WO2023284875A1 - Récepteur antigénique chimérique - Google Patents

Récepteur antigénique chimérique Download PDF

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Publication number
WO2023284875A1
WO2023284875A1 PCT/CN2022/106096 CN2022106096W WO2023284875A1 WO 2023284875 A1 WO2023284875 A1 WO 2023284875A1 CN 2022106096 W CN2022106096 W CN 2022106096W WO 2023284875 A1 WO2023284875 A1 WO 2023284875A1
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cells
binding
region
binding region
car
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Chinese (zh)
Inventor
李宗海
廖朝晖
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Crage Medical Co Ltd
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Crage Medical Co Ltd
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Priority to CN202280042415.8A priority Critical patent/CN117730145A/zh
Publication of WO2023284875A1 publication Critical patent/WO2023284875A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • the present application provides a chimeric antigen receptor (CAR) and an engineered dual-targeted T cell and a preparation method thereof.
  • CAR chimeric antigen receptor
  • the engineered dual-targeted T cells and the composition containing the engineered dual-targeted T cells can be used for disease treatment.
  • Immune cell therapy as cell-based cancer therapy is currently the most promising therapeutic approach, immune cells have the potential to target tumor cells while sparing normal tissues; clinical observations indicate that they have major anticancer activity. There is still an urgent need to improve immune cell therapy by broadening the applicability and enhancing the efficacy of immune cells.
  • the effective activation of CAR-T cells depends heavily on the specificity of antibodies that recognize tumor-associated antigens and the affinity of antigen binding. Therefore, under the current situation that the design of the intracellular signal transduction region of CAR-T cells has matured, the design of the antigen-binding region has become the focus and key of the development of new CAR-T technology.
  • the purpose of the present application is to provide an engineered dual-targeting T cell and a chimeric antigen receptor (CAR), wherein antibody fragments targeting two antigens respectively are expressed in tandem on a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • provided engineered cells exhibit an enhanced immune response, including treatment of cancer, microbial infection, and/or viral infection in a subject.
  • the provided engineered cells not only have the effect of resisting host immune rejection, but also have anti-tumor effect.
  • methods of treating a subject by administering a therapeutically effective amount of the provided engineered T cells.
  • the present application provides a chimeric antigen receptor (CAR), said CAR comprising a bispecific antigen binding domain, said bispecific antigen binding domain comprising an NKG2A antigen binding domain and a tumor antigen binding domain, said The NKG2A antigen binding domain comprises a first binding region L and a first binding region H, the tumor antigen binding domain comprises a second binding region L and a second binding region H, the first binding region L, the first binding region H, The second bonding region L and the second bonding region H are connected in a manner to form a loop structure.
  • CAR chimeric antigen receptor
  • the loop structure is formed in the following manner: the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to the The two ends of the antibody are linked.
  • the antibody comprises in order from the N-terminus to the C-terminus:
  • the binding region H the binding region L.
  • the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.
  • the binding region H of the another antigen-binding domain is connected to the N-terminus of the antibody, and the binding region L of the other antigen-binding domain is connected to the C-terminus of the antibody.
  • connection is that the binding region H of the antibody is the first binding region H
  • the connection is that the binding region L of the antibody is the first binding region L
  • the binding region L of the other antigen-binding domain is is the second binding region L
  • the binding region H of the other antigen-binding domain is the second binding region H.
  • connection is that the binding region H of the antibody is the second binding region H, and the connection is that the binding region L of the antibody is the second binding region L.
  • the binding region L of the other antigen-binding domain is the first binding region L
  • the binding region H of the other antigen-binding domain is the first binding region H.
  • the antibody comprises in order from the N-terminus to the C-terminus:
  • the binding region L the binding region H.
  • the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, and the binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.
  • the binding region H of the another antigen-binding domain is connected to the N-terminus of the antibody, and the binding region L of the other antigen-binding domain is connected to the C-terminus of the antibody.
  • connection is that the binding region H of the antibody is the first binding region H
  • the connection is that the binding region L of the antibody is the first binding region L
  • the binding region L of the other antigen-binding domain is is the second binding region L
  • the binding region H of the other antigen-binding domain is the second binding region H.
  • connection is that the binding region H of the antibody is the second binding region H, and the connection is that the binding region L of the antibody is the second binding region L.
  • the binding region L of the other antigen-binding domain is the first binding region L
  • the binding region H of the other antigen-binding domain is the first binding region H.
  • the first binding region L comprises a first light chain or a first light chain variable region (VL)
  • the first binding region H comprises a first heavy chain or a first heavy chain variable region (VL).
  • Region (VH) said second binding region L comprises a second light chain or a second light chain variable region (VL)
  • said second binding region H comprises a second heavy chain or a second heavy chain variable region ( VH).
  • the first binding region L comprises a first light chain variable region (VL)
  • the first binding region H comprises a first heavy chain variable region (VH)
  • the second binding region Region L comprises a second light chain variable region (VL) and said second binding region H comprises a second heavy chain variable region (VH).
  • the antibody comprises a scFv.
  • said first light chain comprises said first light chain variable region (VL)
  • said first heavy chain comprises said first heavy chain variable region (VH)
  • said second Two light chains comprise said second light chain variable region (VL)
  • said second heavy chain comprises said second heavy chain variable region (VH).
  • the NKG2A antigen binding domain comprises a single domain antibody, and/or the tumor antigen binding domain comprises a single domain antibody.
  • the first light chain variable region comprises a first light chain complementarity determining region (LCDR) as set forth in any one of SEQ ID NOs: 6-8, or a combination thereof.
  • LCDR first light chain complementarity determining region
  • the first heavy chain variable region comprises a first heavy chain complementarity determining region (HCDR) as set forth in any one of SEQ ID NOs: 3-5, or a combination thereof.
  • the first light chain variable region comprises a first light chain complementarity determining region 1 (LCDR1), a first light chain complementarity determining region 2 (LCDR2), a first light chain complementarity determining Region 3 (LCDR3)
  • the first LCDR1 comprises the amino acid sequence shown in SEQ ID NO:6
  • the first LCDR2 comprises the amino acid sequence shown in SEQ ID NO:7
  • the first LCDR3 comprises the amino acid sequence shown in SEQ ID NO:7 Amino acid sequence shown in ID NO:8.
  • the first heavy chain variable region comprises a first heavy chain complementarity determining region 1 (HCDR1), a first heavy chain complementarity determining region 2 (HCDR2), a first heavy chain complementarity determining Region 3 (HCDR3)
  • the first HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3
  • the first HCDR2 comprises the amino acid sequence shown in SEQ ID NO:4
  • the NKG2A antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 3-8.
  • the first heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 1
  • the first light chain variable region (VL) comprises the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 2.
  • the tumor antigen comprises CD19, GPC3, Claudin18.2, WT1, HER2, EGFR, BCMA, or a combination thereof.
  • the tumor antigen comprises BCMA.
  • the tumor antigen binding domain comprises a BCMA antigen binding domain.
  • the second light chain variable region comprises a second light chain complementarity determining region (LCDR) as set forth in any one of SEQ ID NOs: 12-14, or a combination thereof.
  • LCDR second light chain complementarity determining region
  • the second heavy chain variable region comprises a second heavy chain complementarity determining region (HCDR) as set forth in any one of SEQ ID NOs: 9-11, or a combination thereof.
  • HCDR second heavy chain complementarity determining region
  • the second light chain variable region comprises a second light chain complementarity determining region 1 (LCDR1), a second light chain complementarity determining region 2 (LCDR2), a second light chain complementarity determining Region 3 (LCDR3)
  • the second LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 12
  • the second LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 13
  • the second LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13
  • the second heavy chain variable region comprises a second heavy chain complementarity determining region 1 (HCDR1), a second heavy chain complementarity determining region 2 (HCDR2), a second heavy chain complementarity determining region Region 3 (HCDR3)
  • the second HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9
  • the second HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10
  • the second HCDR3 comprises the amino acid sequence shown in SEQ ID NO : Amino acid sequence shown in 11.
  • the BCMA antigen binding domain comprises the amino acid sequence shown in SEQ ID NO: 9-14.
  • the NKG2A antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:3-8
  • the BCMA antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:9-14.
  • the second heavy chain variable region (VH) comprises the amino acid sequence shown in SEQ ID NO: 15
  • the second light chain variable region (VL) comprises the amino acid sequence as shown in SEQ ID NO: The amino acid sequence shown in 16.
  • the second binding region L comprises a light chain variable region in the sequence shown in any one of SEQ ID NO:46-50
  • the second binding region H comprises such as SEQ ID NO The heavy chain variable region in the sequence shown in any one of :46-50.
  • the NKG2A antigen binding domain comprises a first heavy chain variable region as shown in SEQ ID NO: 1 and a first light chain variable region as shown in SEQ ID NO: 2, said The BCMA antigen binding domain comprises a second heavy chain variable region as set forth in SEQ ID NO: 15 and a second light chain variable region as set forth in SEQ ID NO: 16.
  • the bispecific antigen binding domain comprises the following linkages from the N-terminus to the C-terminus:
  • the bispecific antigen binding domain comprises the following linkages from the N-terminus to the C-terminus:
  • the linking includes connecting via a linker, the linker comprising Lin1 or (G4S)n, wherein n is an integer equal to or greater than 1; the Lin1 comprises Lin1 as shown in SEQ ID NO: 18 The amino acid sequence shown.
  • n 1 or 3.
  • the bispecific antigen binding domain comprises the following linkages from the N-terminus to the C-terminus:
  • the bispecific antigen binding domain comprises the following linkages from the N-terminus to the C-terminus:
  • the bispecific antigen binding domain comprises the following linkages from the N-terminus to the C-terminus:
  • the bispecific antigen binding domain comprises the following linkages from the N-terminus to the C-terminus:
  • the antibody comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 39-41.
  • the antibody comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 46-50.
  • the bispecific antigen binding domain comprises the amino acid sequence set forth in any one of SEQ ID Nos: 20, 23 and 26.
  • the CAR comprises a transmembrane domain.
  • the CAR comprises an intracellular signaling domain.
  • the CAR comprises a transmembrane domain, an intracellular signaling domain.
  • the transmembrane domain comprises a CD28 transmembrane domain or a CD8 transmembrane domain.
  • the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:30
  • the CD28 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:31.
  • the intracellular signaling domain comprises a CD3 ⁇ intracellular signaling domain.
  • the CD3 ⁇ intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:34.
  • the CAR further comprises a co-stimulatory signaling domain.
  • the CAR comprises two identical or different co-stimulatory signaling domains.
  • the costimulatory signaling domain comprises a 4-1BB costimulatory signaling domain and/or a CD28 costimulatory signaling domain.
  • the CAR comprises a 4-1BB co-stimulatory signaling domain and a CD3 ⁇ intracellular signaling domain.
  • the 4-1BB co-stimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:33
  • the CD28 costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:32 .
  • the CAR comprises a hinge region.
  • the hinge region comprises a CD8 hinge region.
  • the CD8 hinge region comprises the amino acid sequence shown in SEQ ID NO:35.
  • the CAR comprises a CD8 transmembrane domain and a CD8 hinge domain.
  • the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:30
  • the CD8 hinge domain comprises the amino acid sequence shown in SEQ ID NO:35.
  • the CAR comprises the amino acid sequence shown in any one of SEQ ID Nos: 22, 25 and 28.
  • the present application also provides a nucleic acid molecule encoding the CAR described in the present application.
  • the nucleic acid molecule comprises a nucleotide sequence as described in any one of SEQ ID 21, 24 and 27.
  • the present application also provides a vector comprising the nucleic acid molecule described in the present application.
  • the present application also provides a cell comprising the CAR described in the present application, the nucleic acid molecule described in the present application and/or the vector described in the present application.
  • the cells comprise immune cells.
  • the cells are 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 cells are T cells.
  • the T cells are autologous T cells and/or allogeneic T cells.
  • the T cells are stem cell-derived autologous T cells and/or stem cell-derived allogeneic T cells.
  • the T cells are primary autologous T cells and/or primary allogeneic T cells.
  • the T cells comprise a genetic modification comprising inhibiting or eliminating the expression, activity of at least one endogenous gene involved in responding to self and/or alloantigen recognition polypeptides.
  • the endogenous TCR and MHC of the cells are not expressed or expressed at a low level.
  • the cells comprise:
  • the low or no expression of the endogenous HLA-I protein comprises knocking out the gene encoding endogenous HLA-I
  • the low expression or no expression of the endogenous TCR protein comprises knocking out the gene encoding the endogenous TCR, and/or
  • the low expression or no expression of the endogenous NKG2A molecule includes knocking out the gene encoding endogenous NKG2A.
  • endogenous TRAC and/or B2M are knocked out using gene knockout technology.
  • the gene knockout technology is CRISPR/Cas9 technology.
  • the cells comprise:
  • the gRNA used by the CRISPR/Cas9 technology includes a nucleotide sequence as shown in any one of SEQ ID NO: 36-38 or a combination thereof.
  • the present application also provides a pharmaceutical composition, which comprises the CAR described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application and/or the cell described in the present application, and a pharmaceutically acceptable Accepted carrier.
  • the present application also provides a kit comprising the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, and/or the CAR described in the present application.
  • the pharmaceutical composition comprising the CAR described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, and/or the CAR described in the present application.
  • the present application also provides a method for improving the survival time and/or expansion ability of tumor-targeting immune cells in the presence of host immune cells, including:
  • modifying the immune cells comprising inhibiting or eliminating the expression and activity of at least one endogenous gene involved in responding to self and/or foreign antigen recognition polypeptides;
  • the method in the step c), includes introducing the nucleic acid molecule described herein or the vector described herein into the immune cells.
  • the method further comprises allowing the immune cells to express the CAR described herein.
  • the method comprises modifying the immune cell, the modification comprising inhibiting or eliminating the expression, activity of at least one endogenous gene involved in responding to a self and/or alloantigen recognition polypeptide.
  • the CAR comprises:
  • NKG2A antigen binding domain and tumor antigen binding domain CD28 or CD8 transmembrane domain, CD28 co-stimulatory signaling domain, 4-1BB co-stimulatory signaling domain, and CD3 ⁇ ;
  • NKG2A antigen binding domain and tumor antigen binding domain NKG2A antigen binding domain and tumor antigen binding domain, CD28 or CD8 transmembrane domain, and CD3 ⁇ .
  • the NKG2A antigen-binding domain comprises an amino acid sequence as shown in SEQ ID NO:3-8; or a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:1 and comprising such as The light chain variable region of the amino acid sequence shown in SEQ ID NO:2.
  • the tumor antigen is BCMA
  • the BCMA antigen binding domain comprises the amino acid sequence shown in SEQ ID NO:9-14; or the heavy chain comprising the amino acid sequence shown in SEQ ID NO:15 can be Variable region and light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:20, 23 and 26.
  • the CAR comprises the amino acid sequence shown in any one of SEQ ID NOs: 22, 25 and 28.
  • said step b) comprises:
  • the gRNA used by the CRISPR/Cas9 technology includes a nucleotide sequence as shown in any one of SEQ ID NO: 36-38 or a combination thereof.
  • the immune cells are 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 immune cells are autologous or allogeneic T cells, stem cell-derived T cells, primary T cells, or autologous T cells derived from a human.
  • the methods are used to treat and/or prevent tumors.
  • the tumor comprises a hematological tumor and/or a solid tumor.
  • the hematological tumor comprises multiple myeloma.
  • the methods are performed ex vivo.
  • the present application also provides the CAR described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the cell described in the present application, and the pharmaceutical composition described in the present application.
  • the purposes in, described medicine is used for preventing, alleviating and/or treating tumor.
  • the present application also provides a method for preventing, alleviating and/or treating tumors, which includes administering the cells described in the present application and the pharmaceutical composition described in the present application to a subject in need.
  • the present application also provides the CAR described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the cell described in the present application, and the pharmaceutical composition described in the present application, which are used for Prevention, mitigation and/or treatment of tumors.
  • the tumor comprises a hematological tumor and/or a solid tumor.
  • the hematological tumor comprises multiple myeloma.
  • Figure 1 shows the killing effect of tandem CAR-T cells in this application on NK cells in vitro.
  • Figure 2 shows the effect of the tandem CAR-T cells in this application on killing tumor cells in vitro.
  • Figure 3 shows the in vivo anti-tumor effect of the tandem CAR-T cells in this application.
  • Figure 4 shows the secretion of cytokines after co-incubation of tandem CAR-T cells and tumor cells in this application.
  • Figure 5 shows that the tandem UCAR-T cells in this application resist NK cell killing in vitro.
  • Figure 6 shows the secretion of cytokines after co-incubation of tandem UCAR-T cells and tumor cells in this application.
  • Figure 7 shows the effect of the tandem UCAR-T cells in this application in inhibiting tumor growth in vivo.
  • Figure 8 shows the effect of tandem UCAR-T cells in this application against NK cell killing and tumor cell killing in vitro.
  • Figure 9A shows the effect of tandem UCAR-T cells in this application in inhibiting tumor growth in an orthotopic tumor model under NK cell conditions.
  • Figure 9B shows the effect of tandem UCAR-T cells in this application on the survival of mice in an orthotopic tumor model under NK cell conditions.
  • Figure 10 shows the specific infiltration of tumor tissue by tandem UCAR-T cells in this application.
  • Figure 11 shows that the tandem UCAR-T cells in this application do not cause graft-versus-host reaction.
  • the present application provides a dual-antigen-specific chimeric antigen receptor (CAR) targeting BCMA antigen and NKG2A antigen, and the CAR includes a BCMA antigen-binding domain and an NKG2A antigen-binding domain.
  • CAR dual-antigen-specific chimeric antigen receptor
  • the present application further provides nucleic acids, recombinant expression vectors, host cells, cell groups and pharmaceutical compositions related to the CAR of the present application.
  • the present application also provides methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal. T cells expressing the CAR described in this application have a strong ability to resist NK cells, thereby having better survival and/or proliferation ability and enhancing anti-tumor ability.
  • anti-NKG2A and anti-tumor antigen CAR can achieve the dual purpose of resisting host immune rejection (such as NK cell attack) and anti-tumor with only one carrier, which is conducive to simplifying the preparation of the process and quality inspection and quality control process.
  • 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.
  • Chimeric Antigen Receptor (Chimeric Antigen Receptor, CAR) generally refers to a fusion protein comprising an extracellular domain capable of binding antigen and at least one intracellular domain. It may include an antigen (eg, tumor-associated antigen (TAA)) binding region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. T cells genetically modified to express CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • TAA tumor-associated antigen
  • 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 is generally meant to include immunoglobulin molecules or immunologically active portions of immunological molecules, ie, molecules that contain an antigen binding site that specifically binds ("immunoreacts") with an antigen. They can include whole antibody molecules (also known as immunoglobulins) or fragments of antibody molecules that retain the ability to bind antigen. In some instances, the term “antibody” is used interchangeably with the term “immunoglobulin” and "antigen binding domain”.
  • Antibodies generally include but are not limited to monoclonal antibodies, polyclonal antibodies, natural antibodies, bispecific antibodies, chimeric antibodies, Fv, Fab, Fab', Fab'-SH, F(ab')2, linear antibodies, single chain Antibody molecules (eg scFv), single domain antibodies.
  • an "antibody” may be a single chain polypeptide comprising a light chain or a light chain variable region, a heavy chain or a heavy chain variable region.
  • a single-chain polypeptide consisting of a light chain and a heavy chain for example, a single-chain polypeptide consisting of a light chain variable region and a heavy chain variable region; for example, a single-chain polypeptide consisting of a light chain variable region and a heavy chain
  • CH comprises three domains CH1, CH2, CH3.
  • Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL).
  • CL consists of one domain.
  • VH and VL can be further divided into regions of hypervariability, 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 can consist of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • peptide As used interchangeably to refer to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • the term "bispecific” generally refers to the property of recognizing and/or binding to two different sites.
  • the site may be a certain number of amino acids or a peptide or a group of peptides thereof, such as a protein expressed on the surface of a cell.
  • the bispecific antigen-binding domain in the present application may comprise two different targeting binding domains, NKG2A antigen-binding domain and tumor antigen-binding domain.
  • “bispecific” is used interchangeably with “dual target”.
  • “bispecific CAR” and “tandem CAR” can be used interchangeably, and both are CARs provided by this application.
  • loop structure generally refers to a curved, non-closed peptide segment, which may be a loop structure formed by an NKG2A antigen-binding domain and a tumor antigen-binding domain through a suitable connection; for example, where The binding region H and the binding region L of one binding domain are connected into an antibody, and the binding region H (such as a heavy chain or a heavy chain variable region) and the binding region L (such as a light chain or a light chain variable region) of the other binding domain are respectively Ligated to both ends of the antibody.
  • binding region L generally refers to a polypeptide comprising a light chain functional fragment (eg, light chain, light chain variable region).
  • the binding region L is a light chain, eg, the binding region L is a light chain variable region.
  • the first binding region L is the light chain of an antibody that recognizes NKG2A, for example, the first binding region L is the light chain variable region of an antibody that recognizes NKG2A, for example, the second binding region L is the light chain of an antibody that recognizes BCMA
  • the second binding region L is the light chain variable region of an antibody that recognizes BCMA.
  • binding region H generally refers to a polypeptide comprising a heavy chain functional fragment (eg heavy chain, heavy chain variable region).
  • binding region H is a heavy chain, such as binding region H is a heavy chain variable region.
  • the first binding region H is a heavy chain of an antibody that recognizes NKG2A, for example, the first binding region H is a heavy chain variable region of an antibody that recognizes NKG2A, for example, the second binding region H is a heavy chain of an antibody that recognizes BCMA
  • the second binding region H is a heavy chain variable region of an antibody that recognizes BCMA.
  • C-terminal generally refers to one of the two ends of the polypeptide chain, the amino acid residue at this end carries a free alpha carboxyl group (—COOH).
  • the free alpha carboxyl groups may also be amidated in certain peptide chains.
  • N-terminal generally refers to one of the two ends of a polypeptide chain, the amino acid residue at this end carries a free ⁇ -amino group (—NH2).
  • the free alpha amino group may also be amidated or cyclized.
  • the term "light chain” generally refers to a peptide chain with a small molecular weight in an immunoglobulin, which may include a light chain variable region (VL) and a light chain constant region (CL). According to the difference in structure and antigenicity of the constant region, it can be divided into two categories: “ ⁇ light chain (kappa light chain)” and “lambda light chain (lambda light chain)”.
  • the term "heavy chain” generally refers to a peptide chain with a relatively large molecular weight in an immunoglobulin, which may include a heavy chain variable region (VL) and a heavy chain constant region (CL). According to the antigenicity of their constant regions, they can be divided into five categories: ⁇ , ⁇ , ⁇ , ⁇ and ⁇ .
  • light chain variable region generally refers to the amino-terminal domain of an antibody light chain.
  • the light chain variable region may be referred to as "VL".
  • VL complementarity determining regions
  • HVRs hypervariable regions
  • FRs framework regions
  • heavy chain variable region generally refers to the amino-terminal domain of an antibody heavy chain.
  • the heavy chain variable region may be referred to as "VH".
  • VH complementarity determining regions
  • HVRs hypervariable regions
  • FRs framework regions
  • linker and “linker fragment” are used interchangeably, and usually refer to an amino acid sequence with flexible characteristics.
  • the linker can be used to connect different functional modules in the chimeric polypeptide (or fusion protein, chimeric protein), and its functions can include improving the folding and stability of the chimeric polypeptide, etc.
  • the length of the connecting peptide can vary widely, such as about 1 to about 100, about 3 to about 20, about 5 to about 30, about 5 to about 18, or about 3 to about 8 amino acid glycine/serine linker fragment.
  • the connecting peptide can be (GGGGS)n/(G4S)n, G is glycine, S is serine, and n is an integer greater than or equal to 1; for example, n can be 1, 2, 3, 4 or 5.
  • G is glycine
  • S is serine
  • n is an integer greater than or equal to 1; for example, n can be 1, 2, 3, 4 or 5.
  • Each binding region H or binding region L in this application can be connected by a suitable linker to achieve the purpose of this application.
  • 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 CD28, HLA family.
  • HVGR host-versus-graft reaction
  • graft-versus-host disease generally refers to the recognition of host normal tissues by donor T lymphocytes due to TCR diversity and incompatibility with host HLA molecules The antigen on the cell is amplified and releases a series of cytokines to attack the host cell.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, eg a human patient.
  • the pharmaceutical composition described in the present application may comprise the nucleic acid molecule described in the present application, the carrier described in the present application and/or the cell described in the present application, and optionally a pharmaceutically acceptable adjuvant.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.
  • the vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell.
  • vectors include: plasmids, liposomes, bacteriophages such as lambda phages or M13 phages, and viral vectors.
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but not only.
  • tumor and cancer generally refer to cells that exhibit at least partial loss of control in normal growth and/or development.
  • tumors or cancer cells have often lost contact inhibition and may be invasive and/or have the ability to metastasize.
  • nucleic acid molecule or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, including any nucleotide sequence encoding a polypeptide of interest or a fragment thereof .
  • endogenous means that the nucleic acid molecule or polypeptide comes from the organism itself.
  • exogenous refers to a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or is not expressed at a level sufficient to function when overexpressed; encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as an exogenous , heterologous and overexpressed nucleic acid molecules and polypeptides.
  • mice rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs , goat, cow, horse, ape, monkey.
  • transplantation immune rejection means that after the host has transplanted allogeneic tissues, organs, or cells, the foreign graft is recognized by the host's immune system as a "foreign component" and initiates an attack against the graft. Immunological responses to attack, destroy and clear.
  • 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 refers to, but is 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.
  • MHC histocompatibility complex
  • HLA antigens play an important role in the transplantation response, with rejection mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue.
  • Human leukocyte antigen is the coding gene of the human major histocompatibility complex, which is closely related to the function of the human immune system.
  • HLA includes class I, class II and class III gene portions.
  • HLA class I is a heterodimer consisting of a heavy chain ( ⁇ 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.
  • the present application provides a chimeric antigen receptor (CAR), the CAR comprises a bispecific antigen-binding domain, the bispecific antigen-binding domain includes an NKG2A antigen-binding domain and a tumor antigen-binding domain, and the NKG2A antigen-binding domain
  • the domain comprises a first binding region L and a first binding region H
  • the tumor antigen binding domain comprises a second binding region L and a second binding region H.
  • NKG2A antigen or “NKG2A” generally refers to NKG2A polypeptide, a member of the NKG2 transcriptome, a heterodimer inhibitory receptor CD94/NKG2A formed by NKG2A and CD94, expressed in NK cells, ⁇ T cells, ⁇ T cells and subpopulations of NKT cells on the surface.
  • NKG2A refers to 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 100% homology or identity 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.
  • the human NKG2A polypeptide has the sequence shown in SEQ ID NO:42.
  • the NKG2A polypeptide is a human NKG2A polypeptide comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% of the amino acid sequence shown in SEQ ID No: 42 , at least about 97%, at least about 98%, at least about 99% or 100% homology or identity amino acid sequences or fragments thereof, and/or may optionally include at most one or at most two or at most three conserved Amino acid substitutions.
  • BCMA antigen or “BCMA” generally refers to BCMA polypeptide, which is a B-cell maturation antigen and 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” refers to any variant, derivative or isoform of the BCMA gene or encoded protein.
  • the BCMA polypeptide has 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 that are about 97%, at least about 98%, at least about 99% or 100% homologous or identical, and/or may optionally include up to one or up to two or up to three conservative amino acid substitutions .
  • the human BCMA polypeptide has the sequence shown in SEQ ID NO:43.
  • the BCMA polypeptide is a human BCMA polypeptide, comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96% of the amino acid sequence shown in SEQ ID No:43 , at least about 97%, at least about 98%, at least about 99% or 100% homology or identity amino acid sequences or fragments thereof, and/or may optionally include at most one or at most two or at most three conserved Amino acid substitutions.
  • 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
  • a chimeric antigen 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 CAR of the present application specifically binds to BCMA polypeptide and NKG2A polypeptide. In one example, the CAR binds to the extracellular domain of the NKG2A polypeptide and the BCMA polypeptide.
  • the heavy chain/heavy chain variable region (VH) and/or the light chain/light chain variable region of the tumor antigen binding domain (such as BCMA antibody), tumor antigen binding domain (such as NKG2A antibody) (VH) may be positioned at any suitable location.
  • the NKG2A antigen binding domain comprises a first binding region L and a first binding region H
  • the tumor antigen binding domain comprises a second binding region L and a second binding region H
  • the first binding region L, the second binding region A binding region H, the second binding region L, and the second binding region H are connected in the following manner:
  • the first binding region L or the first binding region H the first binding region H or the first binding region L—the second binding region H or the The second binding region L—the second binding region L or the second binding region H,
  • the first binding region L is connected to the second binding region H to form a first polypeptide
  • the second binding region L is connected to the first binding region H to form a second polypeptide
  • the first The polypeptide is linked to the second polypeptide, wherein the first binding region L is not connected to the first binding region H, and the second binding region L is not connected to the second binding region H.
  • the first binding region L comprises a first light chain or a first light chain variable region (VL).
  • the first binding region H comprises a first heavy chain or a first heavy chain variable region (VH).
  • the second binding region L comprises a second light chain or a second light chain variable region (VL).
  • the second binding region H comprises a second heavy chain or a second heavy chain variable region (VH).
  • the first binding region L is connected to the first binding region H
  • the second binding region L is connected to the second binding region H.
  • the first light chain is linked to the first heavy chain
  • the second light chain is linked to the second heavy chain
  • the first light chain variable region (VL) is connected to the first heavy chain variable region (VH), and the second light chain variable region (VL) ) is connected to the second heavy chain variable region (VH).
  • connection is as follows: NKG2A VL—NKG2A VH—BCMA VH—BCMAVL.
  • connection is as follows: NKG2A VL—(G4S)3—NKG2A VH—(G4S)3—BCMA VH—(G4S)3—BCMA VL.
  • connection is as follows: BCMA VH—BCMA VL—NKG2A VL—NKG2A VH.
  • connection is as follows: BCMA VH—(G4S)3—BCMA VL—(G4S)3—NKG2A VL—(G4S)3—NKG2A VH.
  • the second binding region L the first binding region H—the second binding region H—the first binding region L;
  • the NKG2A antigen binding domain comprises a first binding region L and a first binding region H
  • the tumor antigen binding domain comprises a second binding region L and a second binding region H
  • the first binding region L, the second binding region A bonding region H, the second bonding region L, and the second bonding region H are connected in a manner to form a loop structure.
  • the loop structure is formed in the following manner: the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to the antibody's Connect both ends.
  • the antibody comprises: the binding region H and the binding region L sequentially from the N-terminus to the C-terminus.
  • the antibody comprises sequentially from N-terminus to C-terminus: the binding region H and the binding region L; the binding region L of the other antigen-binding domain is connected to the N-terminus of the antibody, so The binding region H of the other antigen-binding domain is linked to the C-terminus of the antibody.
  • connection is that the binding region H of the antibody is the first binding region H
  • the connection is that the binding region L of the antibody is the first binding region L
  • the binding region L of the other antigen-binding domain is the second binding region
  • the second binding region L, the binding region H of the other antigen-binding domain is the second binding region H.
  • connection is that the binding region H of the antibody is the second binding region H, and the connection is that the binding region L of the antibody is the second binding region L.
  • the binding region L of the other antigen-binding domain is the first binding region L
  • the binding region H of the other antigen-binding domain is the first binding region H.
  • the antibody comprises sequentially from the N-terminal to the C-terminal: the binding region H and the binding region L; the binding region H of the other antigen-binding domain is connected to the N-terminal of the antibody, so The binding region L of the other antigen binding domain is connected to the C-terminus of the antibody.
  • connection is that the binding region H of the antibody is the first binding region H
  • the connection is that the binding region L of the antibody is the first binding region L
  • the binding region L of the other antigen-binding domain is the second binding region
  • the second binding region L, the binding region H of the other antigen-binding domain is the second binding region H.
  • connection is that the binding region H of the antibody is the second binding region H, and the connection is that the binding region L of the antibody is the second binding region L.
  • the binding region L of the other antigen-binding domain is the first binding region L
  • the binding region H of the other antigen-binding domain is the first binding region H.
  • the antibody comprises: the binding region L and the binding region H sequentially from the N-terminus to the C-terminus.
  • the antibody comprises sequentially from the N-terminal to the C-terminal: the binding region L and the binding region H; the binding region L of the other antigen-binding domain is connected to the N-terminal of the antibody, so The binding region H of the other antigen-binding domain is connected to the C-terminus of the antibody.
  • the antibody comprises sequentially from the N-terminus to the C-terminus: the binding region L and the binding region H; the binding region H of the other antigen-binding domain is connected to the N-terminus of the antibody, so The binding region L of the other antigen binding domain is connected to the C-terminus of the antibody.
  • connection is that the binding region H of the antibody is the first binding region H
  • the connection is that the binding region L of the antibody is the first binding region L
  • the binding region L of the other antigen-binding domain is the second binding region
  • the second binding region L, the binding region H of the other antigen-binding domain is the second binding region H.
  • connection is that the binding region H of the antibody is the second binding region H, and the connection is that the binding region L of the antibody is the second binding region L.
  • the binding region L of the other antigen-binding domain is the first binding region L
  • the binding region H of the other antigen-binding domain is the first binding region H.
  • first binding region L, the first binding region H, the second binding region L, and the second binding region H are connected in the following manner:
  • the second binding area L the first binding area H—the first binding area L—the second binding area H.
  • first binding region L, the first binding region H, the second binding region L, and the second binding region H are connected in the following manner:
  • the first binding region L comprises a first light chain or a first light chain variable region (VL).
  • the first binding region H comprises a first heavy chain or a first heavy chain variable region (VH).
  • the second binding region L comprises a second light chain or a second light chain variable region (VL).
  • the second binding region H comprises a second heavy chain or a second heavy chain variable region (VH).
  • the first binding region L comprises a first light chain
  • the first binding region H comprises a first heavy chain
  • the second binding region L comprises a second light chain
  • the second binding region H comprises the second heavy chain
  • the first binding region L comprises a first VL
  • the first binding region H comprises a first VH
  • the second binding region L comprises a second light chain
  • the second binding region H comprises second heavy chain.
  • the first binding region L comprises a first light chain
  • the first binding region H comprises a first heavy chain
  • the second binding region L comprises a second VL
  • the second binding region H comprises a first heavy chain. Contains the second VH.
  • the first binding domain L comprises a first VL
  • the first binding domain H comprises a first VH
  • the second binding domain L comprises a second VL
  • the second binding domain H comprises a second VL. Two VH.
  • the first VL is the VL of the NKG2A antibody
  • the first light chain is the light chain of the NKG2A antibody
  • the first VH is the VH of the NKG2A antibody
  • the first The heavy chain is the heavy chain of the NKG2A antibody
  • the second VL is the VL of the antibody that recognizes the tumor antigen
  • the second light chain is the light chain of the antibody that recognizes the tumor antigen
  • the second VH For the VH of an antibody that recognizes the tumor antigen, the second heavy chain is the heavy chain of an antibody that recognizes the tumor antigen.
  • the following linkages are included from the N-terminus to the C-terminus:
  • the following linkages are included from the N-terminus to the C-terminus:
  • each light chain/VL, heavy chain/VH in the CAR can be linked by a linker.
  • the linker may comprise any suitable amino acid sequence for providing flexibility to the extracellular antigen-binding region as part of the CAR, both in composition and length.
  • the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody through a linker, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to both ends of the antibody through a linker.
  • the linker comprises Lin1 or (G4S)n, wherein n is an integer equal to or greater than 1, and the Lin1 comprises the amino acid sequence shown in SEQ ID NO:18.
  • n in (G4S)n is 1 or 3.
  • the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody through Lin1 or (G4S)3, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to the antibody through a linker. Both ends are connected.
  • the binding region H and the binding region L of one antigen-binding domain are connected to form an antibody through Lin1 or (G4S)3, and the binding region H and the binding region L of the other antigen-binding domain are respectively connected to the two parts of the antibody through G4S. end connection.
  • the antibody comprises the first binding region H and the first binding region L, the N-terminus of the antibody is connected to the second binding region H or the second binding region L through G4S, and the antibody The N-terminus of is connected to the second binding region L or the second binding region H through G4S.
  • the antibody comprises the second binding region H and the second binding region L, the N-terminus of the antibody is connected to the first binding region H or the first binding region L through G4S, and the antibody The N-terminus of is connected to the first binding region L or the first binding region H through G4S.
  • the following linkages are included from the N-terminus to the C-terminus:
  • connection methods from the N-terminal to the C-terminal includes the following connection methods:
  • the CAR comprises the amino acid sequence of any one of SEQ ID Nos: 22, 25 and 28.
  • the second VL is the VL of the BCMA antibody
  • the second light chain is the light chain of the BCMA antibody
  • the second VH is the VH of the BCMA antibody
  • the second heavy chain is the BCMA antibody heavy chain.
  • each light chain/VL and heavy chain/VH of the antibody in the CAR can be linked by a linker.
  • a linker may comprise any suitable amino acid sequence.
  • the linker comprises Lin1 or (G4S)n, wherein n is an integer equal to or greater than 1; the Lin1 comprises the amino acid sequence shown in SEQ ID NO:18.
  • the linker is a glycine/serine linkage of about 1 to about 100, about 3 to about 20, about 5 to about 30, about 5 to about 18, or about 3 to about 8 amino acids in length Fragments, and consist of glycine and/or serine residues in the sequence.
  • the glycine/serine linker is a peptide of formula [GGGGS]n((G4S)n), where n is an integer from 1 to 10, 2 to 8, or 3 to 5. For example, n is 1, 2, 3 or 4.
  • NKG2A antibody VL and VH can be linked by a linker fragment.
  • the NKG2A antibody VL and VH are linked by a linking fragment comprising the sequence shown in SEQ ID NO: 18, or linked by (G4S)3.
  • BCMA antibody VL and VH can be linked by a linker fragment.
  • the BCMA antibody comprises the sequence shown in SEQ ID NO: 9-14, also includes the sequence shown in SEQ ID NO: 18 or (G4S) 3; or includes the sequence shown in SEQ ID NO: 15 and 16 The sequence shown, also includes the sequence shown in SEQ ID NO: 18 or (G4S) 3; Or includes the sequence shown in SEQ ID NO: 39 or 40.
  • an NKG2A antibody comprises VH, VL and linking fragments.
  • the NKG2A antibody comprises the sequence shown in SEQ ID NO: 3-8, also includes the sequence shown in SEQ ID NO: 18 or (G4S) 3; or includes the sequence shown in SEQ ID NO: 1 and 2
  • the sequence shown also includes the sequence shown in SEQ ID NO: 18 or (G4S) 3; Or includes the sequence shown in SEQ ID NO: 41.
  • the BCMA antibody and the NKG2A antibody are linked by a linking fragment.
  • Linking fragments may comprise any suitable amino acid sequence.
  • the linking fragment linking said BCMA antibody and said NKG2A antibody comprises a glycine/serine linker, said glycine/serine linker being a peptide of formula (G4S)n, wherein n is 1 to 10, 2 to 8 or an integer from 3 to 5.
  • G4S glycine/serine linker
  • n is 1,2,3,4.
  • the linking fragment connecting the BCMA antibody and the NKG2A antibody comprises a (G4S)3 sequence (comprising the BCMA antibody heavy chain and light chain and the NKG2A antibody heavy chain and light chain connected by the linking fragment, (G4S)3 being located between the BCMA antibody and the NKG2A antibody between NKG2A antibodies).
  • (G4S)3 being located between the BCMA antibody and the NKG2A antibody between NKG2A antibodies.
  • the CAR comprises (i) an NKG2A antibody light chain located near the amino terminus of the BCMA antibody heavy chain with a linking segment therebetween, (ii) an NKG2A antibody heavy chain located near the amino terminus of the NKG2A antibody light chain , wherein the connecting segment is positioned between them, and (iii) a BCMA antibody light chain positioned near the amino terminus of the NKG2A antibody heavy chain, wherein the connecting segment is positioned between them; said connecting segment optionally includes, as SEQ ID NO : the sequence shown in 18, G4S or (G4S)3.
  • the CAR comprises (i) a BCMA antibody light chain located near the amino terminus of the NKG2A antibody heavy chain with a linking segment therebetween, (ii) a BCMA antibody heavy chain located near the amino terminus of the BCMA antibody light chain , wherein the connecting fragment is positioned between them, and (iii) the NKG2A antibody light chain near the amino terminus of the BCMA antibody heavy chain, wherein the connecting fragment is positioned between them; said connecting fragment optionally includes, as SEQ ID NO: The sequence shown in 18, G4S or (G4S)3.
  • the BCMA antibody can be positioned adjacent to the amino terminus of the NKG2A antibody (with the linking fragment therebetween). In one embodiment, the NKG2A antibody is located near the amino terminus of the BCMA antibody (with the linking fragment in between).
  • the car-t cells expressing the CAR provided by the present application have a better effect of killing NK cells in vitro than the car-t cells only targeting tumor antigens.
  • ucar-t cells expressing the CAR provided by the present application have a better effect of killing NK cells in vitro than ucar-t cells that only target tumor antigens.
  • ucar-t cells expressing the CAR provided by the present application have a better effect of inhibiting tumor growth in vivo than ucar-t cells that only target tumor antigens.
  • ucar-t cells expressing the CAR provided herein have longer survival time and/or expansion ability than ucar-t cells that only target tumor antigens.
  • ucar-t cells expressing the CAR provided by the present application have better anti-NK cell and tumor-killing effects in an NK cell environment in vitro.
  • the ucar-t cells expressing the CAR provided by the present application have better anti-NK cell and tumor growth inhibitory effects in the NK cell environment in vivo.
  • the ucar-t cells expressing the CAR provided by the present application have a better effect of specifically infiltrating into tumor tissue than the ucar-t cells that only target tumor antigens.
  • ucar-t cells expressing the CAR provided herein will not cause graft-versus-host (GVHD) reactions.
  • the NKG2A antigen binding domain comprises a scFv of an antibody that recognizes an NKG2A polypeptide (also referred to as an NKG2A antibody).
  • the NKG2A antigen binding domain comprises a light chain variable region (VL) and/or a heavy chain variable region (VH) of an NKG2A antibody.
  • 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 a sequence as shown in SEQ ID NO: 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 the sequences shown in SEQ ID NO: 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.
  • the NKG2A antibody or NKG2A antigen binding domain comprises the sequences shown in SEQ ID NO: 1 and 2.
  • the BCMA antigen binding domain comprises a scFv of an antibody that recognizes a BCMA polypeptide (also referred to as a BCMA antibody).
  • 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: 9, HCDR2 of the sequence shown in SEQ ID NO: 10, HCDR2 of the sequence shown in SEQ ID NO: HCDR3 of the sequence shown in NO:11.
  • the heavy chain or VH of the BCMA antibody comprises a sequence as shown in SEQ ID NO: 9-11.
  • the BCMA antibody light chain or VL comprises one or more light chain CDRs (LCDR): LCDR1 of the sequence shown in SEQ ID NO: 12, LCDR2 of the sequence shown in SEQ ID NO: 13, LCDR2 of the sequence shown in SEQ ID NO: 13, LCDR3 of the sequence shown in NO:14.
  • the BCMA antibody light chain or VL comprises a sequence as shown in SEQ ID NO: 12-14.
  • the BCMA antibody heavy chain or VH comprises the sequence shown in SEQ ID NO: 15.
  • the BCMA antibody light chain or VL comprises the sequence shown in SEQ ID NO: 16.
  • the BCMA antibody or BCMA antigen binding domain comprises the VH of the sequence shown in SEQ ID NO:15 and/or the VL of the sequence shown in SEQ ID NO:16. In one example, the BCMA antibody or BCMA antigen binding domain comprises the sequences shown in SEQ ID NO: 16 and 17. In one example, the BCMA antibody or BCMA antigen binding domain comprises the sequences shown in SEQ ID NO: 46-50.
  • the CAR comprises all CDR regions of the NKG2A antibody and the BCMA antibody. In one example, the CAR comprises the sequences set forth in SEQ ID NOs: 3-14. In one example, the CAR includes the light chain variable region and the heavy chain variable region of an antibody that recognizes the NKG2A polypeptide and an antibody that recognizes the BCMA polypeptide. In one example, the CAR comprises the sequences set forth in SEQ ID NOs: 1, 2, 15 and 16.
  • 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 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.
  • antibodies or antibody fragments of the present application can be further modified such that they vary in amino acid sequence (e.g., relative to wild-type or a sequence provided herein), but not in the desired activity. no change.
  • 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 may 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 CAR provided by the present application may also include a leader sequence and/or a hinge domain.
  • the antigen binding domain is linked directly to the transmembrane domain or via a hinge.
  • the hinge comprises a CD8 hinge, for example, the CD8 hinge comprises SEQ ID NO: 35 or a sequence having 95-99% identity to SEQ ID NO: 35.
  • the antigen binding domain comprises a leader sequence.
  • the leader sequence can be located at the amino terminus of the BCMA antibody VH (eg, at the amino terminus of the BCMA antibody heavy chain).
  • the leader sequence is amino-terminal to the VL of the BCMA antibody (eg, amino-terminal to the light chain of the BCMA antibody).
  • the leader sequence is amino-terminal to the VL of the NKG2A antibody (e.g., amino-terminal to the light chain of the NKG2A antibody).
  • a leader sequence may comprise any suitable leader sequence.
  • the leader sequence comprises the amino acid sequence of SEQ ID NO: 29.
  • the CAR lacks a leader sequence.
  • the CAR provided herein may also include a transmembrane domain.
  • the transmembrane domain can anchor the CAR to the cell membrane.
  • the transmembrane domain of the CAR of the present application may include a transmembrane domain selected from the following proteins: the transmembrane domain of T cell receptor ⁇ , ⁇ , or ⁇ , 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,
  • the CAR comprises a CD8 transmembrane domain having at least one, two or three modifications, but no more than 20, 10 or 5 modifications of the sequence shown in SEQ ID NO:30, or the same as SEQ ID NO:30
  • the amino acid sequence of NO:30 has a sequence of 95-99% identity.
  • the CD8 transmembrane domain comprises the sequence shown in SEQ ID NO:30.
  • the CAR comprises a CD28 transmembrane domain having at least one, two or three modifications as shown in SEQ ID NO: 31, but no more than 20, 10 or 5 modifications, or the same as SEQ ID NO: : A sequence having 95-99% identity to the amino acid sequence shown in 31.
  • the CD28 transmembrane domain comprises the sequence shown in SEQ ID NO: 31.
  • the CAR provided by the present invention may also include an intracellular signaling domain.
  • the intracellular signaling domain (also referred to as the primary signaling domain) comprises a signaling domain selected from a protein molecule selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a , CD79b, FcyRIIa.
  • the intracellular signaling domain comprises the intracellular signaling domain of CD3 ⁇ .
  • 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: 34, or an amino acid sequence identical to that of SEQ ID NO: 34 The amino acid sequences shown are 95-99% identical to the sequences.
  • the CD3 ⁇ signaling domain comprises the amino acid sequence shown in SEQ ID NO:34.
  • the intracellular signaling domain of the CAR provided by the present invention includes a human CD3 ⁇ signaling domain. In one example, the intracellular signaling domain of the CAR includes a human CD3 ⁇ signaling domain and a CD28 co-stimulatory signaling domain.
  • the CAR provided in the present application may also include a co-stimulatory signaling domain.
  • the co-stimulatory signaling domain comprises a protein function signaling domain selected from the following: 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 specifically binding 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, CD103
  • SLAMF4 (CD244,2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D.
  • the co-stimulatory signal domain 4-1BB 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 shown in SEQ ID NO: 33, or an amino acid sequence with A sequence having 95-99% identity to the amino acid sequence shown in SEQ ID NO:33.
  • the co-stimulatory signaling domain comprises the sequence shown in SEQ ID NO:33.
  • the co-stimulatory signal domain CD28 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 shown in SEQ ID NO: 32, or an amino acid sequence with SEQ ID NO:32
  • the amino acid sequence shown in ID NO:32 has 95-99% identity sequence.
  • the co-stimulatory signaling domain comprises the sequence shown in SEQ ID NO:32.
  • the intracellular signaling domain of the CAR provided by the present invention includes a human CD3 ⁇ signaling domain. In one example, the intracellular signaling domain of the CAR includes a human CD3 ⁇ signaling domain and a CD28 co-stimulatory signaling domain. In one example, the intracellular signaling domain of the CAR includes a human CD3 ⁇ signaling domain and a 4-1BB co-stimulatory signaling domain. In one example, the intracellular signaling domain of CAR includes CD3 ⁇ signaling domain, CD28 and 4-1BB co-stimulatory signaling domain.
  • the CAR of the present application includes the sequence shown in SEQ ID NO: 17, 22, 25 or 28.
  • the CAR of the present application includes any one of the sequences shown in SEQ ID NO: 17, 22, 25 or 28 connected in sequence with any one of the sequences shown in SEQ ID NO: 19, 44 or 45 sequence.
  • the application provides cells comprising the CAR provided herein.
  • Such cells include immune cells derived from stem cells or lymphoid lineages. After the CAR binds to the target antigen, it can activate the immune cells; the CAR (also called tandem CAR) provided in this application comprises a bispecific antigen binding domain, the bispecific antigen binding domain comprises NKG2A antigen binding domain and tumor Antigen binding domain.
  • the CAR provided by this application has been described above, and the cells provided by this application include all technical solutions thereof.
  • the cells of the present application include immune cells (such as T, NKT cells) that recognize NKG2A polypeptides and tumor antigens. In one example, the cells of the present application include immune cells that recognize NKG2A polypeptides and pathogen antigens.
  • the immune cells of the present application have longer survival time and/or expansion ability in the presence of host immune cells (eg, NK cells).
  • host immune cells eg, NK cells
  • the immune cells of the present application exhibit stronger cell killing effects in vivo and in vitro on cells carrying target tumor antigens.
  • the lymphoid lineage including B, T, and natural killer (NK) cells can 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.
  • 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 (eg, 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 are T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • Immune cells 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
  • the subject's own immune cells can be engineered to express the CAR of the present application.
  • immune cells from donors other than the subject (allogeneic) can be engineered to express the CAR of the present application.
  • the immune cells are T cells.
  • the T cells can be CD4+ T cells and/or CD8+ T cells.
  • the immune cells are CD3+ T cells.
  • the cells of the present application include cell populations collected from PBMC cells stimulated by CD3 magnetic beads.
  • 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.
  • the cells of the present application are capable of modulating 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
  • cells expressing tandem CARs also include immune cells with low or no expression of HLA-class I molecules, TCR molecules, NKG2A molecules or combinations thereof.
  • Low expression or no expression of TCR, B2M or NKG2A means that the expression of TCR, B2M 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%, respectively , at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • low expression or no expression of TCR, B2M or NKG2A means that the content of TCR, B2M 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%, respectively. %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%.
  • the expression or content of the protein in cells can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using specific antibodies to TCR, B2M or NKG2A.
  • allogeneic cells such as immune cells expressing tandem CAR
  • the host CD8+-mediated cellular immune rejection can be reduced.
  • the present application provides tandem CARs that recognize NKG2A polypeptides and tumor antigens, and have low or no expression of endogenous B2M immune cells.
  • the present application provides immune cells that recognize NKG2A polypeptides and pathogen antigens in tandem CAR, and have low or no expression of endogenous B2M.
  • immune cells expressing tandem CARs have a longer survival time and/or ability to expand in the presence of host immune cells (eg, NK cells).
  • host immune cells eg, NK cells.
  • immune cells expressing tandem CARs exhibit stronger cell killing effects in vivo and in vitro.
  • the present application provides immune cells that recognize NKG2A polypeptides and tumor antigens and have low or no expression of endogenous TCR; optionally, the immune cells have low or no expression of endogenous B2M.
  • the present application provides an immune cell that recognizes NKG2A polypeptide and tumor antigen tandem CAR, and has low or no expression of endogenous TCR; optionally, the immune cell has low or no expression of endogenous B2M.
  • the present application provides immune cells that recognize a tandem CAR of NKG2A polypeptide and BCMA polypeptide, and have low or no expression of endogenous TCR/B2M.
  • the immune cells expressing the tandem CAR have a longer survival time and/or expansion ability.
  • the above-mentioned immune cells expressing tandem CARs exhibit stronger cell killing effects in vivo and in vitro.
  • the present application provides tandem CARs that recognize NKG2A polypeptides and tumor antigens and have low or no endogenous NKG2A expression; optionally, the immune cells have low or no endogenous B2M expression, Low or no expression of endogenous TCR, low or no expression of endogenous B2M/TCR.
  • the present application provides an immune cell that recognizes a tandem CAR of NKG2A polypeptide and BCMA polypeptide, and has low or no expression of endogenous TCR/B2M/NKG2A.
  • the immune cells expressing the tandem CAR have a longer survival time and/or expansion ability.
  • the above-mentioned immune cells expressing tandem CARs exhibit stronger cell killing effects in vivo and in vitro.
  • gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR, B2M or NKG2A.
  • gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous NKG2A.
  • gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR/B2M.
  • gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR/B2M.
  • gene knockout technology and/or gene silencing technology are used to prepare immune cells with low or no expression of endogenous TCR/B2M/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 Cas (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA, comprising the RNA that Cas 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 Cas), transactivating crRNA (tracrRNA, which binds to crRNA, forms an active complex with Cas), and an optional segment of the DNA repair template (which directs the cellular repair process to allow the insertion of specific DNA sequence of DNA).
  • CRISPR/Cas9 usually uses plasmids or electroporation to deliver nucleic acid fragments to target cells.
  • CRISPR/Cas9 usually uses plasmids or electroporation to deliver a complex comprising nucleic acid fragments and recombinant proteins to target cells, such as ribonucleoprotein complex (RNP) of gRNA and Cas9.
  • RNP ribonucleoprotein complex
  • 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.
  • crRNA and tracrRNA can be combined to form a guide RNA (gRNA).
  • the gRNA sequence of this application can be represented by the gRNA targeting domain sequence.
  • the gRNA sequence is a targeting DNA sequence.
  • the gRNA sequence is a nucleic acid sequence that is completely or partially complementary to the gRNA targeting DNA sequence.
  • the gRNA molecule includes a molecule of a complete Cas9 guide sequence formed by a gRNA sequence and crRNA/TracrRNA.
  • the methods provided herein include delivering one or more gRNA constructs and one or more Cas9 polypeptides or nucleic acid sequences encoding Cas9 polypeptides to a cell.
  • one or more gRNA constructs, one or more Cas9 polypeptides are delivered by vectors (such as AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation Or the nucleic acid sequence encoding Cas9 polypeptide).
  • crRNA and tracrRNA including the gRNA targeting domain are administered alone, or a whole RNA can be administered.
  • CRISPR/Cas9 transgenes can be delivered by vectors (eg, AAV, adenovirus, lentivirus), and/or particles and/or nanoparticles, and/or electroporation.
  • vectors eg, AAV, adenovirus, lentivirus
  • particles and/or nanoparticles e.g., adenovirus, lentivirus
  • electroporation e.g, electroporation
  • the present application also provides nucleic acid molecules encoding one or more exogenous receptors described herein (eg, CAR), and nucleic acid molecules targeting endogenous TCR, B2M or NKG2A nucleic acid inhibitory molecules or gRNA.
  • the gRNA targeting NKG2A includes the sequence shown in SEQ ID NO: 38; the gRNA targeting TRAC includes the sequence shown in SEQ ID NO: 36; the gRNA targeting B2M includes the sequence shown in SEQ ID NO: 37 sequence shown.
  • a tandem CAR encoding a recognition target antigen is introduced into T cells to generate immune cells provided herein, optionally targeting endogenous TCR, B2M and /or NKG2A nucleic acid inhibitory molecules or gRNA nucleic acid molecules are introduced into T cells.
  • nucleic acid molecules of in vitro transcribed tandem CARs, nucleic acid inhibitory molecules or gRNAs targeting endogenous TCR, B2M or NKG2A can be introduced into cells as transient transfections.
  • 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 nucleic acid molecule may encode the chimeric polypeptide described in the present application.
  • the nucleic acid molecule may be an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs isolated from natural environments or artificially synthesized, but may encode the chimeric polypeptides.
  • the nucleic acid molecule may comprise the nucleic acid sequence shown in any one of SEQ ID NO:21, 24 and 27.
  • Immunogenesis of immune cells can be accomplished by transducing a substantially homogeneous population of cells with the nucleic acid molecule or a vector comprising the nucleic acid molecule.
  • retroviral vectors gamma-retroviruses or lentiviruses
  • a polynucleotide encoding a tandem CAR can be cloned into a retroviral vector.
  • Non-viral vectors can also be used.
  • Transduction can use any suitable viral vector or non-viral delivery system.
  • Tandem CARs can be constructed with accessory molecules (such as 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 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.
  • the present application also provides a pharmaceutical composition, which comprises the CAR described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application and/or the cell described in the present application, and a pharmaceutically acceptable carrier.
  • compositions of 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 a selected pH.
  • sterile liquid preparations such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to a 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 immune cells of the present application into the required amount of an appropriate solvent, and incorporating different amounts of other ingredients as needed.
  • 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 cells in the composition to be administered will vary for the subject being treated. More potent 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 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
  • compositions comprising the present application can be provided systemically or directly to the subject to induce and/or enhance the immune response to the antigen and/or treat and/or prevent tumor, pathogenic infection or infectious disease.
  • a composition of the invention is injected directly into an organ of interest (eg, an organ affected by a tumor).
  • the compositions of the invention 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.
  • Cells of the present application may include purified cell populations.
  • One skilled in the art can readily determine the percentage of immune cells of the invention in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Suitable ranges for purity are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70% in a population comprising the immune cells of the present application.
  • 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 immune cells or progenitor cells of the present application 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.).
  • the present application provides the CAR described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application, the cell described in the present application, and the use of the pharmaceutical composition described in the present application in the preparation of medicines.
  • the above-mentioned medicines are used for preventing, alleviating and/or treating tumors.
  • the present application provides a method for preventing, alleviating and/or treating tumors, which includes administering the cells described in the present application and the pharmaceutical composition described in the present application to a subject in need.
  • the present application provides methods for inducing and/or increasing an immune response in a subject in need of the cells or pharmaceutical compositions of the present application.
  • the cells or pharmaceutical compositions of the present application can be used to treat and/or prevent tumors in subjects.
  • the cells or pharmaceutical compositions of the present application can be used to prolong the survival of a subject with a tumor.
  • the cells or pharmaceutical compositions of the present application can also be used to treat and/or prevent pathogenic infections or other infectious diseases, such as in immunocompromised human subjects.
  • Such methods involve administering an effective amount of a cell or pharmaceutical 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.
  • a cell or pharmaceutical composition comprising the present application can be used to treat a subject with tumor cells that express low levels of surface antigens, for example due to relapse of the disease, where the subject has received a 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 cell or pharmaceutical composition comprising the present application can be used to treat a subject with relapsed disease, wherein the subject has received immune cells (e.g., T cells) comprising alone administering a CAR comprising Intracellular signaling domains, including co-stimulatory signaling domains (e.g. 4-1BBz CAR).
  • 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 the cells or pharmaceutical compositions of the present application to achieve a desired effect, alleviate an existing condition or 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 subject's age, sex, and weight, the disease being treated, the severity of the disease, and the form and effective concentration of the administered cells or pharmaceutical compositions.
  • cell doses in the range of about 10 6 -10 10 are typically infused.
  • T cells specific for a specific antigen are induced.
  • the cells or pharmaceutical 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.
  • Non-limiting examples of tumors include hematological tumors (such as leukemia, lymphoma, and myeloma), solid tumors; solid tumors include: ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer Carcinoma, prostate cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcomas and various cancers (including prostate cancer and small cell lung cancer ).
  • hematological tumors such as leukemia, lymphoma, and myeloma
  • solid tumors include: ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer Carcinoma, prostate cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblast
  • 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 cells or pharmaceutical compositions of the present application can be used to treat and/or prevent conventional treatment measures are not suitable or recurrent 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 hematological tumor.
  • the therapeutic goals of the present application may include relieving 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 cell or a pharmaceutical composition of the present application to a subject suffering from a pathogenic infection.
  • a pathogenic infection eg, viral, bacterial, fungal, parasitic, or protozoan infection
  • the method may comprise administering an effective amount of a cell or a pharmaceutical 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.
  • cells expressing the CAR provided by the present application have longer survival time and/or expansion ability, specific infiltration into tumor tissue, and good resistance to NK cell attack and tumor suppression in a mouse subcutaneous tumor model growth effect, showing good function in the treatment of solid tumors.
  • the cells expressing the CAR provided by the present application have longer survival time and/or expansion ability in the mouse orthotopic hematological tumor model, as well as good effects of resisting NK cell attack and inhibiting tumor growth, showing Good function for treating blood tumors.
  • cells expressing the CAR provided herein target tumors that express positive BCMA. In one example, cells expressing a CAR provided herein target multiple myeloma.
  • kits comprising the CAR provided in the present application, the nucleic acid molecule provided in the present application, the vector provided in the present application, the cell provided in the present application, and/or the pharmaceutical composition provided in the present application.
  • the kit provided in this application is used for inducing and/or enhancing immune response and/or treating and/or preventing tumor or pathogen infection in a subject.
  • the kit includes effective amounts of cells and pharmaceutical compositions 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.
  • kits may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug.
  • the kit includes a nucleic acid molecule encoding the CAR of the present application, which recognizes an antigen of interest in an expressible form, and may optionally be included in one or more vectors.
  • instructions are also included.
  • the instructions generally include information about the use of the cells or pharmaceutical composition in the treatment and/or prevention of tumor or pathogenic infection.
  • 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.
  • T cells Primary PBMCs isolated from healthy donors 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.
  • CAR3 polypeptide SEQ ID NO: 22
  • CAR4 polypeptide SEQ ID NO: 25
  • CAR5 polypeptide SEQ ID NO: 25
  • CAR-T3 cells CAR-T4 cells
  • CAR-T5 cells BCMA-CAR-T cells of BCMA-CAR (SEQ ID NO: 17) polypeptide.
  • the tandem fragments of the antigen-binding domain are shown in Table 2, and their amino acid sequences are shown in any one of SEQ ID NO: 20, 23 and 26, respectively.
  • Effector cells (UTD, BCMA-CAR-T, CAR-T3, CAR-T4, CAR-T5) and target cells (multiple myeloma cell line RPMI-8226) were compared according to the effect-to-target ratio of 1:3 and 1:3, respectively.
  • kit instructions (CytoTox Non-Radioactive Cytotoxicity Assay, promega company), the supernatant was taken for lactate dehydrogenase (LDH) content determination, and the target cell lysis efficiency of each group was calculated.
  • LDH lactate dehydrogenase
  • RPMI-8226 cells were subcutaneously inoculated into NPG immunodeficient mice (purchased from Beijing Weitongda Biotechnology Co., Ltd.) (the inoculation diary is D0), and the average tumor volume was about 190mm 3 11 days after inoculation, divided into 5 Groups: UTD cell group, BCMA-CAR-T cell group, CAR-T3 cell group, CAR-T4 cell group, CAR-T5 cell group, 5 rats in each group. 1 ⁇ 10 6 of the above UTD, BCMA-CAR-T, CAR-T3, CAR-T4, and CAR-T5 cells were injected into the tail vein respectively.
  • each group of tandem CAR-T cells can inhibit tumor growth in vivo.
  • CBA cytokine microsphere detection
  • gRNA sequences (SEQ ID NO: 36, 37, 38) targeting TCR, B2M, and NKG2A were synthesized in vitro, and conventional CRISPR/Cas9 technology was used, that is, Cas 9 enzyme and The gRNA was mixed at a ratio of 1:4 to form RNP, incubated at room temperature, and the cells of each group were mixed with the RNP, and the RNP complex was introduced into CAR-T cells using a maxcyte electrotransfer instrument to knock out UTD, BCMA-CAR-T, and CAR-T cells.
  • UTD-DKO, BCMA-UCAR-T, UCAR-T3, UCAR-T4, and UCAR-T5 cells were obtained from the endogenous TRAC/B2M of T3, CAR-T4, and CAR-T5 cells, respectively, and the endogenous TRAC/B2M of CAR-T3 cells was knocked out UCAR-T3-NKG2A KO cells were obtained from TRAC/B2M/NKG2A.
  • the untransfected T cells with endogenous TCR/B2M knockout were named UTD-DKO.
  • Example 7 Resistance function of UCAR-T cells in series against NK cells in vitro
  • RPMI-8226 cells were inoculated subcutaneously in NPG mice, and the average tumor volume was about 350mm 3 12-14 days after inoculation, divided into 4 groups: UTD-DKO cell group, BCMA UCAR-T cell group, UCAR-T3 cell group group, UCAR-T5 cell group, 5 rats in each group. 1.5 ⁇ 10 6 UTD-DKO, BCMA UCAR-T, UCAR-T3 or UCAR-T5 cells were injected into the tail vein respectively. Referring to the method described in Example 4, the tumor growth curve was drawn. The results are shown in Figure 7, each group of tandem UCAR-T cells can inhibit tumor growth in vivo.
  • Example 10 In vitro anti-tumor activity and anti-NK activity of UCAR-T cells in series in the presence of NK cells
  • Example 11 In vivo antigenic tumor activity of tandem UCAR-T cells in the presence of NK cells
  • MM.1S-luciferase cells 2 ⁇ 10 6 multiple myeloma cell line MM.1S-luciferase cells (MM.1S-luc) were inoculated into NPG mice in the tail vein, and the tumor burden was detected by fluorescence imaging at about 2-5 ⁇ 10 4 Radiance 9 days after inoculation (p/s/cm2/sr), divided into 7 groups (UTD-DKO, BCMA UCAR-T, BCMA UCAR-T+NK-1, BCMA UCAR-T+NK-2, UCAR-T3-NKG2A KO and UCAR- T3-NKG2A KO+NK-1, UCAR-T3-NKG2A KO+NK-2), 5 rats in each group.
  • NK cells On D10, 1 ⁇ 10 6 NK cells (marked as NK-1 as above) or 2 ⁇ 10 6 NK cells (marked as NK-2 as above) were injected into the tail vein respectively, and then NK cells were injected every two days, A total of 5 injections were used to simulate the presence of NK cells; after the first NK cell injection, 1 ⁇ 10 6 UCAR-T cells were injected into the tail vein. After the injection, fluorescence imaging was performed once a week to monitor the tumor burden , body weight was measured twice a week (including the day of group administration and euthanasia), the tumor growth curve was drawn according to the tumor load, the difference of tumor growth curve among the groups was compared, and the survival period of the mice was recorded.
  • NK cells significantly weakened the anti-tumor activity of BCMA UCAR-T cells, and reduced the survival period of mice in the BCMA UCAR-T cell group; and in the presence of low-dose and high-dose NK cells, Neither the antitumor activity of UCAR-T3 nor the survival of mice were significantly changed. It shows that tandem UCAR-T cells can resist NK cell killing and inhibit tumor growth in vivo.
  • UCAR-T cells to treat RPMI-8226 subcutaneous tumor-bearing model mice (as described in Example 9), including tumor tissues of UTD-DKO, BCMA UCAR-T, UCAR-T3-NKG2A KO mice, and heart and liver Normal tissue of spleen, lung and kidney. Sections were fixed with paraformaldehyde and stained with anti-CD3e immunohistochemistry. As shown in Figure 10, in the UCAR group, especially in the tandem UCAR-T group, T cell infiltration in the tumor tissue was more than that in the BCMA UCAR-T group; in normal tissues (including heart, liver, spleen, lung and kidney tissues), no obvious T cell infiltration. This shows that tandem UCAR-T cells not only have longer survival time and expansion ability, but also specifically infiltrate into tumor tissue, and have good safety.
  • mice in the UTD group began to lose weight significantly from D25, and began to experience obvious hair loss and activity reduction from D30, showing obvious GVHD symptoms.
  • the serial UCAR-T group was similar to PBS, the mice grew normally and gradually gained weight. This suggests that UCAR-T3 cells do not cause graft-versus-host (GVHD) reactions.

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Abstract

L'invention concerne une cellule CAR-T et un CAR ciblant à la fois des antigènes tumoraux et NKG2A en même temps. Le CAR comprend un domaine de liaison à l'antigène bispécifique, et le domaine de liaison à l'antigène bispécifique comprend un domaine de liaison à l'antigène NKG2A et un domaine de liaison à l'antigène tumoral.
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