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WO2023284875A1 - 嵌合抗原受体 - Google Patents

嵌合抗原受体 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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WIPO (PCT)
Prior art keywords
cells
binding
region
binding region
car
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Application number
PCT/CN2022/106096
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English (en)
French (fr)
Inventor
李宗海
廖朝晖
Original Assignee
克莱格医学有限公司
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Priority to CN202280042415.8A priority Critical patent/CN117730145A/zh
Publication of WO2023284875A1 publication Critical patent/WO2023284875A1/zh

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    • 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 A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—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

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

提供了同时靶向NKG2A和肿瘤抗原的CAR-T细胞及CAR，所述CAR包含双特异性抗原结合域，所述双特异性抗原结合域包括NKG2A抗原结合域和肿瘤抗原结合域。

Description

嵌合抗原受体
相关申请
本专利申请要求于2021年7月16日递交的申请号为202110807344.3的中国专利申请，于2021年8月11日递交的申请号为202110918700.9的中国专利申请，以及于2022年4月26日递交的申请号为202210447726.4的中国专利申请的优先权。
同时提交的序列表文件
下列XML文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(名称：FF00640PCT-sequence listing.xml，日期：20220714，大小：55KB)。
技术领域
本申请提供嵌合抗原受体(CAR)和工程化双靶向T细胞和及其制备方法。所述工程化双靶向T细胞和含有所述工程化双靶向T细胞的组合物可用于疾病治疗。
背景技术
免疫细胞疗法作为基于细胞的癌症疗法是目前最有前景的治疗方法,免疫细胞具有靶向肿瘤细胞同时保留正常组织的潜力；临床观察指示它们具有主要的抗癌活性。现仍迫切需要通过拓宽免疫细胞适用性和增强其功效用于改善免疫细胞疗法。CAR-T细胞的有效激活均严重依赖识别肿瘤相关抗原的抗体的特异性以及抗原结合的亲和力高低等性质。所以在目前CAR-T细胞胞内信号转导区的设计已经趋于成熟的现状下，抗原结合区的设计成为新型CAR-T技术开发的重点和关键。
发明内容
本申请的目的在于提供一种工程化双靶向T细胞和嵌合抗原受体(CAR)，其中分别靶向两个抗原的抗体片段串联表达在一个嵌合抗原受体(CAR)上。在一些实施方式中，所提供的工程化细胞表现出增强的免疫反应，包括对于受试者的癌症、微生物感染和/或病毒感染的治疗。在一些实施例中，所提供的工程化细胞既具有抵抗宿主免疫排斥作用，也具有抗肿瘤作用。还提供了通过给予治疗有效量的所提供的工程化T细胞来治疗受试者的方法。
一方面，本申请提供了一种嵌合抗原受体(CAR)，所述CAR包含双特异性抗原结合域，所述双特异性抗原结合域包括NKG2A抗原结合域和肿瘤抗原结合域，所述NKG2A抗原结合域包含第一结合区L、第一结合区H,所述肿瘤抗原结合域包含第二结合区L、第二结合区H,所述第一结合区L、第一结合区H、第二结合区L、第二结合区H以形成loop结构的方式连接。
在某些实施方式中，所述loop结构通过如下方式形成：其中一个抗原结合域的结合区H和结合区L连接为抗体，另一个抗原结合域的结合区H、结合区L分别与所述抗体的两端连接。 在某些实施方式中，所述抗体自N端至C端依次包含：
所述结合区H、所述结合区L。
在某些实施方式中，所述另一个抗原结合域的结合区L与所述抗体的N端连接，所述另一个抗原结合域的结合区H与所述抗体的C端连接。
在某些实施方式中，所述另一个抗原结合域的结合区H与所述抗体的N端连接，所述另一个抗原结合域的结合区L与所述抗体的C端连接。
在某些实施方式中，所述连接为抗体的结合区H为第一结合区H，所述连接为抗体的结合区L为第一结合区L，所述另一个抗原结合域的结合区L为第二结合区L，所述另一个抗原结合域的结合区H为第二结合区H。
在某些实施方式中，所述连接为抗体的结合区H为第二结合区H，所述连接为抗体的结合区L为第二结合区L。所述另一个抗原结合域的结合区L为第一结合区L，所述另一个抗原结合域的结合区H为第一结合区H。
在某些实施方式中，所述抗体自N端至C端依次包含：
所述结合区L、所述结合区H。
在某些实施方式中，所述另一个抗原结合域的结合区L与所述抗体的N端连接，所述另一个抗原结合域的结合区H与所述抗体的C端连接。
在某些实施方式中，所述另一个抗原结合域的结合区H与所述抗体的N端连接，所述另一个抗原结合域的结合区L与所述抗体的C端连接。
在某些实施方式中，所述连接为抗体的结合区H为第一结合区H，所述连接为抗体的结合区L为第一结合区L，所述另一个抗原结合域的结合区L为第二结合区L，所述另一个抗原结合域的结合区H为第二结合区H。
在某些实施方式中，所述连接为抗体的结合区H为第二结合区H，所述连接为抗体的结合区L为第二结合区L。所述另一个抗原结合域的结合区L为第一结合区L，所述另一个抗原结合域的结合区H为第一结合区H。
在某些实施方式中，所述第一结合区L包含第一轻链或第一轻链可变区(VL)，所述第一结合区H包含第一重链或第一重链可变区(VH),所述第二结合区L包含第二轻链或第二轻链可变区(VL)，所述第二结合区H包含第二重链或第二重链可变区(VH)。
在某些实施方式中，所述第一结合区L包含第一轻链可变区(VL)，所述第一结合区H包含第一重链可变区(VH),所述第二结合区L包含第二轻链可变区(VL)，所述第二结合区H包含第二重链可变区(VH)。
在某些实施方式中，所述抗体包含scFv。
在某些实施方式中，所述第一轻链包含所述第一轻链可变区(VL)，所述第一重链包含 所述第一重链可变区(VH),所述第二轻链包含所述第二轻链可变区(VL)，所述第二重链包含所述第二重链可变区(VH)。
在某些实施方式中，所述NKG2A抗原结合域包含单域抗体，和/或肿瘤抗原结合域包含单域抗体。
在某些实施方式中，所述第一轻链可变区(VL)包含如SEQ ID NO:6-8中任一项所示的第一轻链互补决定区(LCDR)或其组合。
在某些实施方式中，所述第一重链可变区(VH)包含如SEQ ID NO:3-5中任一项所示的第一重链互补决定区(HCDR)或其组合。
在某些实施方式中，所述第一轻链可变区(VL)包含第一轻链互补决定区1(LCDR1)、第一轻链互补决定区2(LCDR2)、第一轻链互补决定区3(LCDR3)，所述第一LCDR1包含如SEQ ID NO:6所示的氨基酸序列，所述第一LCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述第一LCDR3包含如SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述第一重链可变区(VH)包含第一重链互补决定区1(HCDR1)、第一重链互补决定区2(HCDR2)、第一重链互补决定区3(HCDR3)，所述第一HCDR1包含如SEQ ID NO:3所示的氨基酸序列，所述第一HCDR2包含如SEQ ID NO:4所示的氨基酸序列，所述第一HCDR3包含如SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述NKG2A抗原结合域包含如SEQ ID NO:3-8所示的氨基酸序列。在某些实施方式中，所述第一重链可变区(VH)包含如SEQ ID NO:1所示的氨基酸序列，所述第一轻链可变区(VL)包含如SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述肿瘤抗原包括CD19、GPC3、Claudin18.2、WT1、HER2、EGFR、BCMA或其组合。
在某些实施方式中，所述肿瘤抗原包括BCMA。
在某些实施方式中，所述肿瘤抗原结合域包括BCMA抗原结合域。
在某些实施方式中，所述第二轻链可变区(VL)包含如SEQ ID NO:12-14中任一项所示的第二轻链互补决定区(LCDR)或其组合。
在某些实施方式中，所述第二重链可变区(VH)包含如SEQ ID NO:9-11中任一项所示的第二重链互补决定区(HCDR)或其组合。
在某些实施方式中，所述第二轻链可变区(VL)包含第二轻链互补决定区1(LCDR1)、第二轻链互补决定区2(LCDR2)、第二轻链互补决定区3(LCDR3)，所述第二LCDR1的包含如SEQ ID NO:12所示氨基酸序列，所述第二LCDR2包含如SEQ ID NO:13所示氨基酸序列，所述第二LCDR3包含如SEQ ID NO:14所示氨基酸序列。
在某些实施方式中，所述第二重链可变区(VH)包含第二重链互补决定区1(HCDR1)、 第二重链互补决定区2(HCDR2)、第二重链互补决定区3(HCDR3)，所述第二HCDR1包含如SEQ ID NO:9所示氨基酸序列，所述第二HCDR2包含如SEQ ID NO:10所示氨基酸序列，所述第二HCDR3包含如SEQ ID NO:11所示氨基酸序列。
在某些实施方式中，所述BCMA抗原结合域包含如SEQ ID NO:9-14所示的氨基酸序列。在某些实施方式中，所述NKG2A抗原结合域包含如SEQ ID NO:3-8所示的氨基酸序列，所述BCMA抗原结合域包含如SEQ ID NO:9-14所示的氨基酸序列。
在某些实施方式中，所述第二重链可变区(VH)包含如SEQ ID NO:15所示的氨基酸序列，所述第二轻链可变区(VL)包含如SEQ ID NO:16所示的氨基酸序列。
在某些实施方式中，所述第二结合区L包含如SEQ ID NO:46-50中任一项所示序列中的轻链可变区，所述第二结合区H包含如SEQ ID NO:46-50中任一项所示序列中的重链可变区。在某些实施方式中，所述NKG2A抗原结合域包含如SEQ ID NO:1所示的第一重链可变区和如SEQ ID NO:2所示的第一轻链可变区，所述BCMA抗原结合域包含如SEQ ID NO:15所示的第二重链可变区和如SEQ ID NO:16所示的第二轻链可变区。
在某些实施方式中，所述双特异性抗原结合域自N端至C端包含如下连接方式：
BCMA VL—NKG2A VH—NKG2A VL—BCMA VH。
在某些实施方式中，所述双特异性抗原结合域自N端至C端包含如下连接方式：
NKG2A V—BCMA VH—BCMA VL—NKG2A VH。
在某些实施方式中，所述连接包括通过连接子连接，所述连接子包含Lin1或(G4S)n，其中，n是等于或大于1的整数；所述Lin1包含如SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，在所述(G4S)n中，n为1或3。
在某些实施方式中，所述双特异性抗原结合域自N端至C端包含如下连接方式：
BCMA VL—G4S—NKG2A VH—(G4S)3—NKG2A VL—G4S—BCMA VH。
在某些实施方式中，所述双特异性抗原结合域自N端至C端包含如下连接方式：
NKG2A VL—G4S—BCMA VH—(G4S)3—BCMA VL—G4S—NKG2A VH。
在某些实施方式中，所述双特异性抗原结合域自N端至C端包含如下连接方式：
BCMA VL—G4S—NKG2A VH—Lin1—NKG2A VL—G4S—BCMA VH。
在某些实施方式中，所述双特异性抗原结合域自N端至C端包含如下连接方式：
NKG2A VL—G4S—BCMA VH—Lin1—BCMA VL—G4S—NKG2A VH。
在某些实施方式中，所述抗体包含如SEQ ID NO:39-41中任一项所示的氨基酸序列。
在某些实施方式中，所述抗体包含如SEQ ID NO:46-50中任一项所示的氨基酸序列。
在某些实施方式中，所述双特异性抗原结合域包含如SEQ ID NO:20、23和26中任一项所示的氨基酸序列。
在某些实施方式中，所述CAR包含跨膜域。
在某些实施方式中，所述CAR包含胞内信号传导域。
在某些实施方式中，所述CAR包含跨膜域、胞内信号传导域。
在某些实施方式中，所述跨膜域包含CD28跨膜结构域或CD8跨膜域。
在某些实施方式中，所述CD8跨膜域包含如SEQ ID NO:30所示的氨基酸序列，所述CD28跨膜域包含如SEQ ID NO:31所示的氨基酸序列。
在某些实施方式中，所述胞内信号传导域包含CD3ζ胞内信号传导域。
在某些实施方式中，所述CD3ζ胞内信号传导域包含如SEQ ID NO:34所示的氨基酸序列。
在某些实施方式中，所述CAR还包含共刺激信号结构域。
在某些实施方式中，所述CAR包含2个相同或不同的共刺激信号结构域。
在某些实施方式中，所述共刺激信号结构域包含4-1BB共刺激信号结构域和/或CD28共刺激信号结构域。
在某些实施方式中，所述CAR包含包含4-1BB共刺激信号结构域和CD3ζ胞内信号传导域。
在某些实施方式中，所述4-1BB共刺激信号结构域包含如SEQ ID NO:33所示的氨基酸序列,所述CD28共刺激信号结构域包含如SEQ ID NO:32所示的氨基酸序列。
在某些实施方式中，所述CAR包含铰链区。
在某些实施方式中，所述铰链区包含CD8铰链区。
在某些实施方式中，所述CD8铰链区包含如SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述CAR包含CD8跨膜域和CD8铰链域。
在某些实施方式中，所述CD8跨膜结构域包含如SEQ ID NO:30所示的氨基酸序列，所述CD8铰链域包含如SEQ ID NO:35所示的氨基酸序列。
在某些实施方式中，所述CAR包含如SEQ ID NO:22、25和28中任一项所示的氨基酸序列。
另一方面本申请还提供了核酸分子，其编码本申请所述的CAR。
在某些实施方式中，所述核酸分子包含如SEQ ID 21、24和27中任一项所述的核苷酸序列。
另一方面本申请还提供了载体，其包含本申请所述的核酸分子。
另一方面本申请还提供了细胞，其包含本申请所述的CAR、本申请所述的核酸分子和/或本申请所述的载体。
在某些实施方式中，所述细胞包含免疫细胞。
在某些实施方式中，所述细胞选自：T细胞、NK细胞、细胞毒性T细胞、NKT细胞、巨噬细胞、CIK细胞和干细胞衍生的免疫细胞。
在某些实施方式中，所述细胞为T细胞。
在某些实施方式中，所述T细胞为自体T细胞和/或同种异体T细胞。
在某些实施方式中，所述T细胞为干细胞衍生的自体T细胞和/或干细胞衍生的同种异体T细胞。
在某些实施方式中，所述T细胞为原代自体T细胞和/或原代同种异体T细胞。
在某些实施方式中，所述T细胞包含基因修饰，所述基因修饰包括抑制或消除参与响应自体和/或异体抗原识别多肽的至少一种内源基因表达、活性。
在某些实施方式中，所述细胞内源性TCR、MHC不表达或低表达。
在某些实施方式中，所述细胞包括：
a)内源性HLA-I分子低表达或不表达，
b)内源性TCR分子低表达或不表达，和/或
c)内源性NKG2A分子低表达或不表达。
在某些实施方式中，
a)所述内源性HLA-I蛋白低表达或不表达包括敲除编码内源性HLA-I的基因，
b)所述内源性TCR蛋白低表达或不表达包括敲除编码内源性TCR的基因，和/或
c)所述内源性NKG2A分子低表达或不表达包括敲除编码内源性NKG2A的基因。
在某些实施方式中，采用基因敲除技术敲除内源性TRAC和/或B2M。
在某些实施方式中，所述基因敲除技术为CRISPR/Cas9技术。
在某些实施方式中，所述细胞包括：
a)采用CRISPR/Cas9技术敲除内源性B2M，
b)采用CRISPR/Cas9技术敲除内源性B2M、TCR，或
c)采用CRISPR/Cas9技术敲除内源性B2M、TCR、NKG2A。
在某些实施方式中，所述CRISPR/Cas9技术使用的gRNA包括如SEQ ID NO：36-38中任一项所示的核苷酸序列或其组合。
另一方面，本申请还提供了药物组合物，其包含本申请所述的CAR、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及药学上可接受的载体。
另一方面，本申请还提供了一种试剂盒，其包含本申请所述的CAR、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞、和/或本申请所述的药物组合物。
另一方面，本申请还提供了提高靶向肿瘤的免疫细胞在有宿主免疫细胞存在时的存活时间和/或扩增能力的方法，包括：
a)提供免疫细胞；
b)任选地，修饰所述免疫细胞，所述修饰包括抑制或消除参与响应自体和/或异体抗原识别多肽的至少一种内源基因表达、活性；
c)编码包含NKG2A抗原结合域和肿瘤抗原结合域的CAR的多核苷酸来修饰所述免疫细 胞。
在某些实施方式中，在所述步骤c)中，所述方法包括向所述免疫细胞中引入本申请所述的核酸分子或本申请所述的载体。
在某些实施方式中，所述方法还包括使所述免疫细胞表达本申请所述的CAR。
在某些实施方式中，所述方法包括修饰所述免疫细胞，所述修饰包括抑制或消除参与响应自体和/或异体抗原识别多肽的至少一种内源基因表达、活性。
在某些实施方式中，所述CAR包括：
a)NKG2A抗原结合域和肿瘤抗原结合域，CD28或CD8的跨膜域，CD28的共刺激信号结构域，和CD3ζ；和/或
b)NKG2A抗原结合域和肿瘤抗原结合域，CD28或CD8的跨膜域，4-1BB的共刺激信号结构域，和CD3ζ；和/或
c)NKG2A抗原结合域和肿瘤抗原结合域，CD28或CD8的跨膜域，CD28的共刺激信号结构域，4-1BB的共刺激信号结构域，和CD3ζ；
d)NKG2A抗原结合域和肿瘤抗原结合域，CD28或CD8的跨膜域，和CD3ζ。
在某些实施方式中，所述NKG2A抗原结合域包含如SEQ ID NO:3-8所示的氨基酸序列；或包含如SEQ ID NO:1所示的氨基酸序列的重链可变区和包含如SEQ ID NO:2所示的氨基酸序列的轻链可变区。
在某些实施方式中，肿瘤抗原为BCMA，所述BCMA抗原结合域包含如SEQ ID NO:9-14所示的氨基酸序列；或包含如SEQ ID NO:15所示的氨基酸序列的重链可变区和包含如SEQ ID NO:16所示的氨基酸序列的轻链可变区。
在某些实施方式中，所述抗原结合域包含如SEQ ID NO:20、23和26中任一项所示的氨基酸序列。
在某些实施方式中，所述CAR包含如SEQ ID NO:22、25和28中任一项所示的氨基酸序列。在某些实施方式中，所述步骤b)包括：
i)采用CRISPR/Cas9技术敲除内源性B2M，
ii)采用CRISPR/Cas9技术敲除内源性B2M、TCR，或
iii)采用CRISPR/Cas9技术敲除内源性B2M、TCR、NKG2A。
在某些实施方式中，所述CRISPR/Cas9技术使用的gRNA包括如SEQ ID NO：36-38中任一项所示的核苷酸序列或其组合。
在某些实施方式中，所述免疫细胞选自：T细胞、NK细胞、细胞毒性T细胞、NKT细胞、巨噬细胞、CIK细胞和干细胞衍生的免疫细胞。
在某些实施方式中，所述免疫细胞为自体或同种异体T细胞、干细胞衍生的T细胞、原代T 细胞或来源于人的自体T细胞。
在某些实施方式中，所述方法用于治疗和/或预防肿瘤。
在某些实施方式中，所述肿瘤包括血液瘤和/或实体瘤。
在某些实施方式中，所述血液瘤包含多发性骨髓瘤。
在某些实施方式中，所述方法在离体条件下进行。
另一方面，本申请还提供了本申请所述的CAR、本申请所述的核酸分子、权本申请所述的载体、本申请所述的细胞、本申请所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
另一方面，本申请还提供了预防、缓解和/或治疗肿瘤的方法，其包括向有需要的受试者施用本申请所述的细胞、本申请所述的药物组合物。
另一方面，本申请还提供了本申请所述的CAR、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞、本申请所述的药物组合物，其用于预防、缓解和/或治疗肿瘤。
在某些实施方式中，所述肿瘤包括血液瘤和/或实体瘤。
在某些实施方式中，所述血液瘤包含多发性骨髓瘤。
应理解，在本申请范围内中，本申请的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示的是本申请中的串联CAR-T细胞体外对NK细胞的杀伤效果。
图2显示的是本申请中的串联CAR-T细胞体外杀伤肿瘤细胞的效果。
图3显示的是本申请中的串联CAR-T细胞的体内抗肿瘤作用。
图4显示的是本申请中的串联CAR-T细胞与肿瘤细胞共孵育后细胞因子分泌情况。
图5显示的是本申请中的串联UCAR-T细胞体外抵抗NK细胞杀伤的。
图6显示的是本申请中的串联UCAR-T细胞与肿瘤细胞共孵育后后细胞因子分泌情况。
图7显示的是本申请中的串联UCAR-T细胞体内抑制肿瘤生长的效果。
图8显示的是本申请中的串联UCAR-T细胞体外对抗NK细胞杀伤并杀伤肿瘤细胞的效果。
图9A显示的是本申请中的串联UCAR-T细胞在NK细胞条件下的原位肿瘤模型中抑制肿瘤生长的效果。
图9B显示的是本申请中的串联UCAR-T细胞在NK细胞条件下的原位肿瘤模型中对 小鼠生存期的影响。
图10显示的是本申请中的串联UCAR-T细胞特异性浸润肿瘤组织。
图11显示的是本申请中的串联UCAR-T细胞不引起移植物抗宿主反应。
具体实施方式
本申请提供了一种靶向BCMA抗原和NKG2A抗原的双抗原特异性嵌合抗原受体(CAR)，该CAR包含BCMA抗原结合域、NKG2A抗原结合域。本申请进一步提供与本申请的CAR相关的核酸、重组表达载体、宿主细胞、细胞群和药物组合物。本申请还提供了检测哺乳动物中是否存在癌症的方法以及治疗或预防哺乳动物中癌症的方法。表达本申请所述CAR的T细胞具有很强的抵抗NK细胞的能力，从而具有更好存活和/或增殖能力，增强抗肿瘤能力。此外，将抗NKG2A和抗肿瘤抗原的CAR串联，可以只用一个载体就能实现抵抗宿主免疫排斥(如NK细胞攻击)和抗肿瘤的双重目的，有利于简化工艺的制备以及质检、质控流程。
除非专门定义，否则本文所用的所有技术和科学术语具有在基因治疗、生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的那些所有方法和材料都可以在本申请的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容通过引用并入本文。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，否则本申请的材料、方法和实施例仅是说明性的，而并非旨在进行限制。根据本申请内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本申请的精神和范围。本申请在范围上并不受限于本文描述的具体实施方案(其仅预期作为本申请的各方面的举例说明)，并且功能等价的方法和组分在本申请的范围内。本申请包括对本申请的主题进行变型和修改来用于各种用途和条件。
除非另有说明，否则本申请的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells  And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
在本申请中，范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本申请的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。
本文使用的术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。在本文中，“约”可以是在所述技术领域内可以接受的误差范围；。例如，可以是指“约”值或参数的±10％范围内的值或参数，例如，约5uM可包括在4.5uM与5.5uM之间的任何数目。
术语
术语“包含”或“包括”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)通常是指包含能够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。其可以包括抗原(例如，肿瘤相关抗原(tumor-associated antigen，TAA))结合区、跨膜结构域、共刺激结构域和胞内信号结构域。经基因修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。
在本申请中，术语“抗原结合域”是指特异性结合抗原决定簇的分子，包括免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。
在本申请中，术语“抗体”通常是指包括免疫球蛋白分子或免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。其可以包括完整的抗体分子(也称作免疫球蛋白)，也可以是保留抗原结合能力的抗体分子的片段。在一些情况下，术语“抗体”与术语“免疫球蛋白”“抗原结合域”可互换使用。抗体通常包括但不限于单克隆抗体、多克隆抗体、天然抗体、双特异性抗体、嵌合抗体、Fv、Fab、Fab’、Fab’-SH、F(ab’)2、 线性抗体、单链抗体分子(例如scFv)、单域抗体。在某些实施方式中，“抗体”可以是包含轻链或轻链可变区、重链或重链可变区的单链多肽。例如，是由轻链、重链组成的单链多肽；例如，是由轻链可变区、重链可变区组成的单链多肽；例如，是由轻链可变区、重链组成的单链多肽；例如，是由轻链、重链可变区组成的单链多肽；例如，是由重链可变区(VH)和重链恒定区(CH)组成的多肽。例如，CH包含三个结构域CH1、CH2、CH3。每条轻链包含轻链可变区(VL)和轻链恒定区(CL)。CL由一个结构域组成。例如，VH和VL可进一步分为高变区，称为互补决定区(CDR)，和散布其间的更保守的区域，称为框架区(FR)。每个VH和VL可由三个CDR和四个FR组成，从氨基端到羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。
在本申请中，术语“肽”、“多肽”和“蛋白质”可互换使用，是指由通过肽键共价连接的氨基酸残基组成的化合物。
在本申请中，术语“双特异性”，通常是指具有与两个不同位点识别和/或结合的特性。所述位点可以是一定数目的氨基酸或其组成的肽段或肽段组，例如表达在细胞表面的蛋白。例如，本申请中的双特异性抗原结合域可以包含NKG2A抗原结合域、肿瘤抗原结合域两个不同的靶向结合域。通常情况下“双特异性”可与“双靶点”互换使用。在一些实例中，“双特异性CAR”、“串联CAR”可互换使用，均为本申请提供的CAR。
在本申请中，术语“loop结构”，通常是指一段弯曲的、非闭合的肽段，其可以是经合适的连接方式由NKG2A抗原结合域、肿瘤抗原结合域形成的loop结构；例如，其中一个结合域的结合区H和结合区L连接为抗体，另一个结合域的结合区H(例如重链或重链可变区)、结合区L(例如轻链或轻链可变区)分别与所述抗体的两端连接。
在本申请中，术语“结合区L”，通常是指包含轻链功能片段(例如轻链、轻链可变区)的多肽。例如，结合区L为轻链，例如结合区L为轻链可变区。例如，第一结合区L为识别NKG2A的抗体的轻链，例如，第一结合区L为识别NKG2A的抗体的轻链可变区，例如，第二结合区L为识别BCMA的抗体的轻链，例如，第二结合区L为识别BCMA的抗体的轻链可变区。
在本申请中，术语“结合区H”，通常是指包含重链功能片段(例如重链、重链可变区)的多肽。例如，结合区H为重链，例如结合区H为重链可变区。例如，第一结合区H为识别NKG2A的抗体的重链，例如，第一结合区H为识别NKG2A的抗体的重链可变区，例如，第二结合区H为识别BCMA的抗体的重链，例如，第二结合区H为识别BCMA的抗体的重链可变区。
在本申请中，术语“C端”通常是指多肽链的两个末端之一，此末端的氨基酸残基携带游离的α羧基(—COOH)。在某些实施方式中，所述游离的α羧基在某些肽链中也可被酰胺化。
在本申请中，术语“N端”通常是指多肽链的两个末端之一，此末端的氨基酸残基携带游离的α氨基(—NH2)。在某些实施方式中，所述游离的α氨基也可被酰胺化或环化。
在本申请中，术语“轻链”通常是指免疫球蛋白中分子量较小的肽链，可包含轻链可变区(VL)和轻链恒定区(CL)。根据其结构和恒定区抗原性的差异可分为“κ轻链(kappa light chain)”和“λ轻链(lambda light chain)”两种类别。
在本申请中，术语“重链”通常是指免疫球蛋白中分子量较大的肽链，可包含重链可变区(VL)和重链恒定区(CL)。根据其恒定区抗原性的不同可分为μ、γ、α、δ和ε五类。
在本申请中，术语“轻链可变区”通常指抗体轻链的氨基末端结构域。轻链可变区可以称为“VL”。这些结构域通常是抗体轻链中变化最大的部分(相对于相同类型的其它抗体)，可以包括互补决定区(CDR)或高变区(HVR)和框架区(FR)。
在本申请中，术语“重链可变区”通常指抗体重链的氨基末端结构域。重链可变区可以称为“VH”。这些结构域通常是抗体重链中变化最大的部分(相对于相同类型的其它抗体)，可以包括互补决定区(CDR)或高变区(HVR)和框架区(FR)。
在本申请中，术语“连接子”、“连接片段”可互换使用，通常是指一段具备柔性特征的氨基酸序列。例如，所述连接子可以用于在嵌合多肽(或融合蛋白、嵌合蛋白)连接不同的功能模块，其功能可以包括提高嵌合多肽的折叠和稳定性等。例如，连接肽的长度可以有较大的变化范围，例如长度约为1至约100、约为3至约20、约为5至约30、约为5至约18或约为3至约8个氨基酸的甘氨酸/丝氨酸连接片段。例如，所述连接肽可以是(GGGGS)n/(G4S)n，G为甘氨酸，S为丝氨酸，n为大于等于1的整数；例如n可以是1、2、3、4或5。本申请中的各结合区H或结合区L之间可通过合适的连接子连接以实现本申请的目的。
在本申请中，术语“跨膜域”通常是指蛋白质序列中跨越细胞膜的区域。在某些实施方式中，蛋白质序列中跨越细胞膜的区域通常为α-螺旋结构，包含大部分为疏水性的氨基酸。在某些实施方式中，所述跨膜域可获取自天然蛋白质(例如来自于CD8的跨膜域或其功能性衍生序列)，或者，跨膜域可以是合成的非天然存在的蛋白质区段，例如在细胞膜中热力学稳定的疏水性蛋白质区段。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激信号的共刺激分 子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。
在本申请中，术语“胞内信号传导域”也称作“初级信号传导结构域”，通常是指含有称作基于免疫受体酪氨酸的活化基序或ITAM的信号转导序列。例如，衍生自CD3ζ、FcRγ(FCER1G)、FcγRIIa、FcRβ(FcεR1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12的初级信号传导结构域。在某些实施方式中，胞内信号传导域转导效应功能信号并指导细胞进行特化功能。虽然可以使用整个胞内信号传导域，但在许多情况下，不必使用整个链。就使用胞内信号传导域的截短部分而言，此类截短部分可用于代替完整链，只要其能够转导效应子功能信号即可。因此，初胞内信号传导域意在包括足以转导效应子功能信号的胞内信号传导结构域的任一截短部分。胞内信号传导域。
在本申请中，术语“铰链域”通常是指在蛋白质的两个域之间的氨基酸区段，其能够允许蛋白质的柔性和/或一个或两个以上的域相对于彼此的运动。例如来源于IgG家族(例如IgG1和IgG4)、IgD以及其他蛋白分子的铰链域，例如来自于CD28、HLA家族的铰链域。
术语“宿主抗移植物反应(HVGR)”通常是指：由于供体和受体(或称为宿主)之间的免疫遗传学差异，在进行外源供体移植时，作为外源移植物的供体会受到宿主体内的免疫细胞(例如NK细胞)识别和攻击，进而抑制或者清除供体。
术语“移植物抗宿主病(GVHD)”通常是指：由于外源移植供体T淋巴细胞的TCR的多样性，以及与宿主HLA分子的不兼容性，供体T淋巴细胞会识别宿主正常组织上的抗原，经扩增并释放一系列细胞因子，攻击宿主细胞。
在本申请中，术语“药物组合物”通常指涉及适合施用于患者、例如人患者的组合物。例如，本申请所述的药物组合物，其可以包含本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的佐剂。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体包括：质粒、脂质体、噬菌体如λ噬菌体或M13噬菌体及病毒载体等。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“肿瘤”和“癌症”通常是指在正常生长和/或发育中呈现出至少部分失去控制的细胞。例如，常见的肿瘤或癌细胞通常已经失去了接触抑制并可能是入侵性的和/或具有转移的能力。
术语“核酸分子”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物，包括编码目的多肽或其片段的任何核苷酸序列。
在本申请中，术语“内源”，是指核酸分子或多肽等来自生物体自身。
术语“外源”，是指核酸分子或多肽不是内源性存在细胞中的，或表达水平不足以实现过表达时具有的功能；涵盖在细胞中表达的任何重组核酸分子或多肽，例如外源、异源和过表达的核酸分子和多肽。
术语“个体”和“受试者”可互换，包括人或来自其他种属的动物，其包括但不限于人、小鼠、大鼠、仓鼠和豚鼠、兔子、狗、猫、绵羊、猪、山羊、牛、马、猿、猴子。
术语“移植免疫排斥”是指宿主进行同种异体的组织、器官、或细胞等移植物移植后，外源的移植物作为一种“异己成分”被宿主的免疫系统识别，并发起针对移植物的攻击、破坏和清除的免疫学反应。
术语“治疗有效量”、“治疗有效的”、“有效量”或“以有效的量”在本文中可互换地使用，是指如本文中所述有效地实现特定生物学结果的化合物、制剂、物质或组合物、药物组合物的量，例如但不限于足以促进T细胞应答的量或剂量。有效量的免疫细胞，是指但不限于：能使抗肿瘤活性增加、增强或延长的免疫细胞的数量；抗肿瘤免疫细胞数目或活化免疫细胞数目的增加；促进IFN-γ分泌、肿瘤消退、肿瘤缩小、肿瘤坏死的免疫细胞的数量。
术语“MHC”为组织相容性复合物，是所有编码生物相容复合体抗原的基因群一种统称。在人类细胞中MHC称为HLA抗原，在移植反应中发挥重要作用，由对所植入的组织的表面上的组织相容性抗原产生反应的T细胞介导排异。
术语“人类白细胞抗原”(Human leukocyte antigen，HLA)是人类的主要组织相容性复合体的编码基因，与人类的免疫系统功能密切相关。HLA包括有I类、II类和III类基因部分。HLA I类是一个异二聚体，由重链(α链)与轻链β2微球蛋白(B2M)组成。HLA-II类基因包括HLA-D家族，主要有HLA-DP、HLA-DQ和HLA-DR等，主要分布于B淋巴细胞、巨噬细胞和树突状细胞等专职抗原提呈细胞表面。
发明详述
本申请提供了一种嵌合抗原受体(CAR)，所述CAR包含双特异性抗原结合域，所述双特异性抗原结合域包括NKG2A抗原结合域和肿瘤抗原结合域，所述NKG2A抗原结合域包含第一结合区L、第一结合区H,所述肿瘤抗原结合域包含第二结合区L、第二结合区H。
抗原
“NKG2A抗原”或“NKG2A”通常是指NKG2A多肽，是NKG2转录物组的成员，NKG2A与CD94形成的异源二聚体抑制性受体CD94/NKG2A，表达于NK细胞、αβT细胞、γδT细胞和NKT细胞的亚群的表面上。如本文所使用的，“NKG2A”是指NKG2A基因或编码的蛋白的任何变体、衍生物或同种型。NKG2A多肽具有与由NCBI GenBank Gene ID：3821的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。在一实例中，人NKG2A多肽具有SEQ ID NO：42所示序列。
在一实例中，所述NKG2A多肽为人NKG2A多肽,包括与SEQ ID No:42所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。
“BCMA抗原”或“BCMA”通常是指BCMA多肽，是B细胞成熟抗原(B-cell maturation antigen)，属TNF受体超家族。BCMA与其配体结合后，可激活B细胞的增殖和存活。BCMA特异地高表达于浆细胞和多发性骨髓瘤细胞，而在造血干细胞和其他正常组织细胞中均不表达。“BCMA”是指BCMA基因或编码的蛋白的任何变体、衍生物或同种型。BCMA多肽具有与由NCBI GenBank Gene ID：NP_001183.2的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。在一实例中，人BCMA多肽具有SEQ ID NO：43所示序列。
在一实例中，所述BCMA多肽为人BCMA多肽,包括与SEQ ID No:43所示的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。
任何肿瘤抗原均可用于本申请所述的肿瘤相关的实施例(本文中“实例”与“实施例”可互换使用)中。抗原表达为多肽或完整蛋白或其部分。本申请的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌 胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)； 淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
在一实例中，嵌合抗原受体(CAR)识别病原体抗原，例如用于治疗和/或预防病原体感染或其他感染性疾病，例如在免疫受损的受试者中。病原体抗原包括但不限于：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原包括但不限于：巨细胞病毒(CMV)抗原、爱泼斯坦-巴尔病毒(EBV)抗原、人类免疫缺陷病毒(HIV)抗原或流感病毒抗原。
在一实例中，本申请的CAR特异性结合BCMA多肽和NKG2A多肽。在一实例中，CAR结合至NKG2A多肽和BCMA多肽的胞外结构域。
双特异性抗原结合域
在本申请提供的CAR中，肿瘤抗原结合域(例如BCMA抗体)、肿瘤抗原结合域(例如NKG2A抗体)的重链/重链可变区(VH)和/或轻链/轻链可变区(VH)可定位在任何合适的位置上。
在本申请中，NKG2A抗原结合域包含第一结合区L、第一结合区H,肿瘤抗原结合域包含第二结合区L、第二结合区H,所述第一结合区L、所述第一结合区H、所述第二结合区L、所述第二结合区H以如下方式连接：
a)自N端至C端：所述第一结合区L或所述第一结合区H—所述第一结合区H或所述第一结合区L—所述第二结合区H或所述第二结合区L—所述第二结合区L或所述第二结合区H，
b).自N端至C端：所述第二结合区H—所述第二结合区L—所述第一结合区L—所述第一结合区H，或
c)所述第一结合区L与所述第二结合区H连接为第一多肽，所述第二结合区L与所述第一结合区H连接为第二多肽，所述第一多肽与所述第二多肽连接，其中，所述第一结合区L不与所述第一结合区H连接，且所述第二结合区L不与所述第二结合区H连接。
在一实例中，所述第一结合区L包含第一轻链或第一轻链可变区(VL)。
在一实例中，所述第一结合区H包含第一重链或第一重链可变区(VH)。
在一实例中，所述第二结合区L包含第二轻链或第二轻链可变区(VL)。
在一实例中，所述第二结合区H包含第二重链或第二重链可变区(VH)。
在一实例中，在所述方式a中，所述第一结合区L与所述第一结合区H连接，所述第二结合区L与所述第二结合区H连接。
在一实例中，在所述方式a中，所述第一轻链与所述第一重链连接，所述第二轻链与所述第二重链连接。
在一实例中，在所述方式a中，所述第一轻链可变区(VL)与所述第一重链可变 区(VH)连接，所述第二轻链可变区(VL)与所述第二重链可变区(VH)连接。
在一实例中，在所述方式a中，以如下方式连接：NKG2A VL—NKG2A VH—BCMA VH—BCMAVL。
在一实例中，在所述方式a中，以如下方式连接：NKG2A VL—(G4S)3—NKG2A VH—(G4S)3—BCMA VH—(G4S)3—BCMA VL。
在一实例中，在所述方式b中，以如下方式连接：BCMA VH—BCMA VL—NKG2A VL—NKG2A VH。
在一实例中，在所述方式b中，以如下方式连接：BCMA VH—(G4S)3—BCMA VL—(G4S)3—NKG2A VL—(G4S)3—NKG2A VH。
在一实例中，在所述方式c中，自N端至C端以如下方式连接:
c1.所述第二结合区H—所述第一结合区L—所述第二结合区H—所述第一结合区L；
c2.所述第一结合区H—所述第二结合区L—所述第一结合区H—所述第二结合区L；
c3.所述第二结合区L—所述第一结合区H—所述第二结合区H—所述第一结合区L；
c4.所述第一结合区L—所述第二结合区H—所述第一结合区H—所述第二结合区L；
c5.所述第二结合区H—所述第一结合区L—所述第一结合区L—所述第二结合区H；或
c6.所述第一结合区H—所述第二结合区L——所述第二结合区L—所述第一结合区H。
在一实例中，在所述方式c1中，以如下方式连接：
所述第二VH—所述第一VL—所述第二VH—所述第一VL。
在一实例中，在所述方式c2中，以如下方式连接：
所述第一VH—所述第二VL—所述第一VH—所述第二VL。
在一实例中，在所述方式c3中，以如下方式连接：
所述第二VL—所述第一VH—所述第二VH—所述第一VL。
在一实例中，在所述方式c4中，以如下方式连接：
所述第一VL—所述第二VH—所述第一VH—所述第二VL。
在一实例中，在所述方式c5中，以如下方式连接：
所述第二VH—所述第一VL—所述第一VL—所述第二VH。
在一实例中，在所述方式c6中，以如下方式连接：
所述第一VH—所述第二VL—所述第二VL—所述第一VH。
在本申请中，NKG2A抗原结合域包含第一结合区L、第一结合区H,肿瘤抗原结合域包含第二结合区L、第二结合区H,所述第一结合区L、所述第一结合区H、所述第二结合区L、所述第二结合区H以形成loop结构的方式连接。
在一实例中，所述loop结构通过如下方式形成：其中一个抗原结合域的结合区H和结合区L连接为抗体，另一个抗原结合域的结合区H、结合区L分别与所述抗体的两端连接。
在一实例中，所述抗体自N端至C端依次包含：所述结合区H、所述结合区L。
在一实例中，所述抗体自N端至C端依次包含：所述结合区H、所述结合区L；所述另一个抗原结合域的结合区L与所述抗体的N端连接，所述另一个抗原结合域的结合区H与所述抗体的C端连接。
在一实例中，所述连接为抗体的结合区H为第一结合区H，所述连接为抗体的结合区L为第一结合区L，所述另一个抗原结合域的结合区L为第二结合区L，所述另一个抗原结合域的结合区H为第二结合区H。
在一实例中，所述连接为抗体的结合区H为第二结合区H，所述连接为抗体的结合区L为第二结合区L。所述另一个抗原结合域的结合区L为第一结合区L，所述另一个抗原结合域的结合区H为第一结合区H。
在一实例中，所述抗体自N端至C端依次包含：所述结合区H、所述结合区L；所述另一个抗原结合域的结合区H与所述抗体的N端连接，所述另一个抗原结合域的结合区L与所述抗体的C端连接。
在一实例中，所述连接为抗体的结合区H为第一结合区H，所述连接为抗体的结合区L为第一结合区L，所述另一个抗原结合域的结合区L为第二结合区L，所述另一个抗原结合域的结合区H为第二结合区H。
在一实例中，所述连接为抗体的结合区H为第二结合区H，所述连接为抗体的结合区L为第二结合区L。所述另一个抗原结合域的结合区L为第一结合区L，所述另一个抗原结合域的结合区H为第一结合区H。
在一实例中，所述抗体自N端至C端依次包含：所述结合区L、所述结合区H。
在一实例中，所述抗体自N端至C端依次包含：所述结合区L、所述结合区H；所述另一个抗原结合域的结合区L与所述抗体的N端连接，所述另一个抗原结合域的结合区H与所述抗体的C端连接。
在一实例中，所述抗体自N端至C端依次包含：所述结合区L、所述结合区H；所述 另一个抗原结合域的结合区H与所述抗体的N端连接，所述另一个抗原结合域的结合区L与所述抗体的C端连接。
在一实例中，所述连接为抗体的结合区H为第一结合区H，所述连接为抗体的结合区L为第一结合区L，所述另一个抗原结合域的结合区L为第二结合区L，所述另一个抗原结合域的结合区H为第二结合区H。
在一实例中，所述连接为抗体的结合区H为第二结合区H，所述连接为抗体的结合区L为第二结合区L。所述另一个抗原结合域的结合区L为第一结合区L，所述另一个抗原结合域的结合区H为第一结合区H。
在某一实例中，所述第一结合区L、所述第一结合区H、所述第二结合区L、所述第二结合区H以如下方式连接：
所述第二结合区L—所述第一结合区H—所述第一结合区L—所述第二结合区H。
在某一实例中，所述第一结合区L、所述第一结合区H、所述第二结合区L、所述第二结合区H以如下方式连接：
所述第一结合区L—所述第二结合区H—所述第二结合区L—所述第一结合区H。
在一实例中，所述第一结合区L包含第一轻链或第一轻链可变区(VL)。
在一实例中，所述第一结合区H包含第一重链或第一重链可变区(VH)。
在一实例中，所述第二结合区L包含第二轻链或第二轻链可变区(VL)。
在一实例中，所述第二结合区H包含第二重链或第二重链可变区(VH)。
在一实例中，所述第一结合区L包含第一轻链，所述第一结合区H包含第一重链,所述第二结合区L包含第二轻链，所述第二结合区H包含第二重链。
在一实例中，所述第一结合区L包含第一VL，所述第一结合区H包含第一VH,所述第二结合区L包含第二轻链，所述第二结合区H包含第二重链。
在一实例中，所述第一结合区L包含第一轻链，所述第一结合区H包含第一重链,所述第二结合区L包含第二VL，所述第二结合区H包含第二VH。
在一实例中，所述第一结合区L包含第一VL，所述第一结合区H包含第一VH,所述第二结合区L包含第二VL，所述第二结合区H包含第二VH。
在本申请中，所述第一VL为所述NKG2A抗体的VL，所述第一轻链为所述NKG2A抗体的轻链，所述第一VH为所述NKG2A抗体的VH，所述第一重链为所述NKG2A抗体的重链，所述第二VL为识别所述肿瘤抗原的抗体的VL，所述第二轻链为识别所述肿瘤抗原的抗体的轻链，所述第二VH为识别所述肿瘤抗原的抗体的VH，所述第二重链为识别所述肿瘤抗原的抗体的重链。
在一实例中，在所述双特异性抗原结合域中，自N端至C端包含如下连接方式：
BCMA VL—NKG2A VH—NKG2A VL—BCMA VH，
BCMA VH—NKG2A VH—NKG2A VL—BCMA VL，
NKG2A VL—BCMA VH—BCMA VL—NKG2A VH，或
NKG2A VH—BCMA VH—BCMA VL—NKG2A VL，
在一实例中，在所述双特异性抗原结合域中，自N端至C端包含如下连接方式：
BCMA VL—NKG2A VL—NKG2A VH—BCMA VH，
NKG2A VL—BCMA VL—BCMA VH—NKG2A VH，
NKG2A VH—BCMA VL—BCMA VH—NKG2A VL，或
BCMA VH—NKG2A VL—NKG2A VH—BCMA VL，
在一实例中，CAR中的各轻链/VL、重链/VH可通过连接子连接。连接子可包含任何合适的氨基酸序列，用于向细胞外抗原结合区提供柔性的CAR的一部分，其组成和长度都是可调的。
在一实例中，一个抗原结合域的结合区H和结合区L通过连接子连接为抗体，另一个抗原结合域的结合区H、结合区L分别通过连接子与所述抗体的两端连接。
在一实例中，所述连接子包含Lin1或(G4S)n，其中，n是等于或大于1的整数，所述Lin1包含如SEQ ID NO:18所示的氨基酸序列。例如，所述(G4S)n中n为1或3。
在一实例中，一个抗原结合域的结合区H和结合区L通过Lin1或(G4S)3连接为抗体，另一个抗原结合域的结合区H、结合区L分别通过连接子与所述抗体的两端连接。
在一实例中，一个抗原结合域的结合区H和结合区L通过Lin1或(G4S)3连接为抗体，另一个抗原结合域的结合区H、结合区L分别通过G4S与所述抗体的两端连接。
在一实例中，所述抗体包含所述第一结合区H、第一结合区L，所述抗体的N端通过G4S与所述第二结合区H或第二结合区L连接,所述抗体的N端通过G4S与所述第二结合区L或第二结合区H连接。
在一实例中，所述抗体包含所述第二结合区H、第二结合区L，所述抗体的N端通过G4S与所述第一结合区H或第一结合区L连接,所述抗体的N端通过G4S与所述第一结合区L或第一结合区H连接。
在一实例中，在所述双特异性抗原结合域中，自N端至C端包含如下连接方式：
BCMA VL—G4S—NKG2A VH—(G4S)3—NKG2A VL—G4S—BCMA VH；
BCMA VH—G4S—NKG2A VH—(G4S)3—NKG2A VL—G4S—BCMA VL；
NKG2A VL—G4S—BCMA VH—(G4S)3—BCMA VL—G4S—NKG2A VH；
NKG2A VH—G4S—BCMA VH—(G4S)3—BCMA VL—G4S—NKG2A VL；
BCMA VH—G4S—NKG2A VL—(G4S)3—NKG2A VH—G4S—BCMA VL；
BCMA VL—G4S—NKG2A VL—(G4S)3—NKG2A VH—G4S—BCMA VH；
NKG2A VL—G4S—BCMA VL—(G4S)3—BCMA VH—G4S—NKG2A VH；或
NKG2A VH—G4S—BCMA VL—(G4S)3—BCMA VH—G4S—NKG2A VL。
在一实例中，在所述方式a)中，自N端至C端包含如下连接方式：
BCMA VL—G4S—NKG2A VH—Lin1—NKG2A VL—G4S—BCMA VH；
BCMA VH—G4S—NKG2A VH—Lin1—NKG2A VL—G4S—BCMA VL；
NKG2A VL—G4S—BCMA VH—Lin1—BCMA VL—G4S—NKG2A VH；
NKG2A VH—G4S—BCMA VH—Lin1—BCMA VL—G4S—NKG2A VL；
BCMA VH—G4S—NKG2A VL—Lin1—NKG2A VH—G4S—BCMA VL；
BCMA VL—G4S—NKG2A VL—Lin1—NKG2A VH—G4S—BCMA VH；
NKG2A VL—G4S—BCMA VL—Lin1—BCMA VH—G4S—NKG2A VH；或
NKG2A VH—G4S—BCMA VL—Lin1—BCMA VH—G4S—NKG2A VL。
在一实例中，所述CAR包含如SEQ ID NO：22、25和28中任一项所述的氨基酸序列。
在一实例中，所述第二VL为BCMA抗体的VL，所述第二轻链为BCMA抗体的轻链，所述第二VH为BCMA抗体的VH，所述第二重链为BCMA抗体的重链。
在一实例中，CAR中的抗体各轻链/VL、重链/VH可通过连接子连接。连接子可包含任何合适的氨基酸序列。
在一实例中，所述连接子包含Lin1或(G4S)n，其中，n是等于或大于1的整数；所述Lin1包含如SEQ ID NO:18所示的氨基酸序列。
在一实例中，连接子是长度约为1至约100、约为3至约20、约为5至约30、约为5至约18或约为3至约8个氨基酸的甘氨酸/丝氨酸连接片段，并且在序列中由甘氨酸和/或丝氨酸残基组成。
在一实例中，所述甘氨酸/丝氨酸连接体是式[GGGGS]n((G4S)n)的肽，其中n是1到10、2到8或3到5的整数。例如，n是1、2、3或4。
在一实例中，NKG2A抗体VL和VH可通过连接片段连接。在一实例中，NKG2A抗体VL和VH通过包含具有如SEQ ID NO:18所示序列的连接片段连接，或者通过(G4S)3连接。
在一实例中，BCMA抗体VL和VH可通过连接片段连接。在一实例中，BCMA抗体包括如SEQ ID NO:9-14所示的序列，还包括如SEQ ID NO:18所示的序列或(G4S)3；或包括如SEQ ID NO:15和16所示的序列，还包括如SEQ ID NO:18所示的序列或(G4S)3；或包括如SEQ ID NO:39或40所示的序列。
在一实例中，NKG2A抗体包含VH、VL和连接片段。在一实例中，NKG2A抗体包含如SEQ ID NO:3-8所示的序列,还包括如SEQ ID NO:18所示的序列或(G4S)3；或包括如SEQ ID NO:1和2所示的序列，还包括如SEQ ID NO:18所示的序列或(G4S)3；或包括如SEQ ID NO:41所示的序列。
在一实例中，BCMA抗体和NKG2A抗体通过连接片段连接。连接片段可包含任何合适的氨基酸序列。优选地，将所述BCMA抗体和所述NKG2A抗体连接的连接片段包括甘氨酸/丝氨酸连接片段，所述甘氨酸/丝氨酸连接体是式(G4S)n的肽，其中n是1到10、2到8或3到5的整数。优选地，n是1、2、3、4。优选地，将BCMA抗体和NKG2A抗体连接的连接片段包含(G4S)3序列(包含由连接片段连接的BCMA抗体重链和轻链和NKG2A抗体重链和轻链，(G4S)3位于BCMA抗体和NKG2A抗体之间)。
在一实例中，CAR包括(i)位于BCMA抗体重链的氨基末端附近的NKG2A抗体轻链，其中连接片段位于它们之间，(ii)位于NKG2A抗体轻链的氨基末端附近的NKG2A抗体重链，其中连接片段位于它们之间，以及(iii)定位在NKG2A抗体重链的氨基末端附近的BCMA抗体轻链，其中连接片段位于它们之间；所述连接片段任选地包括，如SEQ ID NO:18所示的序列、G4S或(G4S)3。在一实例中，CAR包括(i)位于NKG2A抗体重链的氨基末端附近的BCMA抗体轻链，其中连接片段位于它们之间，(ii)位于BCMA抗体轻链的氨基末端附近的BCMA抗体重链，其中连接片段位于它们之间，以及(iii)位于BCMA抗体重链的氨基末端附近的NKG2A抗体轻链，其中连接片段位于它们之间；所述连接片段任选地包括，如SEQ ID NO:18所示的序列、G4S或(G4S)3。
在一实例中，BCMA抗体可定位在与NKG2A抗体的氨基末端相邻的位置(其中连接片段位于它们之间)。在一实施例中，NKG2A抗体位于BCMA抗体的氨基末端附近(其中连接片段位于它们之间)。
在一实例中,表达本申请提供的CAR的car-t细胞相对于仅靶向肿瘤抗原的car-t细胞具有更好的体外杀伤NK细胞的效果。
在一实例中,表达本申请提供的CAR的ucar-t细胞相对于仅靶向肿瘤抗原的ucar-t细胞具有更好的体外杀伤NK细胞的效果。
在一实例中,表达本申请提供的CAR的ucar-t细胞相对于仅靶向肿瘤抗原ucar-t细胞具有更好的体内抑制肿瘤生长的效果。
在一实例中,表达本申请提供的CAR的ucar-t细胞与仅靶向肿瘤抗原的ucar-t细胞相比，具有更长存活时间和/或扩增能力。
在一实例中,表达本申请提供的CAR的ucar-t细胞与仅靶向肿瘤抗原的ucar-t细胞 相比，在体外NK细胞环境下具有更好的抗NK细胞和杀伤肿瘤细胞的效果。
在一实例中,表达本申请提供的CAR的ucar-t细胞与仅靶向肿瘤抗原的ucar-t细胞相比，在体内NK细胞环境下具有更好的抗NK细胞和抑制肿瘤生长的效果。
在一实例中,表达本申请提供的CAR的ucar-t细胞与仅靶向肿瘤抗原的ucar-t细胞相比，具有更好的特异性向肿瘤组织浸润的效果。
在一实例中,表达本申请提供的CAR的ucar-t细胞不会引起移植物抗宿主(GVHD)反应。
在一实例中，NKG2A抗原结合域包含识别NKG2A多肽的抗体(也称NKG2A抗体)的scFv。
在一实例中，NKG2A抗原结合域包含NKG2A抗体的轻链可变区(VL)和/或重链可变区(VH)。在一实例中，NKG2A抗原结合域包含NKG2A抗体的轻链和/或重链。在一实例中，NKG2A的抗体重链或VH包含一个或多个重链CDR(HCDR)：如SEQ ID NO:3序列所示的HCDR1、如SEQ ID NO:4所示序列的HCDR2、如SEQ ID NO:5所示序列的HCDR3。在一实例中，NKG2A抗体重链或VH包含如SEQ ID NO:3-5所示序列。在一实例中，NKG2A抗体轻链或VL包含一个或多个轻链CDR(LCDR)：如SEQ ID NO:6所示序列的LCDR1、如SEQ ID NO:7所示序列的LCDR2、如SEQ ID NO:8所示序列的LCDR3。在一实例中，NKG2A抗体轻链或VL包含如SEQ ID NO:6-8所示序列。在一实例中，NKG2A抗体重链或VH包含如SEQ ID NO:1所示的序列。在一实例中，NKG2A抗体轻链或VL包含如SEQ ID NO:2所示的序列。在一实例中，NKG2A抗体或NKG2A抗原结合域包含如SEQ ID NO:1所示序列的VH和/或如SEQ ID NO:2所示序列的VL。在一实例中，NKG2A抗体或NKG2A抗原结合域包含如SEQ ID NO:1和2所示序列。
在一实例中，BCMA抗原结合域包含识别BCMA多肽的抗体(也称BCMA抗体)的scFv。在一实例中，BCMA抗原结合域包含BCMA抗体VL和/或VH。在一实例中，BCMA抗原结合域包含BCMA抗体的轻链和/或重链。在一实例中，BCMA抗体重链或VH包含一个或多个重链CDR(HCDR)：如SEQ ID NO:9所示序列的HCDR1、如SEQ ID NO:10所示序列的HCDR2、如SEQ ID NO:11所示序列的HCDR3。在一实例中，BCMA抗体重链或VH包含如SEQ ID NO:9-11所示序列。在一实例中，BCMA抗体轻链或VL包含一个或多个轻链CDR(LCDR)：如SEQ ID NO:12所示序列的LCDR1、如SEQ ID NO:13所示序列的LCDR2、如SEQ ID NO:14所示序列的LCDR3。在一实例中，BCMA抗体轻链或VL包含如SEQ ID NO:12-14所示序列。在一实例中，BCMA抗体重链或VH包含如SEQ ID NO:15所示的序列。在一实例中，BCMA抗体轻链或VL包含如SEQ ID  NO:16所示的序列。在一实例中，BCMA抗体或BCMA抗原结合域包含如SEQ ID NO:15所示序列的VH和/或如SEQ ID NO:16所示序列的VL。在一实例中，BCMA抗体或BCMA抗原结合域包含如SEQ ID NO:16和17所示序列。在一实例中，BCMA抗体或BCMA抗原结合域包含如SEQ ID NO:46-50所示序列。
在一实例中，CAR包含NKG2A抗体和BCMA抗体全部CDR区域。在一实例中，CAR包括如SEQ ID NO:3-14所示的序列。在一实例中，CAR包括识别NKG2A多肽的抗体和识别BCMA多肽的抗体的轻链可变区和重链可变区。在一实例中，CAR包括如SEQ ID NO:1、2、15和16所示的序列。
在一个方面，本申请考虑到产生功能上等同的分子的起始抗体或片段(例如，VH或VL)氨基酸序列的修饰。例如，可修饰CAR中包括的NKG2A抗体或BCMA抗体的VH或VL，保留NKG2A或BCMA抗体如VH或VL至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。
本申请考虑到整个CAR分子的修饰，例如，CAR分子的各个结构域的一个或多个氨基酸序列的修饰，以便产生功能上等同的分子。可修饰CAR分子保留起始CAR分子的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
本领域普通技术人员将会理解，可进一步修饰本申请的抗体或抗体片段，使得它们在氨基酸序列上(例如，相对于野生型或本申请提供的序列)有所变化，但在所需活性上没有变化。例如，可对蛋白质进行另外的核苷酸置换，导致“非必需”氨基酸残基处的氨基酸置换。例如，分子中的非必需氨基酸残基可被来自相同侧链家族的另一个氨基酸残基取代。在另一个实施方案中，氨基酸片段可被结构相似但在顺序和/或组成上与侧链家族成员不同的氨基酸片段取代，例如，可进行保守置换，其中氨基酸残基被具有相似侧链的氨基酸残基所取代。
前导序列和铰链域
本申请提供的CAR还可以包括前导序列和/或铰链域。在一实例中，抗原结合域与跨膜域直接连接或通过铰链连接。在一实例中，所述铰链包括CD8铰链，例如，CD8铰链包含SEQ ID NO:35或与SEQ ID NO:35具有95-99％同一性的的序列。
在一实例中，抗原结合域包含前导序列。在一实例中，前导序列可定位于BCMA抗体VH的氨基末端(例如，在BCMA抗体重链的氨基末端)。在一实例中，前导序列位于BCMA抗体VL的氨基末端(例如，位于BCMA抗体轻链的氨基末端)。在一实 例中，前导序列位于NKG2A抗体VL的氨基末端(例如，位于NKG2A抗体轻链的氨基末端)。前导序列可以包括任何合适的前导序列。在一实例中，前导序列包含如SEQ ID NO:29的氨基酸序列。在一实例中，CAR缺少前导序列。
跨膜域
本申请提供的CAR还可以包括跨膜域。跨膜域可以将CAR锚定在细胞膜上。本申请CAR的跨膜域可以包括选自以下蛋白的跨膜结构域:T细胞受体的α、β、或ζ的跨膜结构域、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D和/或NKG2C的跨膜结构域。
在一实例中，CAR包括CD8跨膜结构域，其具有如SEQ ID NO:30所示序列的至少一个、两个或三个修饰，但不超过20，10或5个修饰,或与SEQ ID NO:30的氨基酸序列具有95-99％同一性的序列。在一个实例中，CD8跨膜结构域包括如SEQ ID NO:30所示的序列。
在一实例中，CAR包括CD28跨膜结构域，其具有如SEQ ID NO:31所示的至少一个、两个或三个修饰，但不超过20，10或5个修饰,或与SEQ ID NO:31所示的氨基酸序列具有95-99％同一性的序列。在一个实例中，CD28跨膜结构域包括如SEQ ID NO:31所示的序列。
胞内信号传导域
本发明提供的CAR还可以包括胞内信号传导域。在一实例中，胞内信号传导域(也称作一级信号结构域)包括选自如下蛋白分子的信号传导域：CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa。
在一实例中，胞内信号传导域包括CD3ζ的胞内信号传导域。CD3ζ胞内信号传导域可包括如SEQ ID NO:34所示的氨基酸序列的至少1、2、或3个修饰但不超过20、10或5个修饰的氨基酸序列，或与SEQ ID NO:34所示的氨基酸序列有95-99％同一性的序列。在一实例中，CD3ζ信号结构域包括如SEQ ID NO:34所示的氨基酸序列。
在一实例中，本发明提供的CAR的胞内信号结构域包括人CD3ζ信号结构域。在一实例中，CAR的胞内信号结构域包括人CD3ζ信号结构域和CD28共刺激信号结构域。
共刺激信号结构域
本申请提供的CAR还可以包括共刺激信号结构域。在一实例中，共刺激信号结构域包括选自如下蛋白功能信号结构域:CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、
SLAMF4(CD244,2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D。
在一实例中，共刺激信号结构域4-1BB包括如SEQ ID NO:33所示氨基酸序列的至少1、2、或3个修饰但不超过20、10或5个修饰的氨基酸序列，或与SEQ ID NO:33所示的氨基酸序列有95-99％同一性的序列。在一实例中，共刺激信号结构域包括如SEQ ID NO:33所示的序列。
在一实例中，共刺激信号结构域CD28包括如SEQ ID NO:32所示的氨基酸序列的至少1、2、或3个修饰但不超过20、10或5个修饰的氨基酸序列，或与SEQ ID NO:32所示的氨基酸序列有95-99％同一性的序列。在一实例中，共刺激信号结构域包括如SEQ ID NO:32所示的序列。
在一实例中，本发明提供的CAR的胞内信号结构域包括人CD3ζ信号结构域。在一实例中，CAR的胞内信号结构域包括人CD3ζ信号结构域和CD28共刺激信号结构域。在一实例中，CAR的胞内信号结构域包括人CD3ζ信号结构域和4-1BB共刺激信号结构域。在一实例中，CAR的胞内信号结构域包括CD3ζ信号结构域、CD28和4-1BB共刺激信号结构域。
示例性，本申请的CAR包括如SEQ ID NO：17、22、25或28所示的序列。
示例性，本申请的CAR包括如SEQ ID NO：17、22、25或28所示序列中的任一项与如SEQ ID NO：19、44或45所示序列中的任一项顺序连接的序列。
细胞
本申请提供了包括本申请提供的CAR的细胞。所述细胞包括衍生自干细胞或淋巴谱系的免疫细胞。CAR与靶抗原结合后能激活所述免疫细胞；本申请所提供的CAR(也称作串联CAR)，其包含双特异性抗原结合域，该双特异性抗原结合域包含NKG2A抗原结合域和肿瘤抗原结合域。本申请提供的CAR已在前文描述本申请提供的细胞包含其全部技术方案。
在一实例中，本申请的细胞包括识别NKG2A多肽和肿瘤抗原的免疫细胞(例如T、NKT细胞)。在一实例中，本申请的细胞包括识别NKG2A多肽和病原体抗原的免疫细胞。
在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，本申请的免疫细胞具有更长存活时间和/或扩增能力。
在一实例中，与包括仅识别肿瘤抗原的CAR的免疫细胞相比，本申请的免疫细胞对携带靶肿瘤抗原的细胞表现出更强的体内外的细胞杀伤作用。
包括B、T和自然杀伤(NK)细胞的淋巴谱系可以提供抗体的产生、细胞免疫系统的调节、血液中外源试剂的检测、宿主外源细胞的检测等。淋巴谱系的免疫细胞的非限制性实例包括T细胞、自然杀伤T(NKT)细胞及其前体，包括胚胎干细胞和多能干细胞(例如，分化成淋巴样细胞的干细胞或多能干细胞)。T细胞可以是在胸腺中成熟的淋巴细胞，主要负责细胞介导的免疫。T细胞参与适应性免疫系统。T细胞可以是任何类型的T细胞，包括但不限于辅助T细胞、细胞毒性T细胞、记忆T细胞(包括中央记忆T细胞、干细胞样记忆T细胞(或干样记忆T细胞)和两种效应记忆T细胞：例如TEM细胞和TEMRA细胞)、调节性T细胞(也称为抑制性T细胞)、自然杀伤T细胞、粘膜相关性不变T细胞、γδT细胞或αβT细胞。细胞毒性T细胞(CTL或杀伤性T细胞)是能够诱导被感染的体细胞或肿瘤细胞死亡的T淋巴细胞。
免疫细胞(例如，T细胞)可以是自体的、非自体的(例如，同种异体的)、或者是体外从工程化的祖细胞或干细胞衍生而来。可从许多来源获得，包括外周血单个核细胞(PBMC)、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。
在某一实例中，受试者自身的免疫细胞可以被工程化改造以表达本申请的CAR。在某一实例中，来自于受试者之外的其他供体(同种异体)的免疫细胞可以被工程化改造以表达本申请的CAR。在一实例中，免疫细胞是T细胞。在一实例中，T细胞可以是CD4+T细胞和/或CD8+T细胞。在一实例中，免疫细胞是CD3+T细胞。在一实例中，本申请的细胞包括由PBMC细胞经CD3磁珠刺激后收集的细胞群。
在本申请的某些方面，可使用本领域技术人员已知的任意数量的技术如Ficoll TM分离 技术从收集自受试者的血液样品中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体的循环血液的细胞。单采血液成分术产物通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个方面，可洗涤通过单采血液成分术收集的细胞以去除血浆部分并将细胞置于适当的缓冲液或培养基中以供后续处理步骤。在本申请的背景下还可使用多轮选择。在某些方面，可能需要进行选择程序并在激活和扩充过程中使用“未选择的”细胞。“未选择的”细胞也可以经受其他轮选择。
本申请的细胞能够调节肿瘤微环境。
未纯化的CTL来源可以是本领域已知的任何来源，例如骨髓、胎儿、新生儿或成年或其它造血细胞来源，例如胎儿肝、外周血或脐带血。可以采用各种技术来分离细胞。例如，阴性选择法可以最初去除非CTL。mAb对于鉴定与特定细胞谱系和/或阳性和阴性选择的分化阶段相关的标志物特别有用。
最初可以通过相对粗略的分离除去大部分末端分化的细胞。例如，最初可以使用磁珠分离来去除大量不相关的细胞。在某些实施方式中，在分离细胞之前将去除总造血细胞的至少约80％，通常至少约70％。
分离的程序包括但不限于密度梯度离心；重沉(resetting)；偶联至改变细胞密度的颗粒；用抗体包被的磁珠进行磁分离；亲和色谱；与mAb结合或结合使用的细胞毒性剂，包括但不限于补体和细胞毒素；并用附着在固体基质(例如板、芯片、淘析)上的抗体淘选或任何其它方便的技术。
分离和分析的技术包括但不限于流式细胞术，其可以具有不同的复杂程度，例如多个颜色通道、低角度和钝角光散射检测通道、阻抗通道。
通过使用与死细胞相关的染料，例如碘化丙啶(PI)，可以针对死细胞选择细胞。在某些实施方式中，将细胞收集在包括2％胎牛血清(FCS)或0.2％牛血清白蛋白(BSA)的培养基或任何其它合适的例如无菌等渗培养基中。
在一实例中，表达串联CAR的细胞还包括HLA-I类分子、TCR分子、NKG2A分子或其组合低表达或不表达的免疫细胞。TCR、B2M或NKG2A低表达或不表达分别是指细胞中TCR、B2M或NKG2A的表达减少至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。更具体而言，TCR、B2M或NKG2A低表达或不表达分别是指细胞中TCR、B2M或NKG2A的含量降低至少1％、至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％。可以通过本领域内已知的任何合适 的方法，如ELISA、免疫组织化学、免疫印迹(Western Blotting)或流式细胞术使用TCR、B2M或NKG2A的特异性抗体测定细胞中蛋白的表达或含量。
HLA-I类分子低表达或不表达
在异体细胞(例如表达串联CAR的免疫细胞)移植中，当异体细胞的HLA-I类分子的缺失，可以降低宿主CD8+介导的细胞免疫排斥作用。
在一实例中，本申请提供识别NKG2A多肽和肿瘤抗原的串联CAR、且内源性B2M低表达或不表达的免疫细胞。在一实例中，本申请提供识别NKG2A多肽和病原体抗原的串联CAR、且内源性B2M低表达或不表达的免疫细胞。在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，表达串联CAR的免疫细胞具有更长存活时间和/或扩增能力。在一实例中，与表达仅识别肿瘤抗原的CAR的免疫细胞相比，表达串联CAR的免疫细胞表现出更强的体内外的细胞杀伤作用。
TCR低表达或不表达
在一实例中，本申请提供包括识别NKG2A多肽和肿瘤抗原、且内源性TCR低表达或不表达的免疫细胞；任选地，所述免疫细胞内源性B2M低表达或不表达。
在一实例中，本申请提供识别NKG2A多肽和肿瘤抗原的串联CAR、且内源性TCR低表达或不表达的免疫细胞；任选地，所述免疫细胞内源性B2M低表达或不表达。在一实例中，本申请提供识别NKG2A多肽和BCMA多肽的串联CAR、且内源性TCR/B2M低表达或不表达的免疫细胞。在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，上述表达串联CAR的免疫细胞具有更长存活时间和/或扩增能力。在一实例中，与表达仅识别肿瘤抗原的CAR的免疫细胞相比，上述表达串联CAR的免疫细胞表现出更强的体内外的细胞杀伤作用。
NKG2A低表达或不表达
在一实例中，本申请提供识别NKG2A多肽和肿瘤抗原的串联CAR、且内源性NKG2A低表达或不表达的免疫细胞；任选地，所述免疫细胞内源性B2M低表达或不表达、内源性TCR低表达或不表达、内源性B2M/TCR低表达或不表达。在一实例中，本申请提供识别NKG2A多肽和BCMA多肽的串联CAR、且内源性TCR/B2M/NKG2A低表达或不表达的免疫细胞。在一实例中，在有宿主免疫细胞(例如NK细胞)存在时，上述表达串联CAR的免疫细胞具有更长存活时间和/或扩增能力。在一实例中，与表达仅识别肿瘤抗原的CAR的免疫细胞相比，上述表达串联CAR的免疫细胞表现出更强的体内外的细胞杀伤作用。
基因敲除
采用基因敲除技术和/或基因沉默技术来制备内源性TCR、B2M或NKG2A低表达或不表达的免疫细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性 NKG2A低表达或不表达的免疫细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M低表达或不表达的免疫细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M低表达或不表达的免疫细胞。在一实例中，采用基因敲除技术和/或基因沉默技术来制备内源性TCR/B2M/NKG2A低表达或不表达的免疫细胞。
基因敲除技术包括Argonaute、CRISPR/Cas9技术、ZFN技术、TALE技术、TALE-CRISPR/Cas9技术、Base Editor技术、引导编辑技术和/或归巢核酸内切酶技术。基因沉默技术包括但不限于：反义RNA、RNA干扰、微小RNA介导的翻译抑制等。
成簇的规律间隔的短回文重复序列(CRISPR)系统用于基因组编辑。该系统包括Cas(一种能够使用crRNA作为其向导来修饰DNA的蛋白质)，CRISPR RNA(crRNA，包含Cas用来引导其到达宿主DNA正确片段的RNA，以及与tracrRNA结合的区域(通常以发夹环形式)，与Cas形成活性复合物)，反式激活crRNA(tracrRNA，与crRNA结合，与Cas形成活性复合物)，以及DNA修复模板的可选片段(可指导细胞修复过程允许插入特定的DNA序列的DNA)。
在一实例中，CRISPR/Cas9通常采用质粒、或电转方式传递核酸片段到靶细胞。在一实例中，CRISPR/Cas9通常采用质粒、或电转方式传递包含核酸片段和重组蛋白的复合物到靶细胞，例如gRNA和Cas9的核糖核蛋白复合物(RNP)。crRNA需要针对每种应用进行设计，因为这是Cas9用来识别并直接结合细胞中靶DNA的序列。crRNA和tracrRNA可以组合在一起以形成指导RNA(gRNA)。本申请gRNA序列可以用gRNA靶向结构域序列来表示。在一实例中，gRNA序列为靶向DNA序列。在一实例中，gRNA序列为与gRNA靶向DNA序列完全或部分互补的核酸序列。在一实例中，gRNA分子包括gRNA序列与crRNA/TracrRNA形成的完整Cas9引导序列的分子。
在一实例中，本文提供的方法包括向细胞递送一种或多种gRNA构建体和一种或多种Cas9多肽或编码Cas9多肽的核酸序列。在一实例中，通过载体(例如AAV、腺病毒、慢病毒)、和/或粒子和/或纳米粒子、和/或电转来递送一种或多种gRNA构建体、一种或多种Cas9多肽或编码Cas9多肽的核酸序列)。在一实例中，包括gRNA靶向结构域的crRNA和tracrRNA单纯施用，也可以施用一条完整RNA。CRISPR/Cas9转基因可以通过载体(例如AAV、腺病毒、慢病毒)、和/或粒子和/或纳米粒子、和/或电转来递送。在本申请中，采用CRISPR/Cas9技术敲除免疫细胞的TCR/B2M基因后，均进行了分选得到TCR/B2M低表达或不表达的免疫细胞。
本申请还提供了编码本文所述的一种或多种外源受体(例如CAR)的核酸分子，和靶向内源性TCR、B2M或NKG2A的核酸抑制分子或gRNA的核酸分子。
示例性的，靶向NKG2A的gRNA包括如SEQ ID NO：38所示的序列；靶向TRAC的gRNA包括SEQ ID NO：36所示的序列；靶向B2M的gRNA包括如SEQ ID NO：37所示的序列。
在一实例中，将编码识别靶抗原(示例性，NKG2A和BCMA肿瘤抗原)的串联CAR引入T细胞中以产生本申请提供的免疫细胞，任选地，将靶向内源性TCR、B2M和/或NKG2A的核酸抑制分子或gRNA的核酸分子引入T细胞。在一个实例中，体外转录的串联CAR的核酸分子、靶向内源性TCR、B2M或NKG2A的核酸抑制分子或gRNA可作为瞬时转染的形式引入细胞中。示例性人工DNA序列是包括连接在一起以形成编码融合蛋白的开放阅读框的基因部分的序列。连接在一起的DNA部分可来自单个生物体或来自多个生物体。
核酸分子、载体及其用途
在本申请中，所述核酸分子可以编码本申请所述的嵌合多肽。例如，所述核酸分子可以为任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从天然环境分离的或人工合成的类似物，但可以编码本申请所述的嵌合多肽。例如，所述的核酸分子可以包含SEQ ID NO:21、24和27中任一项所示的核酸序列。
对免疫细胞(例如，T细胞或NKT细胞)的基因修饰可以通过用所述核酸分子或包含所述核酸分子的载体转导基本上均质的细胞群来完成。在一实例中，逆转录病毒载体(γ-逆转录病毒或慢病毒)用于将核酸分子引入细胞。例如，可以将编码串联CAR的多核苷酸克隆到逆转录病毒载体。也可以使用非病毒载体。转导可以使用任何合适的病毒载体或非病毒递送系统。可以在单个多顺反子表达盒、单个载体的多个表达盒或多个载体中用辅助分子(例如细胞因子)构建串联CAR。产生多顺反子表达盒的元件的实例包括但不限于各种病毒和非病毒内部核糖体进入位点(IRES，例如，FGF-1IRES、FGF-2IRES、VEGF IRES、IGF-II IRES、NF-κB IRES、RUNX1IRES、p53IRES、甲型肝炎IRES、丙型肝炎IRES、瘟病毒IRES、无杆状病毒IRES、小核糖核酸病毒IRES、脊髓灰质炎病毒IRES和脑心肌炎病毒IRES)和可切割的接头(例如2A肽，例如P2A、T2A、E2A和F2A肽)。
可以使用的其它病毒载体包括，例如，腺病毒、慢病毒和与腺相关的病毒载体、牛痘病毒、牛乳头瘤病毒或疱疹病毒，例如爱泼斯坦-巴尔病毒。
非病毒方法也可以用于免疫细胞的修饰。例如，可以通过在脂质转染，脱唾液酸血清类粘蛋白-聚赖氨酸偶联，或手术条件下的微注射将核酸分子引入免疫细胞中。其它非病毒的基因转移方法包括使用脂质体、磷酸钙、DEAE葡聚糖、电穿孔和原生质体融合的体外转染。也可以先将核酸分子转移到可离体培养的细胞类型(例如，自体或同种异体原代细胞或其后代)中，再将经所述核酸分子修饰后的细胞(或其后代)注射到受试者目标组织中 或全身注射。
药物组合物
本申请还提供了药物组合物，其包含本申请所述的CAR、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及药学上可接受的载体。
本申请的药物组合物可以方便地以无菌液体制剂的形式提供，例如等渗水溶液剂、悬浮液、乳剂、分散剂或粘性组合物，其可以缓冲至选定的pH。液体制剂通常比凝胶、其它粘性组合物和固体组合物更容易制备。另外，液体组合物在某种程度上更方便施用，尤其是通过注射。另一方面，可以在适当的粘度范围内配制粘性组合物以提供与特定组织的更长的接触时间。液体或粘性组合物可以包括载体，所述载体可以是溶剂或分散介质，其包括例如水、盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇等)及其合适的混合物。
可以通过将本申请的免疫细胞掺入所需量的适当溶剂中，并根据需要掺入不同量的其它成分来制备无菌注射溶液。这样的组合物可以与合适的载体、稀释剂或赋形剂例如无菌水、生理盐水、葡萄糖、右旋糖等混合。组合物也可以冻干。所述组合物可包括辅助物质，例如润湿剂、分散剂或乳化剂(例如，甲基纤维素)、pH缓冲剂、胶凝剂或增粘剂、防腐剂、矫味剂、颜料等，这取决于给药途径和所需制剂。
可以添加增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。可以通过各种抗细菌和抗真菌剂，例如对羟基苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等来确保防止微生物的作用。可通过使用延迟吸收的试剂例如单硬脂酸铝和明胶来延长可注射药物形式的吸收。然而，所使用的任何媒介物、稀释剂或添加剂将必须与遗传修饰的免疫细胞或其祖细胞相容。
该组合物可以是等渗的，即它们可以具有与血液和/或泪液相同的渗透压。组合物的所需等渗性可以使用氯化钠或其它药学上可接受的试剂例如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。氯化钠可以特别适用于含有钠离子的缓冲剂。
如果需要，可使用药学上可接受的增稠剂将组合物的粘度保持在选定水平。例如，甲基纤维素容易且经济地获得并且易于使用。其它合适的增稠剂包括，例如，黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的浓度可以取决于选择的试剂。重要的是要使用能够达到所选粘度的用量。显然，合适载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型(例如，是否将组合物配制成溶液剂、悬浮液、凝胶剂或其它液体形式，例如定时释放形式或液体填充形式)。
对于所治疗的受试者，要施用的组合物中的细胞数量将有所不同。可以更少的数量施用更有效的细胞。可以根据每个受试者的个体因素，包括其大小、年龄、性别、体重和受 试者的状况，来确定有效剂量的精确确定。本领域技术人员从本申请和本领域知识中可以容易地确定剂量。
本领域技术人员可以容易地确定组合物中和在方法中施用的细胞和任选的添加剂、媒介物和/或载体的量。通常，任何添加剂(除一种或多种活性细胞和/或一种或多种试剂外)在磷酸盐缓冲盐水中的存在量为0.001％至50％(重量)溶液，并且活性成分按微克至毫克的顺序存在，例如约0.0001wt％至约5wt％、约0.0001wt％至约1wt％、约0.0001wt％至约0.05wt％或约0.001wt％至约20wt％、约0.01wt％至约10wt％或约0.05wt％至约5wt％。对于要施用于动物或人的任何组合物，可以确定以下结果：毒性，例如通过在合适的动物模型例如啮齿类动物如小鼠中确定致死剂量(LD)和LD50；组合物的剂量，其中的组分浓度和施用组合物的时间，引起合适的反应。
可以将包括本申请的药物组合物系统地或直接提供给受试者，以诱导和/或增强对抗原的免疫应答和/或治疗和/或预防肿瘤、病原体感染或感染性疾病。在一实例中，将本发明的组合物直接注射到目的器官(例如，受肿瘤影响的器官)中。或者，例如通过向循环系统(例如，静脉、肿瘤脉管系统)给药，将本发明的组合物间接地提供给目的器官。可以在施用组合物之前、同时或之后提供扩增和分化剂，以增加体外或体内T细胞、NKT细胞或CTL细胞的产生。
本申请的细胞可以包括纯化的细胞群。本领域技术人员可以使用各种众所周知的方法，例如荧光激活细胞分选(FACS)，容易地确定群体中本发明的免疫细胞的百分比。在包括本申请的免疫细胞的群体中，纯度的合适范围是约50％至约55％、约5％至约60％、以及约65％至约70％。在某些实施方式中，纯度为约70％至约75％、约75％至约80％或约80％至约85％。在某些实施方式中，纯度为约85％至约90％，约90％至约95％以及约95％至约100％。剂量可以由本领域技术人员容易地调节(例如，纯度降低可能需要增加剂量)。可以通过注射、导管等引入细胞。
本申请的组合物可以是包括本申请的免疫细胞或其祖细胞和药学上可接受的载体的药物组合物。给药可以是自体的或异体的。例如，可以从一个受试者获得免疫细胞或祖细胞，并将其施用于相同受试者或不同的相容受试者。外周血来源的免疫细胞或其后代(例如，体内、离体或体外来源)可通过局部注射施用，包括导管给药、全身注射、局部注射、静脉内注射或肠胃外给药。当施用本申请的组合物时，可以将其配制成单位剂量可注射形式(溶液剂、悬浮剂、乳剂等)。
8.用途或方法
本申请提供了本申请所述的CAR、本申请所述的核酸分子、权本申请所述的载体、本申请所述的细胞、本申请所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解 和/或治疗肿瘤。
本申请提供了预防、缓解和/或治疗肿瘤的方法，其包括向有需要的受试者施用本申请所述的细胞、本申请所述的药物组合物。
本申请提供了用于在需要本申请的细胞或药物组合物的受试者中诱导和/或增加免疫应答的方法。
本申请的细胞或药物组合物可以用于治疗和/或预防受试者的肿瘤。本申请细胞或药物组合物可以用于延长患有肿瘤的受试者的存活。本申请的细胞或药物组合物也可以用于治疗和/或预防诸如免疫功能低下的人受试者的病原体感染或其它感染性疾病。这种方法包括施用有效量的本申请的细胞或药物组合物以达到期望的效果，无论是减轻现有病症还是预防复发。为了治疗，施用的量是有效产生所需效果的量。可以一次或多次给药来提供有效量。可以大剂量或通过连续灌注来提供有效量。
在一实例中，包括本申请的细胞或药物组合物可以用于治疗具有表面抗原表达水平低的肿瘤细胞的受试者，例如由于疾病的复发，其中受试者接受过导致残留肿瘤细胞的治疗。在某些实施方式中，肿瘤细胞在肿瘤细胞表面上具有低密度的靶分子。
在一实例中，包括本申请的细胞或药物组合物可用于治疗患有疾病复发的受试者，其中该受试者接受过包括单独施用CAR的免疫细胞(例如，T细胞)，该CAR包括细胞内信号结构域，其包括含有共刺激性信号结构域(例如4-1BBz CAR)。在一实例中，肿瘤细胞在肿瘤细胞表面上具有低密度的肿瘤特异性抗原。在一实例中，该疾病是BCMA阳性肿瘤。在一实例中，肿瘤细胞在肿瘤细胞上具有低密度的BCMA。这种方法包括施用有效量的本申请的细胞或药物组合物以达到期望的效果，缓解现有病症或预防复发。
“有效量”(或“治疗有效量”)是足以在治疗后产生有益或期望的临床结果的量。可以以一剂或多剂剂量将有效量施用于受试者。就治疗而言，有效量是足以缓解、改善、稳定、逆转或减慢疾病进展或以其它方式减少疾病病理后果的量。有效量通常由医师根据具体情况确定，并且在本领域技术人员的能力范围内。当确定合适的剂量以达到有效量时，通常要考虑几个因素。这些因素包括受试者的年龄、性别和体重、所治疗的疾病、疾病的严重程度以及所施用的本申请的细胞或药物组合物的形式和有效浓度。
对于使用抗原特异性T细胞的过继免疫疗法，通常输注约10 6-10 10范围内的细胞剂量。在将本申请的免疫细胞施用于宿主并随后分化后，诱导特异性针对特定抗原的T细胞。本申请的细胞或药物组合物可以通过本领域已知的任何方法施用，包括但不限于静脉内、皮下、结内、肿瘤内、鞘内、胸膜内、腹膜内和直接向胸腺施用。
肿瘤的非限制性实例包括血液瘤(例如白血病、淋巴瘤和骨髓瘤)、实体瘤；实体瘤包括：卵巢癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、前列腺癌、 皮肤癌、胃癌、胶质母细胞瘤、喉癌、黑素瘤、神经母细胞瘤、腺癌、神经胶质瘤、软组织肉瘤和各种癌(包括前列腺癌和小细胞肺癌)。肿瘤的非限制性实例包括但不限于星形细胞瘤、纤维肉瘤、粘液肉瘤、脂肪肉瘤、少突胶质细胞瘤、室管膜瘤、髓母细胞瘤、原始神经外胚层肿瘤(PNET)、软骨肉瘤、成骨肉瘤、胰腺导管腺癌、小细胞和大细胞肺腺癌、脊索瘤、血管肉瘤、内皮肉瘤、鳞状细胞癌、支气管肺泡癌、上皮腺癌及其肝转移灶、淋巴管肉瘤、淋巴管内皮肉瘤、肝癌、胆管癌、滑膜瘤、间皮瘤、尤文氏瘤、横纹肌肉瘤、结肠癌、基底细胞癌、汗腺癌、乳头状癌、皮脂腺癌、状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、胆小管癌、绒毛膜癌、精原细胞瘤、胚胎癌、Wilms’肿瘤、睾丸肿瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质细胞瘤、脑膜瘤、神经母细胞瘤、视网膜母细胞瘤、白血病、多发性骨髓瘤、Waldenstrom’s巨球蛋白血症和重链疾病、诸如导管和小叶腺癌的乳腺肿瘤、子宫颈的鳞状和腺癌、子宫和卵巢上皮癌、前列腺腺癌、膀胱移行鳞状细胞癌、B和T细胞淋巴瘤(结节性和弥漫性)浆细胞瘤、急慢性白血病、恶性黑色素瘤、软组织肉瘤和平滑肌肉瘤。在某些实施方式中，肿瘤选自血液癌症(例如白血病、淋巴瘤和骨髓瘤)、卵巢癌、前列腺癌、乳腺癌、膀胱癌、脑癌、结肠癌、肠癌、肝癌、肺癌、胰腺癌、前列腺癌、皮肤癌、胃癌、胶质母细胞瘤和喉癌。在一实例中，本申请的细胞或药物组合物可以用于治疗和/或预防常规治疗措施不适合或复发难治性实体瘤，例如肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌。在一实例中，肿瘤是血液肿瘤。
本申请的治疗目标可以包括缓解或逆转疾病进展和/或减轻副作用、或治疗目标包括降低或延迟复发风险。
本申请提供用于在例如免疫受损的受试者中治疗和/或预防病原体感染(例如病毒感染、细菌感染、真菌感染、寄生虫感染或原生动物感染)的方法。该方法可以包括向患有病原体感染的受试者施用有效量的本申请的细胞或药物组合物。易于治疗的示例性病毒感染包括但不限于巨细胞病毒、爱泼斯坦-巴尔病毒、人免疫缺陷病毒和流感病毒感染。
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强的应答可以包括应答性中5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％更多。
在一实例中，表达本申请提供的CAR的细胞在小鼠皮下肿瘤模型中具有更长存活时 间和/或扩增能力、特异性的向肿瘤组织浸润、以及良好的抗NK细胞攻击和抑制肿瘤生长的效果，显示出治疗实体瘤的良好功能。
在一实例中，表达本申请提供的CAR的细胞在小鼠原位血液肿瘤模型中具有更长存活时间和/或扩增能力、以及良好的抗NK细胞攻击和抑制肿瘤生长的效果，显示出治疗血液瘤的良好功能。
在一实例中，表达本申请提供的CAR的细胞靶向BCMA表达阳性的肿瘤。在一实例中，表达本申请提供的CAR的细胞靶向多发性骨髓瘤。
9.试剂盒
本申请提供一种试剂盒，其包含本申请提供的CAR、本申请提供的的核酸分子、本申请提供的载体、本申请提供的细胞、和/或权本申请提供的的药物组合物。本申请提供的试剂盒用于在受试者中诱导和/或增强免疫应答和/或治疗和/或预防肿瘤或病原体感染。在一实例中，试剂盒包括有效量的本申请的细胞和药物组合物。在一实例中，试剂盒包括无菌容器；这样的容器可以是盒子、安瓿、瓶、小瓶、管、袋、小袋、泡罩包装或本领域已知的其它合适的容器形式。这样的容器可以由塑料、玻璃、层压纸、金属箔或其它适合于容纳药物的材料制成。在一实例中，试剂盒包括编码本申请的CAR的核酸分子，其以可表达的形式识别目的抗原，可以任选地包括在一种或多种载体中。
在一实例中，还包括说明书。说明书通常包括有关细胞或药物组合物用于治疗和/或预防肿瘤或病原体感染的信息。在一实例中，说明书包括以下至少一项：治疗剂的描述；用于治疗或预防肿瘤、病原体感染或免疫疾病或其症状的剂量表和给药；注意事项；警告；适应症；不适应症；用药信息；不良反应；动物药理学；临床研究；和/或参考。这些说明书可以直接打印在容器上，或者作为粘贴在容器上的标签，或者作为单独的纸页、小册子、卡片或文件夹提供在容器内或与容器一起。
本申请包括，例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN109908176A、CN109880803A、CN110055275A、CN110123837A、CN110438082A、CN110468105A以及例如国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、 WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的那些CAR-T细胞及其制备方法、抗体。
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。本说明书中提到的所有出版物、专利和专利申请均通过引用并入本文，其程度如同特别地且单独地指出每一个单独的出版物、专利或专利申请均通过引用而并入本文。
本申请实施例中所用生物材料如表1所示。
表1
名称 来源
T细胞 分离自健康供体原代PBMC
NK细胞 分离自健康供体原代PBMC
单核细胞 分离自健康供体原代PBMC
细胞系RPMI-8226 购自ATCC
MM.1S肿瘤细胞 购自ATCC
NPG免疫缺陷的小鼠 购自北京维通达生物技术有限公司
实施例1靶向BCMA和NKG2A的串联CAR以及CAR-T细胞的制备。
采用本领域常规分子生物学方法，以BCMA和NKG2A为靶点，使用载体PRRLsin，分别构建表达CAR3多肽(SEQ ID NO：22)、CAR4多肽(SEQ ID NO：25)、CAR5多肽(SEQ ID NO：28)、BCMA-CAR(SEQ ID NO：17)多肽的CAR-T3细胞、CAR-T4细胞、CAR-T5细胞、BCMA-CAR-T细胞。其中抗原结合域的串联片段如表2所示，其氨基酸序列分别如SEQ ID NO：20、23和26中任一项所示。
表2.
片段 组成
串联片段3 BCMAVL-G4S-NKG2AVH-(G4S)3-NKG2AVL-G4S-BCMAVH
串联片段4 NKG2AVL-G4S-BCMAVH-(G4S)3-BCMAVL-G4S-NKG2AVH
串联片段5 NKG2AVL-G4S-BCMAVH-linker1-BCMAVL-G4S-NKG2AVH
实施例2串联CAR-T体外杀伤NK细胞
取5×10 4个UTD细胞(即没有进行病毒载体转染的T细胞组)，以及由实施例1制备的BCMA-CAR-T、CAR-T3、CAR-T4、CAR-T5细胞，体外与活化扩增的NK细胞 (aNK)分别按效靶比E：T＝1：3、1：1、3：1进行共孵育18hr，按试剂盒说明书(CytoTox
Figure PCTCN2022106096-appb-000001
Non-Radioactive Cytotoxicity Assay，promega公司)，取上清进行乳酸脱氢酶(LDH)含量测定，并计算各组中aNK的裂解效率。如图1所示，各组串联CAR-T细胞均能杀伤NK细胞。
实施例3串联CAR-T体外杀伤肿瘤细胞
将效应细胞(UTD、BCMA-CAR-T、CAR-T3、CAR-T4、CAR-T5)与靶细胞(多发性骨髓瘤细胞系RPMI-8226)，分别按效靶比1：3、1：1、3：1共孵育18小时，检测体外肿瘤细胞杀伤。按试剂盒说明书(CytoTox
Figure PCTCN2022106096-appb-000002
Non-Radioactive Cytotoxicity Assay，promega公司)，取上清进行乳酸脱氢酶(LDH)含量测定，并计算各组靶细胞裂解效率。结果如图2所示，各组串联CAR-T细胞均能杀伤肿瘤细胞。
实施例4串联CAR-T体内抗肿瘤作用
皮下接种5×10 6个RPMI-8226细胞于NPG免疫缺陷小鼠(购买自北京维通达生物技术有限公司)中(接种日记为D0)，接种11天后瘤平均体积为约190mm 3，分为5组：UTD细胞组、BCMA-CAR-T细胞组、CAR-T3细胞组、CAR-T4细胞组、CAR-T5细胞组，每组5只。尾静脉分别注射1×10 6个上述UTD、BCMA-CAR-T、CAR-T3、CAR-T4、CAR-T5细胞，注射后，每周2次测量体重(包括分组给药及安乐死当天)，并用游标卡尺测量并记录肿瘤长径、短径，计算肿瘤体积，根据肿瘤体积绘制肿瘤生长曲线，并比较各组间肿瘤生长曲线的差异(肿瘤体积：V＝1/2×长径×短径 2)。如图3所示，各组串联CAR-T细胞均能抑制体内肿瘤生长。
实施例5串联CAR-T细胞体外细胞因子分泌
取UTD、BCMA-CAR-T、CAR-T3、CAR-T5细胞，以RPMI-8226细胞作为靶细胞，按T细胞：靶细胞＝1：1或按T细胞：靶细胞：自体来源的单核细胞＝1：1：1进行共孵育，24hr后取上清进行细胞因子微球检测技术(CBA)监测(如图4所示)。
实施例6串联UCAR-T细胞制备
按照试剂说明书(GeneArt TM Precision gRNA Synthesis Kit，Thermo Fisher)体外合成靶向TCR、B2M、NKG2A的gRNA序列(SEQ ID NO：36、37、38)，采用常规CRISPR/Cas9技术，即将Cas 9酶和gRNA按1：4比例进行混合形成RNP，室温孵育，将各组细胞与RNP进行混合,利用maxcyte电转仪将RNP复合物导入到CAR-T细胞，敲除UTD、BCMA-CAR-T、CAR-T3、CAR-T4、CAR-T5细胞内源性TRAC/B2M分别得到UTD-DKO、BCMA-UCAR-T、UCAR-T3、UCAR-T4、UCAR-T5细胞，敲除CAR-T3细胞内源性TRAC/B2M/NKG2A得到UCAR-T3-NKG2A KO细胞。未转染病毒的且内源性TCR/B2M敲除的T细胞命名为UTD-DKO。
实施例7：串联UCAR-T细胞体外对NK细胞的抵抗功能
取5×10 4个由实施例6制备的UTD-DKO、BCMA UCAR-T、UCAR-T3、UCAR-T4、UCAR-T5细胞，体外与活化扩增的NK细胞(aNK)分别按1：1、2：1的比例进行共孵育，在0hr，4hr,24hr和48hr进行流式染色，检测共培养体系中UCAR-T细胞和NK细胞的比例。如图5所示，串联UCAR-T均能抵抗NK细胞的杀伤。
实施例8.串联UCAR-T细胞体外细胞因子分泌
按照实施例5所述方法检测串联UCAR-T细胞体外细胞因子分泌，结果如图6所示。
实施例9.串联UCAR-T细胞的体内抗肿瘤功能
皮下接种5×10 6个RPMI-8226细胞于NPG小鼠，接种12-14天后瘤平均体积为约350mm 3，分4组：UTD-DKO细胞组，BCMA UCAR-T细胞组、UCAR-T3细胞组、UCAR-T5细胞组，每组5只。尾静脉分别注射1.5×10 6个UTD-DKO、BCMA UCAR-T、UCAR-T3或UCAR-T5细胞。参照实施例4所记载的方法绘制肿瘤生长曲线。结果如图7所示，各组串联UCAR-T细胞均能抑制体内肿瘤生长。
实施例10：串联UCAR-T细胞在NK细胞存在下体外抗肿瘤活性以及抗NK活性
取2×10 5个经CFSE染色和标记后的RPMI-8226细胞，按肿瘤细胞：T细胞：aNK细胞＝1：2：1的比例，接种到96孔板，培养5天，进行细胞计数和流式染色(图8)。取2×10 5个经CFSE染色和标记后的多发性骨髓瘤细胞系MM.1S-GFP，按肿瘤细胞：T细胞：aNK细胞＝2：2：0、2：2：1、2：2：2的比例，接种到96孔板培养5天，进行细胞计数和流式染色(图8)。如图8所示，串联UCAR-T细胞具有更长存活时间和/或扩增能力，既能靶向杀伤肿瘤细胞、又能杀伤NK细细胞。
实施例11.串联UCAR-T细胞在NK细胞存在下体内抗原位肿瘤活性
尾静脉接种2×10 6个多发性骨髓瘤细胞系MM.1S-luciferase细胞(MM.1S-luc)于NPG小鼠中，接种9天后荧光成像检测肿瘤负荷在约2-5×10 4Radiance(p/s/cm2/sr)，分7组(UTD-DKO，BCMA UCAR-T、BCMA UCAR-T+NK-1、BCMA UCAR-T+NK-2、UCAR-T3-NKG2A KO和UCAR-T3-NKG2A KO+NK-1、UCAR-T3-NKG2A KO+NK-2)，每组5只。在D10,尾静脉分别注射1×10 6个NK细胞(如上标记为NK-1)或者2×10 6个NK细胞(如上标记为NK-2)，后续每隔两天注射1次NK细胞，一共注射5次，用于模拟NK细胞的存在；第1次NK细胞注射完后，再尾静脉注射1×10 6个UCAR-T细胞，注射后，每周1次进行荧光成像，监测肿瘤负荷，每周2次测量体重(包括分组给药及安乐死当天)，根据肿瘤负荷绘制肿瘤生长曲线，比较各组间肿瘤生长曲线的差异，并记录小鼠的生存期。结果如图9A-B所示，NK细胞明显减弱了BCMA UCAR-T细胞的抗肿瘤活性，降低了BCMA UCAR-T细胞组的小鼠生存期；而低剂量 以及高剂量的NK细胞存在下，UCAR-T3的抗肿瘤活性和小鼠的生存期均未受到明显改变。表明串联UCAR-T细胞在体内可以抵抗NK细胞杀伤，并抑制肿瘤生长。
实施例12.串联UCAR-T细胞的特异性向肿瘤组织浸润
取UCAR-T细胞治疗RPMI-8226皮下荷瘤模型小鼠(如实施例9中记载)，包括UTD-DKO，BCMA UCAR-T，UCAR-T3-NKG2A KO三组小鼠的肿瘤组织，以及心肝脾肺肾正常组织。经多聚甲醛固定后进行切片和抗CD3e免疫组化染色。如图10所示，在UCAR组中，尤其是串联UCAR-T组的肿瘤组织中T细胞浸润多于BCMA UCAR-T组；正常组织内(包括心肝脾肺肾组织)，均未检测到明显的T细胞浸润。这表明串联UCAR-T细胞不仅具有更长存活时间、扩增能力，并且特异性向肿瘤组织浸润，安全性好。
实施例13.串联UCAR-T细胞不引起移植物抗宿主反应
分别取1×10 7个UTD、BCMA UCAR-T、UCAR-T3-NKG2A KO细胞注射到NPG小鼠中，PBS组作为阴性对照，每周2次测量体重，观察小鼠的毛发及基础情况。
如图11所示，UTD组小鼠从D25开始发生明显的体重下降，从D30开始出现明显的脱毛、活动性降低，表现出明显的GVHD症状。串联UCAR-T组与PBS类似，小鼠正常生长，且体重逐渐增加。这表明UCAR-T3细胞不会引起移植物抗宿主(GVHD)反应。
本申请所述实施例包括将该实施例作为任何单一实施例或与任何其他实施例或其部分相结合。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列信息
Figure PCTCN2022106096-appb-000003
Figure PCTCN2022106096-appb-000004
Figure PCTCN2022106096-appb-000005
Figure PCTCN2022106096-appb-000006
Figure PCTCN2022106096-appb-000007
Figure PCTCN2022106096-appb-000008

Claims (33)

  1. 嵌合抗原受体(CAR)，其特征在于，所述CAR包含双特异性抗原结合域，所述双特异性抗原结合域包括NKG2A抗原结合域和肿瘤抗原结合域，所述NKG2A抗原结合域包含第一结合区L、第一结合区H,所述肿瘤抗原结合域包含第二结合区L、第二结合区H，所述第一结合区L、所述第一结合区H、所述第二结合区L、所述第二结合区H以形成loop结构的方式连接。
  2. 如权利要求1中所述的CAR,其特征在于，所述loop结构通过如下方式形成：其中一个抗原结合域的结合区H和结合区L连接为抗体，另一个抗原结合域的结合区H、结合区L分别与所述抗体的两端连接。
  3. 如权利要求2中所述的CAR,其特征在于，所述抗体自N端至C端依次包含：
    所述结合区H、所述结合区L。
  4. 如权利要求3中所述的CAR,其特征在于，所述另一个抗原结合域的结合区L与所述抗体的N端连接，所述另一个抗原结合域的结合区H与所述抗体的C端连接。
  5. 如权利要求4中所述的CAR,其特征在于，所述连接为抗体的结合区H为第一结合区H，所述连接为抗体的结合区L为第一结合区L，所述另一个抗原结合域的结合区L为第二结合区L，所述另一个抗原结合域的结合区H为第二结合区H。
  6. 如权利要求4中所述的CAR,其特征在于，所述连接为抗体的结合区H为第二结合区H，所述连接为抗体的结合区L为第二结合区L。所述另一个抗原结合域的结合区L为第一结合区L，所述另一个抗原结合域的结合区H为第一结合区H。
  7. 如权利要求5或6中所述的CAR,其特征在于，所述第一结合区L包含第一轻链可变区(VL)，所述第一结合区H包含第一重链可变区(VH),所述第二结合区L包含第二轻链可变区(VL)，所述第二结合区H包含第二重链可变区(VH)。
  8. 如权利要求7所述的CAR,其特征在于，所述第一轻链可变区(VL)包含如SEQ ID NO:6-8中任一项所示的第一轻链互补决定区(LCDR)或其组合。
  9. 如权利要求7或8所述的CAR,其特征在于，所述第一重链可变区(VH)包含如SEQ ID NO:3-5中任一项所示的第一重链互补决定区(HCDR)或其组合。
  10. 如权利要求7-9中任一项所述的CAR,其特征在于，所述第一轻链可变区(VL)包含第一轻链互补决定区1(LCDR1)、第一轻链互补决定区2(LCDR2)、第一轻链互补决定区3(LCDR3)，所述第一LCDR1包含如SEQ ID NO:6所示的氨基酸序列，所述第一LCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述第一LCDR3包含如SEQ ID NO:8所示的氨基酸序列。
  11. 如权利要求7-10中任一项所述的CAR,其特征在于，所述第一重链可变区(VH)包含第一重链互补决定区1(HCDR1)、第一重链互补决定区2(HCDR2)、第一重链互补决定区3(HCDR3)，所述第一HCDR1包含如SEQ ID NO:3所示的氨基酸序列，所述第一HCDR2包含如SEQ ID NO:4所示的氨基酸序列，所述第一HCDR3包含如SEQ ID NO:5所示的氨基酸序列。
  12. 如权利要求1-11中任一项所述的CAR，其特征在于，所述肿瘤抗原选自CD19、GPC3、Claudin18.2、WT1、HER2、EGFR、BCMA或它们的组合。
  13. 如权利要求7-12中任一项所述的CAR,其特征在于，所述第二轻链可变区(VL)包含如SEQ ID NO:12-14中任一项所示的第二轻链互补决定区(LCDR)或其组合。
  14. 如权利要求7-13中任一项所述的CAR,其特征在于，所述第二重链可变区(VH)包含如SEQ ID NO:9-11中任一项所示的第二重链互补决定区(HCDR)或其组合。
  15. 如权利要求7-14中任一项所述的CAR,其特征在于，所述第二轻链可变区(VL)包含第二轻链互补决定区1(LCDR1)、第二轻链互补决定区2(LCDR2)、第二轻链互补决定区3(LCDR3)，所述第二LCDR1包含如SEQ ID NO:12所示的氨基酸序列，所述第二LCDR2包含如SEQ ID NO:13所示的氨基酸序列，所述第二LCDR3包含如SEQ ID NO:14所示的氨基酸序列。
  16. 如权利要求7-15中任一项所述的CAR,其特征在于，所述第二重链可变区(VH)包含第二重链互补决定区1(HCDR1)、第二重链互补决定区2(HCDR2)、第二重链互补决定区3(HCDR3)，所述第二HCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述第二HCDR2包含如SEQ ID NO:10所示的氨基酸序列，所述第二HCDR3包含如SEQ ID NO:11所示的氨基酸序列。
  17. 如权利要求1-16中任一项所述的CAR，其特征在于，所述连接包括通过连接子连接，所述连接子选自Lin1或(G4S)n，其中，n是1或3；所述Lin1包含如SEQ ID NO:18所示的氨基酸序列。
  18. 核酸分子，其编码权利要求1-18中任一项所述的CAR。
  19. 载体，其包含权利要求19所述的核酸分子。
  20. 细胞，其包含权利要求1-18中任一项所述的CAR、权利要求19所述的核酸分子和/或权利要求20所述的载体。
  21. 如权利要求20所述的细胞，其特征在于，所述细胞选自：T细胞、NK细胞、NKT细胞、巨噬细胞、CIK细胞和干细胞衍生的免疫细胞。
  22. 如权利要求20-21中任一项所述的细胞，其特征在于，所述细胞包括：
    a)内源性HLA-I分子低表达或不表达，
    b)内源性TCR分子低表达或不表达，和/或
    c)内源性NKG2A分子低表达或不表达。
  23. 如权利要求22所述的细胞，其特征在于，
    a)所述内源性HLA-I蛋白低表达或不表达包括编码内源性HLA-I的基因的敲除，
    b)所述内源性TCR蛋白低表达或不表达包括编码内源性TCR的基因的敲除，和/或
    c)所述内源性NKG2A分子低表达或不表达包括编码内源性NKG2A的基因的敲除。
  24. 药物组合物，其包含如权利要求1-17中任一项所述的CAR、权利要求18中所述的核酸分子、权利要求19中所述的载体和/或权利要求20-23中任一项所述的细胞，以及药学上可接受的载体。
  25. 一种试剂盒，其包含如权利要求1-17中任一项所述的CAR、权利要求18中所述的核酸分子、权利要求19中所述的载体、权利要求20-23中任一项所述的细胞、和/或权利要求24所述的药物组合物。
  26. 提高靶向肿瘤的免疫细胞在有宿主免疫细胞存在时的存活时间和/或扩增能力的方法，其包括向所述免疫细胞中引入如权利要求18中所述的核酸分子或权利要求19中所述的载体。
  27. 如权利要求27所述的方法，其还包括使所述免疫细胞表达如权利要求1-17中任一项所述的CAR。
  28. 如权利要求26-27中任一项所述的方法，其还包括对所述免疫细胞进行修饰，所述修饰包括抑制或消除参与响应自体和/或异体抗原识别多肽的至少一种内源基因表达、活性。
  29. 如权利要求1-17中任一项所述的CAR、权利要求18中所述的核酸分子、权利要求19中所述的载体、权利要求20-23中任一项所述的细胞、权利要求24所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
  30. 预防、缓解和/或治疗肿瘤的方法，其包括向有需要的受试者施用如权利要求20-23中任一项所述的细胞、权利要求24所述的药物组合物。
  31. 权利要求1-17中任一项所述的CAR、权利要求18中所述的核酸分子、权利要求19中所述的载体、权利要求20-23中任一项所述的细胞、权利要求24所述的药物组合物，其用于预防、缓解和/或治疗肿瘤。
  32. 如权利要求29所述的用途、权利要求30所述的方法、权利要求31所述的CAR、核酸分子、载体、细胞、药物组合物，其中所述肿瘤包括血液瘤和/或实体瘤。
  33. 如权利要求29所述的用途、权利要求30所述的方法、权利要求31所述的CAR、核酸分子、载体、细胞、药物组合物，其中所述血液瘤包括多发性骨髓瘤。
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