLs can be any source known in the art, such as bone marrow, fetal, neonatal or adult or other source of hematopoietic cells, such as fetal liver, peripheral blood or umbilical cord blood. Cells can be isolated using various techniques. For example, negative selection can initially remove non-CTLs. mAbs are particularly useful for identifying markers associated with specific cell lineages and/or differentiation stages of positive and negative selection.
最初可以通过相对粗略的分离除去大部分末端分化的细胞。例如，最初可以使用磁珠分离来去除大量不相关的细胞。在某些实施方式中，在分离细胞之前将去除总造血细胞的至少约80％，通常至少约70％。Most of the terminally differentiated cells can be removed initially by relatively rough dissection. For example, magnetic bead separation can be used initially to remove large numbers of irrelevant cells. In certain embodiments, at least about 80%, usually at least about 70%, of the total hematopoietic cells will be removed prior to isolating the cells.
分离的程序包括但不限于密度梯度离心；重沉(resetting)；偶联至改变细胞密度的颗粒；用抗体包被的磁珠进行磁分离；亲和色谱；与mAb结合或结合使用的细胞毒性剂，包 括但不限于补体和细胞毒素；并用附着在固体基质(例如板、芯片、淘析)上的抗体淘选或任何其它方便的技术。Separation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; agents, including but not limited to complement and cytotoxins; and panning with antibodies attached to a solid substrate (eg, plate, chip, elutriation) or any other convenient technique.
分离和分析的技术包括但不限于流式细胞术，其可以具有不同的复杂程度，例如多个颜色通道、低角度和钝角光散射检测通道、阻抗通道。Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low- and obtuse-angle light-scattering detection channels, impedance channels.
通过使用与死细胞相关的染料，例如碘化丙啶(PI)，可以针对死细胞选择细胞。在某些实施方式中，将细胞收集在包括2％胎牛血清(FCS)或0.2％牛血清白蛋白(BSA)的培养基或任何其它合适的例如无菌等渗培养基中。Cells can be selected for dead cells by using dyes associated with dead cells, such as propidium iodide (PI). In certain embodiments, cells are harvested in medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, eg, sterile isotonic medium.
在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，本申请的组合物中的免疫细胞具有更长存活时间和/或扩增能力。在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，本申请的组合物中的第二免疫细胞具有更长存活时间和/或扩增能力。在一实例中，与第一免疫细胞或第二免疫细胞相比，包括第一免疫细胞和第二免疫细胞的的组合物对携带靶肿瘤抗原的细胞表现出更强的体内外的细胞杀伤作用。在一实例中，第一免疫细胞和/或第二免疫细胞中参与响应自体和非自体抗原识别多肽的至少一种内源基因表达、活性和/或信号被降低或抑制；包含所述第一和第二免疫细胞的细胞组合物不引起移植物抗宿主反应。In one example, in the presence of host immune cells (eg, NK cells), the immune cells in the composition of the present application have a longer survival time and/or ability to expand. In one example, the second immune cells in the composition of the present application have longer survival time and/or expansion ability in the presence of host immune cells (eg, NK cells). In one example, compared with the first immune cell or the second immune cell, the composition comprising the first immune cell and the second immune cell exhibits a stronger cell killing effect in vivo and in vitro on cells carrying a target tumor antigen . In one example, the expression, activity and/or signaling of at least one endogenous gene involved in the response to self and non-self antigen recognition polypeptides in the first immune cell and/or the second immune cell is reduced or inhibited; comprising said first The cellular composition of the second immune cell does not cause a graft-versus-host reaction.
本申请的第一免疫细胞和第二免疫细胞的种类可以是相同的也可以是不同的。第一免疫细胞或第二免疫细胞可以是已知的任何合适的免疫细胞，第一免疫细胞的种类不影响第二免疫细胞的种类。例如，第一免疫细胞可以是T、NKT细胞，第二免疫细胞可以是T、NKT细胞或者其他任何合适的免疫细胞；例如，第二免疫细胞可以是T、NKT细胞，第二免疫细胞可以是T、NKT细胞或者其他任何合适的免疫细胞；在一实例中，第一免疫细胞、第二免疫细胞包含T、NKT细胞。The types of the first immune cell and the second immune cell in this application may be the same or different. The first immune cell or the second immune cell may be any known suitable immune cell, and the type of the first immune cell does not affect the type of the second immune cell. For example, the first immune cell can be T, NKT cell, and the second immune cell can be T, NKT cell or any other suitable immune cell; for example, the second immune cell can be T, NKT cell, and the second immune cell can be T, NKT cells or any other suitable immune cells; in one example, the first immune cells and the second immune cells include T, NKT cells.
本申请的第一免疫细胞和第二免疫细胞的来源/制备方法可以是相同的也可以是不同的。第一免疫细胞或第二免疫细胞可以是通过已知的任何方法制备获得的免疫细胞，第一免疫细胞的制备方法不影响第二免疫细胞的制备方法。例如，第一免疫细胞可以是分离自供体的天然免疫细胞，第二免疫细胞可以是分离自供体的天然免疫细胞或者通过其他任何已知的方法制备所得的免疫细胞(例如衍生自神经元、上皮细胞、内皮细胞或干细胞的免疫细胞)。例如，第二免疫细胞可以是分离自供体的天然免疫细胞，第一免疫细胞可以是分离自供体的天然免疫细胞或者通过其他任何已知的方法制备所得的免疫细胞(例如衍生自神经元、上皮细胞、内皮细胞或干细胞的免疫细胞)。The source/preparation method of the first immune cell and the second immune cell of the present application may be the same or different. The first immune cell or the second immune cell may be prepared by any known method, and the preparation method of the first immune cell does not affect the preparation method of the second immune cell. For example, the first immune cell can be an innate immune cell isolated from a donor, and the second immune cell can be an innate immune cell isolated from a donor or an immune cell prepared by any other known method (for example, derived from a neuron , epithelial cells, endothelial cells or stem cells of immune cells). For example, the second immune cell can be an innate immune cell isolated from a donor, and the first immune cell can be an innate immune cell isolated from a donor or an immune cell prepared by any other known method (for example, derived from a neuron). , epithelial cells, endothelial cells or stem cells of immune cells).
第一免疫细胞或第二免疫细胞可以是已知的任何来源的免疫细胞，第一免疫细胞的来源不影响第二免疫细胞的来源。例如，第一免疫细胞可以是自体免疫细胞，第二免疫细胞可以是自体细胞或者其他任何合适来源(例如，同种异体的)的免疫细胞；例如，第二免 疫细胞可以是自体免疫细胞，第一免疫细胞可以是自体细胞或者其他任何合适来源(例如，同种异体的)的免疫细胞；在一实例中，第一免疫细胞、第二免疫细胞包含同种异体的免疫细胞。The first immune cell or the second immune cell can be any known source of immune cells, and the source of the first immune cell does not affect the source of the second immune cell. For example, the first immune cell can be an autologous immune cell, and the second immune cell can be an autologous cell or an immune cell of any other suitable source (e.g., allogeneic); for example, the second immune cell can be an autologous immune cell, and the second immune cell can be an autologous immune cell. An immune cell can be an autologous cell or an immune cell from any other suitable source (eg, allogeneic); in one example, the first immune cell and the second immune cell include allogeneic immune cells.
本申请的细胞组合物中的免疫细胞还可以包括移植物抗宿主病(GVHD)和/或宿主抗移植物反应(HVGR)相关内源性基因的基因修饰。例如所述基因修饰可以包括HLA-I类分子、TCR和/或HLA-II类分子低表达或不表达；例如所述基因修饰还可以包括NKG2A分子低表达或不表达。低表达或不表达分别是指细胞中TCR、B2M、HLA-II或NKG2A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，TCR、B2M、HLA-II或NKG2A低表达或不表达分别是指细胞中TCR、B2M、HLA-II或NKG2A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TCR、B2M、HLA-II或NKG2A的特异性抗体测定细胞中蛋白的表达或含量。The immune cells in the cell composition of the present application may also include genetic modification of endogenous genes related to graft-versus-host disease (GVHD) and/or host-versus-graft reaction (HVGR). For example, the genetic modification may include low or no expression of HLA-I molecules, TCR and/or HLA-II molecules; for example, the genetic modification may also include low or no expression of NKG2A molecules. Low expression or no expression means that the expression of TCR, B2M, HLA-II or NKG2A in cells 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, low or no expression of TCR, B2M, HLA-II or NKG2A means that the content of TCR, B2M, HLA-II or NKG2A in cells is reduced by at least 1%, at least 5%, at least 10%, at least 20%, respectively. %, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The amount 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 antibodies specific for TCR, B2M, HLA-II, or NKG2A. expression or content.
第一免疫细胞与第二免疫细胞的基因修饰不必是相同的，其各自独立的进行内源性基因修饰，例如第一免疫细胞包含TCR的修饰，第二免疫细胞包含B2M的修饰，或同时包括两者的修饰。包含TCR、B2M、HLA-II或NKG2A的任意组合的基因修饰均在本申请范围内。The genetic modification of the first immune cell and the second immune cell need not be the same, and they are independently endogenously modified, for example, the first immune cell contains the modification of TCR, and the second immune cell contains the modification of B2M, or both Modifications for both. Genetic modifications comprising any combination of TCR, B2M, HLA-II or NKG2A are within the scope of this application.
HLA-I类分子低表达或不表达Low expression or no expression of HLA class I molecules
由于供体和受体(或称为宿主)之间的免疫遗传学差异，在进行外源供体移植时，作为外源移植物的供体会受到宿主体内的免疫细胞(例如NK细胞)识别和攻击，进而抑制或者清除供体(例如第一免疫细胞、第二免疫细胞)，产生宿主抗移植物反应(HVGR)。如在异体细胞(例如第一免疫细胞、第二免疫细胞)移植中，当异体细胞的HLA-I类分子的缺失，可以降低宿主CD8+介导的细胞免疫排斥作用。Due to the immunogenetic differences between the donor and the recipient (or host), when exogenous donor transplantation is performed, the donor as an exogenous graft will be recognized and recognized by immune cells (such as NK cells) in the host. Attack, and then inhibit or eliminate the donor (such as the first immune cell, the second immune cell), resulting in host-versus-graft response (HVGR). For example, in the transplantation of allogeneic cells (such as the first immune cell and the second immune cell), when the HLA-I molecules of the allogeneic cells are deleted, the host CD8+-mediated cellular immune rejection can be reduced.
在一实例中，本申请提供细胞组合物：包括识别NKG2A且内源性B2M低表达或不表达的第一免疫细胞，和/或识别肿瘤和/或病原体抗原且内源性B2M低表达或不表达的第二免疫细胞。在一实例中，本申请提供细胞组合物：包括识别NKG2A多肽和肿瘤抗原、且内源性B2M低表达或不表达的第一免疫细胞，和/或识别肿瘤抗原且内源性B2M低表达或不表达的第二免疫细胞。In one example, the present application provides a cell composition: comprising a first immune cell that recognizes NKG2A and has low or no endogenous B2M expression, and/or recognizes tumor and/or pathogen antigens and has low or no endogenous B2M expression Expressed secondary immune cells. In one example, the present application provides a cell composition: including first immune cells that recognize NKG2A polypeptides and tumor antigens and have low or no expression of endogenous B2M, and/or recognize tumor antigens and have low or no expression of endogenous B2M Secondary immune cells that do not express.
TCR低表达或不表达TCR low expression or no expression
移植物抗宿主病(GVHD)是由于外源移植供体T淋巴细胞的TCR的多样性，以及 与宿主HLA分子的不兼容性，供体T淋巴细胞会识别宿主正常组织上的抗原，经扩增并释放一系列细胞因子，大大增强了移植物对宿主抗原的免疫反应，攻击宿主细胞。在一实例中，本申请采用CRISPR***敲除内源性TCR的α链的基因TRAC制备得到内源性TCR低表达或不表达的细胞。Graft-versus-host disease (GVHD) is due to the diversity of TCR of exogenously transplanted donor T lymphocytes and the incompatibility with host HLA molecules. It increases and releases a series of cytokines, which greatly enhances the immune response of the graft to host antigens and attacks the host cells. In one example, the present application uses the CRISPR system to knock out the gene TRAC of the α chain of the endogenous TCR to prepare cells with low or no expression of the endogenous TCR.
在一实例中，本申请提供细胞组合物：包括识别NKG2A、且内源性TCR低表达或不表达的第一免疫细胞,和/或识别肿瘤和/或病原体抗原、且内源性TCR低表达或不表达的第二免疫细胞；任选地，所述第一和/或第二免疫细胞内源性B2M低表达或不表达。在一实例中，本申请提供细胞组合物：包括识别NKG2A和肿瘤抗原、且内源性TCR低表达或不表达的第一免疫细胞，和/或识别肿瘤抗原、且内源性TCR低表达或不表达的第二免疫细胞；任选地，所述第一和/或第二免疫细胞内源性B2M低表达或不表达。In one example, the present application provides a cell composition: including a first immune cell that recognizes NKG2A and has low or no expression of endogenous TCR, and/or recognizes tumor and/or pathogen antigens and has low expression of endogenous TCR or non-expressing second immune cells; optionally, the first and/or second immune cells have low or no expression of endogenous B2M. In one example, the present application provides a cell composition: including first immune cells that recognize NKG2A and tumor antigens and have low or no endogenous TCR expression, and/or recognize tumor antigens and have low or no endogenous TCR expression A second immune cell that does not express; optionally, the first and/or second immune cell expresses low or no endogenous B2M.
HLA-II低表达或不表达Low or no expression of HLA-II
HLA-II基因编码HLA-II类抗原，将细胞外抗原呈递给CD4+T细胞，促进CD4+T细胞增殖，进而刺激B细胞产生抗原特异性抗体,主要诱导体液免疫进行免疫排斥。MHC-Ⅱ反式激活蛋白(CIITA)是一类HLA-II的正调控因子，可协调多种转录因子与HLA-Ⅱ基因启动子作用，调控HLA-II的表达。The HLA-II gene encodes HLA-II antigens, presents extracellular antigens to CD4+ T cells, promotes the proliferation of CD4+ T cells, and then stimulates B cells to produce antigen-specific antibodies, mainly inducing humoral immunity for immune rejection. MHC-II transactivator (CIITA) is a kind of positive regulator of HLA-II, which can coordinate the action of various transcription factors and HLA-II gene promoter to regulate the expression of HLA-II.
在一实例中，本申请提供细胞组合物：包括识别NKG2A、且内源性HLA-II低表达或不表达的第一免疫细胞，和/或识别肿瘤和/或病原体抗原、且内源性HLA-II低表达或不表达的第二免疫细胞；任选地，所述第一和/或第二免疫细胞内源性B2M低表达或不表达、内源性TCR低表达或不表达、或内源性B2M/TCR低表达或不表达。在一实例中，本申请提供细胞组合物：包括识别NKG2A和肿瘤抗原、且内源性HLA-II低表达或不表达的第一免疫细胞，和/或识别肿瘤抗原、且内源性HLA-II低表达或不表达的第二免疫细胞；任选地，所述第一和/或第二免疫细胞内源性B2M低表达或不表达、内源性TCR低表达或不表达、或内源性B2M/TCR低表达或不表达。In one example, the present application provides a cell composition: including first immune cells that recognize NKG2A and have low or no expression of endogenous HLA-II, and/or recognize tumor and/or pathogen antigens, and endogenous HLA-II -Second immune cells with low or no expression of II; optionally, the first and/or second immune cells have low or no expression of endogenous B2M, low or no expression of endogenous TCR, or endogenous TCR Low expression or no expression of derived B2M/TCR. In one example, the present application provides a cell composition: including first immune cells that recognize NKG2A and tumor antigens, and endogenous HLA-II is low or not expressed, and/or recognize tumor antigens, and endogenous HLA-II Second immune cells with low or no expression of II; optionally, the first and/or second immune cells have low or no expression of endogenous B2M, low or no expression of endogenous TCR, or endogenous Sexual B2M/TCR low expression or no expression.
NKG2A低表达或不表达Low or no expression of NKG2A
在靶细胞(例如表达靶抗原的肿瘤细胞)反复刺激下，外源移植物的供体免疫细胞中内源性NKG2A表达上调，会被本申请细胞组合物中识别NKG2A的免疫细胞杀伤。此外，NKG2A低表达或不表达可能解除免疫细胞本身的抑制作用，从而发挥更强的抗肿瘤能力。Under repeated stimulation of target cells (such as tumor cells expressing target antigens), the expression of endogenous NKG2A in donor immune cells of exogenous grafts is up-regulated, and will be killed by immune cells that recognize NKG2A in the cell composition of the present application. In addition, low expression or no expression of NKG2A may release the inhibitory effect of immune cells themselves, thus exerting stronger anti-tumor ability.
在一实例中，本申请提供细胞组合物：包括识别NKG2A、且内源性NKG2A低表达或不表达的第一免疫细胞,和/或识别肿瘤和/或病原体抗原、且内源性NKG2A低表达或不表达的第二免疫细胞；任选地，所述第一和/或第二免疫细胞内源性B2M低表达或不表达、内源性TCR低表达或不表达、内源性HLA-II低表达或不表达、内源性B2M/TCR低表达或 不表达、内源性B2M/HLA-II低表达或不表达、内源性TCR/HLA-II低表达或不表达、或内源性B2M/TCR/HLA-II低表达或不表达。在一实例中，本申请提供细胞组合物：包括识别NKG2A和肿瘤抗原、且内源性NKG2A低表达或不表达的第一免疫细胞,和/或识别肿瘤和/或病原体抗原、且内源性NKG2A低表达或不表达的第二免疫细胞；任选地，所述第一和/或第二免疫细胞内源性B2M低表达或不表达、内源性TCR低表达或不表达、内源性HLA-II低表达或不表达、内源性B2M/TCR低表达或不表达、内源性B2M/HLA-II低表达或不表达、内源性TCR/HLA-II低表达或不表达、或内源性B2M/TCR/HLA-II低表达或不表达。In one example, the present application provides a cell composition: comprising a first immune cell that recognizes NKG2A and has low or no expression of endogenous NKG2A, and/or recognizes tumor and/or pathogen antigens and has low expression of endogenous NKG2A or non-expressing second immune cells; optionally, the first and/or second immune cells have low or no expression of endogenous B2M, low or no expression of endogenous TCR, endogenous HLA-II Low or no expression, low or no expression of endogenous B2M/TCR, low or no expression of endogenous B2M/HLA-II, low or no expression of endogenous TCR/HLA-II, or endogenous Low expression or no expression of B2M/TCR/HLA-II. In one example, the present application provides a cell composition: including first immune cells that recognize NKG2A and tumor antigens, and endogenous NKG2A low expression or no expression, and/or recognize tumor and/or pathogen antigens, and endogenous The second immune cell with low or no expression of NKG2A; optionally, the first and/or second immune cell has low or no expression of endogenous B2M, low or no expression of endogenous TCR, endogenous Low or no expression of HLA-II, low or no expression of endogenous B2M/TCR, low or no expression of endogenous B2M/HLA-II, low or no expression of endogenous TCR/HLA-II, or Low expression or no expression of endogenous B2M/TCR/HLA-II.
载体carrier
对组合物中免疫细胞(例如，T细胞或NKT细胞)的遗传修饰可以通过用重组核酸分子转导基本上均质的细胞群来完成。在一实例中，逆转录病毒载体(γ-逆转录病毒或慢病毒)用于将核酸分子引入细胞。例如，可以将编码外源受体(例如CAR)的多核苷酸克隆到逆转录病毒载体。也可以使用非病毒载体。转导可以使用任何合适的病毒载体或非病毒递送***。可以在单个多顺反子表达盒、单个载体的多个表达盒或多个载体中用辅助分子(例如细胞因子)构建CAR。产生多顺反子表达盒的元件的实例包括但不限于各种病毒和非病毒内部核糖体进入位点(IRES，例如，FGF-1IRES、FGF-2IRES、VEGF IRES、IGF-II IRES、NF-κB IRES、RUNX1IRES、p53IRES、甲型肝炎IRES、丙型肝炎IRES、瘟病毒IRES、无杆状病毒IRES、小核糖核酸病毒IRES、脊髓灰质炎病毒IRES和脑心肌炎病毒IRES)和可切割的接头(例如2A肽，例如P2A、T2A、E2A和F2A肽)。Genetic modification of immune cells (eg, T cells or NKT cells) in the composition can be accomplished by transducing a substantially homogeneous population of cells with a recombinant nucleic acid molecule. In one example, retroviral vectors (gamma-retroviruses or lentiviruses) are used to introduce nucleic acid molecules into cells. For example, a polynucleotide encoding a foreign receptor (eg, CAR) can be cloned into a retroviral vector. Non-viral vectors can also be used. Transduction can use any suitable viral vector or non-viral delivery system. CARs can be constructed with accessory molecules (eg, cytokines) in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF- κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, abaculovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers ( For example 2A peptides such as P2A, T2A, E2A and F2A peptides).
可以使用的其它病毒载体包括，例如，腺病毒、慢病毒和与腺相关的病毒载体、牛痘病毒、牛***瘤病毒或疱疹病毒，例如爱泼斯坦-巴尔病毒。Other viral vectors that may be used include, for example, adenovirus, lentivirus and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus or herpes viruses such as Epstein-Barr virus.
非病毒方法也可以用于免疫细胞的遗传修饰。例如，可以通过在脂质转染，脱唾液酸血清类粘蛋白-聚赖氨酸偶联，或手术条件下的微注射将核酸分子引入免疫细胞中。其它非病毒的基因转移方法包括使用脂质体、磷酸钙、DEAE葡聚糖、电穿孔和原生质体融合的体外转染。也可以先将核酸分子转移到可离体培养的细胞类型(例如，自体或同种异体原代细胞或其后代)中，再将经所述核酸分子修饰后的细胞(或其后代)注射到受试者目标组织中或全身注射。Non-viral methods can also be used for the genetic modification of immune cells. For example, nucleic acid molecules can be introduced into immune cells by microinjection under lipofection, asialomucoid-polylysine coupling, or surgical conditions. Other non-viral methods of gene transfer include in vitro transfection using liposomes, calcium phosphate, DEAE-dextran, electroporation and protoplast fusion. It is also possible to first transfer the nucleic acid molecule into a cell type that can be cultured in vitro (for example, an autologous or allogeneic primary cell or its progeny), and then inject the cell (or its progeny) modified by the nucleic acid molecule into Subject target tissue or systemic injection.
基因敲除gene knockout
采用基因敲除技术和/或基因沉默技术来制备内源性TCR、B2M、HLA-II或NKG2A低表达或不表达的工程细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性NKG2A低表达或不表达的工程细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M低表达或不表达的工程细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M/HLA-II低表达或不表达的工程细胞。 在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M/NKG2A低表达或不表达的工程细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M/HLA-II/NKG2A低表达或不表达的工程细胞。Use gene knockout technology and/or gene silencing technology to prepare engineered cells with low or no expression of endogenous TCR, B2M, HLA-II or NKG2A. In one example, gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous NKG2A. In one example, gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M. In one example, gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M/HLA-II. In one example, gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M/NKG2A. In one example, gene knockout technology and/or gene silencing technology are used to prepare engineered cells with low or no expression of endogenous TCR/B2M/HLA-II/NKG2A.
基因敲除技术包括Argonaute、CRISPR/Cas9技术、ZFN技术、TALE技术、TALE-CRISPR/Cas9技术、Base Editor技术、引导编辑技术和/或归巢核酸内切酶技术。基因沉默技术包括但不限于：反义RNA、RNA干扰、微小RNA介导的翻译抑制等。Gene knockout technologies include Argonaute, CRISPR/Cas9 technology, ZFN technology, TALE technology, TALE-CRISPR/Cas9 technology, Base Editor technology, guided editing technology and/or homing endonuclease technology. Gene silencing techniques include, but are not limited to: antisense RNA, RNA interference, microRNA-mediated translational inhibition, etc.
成簇的规律间隔的短回文重复序列(CRISPR)***用于基因组编辑。该***包括Cas9(一种能够使用crRNA作为其向导来修饰DNA的蛋白质)，CRISPR RNA(crRNA，包含Cas9用来引导其到达宿主DNA正确片段的RNA，以及与tracrRNA结合的区域(通常以发夹环形式)，与Cas9形成活性复合物)，反式激活crRNA(tracrRNA，与crRNA结合，与Cas9形成活性复合物)，以及DNA修复模板的可选片段(可指导细胞修复过程允许***特定的DNA序列的DNA)。CRISPR/Cas9通常采用质粒、或电转方式传递核酸片段到靶细胞。crRNA需要针对每种应用进行设计，因为这是Cas9用来识别并直接结合细胞中靶DNA的序列。多个crRNA和tracrRNA可以包装在一起以形成指导RNA(gRNA)。该gRNA可以与Cas9基因连接在一起并制成质粒，以便被转染到细胞中。本发明凡涉及gRNA的序列时，其可以为靶向的DNA序列，亦可以为所述DNA对应的核糖核苷酸与crRNA、TracrRNA形成的完整Cas9引导序列。在进行基因编辑时，施用的gRNA、tracr配对序列及tracr序列可以单独施用，也可以一条完整的RNA序列施用。CRISPR/Cas9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。The clustered regularly interspaced short palindromic repeat (CRISPR) system is used for genome editing. The system consists of Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, comprising the RNA that Cas9 uses to guide it to the correct segment of host DNA, and a region (usually in the form of a hairpin) that binds to tracrRNA. loop form), which forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA, forms an active complex with Cas9), and an optional segment of the DNA repair template (which directs the cellular repair process to allow the insertion of specific DNA sequence of DNA). CRISPR/Cas9 usually uses plasmids or electroporation to deliver nucleic acid fragments to target cells. crRNA needs to be designed for each application because this is the sequence that Cas9 uses to recognize and directly bind to target DNA in cells. Multiple crRNAs and tracrRNAs can be packaged together to form guide RNAs (gRNAs). This gRNA can be ligated with the Cas9 gene and made into a plasmid to be transfected into cells. When the present invention relates to the sequence of gRNA, it may be a targeted DNA sequence, or it may be a complete Cas9 guide sequence formed by ribonucleotides corresponding to the DNA, crRNA and TracrRNA. When performing gene editing, the administered gRNA, tracr paired sequence and tracr sequence can be administered alone, or a complete RNA sequence can be administered. CRISPR/Cas9 transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
锌指核酸酶(ZFN)是一种人工限制性酶，通过将锌指DNA结合结构域与DNA切割结构域结合而产生。锌指结构域可以被工程化以靶向特定的DNA序列，其允许锌指核酸酶靶向基因组内的靶序列。Zinc finger nuclease (ZFN) is an artificial restriction enzyme produced by combining a zinc finger DNA binding domain with a DNA cleavage domain. Zinc finger domains can be engineered to target specific DNA sequences that allow zinc finger nucleases to target sequences within the genome.
转录激活因子样效应物核酸酶(TALEN)是限制性酶，可以工程化为切割DNA的特定序列。TALEN***的工作原理几乎与ZFN相同。它们是通过将转录激活因子样效应物DNA结合结构域与DNA切割结构域结合而产生的。Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. The TALEN system works almost the same as the ZFN. They are produced by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain.
本申请还提供了编码本文所述的一种或多种外源受体(例如CAR)的核酸分子，和靶向内源性TCR、B2M、CIITA或NKG2A的核酸抑制分子或gRNA的核酸分子。The present application also provides nucleic acid molecules encoding one or more exogenous receptors described herein (such as CAR), and nucleic acid molecules targeting endogenous TCR, B2M, CIITA or NKG2A nucleic acid inhibitory molecules or gRNA.
示例性，靶向NKG2A、TRAC、B2M、CIITA的gRNA分别包括SEQ ID NO：10、46、47、48所示序列。在一实例中，将编码分别识别NKG2A、靶抗原(示例性，BCMA)的CAR引入T细胞中以产生本申请组合物中的工程细胞，任选地，将靶向内源性TCR、B2M、CIITA和/或NKG2A的核酸抑制分子或gRNA的核酸分子引入工程细胞。在一个实例 中，体外转录的CAR核酸分子、靶向内源性TCR、B2M、CIITA或NKG2A的核酸抑制分子或gRNA可作为瞬时转染的形式引入细胞中。示例性人工DNA序列是包括连接在一起以形成编码融合蛋白的开放阅读框的基因部分的序列。连接在一起的DNA部分可来自单个生物体或来自多个生物体。Exemplarily, gRNAs targeting NKG2A, TRAC, B2M, and CIITA include sequences shown in SEQ ID NO: 10, 46, 47, and 48, respectively. In one example, CARs encoding NKG2A and target antigens (exemplary, BCMA) are introduced into T cells to generate engineered cells in the composition of the present application. Optionally, targeting endogenous TCR, B2M, CIITA and/or NKG2A nucleic acid inhibitory molecules or gRNA nucleic acid molecules are introduced into engineered cells. In one example, in vitro transcribed CAR nucleic acid molecules, nucleic acid inhibitory molecules or gRNAs targeting endogenous TCR, B2M, CIITA, or NKG2A can be introduced into cells as a transient transfection. An exemplary artificial DNA sequence is a sequence comprising portions of a gene joined together to form an open reading frame encoding a fusion protein. The DNA portions joined together can be from a single organism or from multiple organisms.
药物组合物pharmaceutical composition
本申请提供了药物组合物，其包括有效量的本申请提供的细胞组合物和药学上可接受的佐剂。所述药物组合物可以是混合物的形式也可以是不同的细胞活性成分(例如第一工程细胞、第二工程细胞)分开制备、存储、放置的形式。The application provides a pharmaceutical composition, which includes an effective amount of the cell composition provided by the application and a pharmaceutically acceptable adjuvant. The pharmaceutical composition may be in the form of a mixture or in the form of separate preparation, storage and placement of different cell active components (eg first engineered cells, second engineered cells).
在一实例中，所述药物组合物包含第一制剂和第二制剂，所述第一制剂包含所述识别NKG2A的第一工程细胞和药学上可接受的第一佐剂，所述第二制剂包含所述识别非NKG2A的抗原的第二工程细胞和药学上可接受的第二佐剂。例如，所述第一制剂与所述第二制剂可以是相同的剂型可以是不同的剂型。例如，所述第一制剂和第二制剂可以分别置于不同的容器中。在一实例中，所述药物组合物包含药物混合物，所述药物混合物包含所述识别NKG2A的第一工程细胞与所述识别非NKG2A的抗原的第二工程细胞。包括本申请的药物组合物可以方便地以无菌液体制剂的形式提供，例如等渗水溶液剂、悬浮液、乳剂、分散剂或粘性组合物，其可以缓冲至选定的pH。液体制剂通常比凝胶、其它粘性组合物和固体组合物更容易制备。另外，液体组合物在某种程度上更方便施用，尤其是通过注射。另一方面，可以在适当的粘度范围内配制粘性组合物以提供与特定组织的更长的接触时间。液体或粘性组合物可以包括载体，所述载体可以是溶剂或分散介质，其包括例如水、盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇等)及其合适的混合物。In one example, the pharmaceutical composition comprises a first preparation and a second preparation, the first preparation comprises the first engineered cells recognizing NKG2A and a pharmaceutically acceptable first adjuvant, the second preparation The second engineered cell comprising the antigen recognizing non-NKG2A and the second pharmaceutically acceptable adjuvant. For example, the first formulation and the second formulation may be in the same dosage form or in different dosage forms. For example, the first formulation and the second formulation may be placed in different containers, respectively. In one example, the pharmaceutical composition comprises a pharmaceutical mixture, and the pharmaceutical mixture comprises the first engineered cells recognizing NKG2A and the second engineered cells recognizing antigens other than NKG2A. Pharmaceutical compositions comprising the present application may conveniently be presented in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to the selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable suitable ones. mixture.
可以通过将本申请药物组合物中的工程细胞掺入所需量的适当溶剂中，并根据需要掺入不同量的其它成分来制备无菌注射溶液。这样的组合物可以与合适的载体、稀释剂或赋形剂例如无菌水、生理盐水、葡萄糖、右旋糖等混合。组合物也可以冻干。所述组合物可包括辅助物质，例如润湿剂、分散剂或乳化剂(例如，甲基纤维素)、pH缓冲剂、胶凝剂或增粘剂、防腐剂、矫味剂、颜料等，这取决于给药途径和所需制剂。Sterile injectable solutions can be prepared by mixing the engineered cells in the pharmaceutical composition of the present application into a required amount of an appropriate solvent, and adding different amounts of other ingredients as required. Such compositions can be mixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, glucose, dextrose and the like. Compositions can also be lyophilized. The composition may include auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-increasing agents, preservatives, flavoring agents, pigments, etc., This depends on the route of administration and formulation desired.
可以添加增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。可以通过各种抗细菌和抗真菌剂，例如对羟基苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等来确保防止微生物的作用。可通过使用延迟吸收的试剂例如单硬脂酸铝和明胶来延长可注射药物形式的吸收。然而，所使用的任何媒介物、稀释剂或添加剂将必须与遗传修饰的免疫细胞或其祖细胞相容。Various additives can be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical forms can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immune cells or progenitors thereof.
该组合物可以是等渗的，即它们可以具有与血液和/或泪液相同的渗透压。组合物的 所需等渗性可以使用氯化钠或其它药学上可接受的试剂例如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。氯化钠可以特别适用于含有钠离子的缓冲剂。The compositions may be isotonic, ie they may have the same osmotic pressure as blood and/or tear fluid. The desired isotonicity of the compositions can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride may be particularly useful for buffers containing sodium ions.
如果需要，可使用药学上可接受的增稠剂将组合物的粘度保持在选定水平。例如，甲基纤维素容易且经济地获得并且易于使用。其它合适的增稠剂包括，例如，黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的浓度可以取决于选择的试剂。重要的是要使用能够达到所选粘度的用量。显然，合适载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型(例如，是否将组合物配制成溶液剂、悬浮液、凝胶剂或其它液体形式，例如定时释放形式或液体填充形式)。If desired, a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level. For example, methylcellulose is readily and economically available and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The concentration of the thickener can depend on the agent chosen. It is important to use the amount that will achieve the chosen viscosity. Obviously, the selection of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or other liquid form, e.g. time-release or liquid-filled form).
对于所治疗的受试者，要施用的组合物中的工程细胞数量将有所不同。可以更少的数量施用更有效的工程细胞。可以根据每个受试者的个体因素，包括其大小、年龄、性别、体重和受试者的状况，来确定有效剂量的精确确定。本领域技术人员从本申请和本领域知识中可以容易地确定剂量。The number of engineered cells in the composition to be administered will vary for the subject being treated. More effective engineered cells can be administered in smaller numbers. The precise determination of an effective dose can be determined according to each subject's individual factors, including its size, age, sex, weight and the condition of the subject. Dosages can be readily determined by those skilled in the art from this application and knowledge in the art.
本领域技术人员可以容易地确定组合物中和在方法中施用的工程细胞和任选的添加剂、媒介物和/或载体的量。通常，任何添加剂(除一种或多种活性细胞和/或一种或多种试剂外)在磷酸盐缓冲盐水中的存在量为0.001％至50％(重量)溶液，并且活性成分按微克至毫克的顺序存在，例如约0.0001wt％至约5wt％、约0.0001wt％至约1wt％、约0.0001wt％至约0.05wt％或约0.001wt％至约20wt％、约0.01wt％至约10wt％或约0.05wt％至约5wt％。对于要施用于动物或人的任何组合物，可以确定以下结果：毒性，例如通过在合适的动物模型例如啮齿类动物如小鼠中确定致死剂量(LD)和LD50；组合物的剂量，其中的组分浓度和施用组合物的时间，引起合适的反应。A person skilled in the art can readily determine the amount of engineered cells and optional additives, vehicles and/or carriers to be administered in the composition and in the methods. Typically, any additives (other than one or more active cells and/or one or more reagents) are present in 0.001% to 50% by weight solution in phosphate-buffered saline, and the active ingredient is present in micrograms to The order of milligrams is present, for example from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.05 wt%, or from about 0.001 wt% to about 20 wt%, from about 0.01 wt% to about 10 wt% % or from about 0.05 wt% to about 5 wt%. For any composition to be administered to animals or humans, the following results can be determined: toxicity, for example by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g. rodents such as mice; the dose of the composition, wherein The concentration of the components and the time of application of the composition elicit an appropriate response.
方法或用途method or use
本申请提供了本申请的细胞组合物的用途、及所述细胞组合物在制备药物中的用途，所述细胞组合物或所述药物用于预防和/或治疗实体瘤、血液瘤、自身免疫性疾病或其组合。本申请提供的细胞组合物已在前文描述，本申请提供的细胞组合物在制备药物中的用途包括其全部技术方案。The application provides the use of the cell composition of the application, and the use of the cell composition in the preparation of medicines, and the cell composition or the medicine is used for the prevention and/or treatment of solid tumors, blood tumors, autoimmune disease or a combination thereof. The cell composition provided by this application has been described above, and the use of the cell composition provided by this application in the preparation of medicine includes all technical solutions thereof.
本申请提供了预防、缓解和/或***的方法，其包括向有需要的受试者施用所述的细胞组合物、所述的药物组合物。本申请提供的细胞组合物、药物组合物已在前文描述，本申请提供的预防、缓解和/或***的方法包括其全部技术方案。The present application provides a method for preventing, alleviating and/or treating tumors, which includes administering the cell composition and the pharmaceutical composition to a subject in need. The cell composition and pharmaceutical composition provided in this application have been described above, and the method for preventing, alleviating and/or treating tumors provided in this application includes all technical solutions thereof.
本申请提供了预防、缓解和/或***的方法，其包括向有需要的受试者施用本申请所述的第一工程细胞和第二工程细胞，第一工程细胞和第二工程细胞可以先后或同时施用。例如先施用第一工程细胞，然后施用第二工程细胞，或者例如先施用第二工程细胞，然后施用第一工程细胞。或者同时向受试者施用第一工程细胞和第二工程细胞。本申请第 一工程细胞和第二工程细胞已在前文描述，本申请提供的预防、缓解和/或***的方法包括其全部技术方案。The present application provides a method for preventing, alleviating and/or treating tumors, which includes administering the first engineered cell and the second engineered cell described in the present application to a subject in need, and the first engineered cell and the second engineered cell can be administered sequentially or simultaneously. For example, the first engineered cells are administered first, and then the second engineered cells are administered, or, for example, the second engineered cells are administered first, and then the first engineered cells are administered. Or the first engineered cell and the second engineered cell are administered to the subject at the same time. The first engineered cell and the second engineered cell of this application have been described above, and the method for preventing, alleviating and/or treating tumors provided by this application includes all technical solutions thereof.
本申请提供的预防、缓解和/或***的方法包括用于在需要本申请的细胞组合物的受试者中诱导和/或增加免疫应答的方法。本申请的组合物可以用于治疗和/或预防受试者的肿瘤。本申请的细胞组合物可以用于延长患有肿瘤的受试者的存活。本申请的细胞组合物也可以用于治疗和/或预防诸如免疫功能低下的受试者的病原体感染或其它感染性疾病。这种方法包括施用有效量的本申请的细胞组合物以达到期望的效果，无论是减轻现有病症还是预防复发。为了治疗，施用的量是有效产生所需效果的量。可以一次或多次给药来提供有效量。可以大剂量或通过连续灌注来提供有效量。The methods for preventing, alleviating and/or treating tumors provided in the present application include methods for inducing and/or increasing immune response in a subject in need of the cell composition of the present application. The composition of the present application can be used to treat and/or prevent tumors in a subject. The cell compositions of the present application can be used to prolong the survival of a subject with a tumor. The cell composition of the present application can also be used to treat and/or prevent pathogen infection or other infectious diseases such as immunocompromised subjects. Such methods involve administering an effective amount of a cellular composition of the present application to achieve a desired effect, whether alleviating an existing condition or preventing relapse. For treatment, the amount administered is that effective to produce the desired effect. An effective amount may be provided in one or more administrations. Effective amounts can be provided in boluses or by continuous infusion.
在一实例中，包括本申请的细胞组合物可以用于治疗具有表面抗原表达水平低的肿瘤细胞的受试者，例如由于疾病的复发，其中受试者接受过导致残留肿瘤细胞的治疗。在某些实施方式中，肿瘤细胞在肿瘤细胞表面上具有低密度的靶分子。In one example, compositions comprising cells of the present application can be used to treat a subject having tumor cells with low expression of surface antigens, eg, due to relapse of the disease, where the subject has received treatment that resulted in residual tumor cells. In certain embodiments, the tumor cell has a low density of the target molecule on the surface of the tumor cell.
在一实例中，包括本申请的细胞组合物可用于治疗患有疾病复发的受试者，其中该受试者接受过包括单独施用CAR的免疫细胞(例如，T细胞)。在一实例中，肿瘤细胞在肿瘤细胞表面上具有低密度的肿瘤特异性抗原。在一实例中，该疾病是BCMA阳性肿瘤。在一实例中，肿瘤细胞在肿瘤细胞上具有低密度的BCMA。这种方法包括施用有效量的本申请的细胞组合物以达到期望的效果，缓解现有病症或预防复发。In one example, a composition comprising cells of the present application can be used to treat a subject with relapsed disease, wherein the subject has received immune cells (eg, T cells) comprising administration of a CAR alone. In one example, the tumor cells have a low density of tumor-specific antigens on the surface of the tumor cells. In one example, the disease is a BCMA positive tumor. In one example, the tumor cells have a low density of BCMA on the tumor cells. Such methods include administering an effective amount of a cellular composition of the present application to achieve a desired effect, to alleviate an existing condition or to prevent relapse.
“有效量”(或“治疗有效量”)是足以在治疗后产生有益或期望的临床结果的量。可以以一剂或多剂剂量将有效量施用于受试者。就治疗而言，有效量是足以缓解、改善、稳定、逆转或减慢疾病进展或以其它方式减少疾病病理后果的量。有效量通常由医师根据具体情况确定，并且在本领域技术人员的能力范围内。当确定合适的剂量以达到有效量时，通常要考虑几个因素。这些因素包括受试者的年龄、性别和体重、所治疗的疾病、疾病的严重程度以及所施用的本申请的细胞组合物的形式和有效浓度。An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce beneficial or desired clinical results following treatment. An effective amount can be administered to a subject in one or more doses. For treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse or slow the progression of the disease or otherwise reduce the pathological consequences of the disease. Effective amounts are generally determined by a physician on a case-by-case basis and are within the capabilities of those skilled in the art. Several factors are generally considered when determining a suitable dosage to achieve an effective amount. These factors include the age, sex and weight of the subject, the disease being treated, the severity of the disease, and the form and effective concentration of the cellular composition of the application administered.
在一实例中，所述识别NKG2A的第一工程细胞与所述识别非NKG2A的抗原的第二工程细胞被配置为同时向受试者施用。例如，所述同时向受试者施用可以包括所述第一工程细胞、第二工程细胞向受试者施用的时间间隔不超过1小时。例如，所述时间间隔为60分钟、45分钟、30分钟、15分钟、1分钟，或者以混合的形式一起施用。In one example, the first engineered cell recognizing NKG2A and the second engineered cell recognizing an antigen other than NKG2A are configured to be administered to the subject at the same time. For example, the simultaneous administration to the subject may include that the time interval between the administration of the first engineered cell and the second engineered cell to the subject is no more than 1 hour. For example, the time interval is 60 minutes, 45 minutes, 30 minutes, 15 minutes, 1 minute, or administered together in admixture.
在一实例中，所述识别NKG2A的第一工程细胞与所述识别非NKG2A的抗原的第二工程细胞被配置为分别向受试者施用。例如，所述分别向受试者施用可以包括第一工程细胞、第二工程细胞向受试者施用的时间间隔大于1小时。例如，所述时间间隔为、5小时、10小时、15小时、24小时、3天、5天、7天，或更长时间。在将本申请的工程细胞施用于宿主并随后分化后，诱导特异性针对特定抗原的T细胞。本申请的细胞组合物可以通过本领 域已知的任何方法施用，包括但不限于静脉内、皮下、结内、肿瘤内、鞘内、胸膜内、腹膜内和直接向胸腺施用。In one example, the first engineered cell recognizing NKG2A and the second engineered cell recognizing an antigen other than NKG2A are configured to be administered to a subject separately. For example, the separate administration to the subject may include the time interval between administering the first engineered cell and the second engineered cell to the subject greater than 1 hour. For example, the time interval is 5 hours, 10 hours, 15 hours, 24 hours, 3 days, 5 days, 7 days, or longer. After the engineered cells of the present application are administered to a host and then differentiated, T cells specific for a specific antigen are induced. The cellular compositions of the present application can be administered by any method known in the art, including but not limited to intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and direct administration to the thymus.
本申请提供用于治疗和/或预防受试者中的肿瘤的方法。该方法可以包括向患有肿瘤的受试者施用有效量的本申请的细胞组合物。The present application provides methods for treating and/or preventing tumors in a subject. The method may comprise administering an effective amount of a cell composition of the present application to a subject having a tumor.
在一实例中，所述肿瘤包括实体瘤和血液瘤。肿瘤的非限制性实例包括血液瘤(例如白血病、淋巴瘤和骨髓瘤)、卵巢癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、***癌、皮肤癌、胃癌、胶质母细胞瘤、喉癌、黑素瘤、神经母细胞瘤、腺癌、神经胶质瘤、软组织肉瘤和各种癌(包括***癌和小细胞肺癌)。肿瘤的非限制性实例包括但不限于星形细胞瘤、纤维肉瘤、粘液肉瘤、脂肪肉瘤、少突胶质细胞瘤、室管膜瘤、髓母细胞瘤、原始神经外胚层肿瘤(PNET)、软骨肉瘤、成骨肉瘤、胰腺导管腺癌、小细胞和大细胞肺腺癌、脊索瘤、血管肉瘤、内皮肉瘤、鳞状细胞癌、支气管肺泡癌、上皮腺癌及其肝转移灶、***肉瘤、***内皮肉瘤、肝癌、胆管癌、滑膜瘤、间皮瘤、尤文氏瘤、横纹肌肉瘤、结肠癌、基底细胞癌、汗腺癌、***状癌、皮脂腺癌、状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、胆小管癌、绒毛膜癌、***瘤、胚胎癌、Wilms’肿瘤、睾丸肿瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质细胞瘤、脑膜瘤、神经母细胞瘤、视网膜母细胞瘤、白血病、多发性骨髓瘤、Waldenstrom’s巨球蛋白血症和重链疾病、诸如导管和小叶腺癌的乳腺肿瘤、子宫颈的鳞状和腺癌、子宫和卵巢上皮癌、***腺癌、膀胱移行鳞状细胞癌、B和T细胞淋巴瘤(结节性和弥漫性)浆细胞瘤、急慢性白血病、恶性黑色素瘤、软组织肉瘤和平滑肌肉瘤。在某些实施方式中，肿瘤选自血液癌症(例如白血病、淋巴瘤和骨髓瘤)、卵巢癌、***癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、***癌、皮肤癌、胃癌、胶质母细胞瘤和喉癌。在一实例中，本申请的组合物可以用于治疗和/或预防常规治疗措施不适合或复发难治性实体瘤，例如肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌。在一实例中，肿瘤是血液瘤。在一实例中，所述血液瘤包括多发性骨髓瘤。In one example, the tumors include solid tumors and hematological tumors. Non-limiting examples of tumors include hematological tumors (such as leukemia, lymphoma, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer , gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcomas, and various carcinomas (including prostate cancer and small cell lung cancer). Non-limiting examples of tumors include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), Chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphatic Sarcoma, lymphangioendothelial sarcoma, liver cancer, cholangiocarcinoma, synovial tumor, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, thyroid carcinoma, cyst gland Carcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependyma tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and severe breast tumors such as ductal and lobular adenocarcinomas, squamous and adenocarcinomas of the cervix, epithelial carcinomas of the uterus and ovaries, adenocarcinomas of the prostate, transitional squamous cell carcinomas of the bladder, B and T-cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma. In certain embodiments, the tumor is selected from hematological cancers (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer , prostate, skin, stomach, glioblastoma, and throat cancers. In one example, the composition of the present application can be used for the treatment and/or prevention of unsuitable or relapsed refractory solid tumors, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer. In one example, the tumor is a hematoma. In one example, the hematological tumor comprises multiple myeloma.
本申请的组合物治疗目标可以包括缓解或逆转疾病进展和/或减轻副作用、或治疗目标包括降低或延迟复发风险。The therapeutic goals of the composition of the present application may include alleviating or reversing disease progression and/or alleviating side effects, or the therapeutic goals may include reducing or delaying the risk of relapse.
本申请提供用于在例如免疫受损的受试者中治疗和/或预防病原体感染(例如病毒感染、细菌感染、真菌感染、寄生虫感染或原生动物感染)的方法。该方法可以包括向患有病原体感染的受试者施用有效量的本申请的组合物。易于治疗的示例性病毒感染包括但不限于巨细胞病毒、爱泼斯坦-巴尔病毒、人免疫缺陷病毒和流感病毒感染。The present application provides methods for treating and/or preventing a pathogenic infection (eg, viral, bacterial, fungal, parasitic, or protozoan infection) in, eg, an immunocompromised subject. The method may comprise administering an effective amount of a composition of the present application to a subject suffering from a pathogenic infection. Exemplary viral infections that are amenable to treatment include, but are not limited to, cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and influenza virus infections.
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强 的应答可以包括应答性中5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％更多。The term "enhancing" refers to allowing a subject or a tumor cell to improve its ability to respond to the treatments disclosed herein. For example, enhanced response can include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% in responsiveness %, 75%, 80%, 85%, 90%, 95%, or 98% or more increase. As used herein, "enhancing" can also refer to increasing the number of subjects who respond to treatment, eg, immune cell therapy. For example, an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% more.
在一实例中，细胞组合物用于治疗BCMA表达阳性的肿瘤。在一实例中，组合物用于治疗多发性骨髓瘤。In one example, the cell composition is used to treat BCMA-positive tumors. In one example, the composition is used to treat multiple myeloma.
可以将包括本申请的组合物***地或直接提供给受试者，以诱导和/或增强对抗原的免疫应答和/或治疗和/或预防肿瘤、病原体感染或感染性疾病。在一实例中，将本申请的组合物直接注射到目的器官(例如，受肿瘤影响的器官)中。或者，例如通过向循环***(例如，静脉、肿瘤脉管***)给药，将本申请的组合物间接地提供给目的器官。可以在施用组合物之前、同时或之后提供扩增和分化剂，以增加体外或体内T细胞、NKT细胞或CTL细胞的产生。The composition comprising the present application can be provided systemically or directly to a subject to induce and/or enhance an immune response to an antigen and/or treat and/or prevent tumors, pathogenic infections or infectious diseases. In one example, a composition of the present application is injected directly into an organ of interest (eg, an organ affected by a tumor). Alternatively, the compositions of the present application are provided to the organ of interest indirectly, eg, by administration to the circulatory system (eg, vein, tumor vasculature). Expansion and differentiation agents can be provided before, simultaneously with or after administration of the composition to increase the production of T cells, NKT cells or CTL cells in vitro or in vivo.
本申请的组合物中的工程细胞可以包括纯化的细胞群。本领域技术人员可以使用各种众所周知的方法，例如荧光激活细胞分选(FACS)，容易地确定群体中本申请的工程细胞的百分比。在包括本申请的工程细胞的群体中，纯度的合适范围是约50％至约55％、约5％至约60％、以及约65％至约70％。在某些实施方式中，纯度为约70％至约75％、约75％至约80％或约80％至约85％。在某些实施方式中，纯度为约85％至约90％，约90％至约95％以及约95％至约100％。剂量可以由本领域技术人员容易地调节(例如，纯度降低可能需要增加剂量)。可以通过注射、导管等引入细胞。Engineered cells in the compositions of the present application may include purified cell populations. One skilled in the art can readily determine the percentage of engineered cells of the present application in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Suitable ranges for purity in a population comprising engineered cells of the present application are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In certain embodiments, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art (eg, decreased purity may require increased dosages). Cells can be introduced by injection, catheter, and the like.
本申请的组合物可以是包括本申请的细胞或其祖细胞和药学上可接受的载体的药物组合物。给药可以是自体的或异体的。例如，可以从一个受试者获得免疫细胞或祖细胞，并将其施用于相同受试者或不同的相容受试者。外周血来源的免疫细胞或其后代(例如，体内、离体或体外来源)可通过局部注射施用，包括导管给药、全身注射、局部注射、静脉内注射或肠胃外给药。当施用本申请的组合物时，可以将其配制成单位剂量可注射形式(溶液剂、悬浮剂、乳剂等)。The composition of the present application may be a pharmaceutical composition comprising the cells of the present application or their progenitor cells and a pharmaceutically acceptable carrier. Administration can be autologous or allogeneic. For example, immune cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject. Peripheral blood-derived immune cells or their progeny (eg, in vivo, ex vivo, or in vitro sources) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering the compositions of the present application, they may be formulated in unit dose injectable forms (solutions, suspensions, emulsions, etc.).
试剂盒Reagent test kit
本申请提供一种试剂盒，其包本申请的细胞组合物或者本申请的药物组合物。所述试剂盒用于在受试者中诱导和/或增强免疫应答和/或治疗和/或预防肿瘤或病原体感染的试剂盒。在一实例中，试剂盒包括有效量的本申请的细胞组合物或药物组合物或本申请的第一工程细胞和第二工程细胞。在一实例中，试剂盒包括无菌容器；这样的容器可以是盒子、 安瓿、瓶、小瓶、管、袋、小袋、泡罩包装或本领域已知的其它合适的容器形式。这样的容器可以由塑料、玻璃、层压纸、金属箔或其它适合于容纳药物的材料制成。The present application provides a kit comprising the cell composition of the present application or the pharmaceutical composition of the present application. The kit is a kit for inducing and/or enhancing immune response and/or treating and/or preventing tumor or pathogen infection in a subject. In one example, the kit includes an effective amount of the cell composition or pharmaceutical composition of the present application or the first engineered cell and the second engineered cell of the present application. In one example, kits include sterile containers; such containers can be in the form of boxes, ampoules, bottles, vials, tubes, bags, sachets, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug.
在一实例中，将本申请的细胞组合物施用于患有肿瘤或病原体或免疫疾病或有发展成肿瘤或病原体或免疫疾病的受试者的说明书一起提供。说明书通常包括有关组合物用于治疗和/或预防肿瘤或病原体感染的信息。在一实例中，说明书包括以下至少一项：治疗剂的描述；用于治疗或预防肿瘤、病原体感染或免疫疾病或其症状的剂量表和给药；注意事项；警告；适应症；不适应症；用药信息；不良反应；动物药理学；临床研究；和/或参考。这些说明书可以直接打印在容器上，或者作为粘贴在容器上的标签，或者作为单独的纸页、小册子、卡片或文件夹提供在容器内或与容器一起。In one example, instructions for administering the cell composition of the present application to a subject suffering from or developing a tumor or pathogenic or immune disease are provided together. The instructions generally include information about the use of the composition in the treatment and/or prophylaxis of tumors or pathogenic infections. In one example, the instructions include at least one of the following: a description of the therapeutic agent; a dosage form and administration for the treatment or prevention of tumors, pathogenic infections, or immune diseases or symptoms thereof; precautions; warnings; indications; incompatibility ; Medication Information; Adverse Reactions; Animal Pharmacology; Clinical Studies; and/or References. These instructions may be printed directly on the container, or as a label affixed to the container, or provided within or with the container as separate sheets, booklets, cards or file folders.
本申请包括，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN109908176A、CN109880803A、CN110055275A、CN110123837A、CN110438082A、CN110468105A以及例如国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的那些CAR-T细胞及其制备方法、抗体。本申请包括，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、 CN109908176A、CN109880803A、CN110055275A、CN110123837A、CN110438082A、CN110468105A以及例如国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018 /219299, WO2018/210279, WO2019/024933, WO2019/114751, WO2019/114762, WO2019/141270, WO2019/149279, WO2019/170147A1, WO2019/210863, WO2019/219029 methods, antibodies.
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。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。本说明书中提到的所有出版物、专利和专利申请均通过引用并入本文，其程度如同特别地且单独地指出每一个单独的出版物、专利或专利申请均通过引用而并入本文。The present application will be further elaborated below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. Experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
本申请实施例中所用生物材料如表1所示。The biological materials used in the examples of this application are shown in Table 1.
表1Table 1
名称name 来源source
T细胞T cell 分离自健康供体原代PBMCPrimary PBMCs isolated from healthy donors
NK细胞NK cells 分离自健康供体原代PBMCPrimary PBMCs isolated from healthy donors
单核细胞monocytes 分离自健康供体原代PBMCPrimary PBMCs isolated from healthy donors
细胞系RPMI-8226Cell line RPMI-8226 购自ATCCPurchased from ATCC
MM.1S肿瘤细胞MM.1S tumor cells 购自ATCCPurchased from ATCC
NPG免疫缺陷的小鼠NPG immunodeficient mice 购自北京维通达生物技术有限公司Purchased from Beijing Weitongda Biotechnology Co., Ltd.
实施例1、CAR-T细胞的制备Example 1. Preparation of CAR-T cells
采用本领域常规分子生物学方法，分别构建表达BCMA-CAR、NKG2A-CAR、BCMA-NKG2A-CAR的BCMA CAR-T细胞、NKG2A CAR-T细胞和BCMA-NKG2A CAR-T细胞。BCMA CAR-T cells, NKG2A CAR-T cells, and BCMA-NKG2A CAR-T cells expressing BCMA-CAR, NKG2A-CAR, and BCMA-NKG2A-CAR were respectively constructed using conventional molecular biology methods in the field.
BCMA-CAR依次包括BCMA的单链抗体(VH的氨基酸序列如SEQ ID NO：19所示，VL的氨基酸序列如SEQ ID NO：20所示)、CD8hinge、CD8跨膜结构域、4-1BB共刺激因子和CD3ζ。BCMA-CAR includes the single-chain antibody of BCMA in turn (the amino acid sequence of VH is shown in SEQ ID NO: 19, and the amino acid sequence of VL is shown in SEQ ID NO: 20), CD8hinge, CD8 transmembrane domain, 4-1BB co- Stimulatory factors and CD3ζ.
NKG2A-CAR依次包括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)、CD8hinge、CD28跨膜结构域、CD28共刺激因子和CD3ζ。NKG2A-CAR includes NKG2A single-chain antibody in turn (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), CD8hinge, CD28 transmembrane domain, and CD28 co-stimulatory factor and CD3ζ.
BCMA-NKG2A-CAR依次包括BCMA和NKG2A的串联单链抗体(氨基酸序列如SEQ ID NO：36所示)、CD8hinge、CD8跨膜结构域、4-1BB共刺激因子和CD3ζ。BCMA-NKG2A-CAR sequentially includes the tandem single-chain antibody of BCMA and NKG2A (the amino acid sequence is shown in SEQ ID NO: 36), CD8hinge, CD8 transmembrane domain, 4-1BB co-stimulatory factor and CD3ζ.
上述CAR中CD8hinge序列如SEQ ID NO：30所示，CD8跨膜结构域序列如SEQ ID NO：31所示，CD28跨膜结构域序列如SEQ ID NO：32所示，4-1BB共刺激因子序列如SEQ ID NO：34所示，CD28共刺激因子序列如SEQ ID NO：33所示，CD3ζ序列如SEQ ID NO：35所示。The CD8hinge sequence in the above CAR is shown in SEQ ID NO: 30, the CD8 transmembrane domain sequence is shown in SEQ ID NO: 31, the CD28 transmembrane domain sequence is shown in SEQ ID NO: 32, 4-1BB co-stimulatory factor The sequence is shown in SEQ ID NO: 34, the CD28 costimulator sequence is shown in SEQ ID NO: 33, and the CD3ζ sequence is shown in SEQ ID NO: 35.
实施例2、UCAR-T细胞的制备 Embodiment 2, the preparation of UCAR-T cell
按照试剂说明书(GeneArt TM Precision gRNA Synthesis Kit，Thermo Tisher)体外分别合成靶向TRAC、B2M和NKG2A的gRNA序列(SEQ ID NO：46、47、10)。分别对实施例1中所得的BCMA CAR-T细胞、NKG2A CAR-T和BCMA-NKG2A CAR-T细胞进行TCR/B2M/NKG2A的三敲除。 The gRNA sequences targeting TRAC, B2M and NKG2A (SEQ ID NO: 46, 47, 10) were synthesized in vitro according to the reagent instructions (GeneArt ™ Precision gRNA Synthesis Kit, Thermo Tisher). The triple knockout of TCR/B2M/NKG2A was performed on the BCMA CAR-T cells, NKG2A CAR-T and BCMA-NKG2A CAR-T cells obtained in Example 1, respectively.
Cas 9酶和gRNA按1:4比例进行室温孵育，将细胞与Cas9酶和gRNA复合物(RNP)进行混合(Cas 9酶的终浓度为3uM),将RNP复合物分别电转入BCMA CAR-T细胞、NKG2A CAR-T、BCMA-NKG2A CAR-T细胞中，得到TCR/B2M/NKG2A敲除的CAR-T细胞，并通过去除TCR/B2M阳性细胞，得到并分别命名为BCMA UCAR-T-NKG2A KO(或称为BCMA UCAR-T-TKO)、NKG2A UCAR-T-NKG2A KO(或称为NKG2A UCAR-T-TKO)和BCMA-NKG2A UCAR-T-NKG2A KO(或称为 BCMA-NKG2A UCAR-T-TKO)。以同样敲除TCR/B2M/NKG2A但未转染病毒的UTD-TKO细胞作为阴性对照。 Cas 9 enzyme and gRNA were incubated at room temperature at a ratio of 1:4, cells were mixed with Cas9 enzyme and gRNA complex (RNP) (the final concentration of Cas 9 enzyme was 3uM), and the RNP complex was electrotransferred into BCMA CAR- From T cells, NKG2A CAR-T, and BCMA-NKG2A CAR-T cells, TCR/B2M/NKG2A knockout CAR-T cells were obtained, and by removing TCR/B2M positive cells, they were obtained and named BCMA UCAR-T- NKG2A KO (or BCMA UCAR-T-TKO), NKG2A UCAR-T-NKG2A KO (or NKG2A UCAR-T-TKO) and BCMA-NKG2A UCAR-T-NKG2A KO (or BCMA-NKG2A UCAR -T-TKO). UTD-TKO cells that also knocked out TCR/B2M/NKG2A but were not transfected with virus were used as negative controls.
实施例3.识别NKG2A的UCAR-T细胞能促进UCAR-T细胞的体外存活和/或扩增Example 3. UCAR-T cells that recognize NKG2A can promote the survival and/or expansion of UCAR-T cells in vitro
利用NK细胞分离试剂盒(购自美天旎公司)从外周血单核细胞中分离原代NK细胞，采用含IL-2的NK细胞培养基体外扩增培养14天。Primary NK cells were isolated from peripheral blood mononuclear cells using an NK cell isolation kit (purchased from Miltenyi), and cultured in vitro for 14 days using NK cell medium containing IL-2.
靶细胞：多发性骨髓瘤RPMI-8226细胞；Target cells: multiple myeloma RPMI-8226 cells;
效应细胞一：原代培养的NK细胞；效应细胞二：NKG2A UCAR-T-TKO、BCMAEffector cell 1: primary cultured NK cells; Effector cell 2: NKG2A UCAR-T-TKO, BCMA
UCAR-T-TKO细胞。UCAR-T-TKO cells.
具体过程：取CFSE标记的3×10 5个RPMI-8226细胞/孔接种，按靶细胞：BCMA UCAR-T-TKO细胞：NKG2A UCAR-T-TKO细胞(或UTD-TKO细胞)：原代NK细胞＝2:2:2:1，2:2:2:2，2:2:2:4三个比例进行接种，共孵育5天后，利用流式染色鉴定肿瘤细胞(RPMI-8226细胞)，NK细胞和UCAR-T细胞，CFSE+HLA-ABC+代表肿瘤细胞，CFSE-HLA-ABC+代表NK细胞，CFSE-HLA-ABC-代表检测体系中存在的UCAR-T细胞，并用绝对计数对UCAR-T细胞进行定量分析，检测UCAR-T细胞体外存活和/或扩增情况，数量的统计情况显示在图1中。 Specific process: Inoculate 3×10 5 RPMI-8226 cells/well labeled with CFSE, according to target cells: BCMA UCAR-T-TKO cells: NKG2A UCAR-T-TKO cells (or UTD-TKO cells): primary NK Cells=2:2:2:1, 2:2:2:2, and 2:2:2:4 were inoculated at three ratios. After co-incubating for 5 days, the tumor cells (RPMI-8226 cells) were identified by flow staining, NK cells and UCAR-T cells, CFSE+HLA-ABC+ represents tumor cells, CFSE-HLA-ABC+ represents NK cells, CFSE-HLA-ABC- represents UCAR-T cells in the detection system, and the absolute count is used to compare UCAR-T Quantitative analysis of cells was performed to detect the survival and/or expansion of UCAR-T cells in vitro, and the statistics of the number are shown in Figure 1.
结果如图1所示：有NK细胞存在下，与UTD-TKO组相比，识别NKG2A的UCAR-T细胞能促进包含BCMA-UCAR-T的细胞组合物的UCAR-T细胞总量显著升高。The results are shown in Figure 1: in the presence of NK cells, compared with the UTD-TKO group, UCAR-T cells that recognize NKG2A can promote a significant increase in the total amount of UCAR-T cells in the cell composition containing BCMA-UCAR-T .
实施例4.识别NKG2A的UCAR-T细胞的体内协同抗肿瘤作用Example 4. In vivo synergistic anti-tumor effect of UCAR-T cells recognizing NKG2A
皮下接种5×10 6个RPMI-8226细胞于NPG免疫缺陷的小鼠中，接种10天后平均瘤体积为200mm 3左右，将小鼠分4组(UTD-TKO，NKG2A UCAR-T-TKO，BCMA UCAR-T-TKO+UTD-TKO，BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO)，每组5只。 5×10 6 RPMI-8226 cells were inoculated subcutaneously in NPG immunodeficient mice. The average tumor volume was about 200 mm 3 10 days after inoculation. The mice were divided into 4 groups (UTD-TKO, NKG2A UCAR-T-TKO, BCMA UCAR-T-TKO+UTD-TKO, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO), 5 rats in each group.
尾静脉分别注射1×10 6个UTD-TKO、1×10 6个NKG2A UCAR-T-TKO、0.6×10 6个BCMA UCAR-T-TKO+1×10 6个UTD-TKO、0.6×10 6个BCMA UCAR-T-TKO+1×10 6个NKG2A UCAR-TKO T细胞，4小时后尾静脉注射1×10 6个NK细胞，后续D14和D17分别再次注射1×10 6个NK细胞。T细胞注射后，每周2次测量体重(包括分组给药及安乐死当天)，并测量肿瘤长径、短径，计算肿瘤体积，根据肿瘤体积绘制肿瘤生长曲线，并比较各组间肿瘤生长曲线的差异(肿瘤体积：V＝1/2×长径×短径 2)。D24天，取小鼠外周血，用抗人-CD45抗体以及抗人-BCMA抗体进行流式染色和绝对定量，检测小鼠外周血中BCMA UCAR-T细胞的数量。 1×10 6 UTD-TKO, 1×10 6 NKG2A UCAR-T-TKO, 0.6×10 6 BCMA UCAR-T-TKO+1×10 6 UTD-TKO, 0.6×10 6 BCMA UCAR-T-TKO + 1×10 6 NKG2A UCAR-TKO T cells, 1×10 6 NK cells were injected into the tail vein 4 hours later, and 1×10 6 NK cells were injected again on D14 and D17 respectively. After T cell injection, body weight was measured twice a week (including group administration and the day of euthanasia), and the long and short diameters of the tumor were measured to calculate the tumor volume, draw the tumor growth curve according to the tumor volume, and compare the tumor growth curves among the groups The difference (tumor volume: V=1/2×long diameter×short diameter 2 ). On day D24, the peripheral blood of the mice was collected, and flow staining and absolute quantification were performed with anti-human-CD45 antibody and anti-human-BCMA antibody to detect the number of BCMA UCAR-T cells in the peripheral blood of the mice.
检测结果如图2A所示，在NK细胞存在下，识别NKG2A的UCAR-T细胞发挥体内 协同BCMA-UCAR-T细胞的抗肿瘤作用。如图2B所示，与UTD-TKO相比，识别NKG2A的UCAR-T细胞能显著增加体内BCMA UCAR-T细胞数量。The test results are shown in Figure 2A. In the presence of NK cells, UCAR-T cells that recognize NKG2A exert the anti-tumor effect of synergistic BCMA-UCAR-T cells in vivo. As shown in Figure 2B, UCAR-T cells recognizing NKG2A significantly increased the number of BCMA UCAR-T cells in vivo compared with UTD-TKO.
实施例5.识别NKG2A的UCAR-T细胞协同UCAR-T细胞特异性向肿瘤组织浸润Example 5. UCAR-T cells that recognize NKG2A cooperate with UCAR-T cells to specifically infiltrate tumor tissue
实施例4中动物实验结束后，取UTD-TKO、BCMA UCAR-T-TKO+UTD-TKO、BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO三组小鼠的肿瘤组织，以及心肝脾肺肾正常组织。经多聚甲醛固定后进行切片和抗人-CD45免疫组化染色，以检测T细胞在肿瘤细胞以及正常组织中的浸润情况。如图2C所示，深色点状或片状代表抗人-CD45的UCAR-T细胞,在UTD-TKO组的肿瘤组织中未检测到明显的T细胞浸润，而相对于BCMA UCAR-T-TKO+UTD-TKO组，BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO组中能检测到非常明显的T细胞浸润，且明显优于BCMA UCAR-T-TKO+UTD-TKO细胞组。After the animal experiment in Example 4, the tumor tissues, as well as the hearts, liver, spleen and lungs of mice in the three groups of UTD-TKO, BCMA UCAR-T-TKO+UTD-TKO, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO were collected. normal kidney tissue. After being fixed with paraformaldehyde, the sections and anti-human-CD45 immunohistochemical staining were performed to detect the infiltration of T cells in tumor cells and normal tissues. As shown in Figure 2C, dark dots or patches represent anti-human-CD45 UCAR-T cells, and no obvious T cell infiltration was detected in the tumor tissue of the UTD-TKO group, while compared to BCMA UCAR-T- TKO+UTD-TKO group, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO group can detect very obvious T cell infiltration, and it is significantly better than BCMA UCAR-T-TKO+UTD-TKO cell group.
采用HE染色观察了各脏器组织的病理改变。结果如图2D所示，3个治疗组的小鼠心、肝、脾和肾组织形态正常，结构清晰、完整，均无显著病理改变；肺脏组织形态正常，肺泡结构较清晰，细支气管周围见淋巴细胞浸润，符合轻度炎症表现，无明显病理改变。表明NKG2A UCAR-T与BCMA UCAR-T联用在小鼠体内不会产生明显的毒副作用。上述结果表明，NKG2A UCAR-T细胞能协助UCAR-T细胞特异性向肿瘤组织浸润，但不会向正常组织浸润，具有良好的安全性。The pathological changes of various organs and tissues were observed by HE staining. The results are shown in Figure 2D. The tissues of the hearts, livers, spleens and kidneys in the three treatment groups were normal, with clear and complete structures, and no significant pathological changes; Lymphocyte infiltration, consistent with mild inflammation, no obvious pathological changes. It shows that the combination of NKG2A UCAR-T and BCMA UCAR-T will not produce obvious toxic side effects in mice. The above results show that NKG2A UCAR-T cells can assist UCAR-T cells to specifically infiltrate into tumor tissue, but not infiltrate into normal tissue, which has good safety.
实施例6.识别NKG2A的UCAR-T细胞与识别肿瘤抗原的UCAR-T联用不引起移植物抗宿主反应Example 6. The combination of UCAR-T cells recognizing NKG2A and UCAR-T recognizing tumor antigens does not cause graft-versus-host reaction
体外扩增培养BCMA UCAR-T-TKO和NKG2A UCAR-T-TKO细胞，采用未转染病毒的T细胞(UTD)、PBS作对照。分别按1×10 7个UTD、1×10 7个BCMA UCAR-T-TKO、5×10 6个BCMA UCAR-T-TKO+5×10 6个NKG2A UCAR-T-TKO注射到NPG小鼠中，每周2次进行体重称量，并观察小鼠的毛发及基础情况，以判断是否存在移植物抗宿主反应(GVHD)的发生。 BCMA UCAR-T-TKO and NKG2A UCAR-T-TKO cells were expanded and cultured in vitro, and untransfected T cells (UTD) and PBS were used as controls. Inject 1×10 7 UTD, 1×10 7 BCMA UCAR-T-TKO, 5×10 6 BCMA UCAR-T-TKO+5×10 6 NKG2A UCAR-T-TKO into NPG mice , body weight was weighed twice a week, and the hair and basic conditions of the mice were observed to determine whether there was a graft-versus-host reaction (GVHD).
如图2E所示，UTD组的小鼠从D51开始发生明显的体重增长减缓或下降，且表现出现脱毛，活动性降低等明显的GVHD症状。然而BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO细胞组与PBS类似，小鼠均正常生长，体重逐渐增加。上述结果表明，识别NKG2A的UCAR-T细胞与BCMA UCAR-T细胞联合施用不引起GVHD。As shown in FIG. 2E , the mice in the UTD group experienced obvious slowing or decreasing of body weight growth from D51, and showed obvious GVHD symptoms such as hair loss and decreased activity. However, the BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO cell group was similar to PBS, and the mice all grew normally and gradually gained weight. The above results indicated that co-administration of NKG2A-recognizing UCAR-T cells and BCMA UCAR-T cells did not cause GVHD.
实施例7.识别NKG2A和肿瘤抗原的UCAR-T细胞的体内协同抗肿瘤作用Example 7. In vivo synergistic anti-tumor effect of UCAR-T cells that recognize NKG2A and tumor antigens
皮下接种5×10 6个RPMI-8226细胞于NPG小鼠中，接种10天后平均瘤体积为250mm 3左右，将小鼠分4组(UTD-TKO，BCMA UCAR-T-TKO+UTD-TKO,BCMA  UCAR-T-TKO+NKG2A UCAR-T-TKO，BCMA UCAR-T-TKO+BCMA-NKG2A UCAR-T-TKO)，每组5只。D12，D13,D15,按上述分组尾静脉注射1×10 6个NK细胞，一共三次。D12,D14,D16，按上述分组尾静脉分别三次注射0.4×10 6个UTD-TKO、0.4×10 6个BCMA UCAR-T-TKO和0.4×10 6个UTD-TKO、0.4×10 6个BCMA UCAR-T-TKO和0.4×10 6个NKG2A UCAR-T-TKO、0.4×10 6个BCMA UCAR-T-TKO和0.4×10 6个BCMA-NKG2A UCAR-T-TKO细胞。按照实施例4所述方法绘制肿瘤生长曲线。 5×10 6 RPMI-8226 cells were inoculated subcutaneously in NPG mice, and the average tumor volume was about 250mm 3 10 days after inoculation. The mice were divided into 4 groups (UTD-TKO, BCMA UCAR-T-TKO+UTD-TKO, BCMA UCAR-T-TKO+NKG2A UCAR-T-TKO, BCMA UCAR-T-TKO+BCMA-NKG2A UCAR-T-TKO), 5 rats in each group. On D12, D13, and D15, 1×10 6 NK cells were injected into the tail vein according to the above groups, a total of three times. On D12, D14, and D16, 0.4×10 6 UTD-TKO, 0.4×10 6 BCMA UCAR-T-TKO and 0.4×10 6 UTD-TKO, 0.4×10 6 BCMA were injected three times into the tail vein according to the above groups UCAR-T-TKO and 0.4×10 6 NKG2A UCAR-T-TKO, 0.4×10 6 BCMA UCAR-T-TKO and 0.4×10 6 BCMA-NKG2A UCAR-T-TKO cells. The tumor growth curve was drawn according to the method described in Example 4.
结果如图3所示，在NK细胞存在下，识别NKG2A的UCAR-T、或识别NKG2A和BCMA的UCAR-T均能发挥体内协同BCMA-UCAR-T细胞的抗肿瘤作用。The results are shown in Figure 3. In the presence of NK cells, UCAR-T that recognizes NKG2A, or UCAR-T that recognizes NKG2A and BCMA, can exert synergistic anti-tumor effects of BCMA-UCAR-T cells in vivo.
实施例8、识别NKG2A的UCAR-T细胞促进UCAR-T细胞体外存活和/或扩增Example 8. UCAR-T cells that recognize NKG2A promote the survival and/or expansion of UCAR-T cells in vitro
按照实施例2方法制备的TCR/B2M/NKG2A/CIITA四敲除的BCMA UCAR-T-FKO、NKG2A UCAR-T-FKO细胞,其中靶向TRAC、B2M、NKG2A和CIITA的gRNA序列分别如SEQ ID NO：46、47、10、48所示。以同样敲除TCR/B2M/NKG2A/CIITA但未转染病毒的UTD细胞作为阴性对照。TCR/B2M/NKG2A/CIITA quadruple knockout BCMA UCAR-T-FKO and NKG2A UCAR-T-FKO cells prepared according to the method of Example 2, wherein the gRNA sequences targeting TRAC, B2M, NKG2A and CIITA are shown in SEQ ID NO: 46, 47, 10, 48. UTD cells that also knocked out TCR/B2M/NKG2A/CIITA but were not transfected with virus were used as negative controls.
靶细胞：多发性骨髓瘤MM.1S-GFP细胞；Target cells: multiple myeloma MM.1S-GFP cells;
效应细胞一：原代培养的NK细胞；效应细胞二：BCMA UCAR-T-FKO、NKG2A UCAR-T-FKO细胞。Effector cell 1: primary cultured NK cells; Effector cell 2: BCMA UCAR-T-FKO, NKG2A UCAR-T-FKO cells.
具体过程：取3×10 4个MM.1S-GFP细胞接种到96孔板，按靶细胞：BCMA UCAR-T-FKO细胞：NKG2A UCAR-T-FKO细胞(或UTD-FKO细胞)：原代NK细胞＝2:2:2:1，2:2:2:2，2:2:2:4三个比例进行接种，共培养5天后，流式染色检测CD45/HLA-ABC细胞比例，并进行绝对细胞定量。用GFP阳性表示肿瘤细胞，CD45+HLA-ABC+细胞代表NK细胞，CD45+HLA-ABC-细胞代表UCAR-T细胞。 Specific process: Take 3×10 4 MM.1S-GFP cells and inoculate them into 96-well plates, according to target cells: BCMA UCAR-T-FKO cells: NKG2A UCAR-T-FKO cells (or UTD-FKO cells): primary NK cells=2:2:2:1, 2:2:2:2, and 2:2:2:4 were inoculated at three ratios. After co-cultivating for 5 days, the ratio of CD45/HLA-ABC cells was detected by flow cytometry, and Perform absolute cell quantification. GFP positive represents tumor cells, CD45+HLA-ABC+ cells represent NK cells, and CD45+HLA-ABC- cells represent UCAR-T cells.
结果如图4所示，在NK细胞存在下，识别NKG2A的四基因敲除的UCAR-T细胞促进组合物中UCAR-T细胞的体外存活和/或扩增、以及发挥协同BCMA-UCAR-T细胞的抗肿瘤作用。The results are shown in Figure 4, in the presence of NK cells, the four-gene knockout UCAR-T cells that recognize NKG2A promote the in vitro survival and/or expansion of UCAR-T cells in the composition, and exert synergistic BCMA-UCAR-T Antitumor effect of cells.
实施例9、识别NKG2A的UCAR-T细胞发挥体内协同抗肿瘤能力Example 9: UCAR-T cells that recognize NKG2A exert synergistic anti-tumor ability in vivo
皮下接种5×10 6个RPMI-8226细胞于NPG小鼠中，接种13天后平均瘤体积为250mm 3左右，将小鼠分4组(UTD-FKO，BCMA UCAR-T-FKO，BCMA UCAR-T-FKO+UTD+NK，BCMA UCAR-T-FKO+NKG2A UCAR-T-FKO+NK)，每组5只。分组后，尾静脉分别注射1×10 6个UTD-FKO、1×10 6个BCMA UCAR-T-FKO细胞、1×10 6个BCMA UCAR-T-FKO和1×10 6个UTD-FKO、1×10 6个BCMA UCAR-T-FKO和1×10 6个NKG2A UCAR-T-FKO。D13，D15,D18,D20,D22按上述分组尾静脉注射1×10 6个的 NK细胞，一共5次。参照实施例4所述方法绘制肿瘤生长曲线。 5×10 6 RPMI-8226 cells were inoculated subcutaneously in NPG mice. The average tumor volume was about 250mm 3 13 days after inoculation. The mice were divided into 4 groups (UTD-FKO, BCMA UCAR-T-FKO, BCMA UCAR-T -FKO+UTD+NK, BCMA UCAR-T-FKO+NKG2A UCAR-T-FKO+NK), 5 rats in each group. After grouping, 1×10 6 UTD-FKO, 1×10 6 BCMA UCAR-T-FKO cells, 1×10 6 BCMA UCAR-T-FKO and 1×10 6 UTD-FKO, 1×10 6 BCMA UCAR-T-FKO and 1×10 6 NKG2A UCAR-T-FKO. On D13, D15, D18, D20, and D22, 1×10 6 NK cells were injected into the tail vein according to the above grouping, a total of 5 times. The tumor growth curve was drawn according to the method described in Example 4.
结果如图5所示，在有NK细胞存在的情况下，识别NKG2A且四敲除的UCAR-T与识别BCMA且四敲除的UCAR-T细胞联用具有体内协同抗肿瘤作用。The results are shown in Figure 5. In the presence of NK cells, the combination of UCAR-T that recognizes NKG2A and four knockouts and UCAR-T cells that recognize BCMA and four knockouts has a synergistic anti-tumor effect in vivo.
本申请所述实施例包括将该实施例作为任何单一实施例或与任何其他实施例或其部分相结合。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。Embodiments described herein include that embodiment as any single embodiment or in combination with any other embodiment or portion thereof. In addition, it should be understood that after reading the above teaching content of the application, those skilled in the art can make various changes or modifications to the application, and these equivalent forms also fall within the scope defined by the appended claims of the application.
序列信息sequence information
Figure PCTCN2022106095-appb-000001
Figure PCTCN2022106095-appb-000001
Figure PCTCN2022106095-appb-000002
Figure PCTCN2022106095-appb-000002
Figure PCTCN2022106095-appb-000003
Figure PCTCN2022106095-appb-000003
Figure PCTCN2022106095-appb-000004
Figure PCTCN2022106095-appb-000004
Figure PCTCN2022106095-appb-000005
Figure PCTCN2022106095-appb-000005
Figure PCTCN2022106095-appb-000006
Figure PCTCN2022106095-appb-000006

Claims (32)

  1. 靶向NKG2A的第一工程细胞与靶向靶抗原的第二工程细胞的联合使用在疾病治疗或在制备用于治疗疾病的药物中的用途，其特征在于，所述第一工程细胞包括识别NKG2A的第一外源受体，所述第二工程细胞包含靶向肿瘤抗原和/或病原体抗原的第二外源受体。Use of the combined use of the first engineered cell targeting NKG2A and the second engineered cell targeting the target antigen in the treatment of diseases or in the preparation of medicines for treating diseases, characterized in that the first engineered cells include NKG2A-recognizing The second engineered cell comprises a second exogenous receptor targeting a tumor antigen and/or a pathogen antigen.
  2. 如权利要求1所述用途，其特征在于，所述第一外源受体还识别肿瘤抗原和/或病原体抗原。The use according to claim 1, characterized in that the first exogenous receptor also recognizes tumor antigens and/or pathogen antigens.
  3. 如权利要求1或2所述用途，其特征在于，所述第二外源受体识别肿瘤抗原和/或病原体抗原，所述疾病选自：实体瘤、血液瘤、自身免疫性疾病或其组合。The use according to claim 1 or 2, characterized in that the second exogenous receptor recognizes tumor antigens and/or pathogen antigens, and the disease is selected from: solid tumors, blood tumors, autoimmune diseases or combinations thereof .
  4. 如权利要求1-3任一所述用途，其特征在于，所述第一外源受体和/或第二外源受体各自独立地选自：嵌合抗原受体(CAR)、嵌合T细胞受体和T细胞抗原耦合器(TAC)。The use according to any one of claims 1-3, wherein the first exogenous receptor and/or the second exogenous receptor are each independently selected from: chimeric antigen receptor (CAR), chimeric T cell receptor and T cell antigen coupler (TAC).
  5. 如权利要求1-4任一所述用途，其特征在于，所述第一工程细胞和/或第二工程细胞各自独立地选自：免疫细胞、神经元、上皮细胞、内皮细胞或干细胞。The use according to any one of claims 1-4, wherein the first engineered cells and/or the second engineered cells are each independently selected from: immune cells, neurons, epithelial cells, endothelial cells or stem cells.
  6. 如权利要求5所述用途，其特征在于，所述免疫细胞选自:B细胞、单核细胞、自然杀伤细胞、嗜碱性粒细胞、嗜酸性粒细胞、中性粒细胞、树突状细胞、巨噬细胞、T细胞或其组合。purposes as claimed in claim 5, is characterized in that, described immune cell is selected from: B cell, monocyte, natural killer cell, basophil, eosinophil, neutrophil, dendritic cell , macrophages, T cells, or combinations thereof.
  7. 如权利要求6所述用途，其特征在于，所述T细胞选自：同种异体T细胞或自体T细胞。The use according to claim 6, characterized in that the T cells are selected from: allogeneic T cells or autologous T cells.
  8. 如权利要求1-7任一所述用途，其特征在于，所述第一工程细胞和/或第二工程细胞为包括如下a)～d)中任一项或两项或三项或四项的第一工程细胞和/或第二工程细胞：The use according to any one of claims 1-7, wherein the first engineered cell and/or the second engineered cell includes any one or two or three or four of the following a) to d) The first engineered cell and/or the second engineered cell:
    a)内源性HLA-I分子低表达或不表达；a) Low expression or no expression of endogenous HLA-I molecules;
    b)内源性TCR分子低表达或不表达；b) low or no expression of endogenous TCR molecules;
    c)内源性HLA-II分子低表达或不表达；和/或c) low or no expression of endogenous HLA-II molecules; and/or
    d)内源性NKG2A分子低表达或不表达。d) Low expression or no expression of endogenous NKG2A molecules.
  9. 如权利要求8所述的用途，其特征在于，所述第一工程细胞和/或第二工程细胞为包括如下a)～d)中任一项或两项或三项或四项的第一工程细胞和/或第二工程细胞：The use according to claim 8, characterized in that, the first engineered cell and/or the second engineered cell is a first engineered cell comprising any one or two or three or four of the following a) to d). Engineered cells and/or second engineered cells:
    a)内源性HLA-I分子低表达或不表达包括编码HLA-I蛋白的基因的敲除；a) Low or no expression of endogenous HLA-I molecules including knockout of genes encoding HLA-I proteins;
    b)内源性TCR分子低表达或不表达包括编码TCR蛋白的基因的敲除；b) Low or no expression of endogenous TCR molecules including knockout of genes encoding TCR proteins;
    c)内源性HLA-II分子低表达或不表达包括编码HLA-II蛋白的基因的敲除；和/或c) low or no expression of endogenous HLA-II molecules including knockout of genes encoding HLA-II proteins; and/or
    d)内源性NKG2A分子低表达或不表达包括编码NKG2A蛋白的基因的敲除。d) Low or no expression of endogenous NKG2A molecules including knockout of the gene encoding the NKG2A protein.
  10. 如权利要求9所述的用途，其特征在于，所述第一工程细胞和/或第二工程细胞为包括a)～e)中任一项的第一工程细胞和/或第二工程细胞：The use according to claim 9, wherein the first engineered cell and/or the second engineered cell is the first engineered cell and/or the second engineered cell comprising any one of a) to e):
    a)采用CRISPR技术敲除内源性B2M；a) Using CRISPR technology to knock out endogenous B2M;
    b)采用CRISPR技术敲除内源性B2M/TCR；b) Using CRISPR technology to knock out endogenous B2M/TCR;
    c)采用CRISPR技术敲除内源性B2M/TCR/CIITA；c) Using CRISPR technology to knock out endogenous B2M/TCR/CIITA;
    d)采用CRISPR技术敲除内源性B2M/TCR/NKG2A；或d) CRISPR technology is used to knock out endogenous B2M/TCR/NKG2A; or
    e)采用CRISPR技术敲除内源性B2M/TCR/CIITA/NKG2A。e) CRISPR technology was used to knock out endogenous B2M/TCR/CIITA/NKG2A.
  11. 如权利要求1-10任一所述的用途，其特征在于，所述第一外源受体包括NKG2A抗原结合域，所述NKG2A抗原包含如SEQ ID NO:11所示的氨基酸序列；和/或所述第二外源受体包括肿瘤抗原结合域。The use according to any one of claims 1-10, wherein the first exogenous receptor comprises an NKG2A antigen-binding domain, and the NKG2A antigen comprises an amino acid sequence as shown in SEQ ID NO: 11; and/ Or the second exogenous receptor comprises a tumor antigen binding domain.
  12. 如权利要求13所述的用途，其特征在于，所述抗原结合域选自：抗体、受体、细胞黏附分子、非抗体分子支架或其组合，其中所述抗体选自：单克隆抗体、多克隆抗体、天然抗体、双特异性抗体、嵌合抗体、Fv、Fab、Fab’、Fab’-SH、F(ab’)2、线性抗体、单链抗体(例如scFv)、单域抗体或其组合。The use according to claim 13, wherein the antigen binding domain is selected from: antibodies, receptors, cell adhesion molecules, non-antibody molecular scaffolds or combinations thereof, wherein the antibodies are selected from: monoclonal antibodies, polyclonal antibodies Clonal antibodies, natural antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single chain antibodies (e.g. scFv), single domain antibodies or combination.
  13. 如权利要求11-12任一所述的用途，其特征在于，所述第一外源受体包含：识别NKG2A的抗体和识别肿瘤抗原的抗体，其中，识别NKG2A的抗体和识别肿瘤抗原的抗体的连接方式是：The use according to any one of claims 11-12, wherein the first exogenous receptor comprises: an antibody recognizing NKG2A and an antibody recognizing a tumor antigen, wherein the antibody recognizing NKG2A and the antibody recognizing a tumor antigen The way to connect is:
    (1)识别NKG2A的抗体的轻链/重链(或轻链可变区/重链可变区)—识别NKG2A的抗体的重链/轻链(或重链可变区/轻链可变区)—识别肿瘤抗原的抗体的重链/轻链(或重链可变区/轻链可变区)—识别肿瘤抗原的抗体的轻链/重链(或轻链可变区/重链可变区)；(1) Light chain/heavy chain (or light chain variable region/heavy chain variable region) of an antibody that recognizes NKG2A—heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes NKG2A Region)—heavy chain/light chain (or heavy chain variable region/light chain variable region) of an antibody that recognizes a tumor antigen—light chain/heavy chain (or light chain variable region/heavy chain) of an antibody that recognizes a tumor antigen variable region);
    (2)识别肿瘤抗原的抗体的轻链(或轻链可变区)—识别NKG2A的抗体的重链(或重链可变区)—识别NKG2A的抗体的轻链(或轻链可变区)—识别肿瘤抗原的抗体的重链(或重链可变区)；和/或(2) The light chain (or light chain variable region) of an antibody that recognizes a tumor antigen—the heavy chain (or heavy chain variable region) of an antibody that recognizes NKG2A—the light chain (or light chain variable region) of an antibody that recognizes NKG2A )—the heavy chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen; and/or
    (3)识别NKG2A的抗体的轻链(或轻链可变区)—识别肿瘤抗原的抗体的重链(或重链可变区)—识别肿瘤抗原的抗体的轻链(或轻链可变区)—识别NKG2A的抗体的重链(或重链可变区)。(3) The light chain (or light chain variable region) of an antibody that recognizes NKG2A—the heavy chain (or heavy chain variable region) of an antibody that recognizes a tumor antigen—the light chain (or light chain variable region) of an antibody that recognizes a tumor antigen region) - the heavy chain (or heavy chain variable region) of an antibody that recognizes NKG2A.
  14. 如权利要求11-13任一所述的用途，其特征在于，所述NKG2A抗原结合域包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2、SEQ ID NO:5所示的HCDR3、SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2、和/或SEQ ID NO:8所示的LCDR3；或所述NKG2A抗原结合域包含SEQ ID NO:1所述的重链可变区和/或SEQ ID NO:2所示的轻链可变区；或所述NKG2A抗原结合域包含SEQ ID NO:36、37、38、39、40或52所示的序列。The use according to any one of claims 11-13, wherein the NKG2A antigen-binding domain comprises HCDR1 shown in SEQ ID NO:3, HCDR2 shown in SEQ ID NO:4, and HCDR2 shown in SEQ ID NO:5. HCDR3 shown in , LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and/or LCDR3 shown in SEQ ID NO:8; or the NKG2A antigen binding domain comprises SEQ ID NO:1 The heavy chain variable region and/or the light chain variable region shown in SEQ ID NO: 2; or the NKG2A antigen-binding domain comprises SEQ ID NO: 36, 37, 38, 39, 40 or 52 the sequence of.
  15. 如权利要求1-14任一所述的用途，其特征在于，所述第一外源受体识别肿瘤抗原；优 选地，所述肿瘤抗原选自：WT1、BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRⅧ或其组合。The use according to any one of claims 1-14, wherein the first exogenous receptor recognizes a tumor antigen; preferably, the tumor antigen is selected from: WT1, BCMA, CD19, GPC3, Claudin18.2 , EGFR, EGFR VIII or combinations thereof.
  16. 如权利要求15所述的用途，其特征在于，所述第一外源受体还包含识别BCMA的抗体；优选地，所述识别BCMA的抗体包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2、和/或SEQ ID NO:18所示的LCDR3；或所述识别BCMA的抗体包含SEQ ID NO:19所示的重链可变区和/或SEQ ID NO:20所示的轻链可变区；或所述识别BCMA的抗体含有SEQ ID NO:21、22、23、24或25所示序列。The use according to claim 15, characterized in that, the first exogenous receptor also comprises an antibody that recognizes BCMA; preferably, the antibody that recognizes BCMA comprises HCDR1 shown in SEQ ID NO: 13, SEQ ID HCDR2 shown in NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and/or LCDR3 shown in SEQ ID NO:18 or the antibody that recognizes BCMA comprises the heavy chain variable region shown in SEQ ID NO:19 and/or the light chain variable region shown in SEQ ID NO:20; or the antibody that recognizes BCMA contains SEQ ID NO : the sequence shown in 21, 22, 23, 24 or 25.
  17. 如权利要求1-16任一所述的用途，其特征在于，所述第二外源受体识别肿瘤抗原；优选地，所述肿瘤抗原选自：WT1、BCMA、CD19、GPC3、Claudin18.2、EGFR、EGFRⅧ或其组合。The use according to any one of claims 1-16, wherein the second exogenous receptor recognizes a tumor antigen; preferably, the tumor antigen is selected from: WT1, BCMA, CD19, GPC3, Claudin18.2 , EGFR, EGFR VIII or combinations thereof.
  18. 如权利要求17所述的用途，其特征在于，所述第二外源受体包含识别BCMA的抗体；优选地，所述识别BCMA的抗体包含SEQ ID NO:13所示的HCDR1、SEQ ID NO:14所示的HCDR2、SEQ ID NO:15所示的HCDR3、SEQ ID NO:16所示的LCDR1、SEQ ID NO:17所示的LCDR2、和/或SEQ ID NO:18所示的LCDR3；或所述识别BCMA的抗体包含SEQ ID NO:19所示的重链可变区和/或SEQ ID NO:20所示的轻链可变区；或所述识别BCMA的抗体含有SEQ ID NO:21、22、23、24或25所示序列。The use according to claim 17, wherein the second foreign receptor comprises an antibody that recognizes BCMA; preferably, the antibody that recognizes BCMA comprises HCDR1, SEQ ID NO shown in SEQ ID NO:13 HCDR2 shown in :14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and/or LCDR3 shown in SEQ ID NO:18; Or the antibody that recognizes BCMA comprises the heavy chain variable region shown in SEQ ID NO:19 and/or the light chain variable region shown in SEQ ID NO:20; or the antibody that recognizes BCMA contains SEQ ID NO: The sequence shown in 21, 22, 23, 24 or 25.
  19. 如权利要求1-18任一所述的用途，其特征在于，所述第一外源受体包含SEQ ID NO：9、41、42、43、44、45或53中任一项所示的序列，或SEQ ID NO：36、37、38、39、40或52分别与EQ ID NO：49、50或51顺序连接而成的序列；和/或第二外源受体包含：如SEQ ID NO:26、27或28中任一项所示的序列，或SEQ ID NO：21、22、23、24或25分别与EQ ID NO：49、50或51顺序连接而成的序列。The use according to any one of claims 1-18, wherein the first exogenous receptor comprises any one of SEQ ID NO: 9, 41, 42, 43, 44, 45 or 53 Sequence, or the sequence formed by connecting SEQ ID NO: 36, 37, 38, 39, 40 or 52 with EQ ID NO: 49, 50 or 51 respectively; and/or the second foreign receptor comprises: such as SEQ ID The sequence shown in any one of NO: 26, 27 or 28, or the sequence formed by sequentially linking SEQ ID NO: 21, 22, 23, 24 or 25 with EQ ID NO: 49, 50 or 51 respectively.
  20. 如权利要求4-19任一所述的用途，其特征在于，所述第一外源受体是第一CAR，所述第二外源受体是第二CAR。The use according to any one of claims 4-19, wherein the first exogenous receptor is a first CAR, and the second exogenous receptor is a second CAR.
  21. 如权利要求20任一所述的用途，其特征在于，所述第一CAR包括：The use according to any one of claim 20, wherein the first CAR comprises:
    (1)识别NKG2A的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD3ζ；(1) recognize the antigen-binding domain of NKG2A, optionally recognize the antigen-binding domain of tumor and/or pathogen antigen, the transmembrane region of CD28 or CD8, CD3ζ;
    (2)识别NKG2A的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD28的共刺激信号结构域和CD3ζ；(2) recognize the antigen-binding domain of NKG2A, optionally recognize the antigen-binding domain of tumor and/or pathogen antigen, the transmembrane region of CD28 or CD8, the co-stimulatory signal domain of CD28 and CD3ζ;
    (3)识别NKG2A的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD137的共刺激信号结构域和CD3ζ；和/或(3) an antigen-binding domain that recognizes NKG2A, optionally an antigen-binding domain that recognizes tumor and/or pathogen antigens, a transmembrane region of CD28 or CD8, a co-stimulatory signaling domain of CD137, and CD3ζ; and/or
    (4)识别NKG2A的抗原结合域、任选地识别肿瘤和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区，CD28的共刺激信号结构域，CD137的共刺激信号结构域和CD3ζ；(4) Antigen-binding domains that recognize NKG2A, optionally recognize antigen-binding domains of tumor and/or pathogen antigens, transmembrane regions of CD28 or CD8, costimulatory signal domains of CD28, costimulatory signal domains of CD137 and CD3ζ ;
    和/或and / or
    所述第二CAR包括：The second CAR includes:
    (1)识别肿瘤抗原和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区、CD3ζ；(1) Antigen-binding domains that recognize tumor antigens and/or pathogen antigens, transmembrane regions of CD28 or CD8, and CD3ζ;
    (2)识别肿瘤抗原和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；(2) Antigen-binding domains that recognize tumor antigens and/or pathogen antigens, CD28 or CD8 transmembrane regions, CD28 co-stimulatory signal domains and CD3ζ;
    (3)识别肿瘤抗原和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或(3) Antigen-binding domains that recognize tumor antigens and/or pathogen antigens, transmembrane regions of CD28 or CD8, co-stimulatory signal domains of CD137, and CD3ζ; and/or
    (4)识别肿瘤抗原和/或病原体抗原的抗原结合域，CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。(4) Antigen-binding domains that recognize tumor antigens and/or pathogen antigens, CD28 or the transmembrane region of CD8, costimulatory signal domains of CD28, costimulatory signal domains of CD137 and CD3ζ.
  22. 如权利要求10-20任一所述用途，其特征在于，所述CRISPR技术使用的gRNA包括如SEQ ID NO：10、46、47、48中任一项所示的序列或其组合。The use according to any one of claims 10-20, wherein the gRNA used by the CRISPR technology comprises a sequence as shown in any one of SEQ ID NO: 10, 46, 47, 48 or a combination thereof.
  23. 如权利要求1-22任一所述用途，其特征在于，所述第一工程细胞与所述第二工程细胞各自存在于不同的容器中。The use according to any one of claims 1-22, characterized in that the first engineered cells and the second engineered cells are respectively present in different containers.
  24. 如权利要求1-22任一所述用途，其特征在于，第一工程细胞和第二工程细胞存在于细胞混合物中，所述细胞混合物包含第一工程细胞与第二工程细胞。The use according to any one of claims 1-22, characterized in that the first engineered cells and the second engineered cells are present in a cell mixture, and the cell mixture comprises the first engineered cells and the second engineered cells.
  25. 一种细胞组合物，其特征在于，包括第一工程细胞和第二工程细胞，其中第一工程细胞和第二工程细胞如权利要求1-24中任一项所定义，任选所述组合物还包括药学上可接受的佐剂。A cell composition, characterized in that it comprises a first engineered cell and a second engineered cell, wherein the first engineered cell and the second engineered cell are as defined in any one of claims 1-24, and the composition is optionally Also included are pharmaceutically acceptable adjuvants.
  26. 预防、缓解和/或***的方法，其包括向有需要的受试者施用如权利要求25所述的细胞组合物或者向有需要的受试者同时或先后施用权利要求1-24中任一项所定义的第一工程细胞和第二工程细胞，所述靶抗原包括肿瘤抗原。A method for preventing, alleviating and/or treating tumors, comprising administering the cell composition according to claim 25 to a subject in need or administering any of claims 1-24 simultaneously or sequentially to a subject in need In the first engineered cell and the second engineered cell as defined, the target antigen includes a tumor antigen.
  27. 如权利要求1-24任一所述的用途、权利要求26所述的方法、权利要求25所述的细胞组合物，其特征在于，所述第一工程细胞与所述第二工程细胞被配置为同时向受试者施用、或分别向受试者施用。The use according to any one of claims 1-24, the method according to claim 26, and the cell composition according to claim 25, wherein the first engineered cells and the second engineered cells are configured For simultaneous administration to the subject, or separate administration to the subject.
  28. 一种试剂盒，其包括权利要求25所述的细胞组合物或者如权利要求1-24中任一项所定义的第一工程细胞和第二工程细胞。A kit comprising the cell composition of claim 25 or the first engineered cell and the second engineered cell as defined in any one of claims 1-24.
  29. 如权利要求28所述的试剂盒，其还包括用于治疗和/或预防肿瘤、病原体感染、自身免疫性疾病或同种异体移植的书面说明书。The kit according to claim 28, further comprising written instructions for treating and/or preventing tumors, pathogenic infections, autoimmune diseases or allogeneic transplantation.
  30. 增加识别NKG2A抗原的第一工程细胞和识别肿瘤抗原和/或病原体抗原的第二工程细 胞在有宿主免疫细胞存在时的存活时间和/或扩增能力的方法，包括：The method for increasing the survival time and/or expansion ability of the first engineered cell recognizing NKG2A antigen and the second engineered cell recognizing tumor antigen and/or pathogen antigen in the presence of host immune cells, comprising:
    a)同时或先后提供第一工程细胞和第二工程细胞；a) providing the first engineered cell and the second engineered cell simultaneously or successively;
    b)任选地，通过编码参与响应自体和非自体抗原识别多肽的至少一种内源基因表达、活性和/或信号被降低或抑制来修饰所述第一工程细胞和/或第二工程细胞；b) Optionally, modifying said first engineered cell and/or second engineered cell by reducing or inhibiting expression, activity and/or signaling of at least one endogenous gene encoding a polypeptide involved in responding to self and non-self antigen recognition ;
    c)编码识别NKG2A抗原的第一外源受体的多核苷酸来修饰所述第一工程细胞；c) modifying the first engineered cell with a polynucleotide encoding a first exogenous receptor that recognizes an NKG2A antigen;
    d)编码识别非NKG2A抗原的第二外源受体的多核苷酸来修饰所述第二工程细胞。d) modifying the second engineered cell with a polynucleotide encoding a second exogenous receptor that recognizes an antigen other than NKG2A.
  31. 如权利要求30所述方法，其特征在于，所述第一工程细胞和/或第二工程细胞选自：免疫细胞、神经元、上皮细胞、内皮细胞或干细胞；优选地，所述第一工程细胞和/或第二工程细胞选自T或NKT细胞。The method according to claim 30, wherein the first engineered cells and/or the second engineered cells are selected from: immune cells, neurons, epithelial cells, endothelial cells or stem cells; preferably, the first engineered cells The cells and/or second engineered cells are selected from T or NKT cells.
  32. 如权利要求31所述方法，其特征在于，步骤b)中，通过CRISPR技术敲除第一工程细胞和/或第二工程细胞的内源性B2M、B2M/TCR、B2M/TCR/CIITA、B2M/TCR/NKG2A或B2M/TCR/CIITA/NKG2A。The method according to claim 31, wherein in step b), the endogenous B2M, B2M/TCR, B2M/TCR/CIITA, B2M of the first engineering cell and/or the second engineering cell are knocked out by CRISPR technology /TCR/NKG2A or B2M/TCR/CIITA/NKG2A.
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