WO2023284874A1 - Composition et procédé pour l'immunologie tumorale - Google Patents

Composition et procédé pour l'immunologie tumorale Download PDF

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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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English (en)
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/zh
Publication of WO2023284874A1 publication Critical patent/WO2023284874A1/fr

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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.

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Abstract

La présente invention concerne une composition comprenant une première cellule immunitaire identifiant un polypeptide NKG2A et une seconde cellule immunitaire identifiant un antigène tumoral et/ou pathogène, ainsi qu'une utilisation de celle-ci dans le traitement de maladies.
PCT/CN2022/106095 2021-07-16 2022-07-15 Composition et procédé pour l'immunologie tumorale WO2023284874A1 (fr)

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