WO2022007795A1 - 一种嵌合受体及其应用 - Google Patents

一种嵌合受体及其应用 Download PDF

Info

Publication number
WO2022007795A1
WO2022007795A1 PCT/CN2021/104764 CN2021104764W WO2022007795A1 WO 2022007795 A1 WO2022007795 A1 WO 2022007795A1 CN 2021104764 W CN2021104764 W CN 2021104764W WO 2022007795 A1 WO2022007795 A1 WO 2022007795A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cancer
tumor
antibody
domain
Prior art date
Application number
PCT/CN2021/104764
Other languages
English (en)
French (fr)
Inventor
徐建青
张晓燕
廖启彬
丁相卿
Original Assignee
上海鑫湾生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海鑫湾生物科技有限公司 filed Critical 上海鑫湾生物科技有限公司
Publication of WO2022007795A1 publication Critical patent/WO2022007795A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/463—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
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • 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
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • A61K39/464838—Viral antigens
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group 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
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/55—Lung
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the technical field of biomedicine. Specifically, the present invention relates to a chimeric receptor. The present invention also relates to said chimeric receptor and its use.
  • CAR-T Chimeric antigen receptor T-cell immunotherapy
  • the tumor antigens currently used are mostly tumor-associated antigens, which are highly expressed in tumor cells, but are also expressed in small amounts in normal tissue cells, resulting in the "on-target off effect of targeting non-tumor cells" in CAR-T cell therapy. -tumor), limiting its clinical application in the treatment of solid tumors.
  • CAIX carbonic anhydrase IX
  • CD16 molecule also known as Fc ⁇ RIII, is mainly expressed in natural killer cells (Natural killer cells, NK), neutrophils and macrophages.
  • the CD16 molecule with killing function can bind to the Fc fragment of the antibody, thereby initiating antibody-dependent cell-mediated cytotoxicity (ADCC), among which NK cells are the main ones.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Antibody-dependent cellular cytotoxicity is mediated by genetically modified antigen-specific human T lymphocytes[J].Blood.2006, 107(12):4669-4677; Ochi F, Fujiwara H, Tanimoto K, et al. Gene-modified human ⁇ / ⁇ -T cells expressing a chimeric CD16-CD3 ⁇ receptor as adoptively transferable effector cells for anticancer monoclonal antibody therapy[J]. Cancer Immunol Res. 2014, 2(3):249-262).
  • a chimeric receptor that specifically binds to Fc fragments, especially Fc high-affinity, cellular protease-resistant CD16 (High-affinity, protease-resistant CD16, hrCD16) chimeric receptor .
  • the purpose of the present invention is to provide a chimeric receptor.
  • the chimeric receptor provided by the present invention especially the hrCD16 chimeric receptor, has high Fc affinity and cellular protease resistance, and can be introduced into a variety of killer immune cells, such as T cells, by viral/non-viral delivery. , NK cells or NKT cells and other immune cells, and stably expressed on them.
  • a variety of killer immune cells such as T cells
  • NK cells or NKT cells and other immune cells and stably expressed on them.
  • Using the chimeric receptor of the present invention especially the immune cells modified by the hrCD16 chimeric receptor, combined with a variety of tumor antigen-targeting antibodies or viral antigen-targeting antibodies, can kill different types of tumor cells and viruses in a broad spectrum.
  • the chimeric receptor of the present application can only play the role of ADCC when combined with a tumor antigen-targeting antibody or a virus antigen-targeting antibody to kill tumor cells or virus-infected cells
  • the method provided in the present application has the potential to improve the safety of treatment. It has many advantages, such as enhancing the efficacy, avoiding tumor antigen escape or virus escape, and rapidly and flexibly switching therapeutic target antibodies.
  • the present invention provides a chimeric receptor comprising:
  • the chimeric receptor further comprises:
  • the extracellular recognition domain of the Fc fragment is the CD16 extracellular domain, CD32 extracellular domain, CD64 extracellular domain, CD89 extracellular domain, CD23 extracellular domain, Fc ⁇ RI extracellular domain, FcRn extracellular domain, Fc binding antibody that specifically binds to the Fc fragment of the antibody , Protein A, Protein G or its mutants or multiple repeat tandem extracellular domains;
  • the extracellular recognition domain of the Fc fragment is the CD16 extracellular domain
  • the extracellular recognition domain of the Fc fragment is the wild-type CD16 extracellular domain, the F176V mutant CD16 extracellular domain, the S197P mutant CD16 extracellular domain or the F176V and S197P double mutant CD16 extracellular domain, which comprises as SEQ ID NO: The amino acid sequence shown in any one of 1-4; more preferably, the extracellular recognition domain of the Fc fragment is F176V and S197P double mutant CD16 extracellular domain, which comprises the amino acid sequence shown in SEQ ID NO:4 .
  • Hinge region of immunoglobulin Fc receptors CD64, CD32, CD16, CD89, Fc ⁇ RI, Fc ⁇ RII (CD23) and FcRn;
  • CD28 hinge region CD137 hinge region, CD8 ⁇ hinge region, CD4 hinge region, PD-1 hinge region and CTLA-4 hinge region;
  • the extracellular spacer is a CD8 hinge region; preferably, the extracellular spacer comprises the amino acid sequence shown in SEQ ID NO:5.
  • the hrCD16 chimeric receptor according to the present invention is characterized in that, the transmembrane region includes but is not limited to any one or more of the following: the transmembrane region of the CD3 ⁇ chain of the T cell receptor complex, the CD28 transmembrane region domain, CD137 transmembrane domain, CD8 ⁇ transmembrane domain, CD4 transmembrane domain, PD-1 transmembrane domain, CTLA-4 transmembrane domain, immunoglobulin Fc receptor transmembrane domain and combinations thereof.
  • the transmembrane region is a CD8 transmembrane region; more preferably, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:6.
  • the hrCD16 chimeric receptor according to the present invention is characterized in that the costimulatory signaling domains include but are not limited to any one or more of the following signaling domains: CD2, CD27, CD28, CD30, CD40 , CD40L, CD137(4-1BB), CD134(OX40), CD278(ICOS), GITR, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, Dap10, ICAM-1, LFA-1, Lck, TNFRI, TNFRII, TIM-1, TIM-2, TIM-3, TIM-4, and combinations thereof.
  • the costimulatory signaling domain is a CD137 signaling domain; more preferably, the costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO:7.
  • the chimeric receptor according to the present invention is characterized in that the cytokine receptor signaling domains include but are not limited to any one or more of the following signaling domains: IL-2R, IL-7R, IL -15R, IL-18R, IL-21R, IL-23R, and combinations thereof.
  • the chimeric receptor according to the present invention is characterized in that, the intracellular signaling domain includes but is not limited to any one or more of the following signaling domains: CD3 ⁇ chain of T cell receptor complex, Fc ⁇ RIII , Fc ⁇ RI, Fc receptor intracellular signaling domains, immunoreceptor tyrosine activation motif (ITAM)-bearing signaling domains, and combinations thereof.
  • the intracellular signaling domain includes but is not limited to any one or more of the following signaling domains: CD3 ⁇ chain of T cell receptor complex, Fc ⁇ RIII , Fc ⁇ RI, Fc receptor intracellular signaling domains, immunoreceptor tyrosine activation motif (ITAM)-bearing signaling domains, and combinations thereof.
  • ITAM immunoreceptor tyrosine activation motif
  • the intracellular signaling domain is a CD3 ⁇ chain signaling domain; more preferably, the intracellular signaling domain is shown in SEQ ID NO:8.
  • the receptor consists of a wild-type CD16 ectodomain, a F176V mutant CD16 ectodomain, an S197P mutant CD16 ectodomain, and F176V and S197P comprising the amino acid sequence shown in any one of SEQ ID NOs: 1-4
  • the double mutant CD16 extracellular domain consists of any CD16 extracellular domain, human CD8 hinge region, human CD8 transmembrane region, human CD137 costimulatory signaling domain and CD3 ⁇ chain signaling domain;
  • amino acid sequence of the chimeric receptor is shown in any one of SEQ ID NOs: 9-12.
  • the chimeric receptor according to the present invention is characterized in that, the receptor is composed of F176V and S197P double mutant CD16 extracellular domain, human CD8 hinge comprising the amino acid sequence shown in SEQ ID NO: 4. region, human CD8 transmembrane region, human CD137 costimulatory signaling domain and CD3 ⁇ chain signaling domain.
  • amino acid sequence of the double mutant hrCD16 chimeric receptor is shown in SEQ ID NO: 12.
  • the present invention also provides a polynucleotide encoding the chimeric receptor.
  • polynucleotide sequence is shown in any one of SEQ ID Nos: 13-16;
  • polynucleotide sequence is as shown in SEQ ID NO:16
  • the present invention also provides a vector comprising the polynucleotide.
  • the vector co-expresses cytokines, chemokines, chemokine receptors, immune checkpoint blocking antibodies or a combination thereof;
  • the cytokines include but are not limited to IL-2, IL-7, IL-15, IL-21, IL-12, IL-18, IL-23 and combinations thereof;
  • the chemokines include but are not limited to Not limited to CXCL9, CXCL10, CXCL11, CCL19, CCL20 and CCL21;
  • the chemokine receptors include but are not limited to CCR1, CCR3, CCR9, CXCR1 and CXCR2;
  • the immune checkpoint blocking antibodies include but are not limited to CTLA-4 Blocking antibodies, PD-1 blocking antibodies, PD-L1 blocking antibodies, LAG-3 blocking antibodies, Tim-3 blocking antibodies, TIGIT blocking antibodies, VISTA blocking antibodies, Siglec-15 blocking antibodies and their combination.
  • the present invention also provides a virus comprising the polynucleotide
  • the viruses include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, poxviruses and herpesviruses.
  • the present invention provides an immune cell expressing the chimeric receptor.
  • the immune cells include but are not limited to T cells, natural killer cells (NK), innate lymphoid cells (ILC), hematopoietic stem cells, embryonic stem cells and pluripotent stem cells, etc.;
  • the T cells include but are not limited to unsorted and purified T cells, sorted and purified T cells, sorted and purified PD-1 + T cells, sorted and purified CD137 + T cells, sorted Purified CD160 + T cells, sorted and purified naive T cells (T naive ), sorted and purified central memory T cells (T CM ), sorted and purified effector memory T cells ( TEM ), sorted and purified effector T cells ( TEMRA ), sorted and purified transitional memory T cells (Transitional Memory T cells, TTM ), sorted and purified tissue memory T cells (Tissue residential memory T cells, TRM ) and natural killer T cells (Natural killer T cells, NKT) and so on.
  • T naive sorted and purified central memory T cells
  • T CM sorted and purified effector memory T cells
  • TEMRA sorted and purified effector T cells
  • TTM Transitional Memory T cells
  • tissue memory T cells Tissue residential memory T cells, T
  • the present invention provides a combination comprising the immune cell and a tumor antigen-targeting antibody.
  • the tumor antigens include but are not limited to one or more of the following: CD19, BCMA, CD20, CD22, CD30, CD33, CD38, CD47, CD70, CD117, CD123, CD133, CD138, CD147, CD171, NKG2DL, HER2, MUC1, MUC16, CEA, EpCAM, IL-13R ⁇ 2, EGFR, EGFRvIII, GD2, DR5, EphA2, FR ⁇ , PSCA, PSMA, TARP, cMet, VEGFR2, BCMA, CTLA-4, PD-L1, AFP, GPC3, AXL, ROR1, ROR2, FAP, Mesothelin, DLL3 and CLDN18.
  • the tumor antigens include, but are not limited to, one or more of the following: HER2, EGFR, CD47, AXL and FAP.
  • the present invention provides a combination comprising the immune cell and viral antigen targeting antibodies.
  • the viral antigen is selected from one or more of the following: gp120 of human acquired immunodeficiency virus HIV-1, surface antigen of hepatitis B virus HBV, hemagglutinin or neuraminidase of influenza virus, Ebola virus spike protein, severe acute respiratory syndrome coronavirus SARS-CoV surface spike protein, Middle East respiratory syndrome coronavirus MERS-CoV surface spike protein and novel coronavirus SARS-CoV-2 surface spike protein;
  • the viral antigen is selected from the surface spike protein of the novel coronavirus SARS-CoV-2.
  • the present invention provides the chimeric receptor, immune cell, immune cell and tumor antigen-targeting antibody combination and immune cell and virus antigen-targeting antibody combination described in the present invention in preparation for the treatment of tumors or Use in medicines for viral infectious diseases;
  • the tumor is selected from one or more of the following: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, renal cancer, pancreatic cancer, nasopharyngeal cancer cancer, small bowel, large bowel, colorectal, bladder, bone, prostate, thyroid, brain, rhabdoid and leiomyoma;
  • the viral infectious disease is selected from one or more of the following: Human Acquired Immunodeficiency Syndrome, Hepatitis B, Influenza, Ebola Virus Disease, Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Novel Coronavirus Pneumonia.
  • Human Acquired Immunodeficiency Syndrome Hepatitis B
  • Influenza Influenza
  • Ebola Virus Disease Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Novel Coronavirus Pneumonia.
  • SARS Severe Acute Respiratory Syndrome
  • MERS Middle East Respiratory Syndrome
  • Novel Coronavirus Pneumonia Novel Coronavirus Pneumonia
  • the present invention provides a method of treating a tumor, the method comprising administering to a subject a therapeutically effective amount of a combination of a chimeric receptor, an immune cell, an immune cell and a tumor antigen targeting antibody;
  • the tumor is selected from one or more of the following: lymphoma, neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, renal cancer, pancreatic cancer, nasopharyngeal cancer cancer, small bowel cancer, colorectal cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer, brain cancer, rhabdomyomas, leiomyomas.
  • the present invention provides a method of treating a FAP+ tumor comprising administering to a patient a combination of an immune cell of the present invention and a FAP-targeting antibody.
  • the present invention provides a method of treating an AXL+ tumor comprising administering to a patient a combination of an immune cell of the present invention and an AXL-targeting antibody.
  • the present invention provides a method of treating a CD47+ tumor comprising administering to a patient a combination of an immune cell of the present invention and a CD47-targeting antibody.
  • the present invention provides a method of treating a HER2+ tumor comprising administering to a patient a combination of an immune cell of the present invention and a HER2-targeting antibody.
  • the present invention provides a method of treating an EGFR+ tumor comprising administering to a patient a combination of an immune cell of the present invention and an EGFR-targeting antibody.
  • the present invention provides a method of treating a viral infectious disease, the method comprising administering to a subject a therapeutically effective amount of an immune cell, a combination of an immune cell and a viral antigen targeting antibody;
  • the virus is selected from one or more of the following: human acquired immunodeficiency virus HIV-1, hepatitis B virus HBV, influenza virus, Ebola virus, severe acute respiratory syndrome coronavirus SARS- CoV, Middle East Respiratory Syndrome Coronavirus MERS-CoV and Novel Coronavirus SARS-CoV-2.
  • HIV-1 human acquired immunodeficiency virus HIV-1
  • hepatitis B virus HBV influenza virus
  • Ebola virus severe acute respiratory syndrome coronavirus SARS- CoV
  • Middle East Respiratory Syndrome Coronavirus MERS-CoV Middle East Respiratory Syndrome Coronavirus MERS-CoV
  • Novel Coronavirus SARS-CoV-2 Novel Coronavirus SARS-CoV-2.
  • the present invention provides a method of treating a novel coronavirus SARS-CoV-2 infectious disease, the method comprising administering to a patient the immune cells of the present invention and the SARS-CoV-2 surface spike protein target to the combination of antibodies.
  • the present invention provides a method for preparing immune cells of the present invention, comprising the steps of:
  • the immune cells are T cells.
  • the present invention also provides a method for amplifying T cells expressing chimeric receptors in large quantities, the method comprising: transfecting the T cells with the vector or infecting the T cells with the virus; and additionally adding anti- Human CD3-stimulating antibodies and anti-human CD28-stimulating antibodies, tumor antigen-expressing cells or recombinant tumor antigens, and tumor antigen-targeting antibodies to stimulate the T cells, proliferate to generate large numbers of chimeric receptor-engineered T cells.
  • the present invention has the following advantages:
  • the present invention provides a new chimeric receptor with high Fc affinity and resistance to cellular proteases, which has higher affinity for antibody Fc fragments, and can be used in combination with tumor antigen-targeting antibodies to significantly enhance the effect of ADCC.
  • the chimeric receptor of the present invention also has the ability to resist the cleavage of metalloprotease, to ensure that the chimeric receptor is efficiently expressed on the killer cells, and to avoid the function damage caused by the down-regulation of the chimeric receptor.
  • the chimeric receptor of the present invention is different from the traditional single-target CAR strategy, and can flexibly switch the targeting antibodies of different tumor antigens, thereby realizing the efficacy of broad-spectrum immunotherapy of multiple cancer types.
  • Figure 1 is the map of recombinant lentivirus expression plasmid
  • FIG. 2 is a graph showing the effect of the chimeric receptor binding to the Fc fragment of the antibody according to the present invention.
  • Figure 3 is a graph showing the expression of chimeric receptors according to the present invention on the surface of primary T cell membranes.
  • Figure 4 is a graph showing the killing of tumor cells by T cells expressing chimeric receptors combined with FAP targeting antibodies according to the present invention.
  • Figure 5 is a graph showing the killing of tumor cells by T cells expressing chimeric receptors combined with AXL targeting antibodies according to the present invention.
  • Figure 6 is a graph showing the killing of tumor cells by T cells expressing chimeric receptor combined with CD47 targeting antibody according to the present invention.
  • FIG. 7 is a graph showing the efficiency of killing tumor cells by T cells expressing wild-type/single-point mutation/double-mutation chimeric receptors combined with CD47 targeting antibody according to the present invention and a statistical graph of the luciferase expression level of surviving tumor cells;
  • FIG. 8 is a graph showing the killing efficiency of T cells expressing chimeric receptors combined with HER2-targeting antibodies against tumor cells according to the present invention.
  • FIG. 9 is a graph showing the efficiency of killing tumor cells by T cells expressing wild-type/single-point mutation/double-mutation chimeric receptor combined with HER2-targeted antibody and the statistical graph of luciferase expression level of surviving tumor cells according to the present invention.
  • FIG. 10 is a graph showing the killing efficiency of T cells expressing chimeric receptor combined with EGFR targeting antibody according to the present invention in killing tumor cells.
  • Fig. 11 is a graph showing the efficiency of killing Spike positive A549-Spike cells according to the present invention in combination with SARS-CoV-2 surface spike protein targeting antibody expressing chimeric receptor T cells.
  • Figure 12 is a graph of inhibition of tumor growth by T cells expressing chimeric receptors in combination with CD47 targeting antibodies according to the present invention.
  • DMEM medium and RPMI1640 medium were purchased from Corning Company, and lymphocyte medium X-VIVO 15 was purchased from Lonza Company.
  • T cell growth medium consists of basal medium and cytokines.
  • the basal medium is lymphocyte medium X-VIVO 15, and the cytokines are IL-7 at a final concentration of 5ng/mL, IL-15 at 10ng/mL and 30ng/mL mL of IL-21.
  • cytokines IL-7 and IL-15 were purchased from R&D Company, and IL-21 was purchased from Nearshore Protein Technology Co., Ltd.
  • Fetal bovine serum was purchased from BI Company.
  • Lenti-X lentiviral concentration reagent was purchased from Takara Company.
  • Tumor-targeting antibodies and novel coronavirus SARS-CoV-2 surface spike protein-targeting antibodies were prepared and provided by Shanghai Xinwan Biotechnology Co., Ltd., including HER2-targeting antibodies (XW-HER2-02), EGFR-targeting antibodies ( XW-EGFR-02), CD47 (XW-CD47-02), GPC3 targeting antibody (XW-GPCR-02), AXL targeting antibody (XW-AXL-02), FAP targeting antibody (XW-FAP-02) ) and SARS-CoV-2 surface spike protein targeting antibody (XW-SARS-CoV-2).
  • HER2-targeting antibodies XW-HER2-02
  • EGFR-targeting antibodies XW-EGFR-02
  • CD47 XW-CD47-02
  • GPC3 targeting antibody XW-GPCR-02
  • AXL targeting antibody XW-AXL-02
  • FAP targeting antibody XW-FAP-02
  • the synthetic gene was purchased from Shanghai Jierui Biological Engineering Co., Ltd.
  • the lentiviral expression plasmid pXW-EF1 ⁇ -MCS-P2A-EGFP was provided by Shanghai Xinwan Biotechnology Co., Ltd., and the packaging plasmid psPAX2 and the envelope plasmid PMD2.G were purchased from Addgene Company.
  • Stable 3 chemically competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • Endotoxin-free plasmid mini kit and endotoxin-free plasmid medium kit were purchased from OMEGA Company and Macherey Nagel Company, respectively.
  • the luciferase substrate was purchased from Promega Biotechnology Co., Ltd.
  • HEK293T cells A549 lung cancer cells, NCI-H292 highly metastatic lung cancer cells, U251 glioma cells, MDA-MB-231 breast cancer cells, and HepG2 liver cancer cells were purchased from ATCC in the United States.
  • A549-luc lung cancer cells, NCI-H292-luc highly metastatic lung cancer cells, U25-luc glioma cells, MDA-MB-231-luc breast cancer cells, HepG2-luc liver cancer cells, and A549-FAP cells were purchased from Shanghai Xinwan Biological Technology Co., Ltd.
  • RTCA Real-time label-free cell function analyzer
  • the microplate luminescence detector was purchased from Promega Biotechnology Co., Ltd.
  • B-NDG immunodeficient mice were purchased from Beijing Biositu Gene Biotechnology Co., Ltd.
  • hrCD16 chimeric receptor means F176V and S197P double mutant CD16 chimeric receptor according to the present invention
  • CD16 chimeric receptor means chimeric receptor containing wild-type CD16
  • CD16F176V chimeric receptor A chimeric receptor represents a chimeric receptor containing F176V mutated CD16
  • a CD16S197P chimeric receptor represents a chimeric receptor containing S197P mutated CD16.
  • the wild-type CD16.BBz, F176V mutant CD16.BBz, S197P mutant CD16.BBz and hrCD16.BBz genes (respectively shown in SEQ ID NOs: 13-16) were synthesized by Shanghai Jierui Bioengineering Co., Ltd.
  • the viral expression plasmids (pXW-EF1 ⁇ -MCS-P2A-EGFP) were obtained as pXW-EF1 ⁇ -CD16.BBz-P2A-EGFP, pXW-EF1 ⁇ -CD16F176V.BBz-P2A-EGFP, pXW-EF1 ⁇ -CD16S197P.BBz-P2A- EGFP and pXW-EF1 ⁇ -hrCD16.BBz-P2A-EGFP recombinant lentiviral expression plasmids.
  • the plasmid map is shown in Figure 1.
  • HEK293T cells were seeded on a 12-well flat-bottom cell culture plate at 4 ⁇ 10 5 cells/2 mL/well.
  • pXW-CD16.BBz and XW-hrCD16.BBz lentiviral expression plasmids were transfected with TurboFect transfection reagent respectively, and the total amount of plasmids was 2 ⁇ g/well.
  • Add 4 ⁇ L of TurboFect transfection reagent at a ratio of plasmid amount ( ⁇ g):transfection reagent ( ⁇ L) 1:2, and add the freshly prepared plasmid transfection complex to the above cell culture plate after incubating at room temperature for 15-20 min. Incubate at 37°C, 5% CO 2 for 48 hours, centrifuge at 500 ⁇ g for 5 min at room temperature, discard the supernatant, and collect the cells for later use.
  • Example 2 The method described in Example 2 was used to prepare HEK293T cells expressing wild-type CD16 chimeric receptor (CD16.BBz) and expressing hrCD16 chimeric receptor (hrCD16.BBz), respectively.
  • Figure 2A is a flow chart of HEK293T cells expressing wild-type CD16 chimeric receptor and hrCD16 chimeric receptor binding to the Fc fragment of the EGFR targeting antibody, respectively.
  • HEK293T cells expressing hrCD16 chimeric receptor were much more efficient than HEK293T cells expressing wild-type CD16 chimeric receptor to bind EGFR targeting antibody Fc fragment (percentage: 85.8% vs 40.4 %; fluorescence intensity: 3720 vs 1033).
  • 2B is a statistical graph of the efficiency of HEK293T cells expressing wild-type CD16 chimeric receptor and HEK293T cells expressing hrCD16 chimeric antibody binding to EGFR targeting antibody Fc fragment.
  • Fc fragment binding rate and Fc fragment strength the ability of HEK293T cells expressing hrCD16 chimeric receptor to bind antibody Fc fragment is better than that of HEK293T cells expressing wild-type CD16 chimeric antibody, especially at higher antibody concentration. Especially obvious.
  • Example 4 Packaging, concentration and titer determination of lentivirus
  • HEK293T cell treatment 24 hours before transfection, HEK293T cells in logarithmic growth phase were collected and seeded in 10 cm cell culture dishes (6-8 ⁇ 10 6 cells) in complete DMEM medium containing 10 mL The cells were grown and cultured at 37°C under 5% CO 2 for 18-24 hours, and the cell density reached 70-90% or more before transfection.
  • the viral supernatant collected by centrifugation was filtered with a 0.45 ⁇ m filter, 1/3 of the viral supernatant volume was added with Lenti-X lentivirus concentration reagent, invert and mixed several times, incubated at 4°C overnight, centrifuged at 2000 ⁇ g for 45min at 4°C, A white precipitate can be seen at the bottom of the centrifuge tube, which is the virus. Carefully discard the supernatant, resuspend the white pellet with 1/50-1/100 volume of the original virus supernatant in blank RPMI1640 medium, aliquot and store at -80°C for later use.
  • lentivirus titer Jurkat T cells were seeded on a 96-well U-bottom plate at 1 ⁇ 10 5 cells/well, and the collected lentivirus concentrate was diluted 10-fold. Add 100 ⁇ L of virus dilution solution to the corresponding wells, add protamine sulfate, a pro-infection reagent, and adjust the concentration to 10 ⁇ g/mL,
  • Example 6 The hrCD16 chimeric receptor is efficiently expressed on the surface of primary T cells
  • Example 4 The method described in Example 4 was used to prepare primary T cells modified by expressing hrCD16 chimeric receptor (hrCD16.BBz).
  • Figure 3A is a flow chart of the expression of hrCD16 on primary T cells. All three hrCD16 chimeric receptor-modified T cells expressed hrCD16 normally, with positive rates ranging from 33.9% to 50.6%, and fluorescence intensities ranging from 3021 to 4987 (Fig. 3B).
  • Example 7 Killing of T cells expressing hrCD16 chimeric receptor combined with highly lethal FAP targeting antibody FAP+ tumor cells
  • the tumor cell killing efficiency was detected by Real Time Cellular Analysis (RTCA, Real Time Cellular Analysis).
  • RTCA Real Time Cellular Analysis
  • 1 ⁇ 10 4 A549-FAP FAP-modified human lung cancer cells
  • Cell growth was dynamically monitored for 8-9 hours using the RTCA system.
  • the results are shown in Figure 4:
  • the solid line in Figure 4 is the target cell growth curve of T cells expressing hrCD16 chimeric receptor combined with FAP targeting antibody to kill tumor cells, and the dotted line is the tumor cell killing curve of T cells expressing hrCD16 chimeric receptor.
  • Target cell growth curve the results show that the target cell growth curve of T cells expressing hrCD16 chimeric receptor combined with FAP targeting antibody group is significantly different from the target cell growth curve of T cells expressing hrCD16 chimeric receptor, indicating that the expression of hrCD16 chimeric
  • the receptor's T cells combined with FAP-targeted antibodies can efficiently kill tumor cells, and the tumor cell killing rate is as high as 100%, effectively inhibiting the growth of tumor cells, so that the growth curve of tumor cells decreases rapidly.
  • Example 8 Killing of T cells expressing hrCD16 chimeric receptor combined with highly lethal AXL targeting antibody AXL+ tumor cells
  • RTCA Real Time Cellular Analysis
  • the results are shown in Figure 5:
  • the solid line in Figure 5A is the target cell growth curve of T cells expressing hrCD16 chimeric receptor combined with AXL targeting antibody to kill U251 tumor cells, and the dotted line is the T cell expressing hrCD16 chimeric receptor combined with irrelevant antibodies
  • the target cell growth curve of killing U251 tumor cells the other two curves are the growth curve of U251 tumor cells and the target cell growth curve of T cells expressing hrCD16 chimeric receptor killing tumor cells, the results show that only hrCD16 chimeric receptor
  • the combination of T cells and AXL-targeted antibodies can make the growth curve of U251 tumor cells decrease rapidly, effectively kill tumor cells, and the killing rate is as high as 94.5%
  • the solid line in Figure 5B shows that T cells expressing hrCD16 chimeric receptor combined with AXL-targeted antibodies kill MDA-
  • the target cell growth curve of MB-231 tumor cells the dotted line is the target cell growth curve of T
  • RTCA Real Time Cellular Analysis
  • the results are shown in Figure 6.
  • the solid line in Figure 6A is the target cell growth curve of T cells expressing hrCD16 chimeric receptor combined with CD47 targeting antibody to kill U251 tumor cells, and the dotted line is the T cell expressing hrCD16 chimeric receptor combined with irrelevant antibodies.
  • the target cell growth curve of killing U251 tumor cells the other two curves are the growth curve of U251 tumor cells and the target cell growth curve of T cells expressing hrCD16 chimeric receptor killing tumor cells, the results show that only hrCD16 chimeric receptor
  • the combination of T cells and CD47-targeted antibodies can make the growth curve of U251 tumor cells decrease rapidly, effectively kill tumor cells, and the killing rate is as high as 94.2%; the solid line in Figure 6B shows that T cells expressing hrCD16 chimeric receptor combined with CD47-targeted antibodies kill MDA-
  • the target cell growth curve of MB-231 tumor cells the dotted line is the target cell growth curve of T cells expressing hrCD16 chimeric receptor combined with irrelevant antibodies to kill MDA-MB-231 tumor cells, and the other two curves are MDA-MB-231
  • the growth curve of tumor cells and the target cell growth curve of T cells expressing hrCD16 chimeric receptor killing tumor cells the results show that only hrCD16
  • SKOV3-Luc luciferase gene-modified human ovarian cancer
  • the luciferase activity value of target cells was detected by a microplate luminescence detector.
  • the cell killing rate was calculated as follows:
  • Cell killing rate (%) (luciferase activity value of target cell group-luciferase activity value of experimental group)/luciferase activity value of target cell group ⁇ 100
  • Tumor cell killing efficiency was assessed using a Luciferase-based cytotoxicity assay.
  • 1 ⁇ 10 4 NCI-H292-Luc (luciferase gene-modified human lung cancer cells) or SKOV3-Luc (luciferase gene-modified human ovarian cancer cells) were seeded on a 96-well flat bottom plate, each well 100 ⁇ L of medium, placed in a 37°C, 5% CO 2 cell incubator for 18-20 hours.
  • the cell killing rate was calculated as follows:
  • Cell killing rate (%) (luciferase activity value of target cell group-luciferase activity value of experimental group)/luciferase activity value of target cell group ⁇ 100
  • T cells expressing hrCD16 chimeric receptors have a certain non-specific killing effect on tumor cells, and the killing efficiency of the combined unrelated control antibody group is comparable to that of the T cells expressing hrCD16 chimeric receptor alone. , suggesting that the ADCC effect of the irrelevant control antibody is very weak.
  • HER2 targeting antibody combined with T cells expressing hrCD16 chimeric receptor can effectively kill tumor cells.
  • the killing rates of lung cancer cells (NCI-H292) and ovarian cancer cells (SKOV3) are as high as 92.2% and 89.4%, respectively, thus effectively removing tumor cells. .
  • Example 12 Killing of T cells expressing different chimeric receptors in combination with highly lethal HER2 targeting antibodies HER2+ tumor cells
  • SKOV3-Luc luciferase gene-modified human ovarian cancer
  • the luciferase activity value of target cells was detected by a microplate luminescence detector.
  • the cell killing rate was calculated as follows:
  • Cell killing rate (%) (luciferase activity value of target cell group-luciferase activity value of experimental group)/luciferase activity value of target cell group ⁇ 100
  • Example 13 Killing of T cells expressing hrCD16 chimeric receptor combined with highly lethal EGFR targeting antibody damage tumor cells
  • Tumor cell killing efficiency was assessed using a Luciferase-based cytotoxicity assay.
  • NCI-H292-Luc luciferase gene-modified human lung cancer cells
  • SKOV3-Luc luciferase gene-modified human ovarian cancer cells, EGFR+
  • the cell killing rate was calculated as follows:
  • Cell killing rate (%) (luciferase activity value of target cell group-luciferase activity value of experimental group)/luciferase activity value of target cell group ⁇ 100
  • T cells expressing hrCD16 chimeric receptors have a certain non-specific killing effect on tumor cells, and the killing efficiency of the combined unrelated control antibody group is comparable to that of the T cells expressing hrCD16 chimeric receptor alone. , suggesting that the ADCC effect of the irrelevant control antibody is very weak.
  • the modified highly lethal EGFR-targeting antibody combined with T cells expressing the hrCD16 chimeric receptor can effectively kill tumor cells.
  • the killing rates of lung cancer cells and ovarian cancer cells are as high as 92.2% and 86.1%, respectively, thus effectively removing tumor cells.
  • Example 14 T cells expressing hrCD16 chimeric receptor combined with SARS-CoV-2 spike protein targeting Antibodies kill Spike+ cells
  • the cell killing efficiency was detected by Real Time Cellular Analysis (RTCA, Real Time Cellular Analysis).
  • RTCA Real Time Cellular Analysis
  • 1 ⁇ 10 4 Spike positive A549-Spike cells were seeded on a 16-well E-Plate electrode plate with 100 ⁇ L of medium per well. Cell growth was dynamically monitored for 18-20 hours using the RTCA system.
  • T cells expressing hrCD16 chimeric receptor have a certain non-specific killing effect (40%) against the spike protein-positive A549-Spike.
  • SARS-CoV-2 spike protein targeting antibodies SARS-CoV-2-505-5 and SARS-CoV-2-553-20 combined with T cells expressing the hrCD16 chimeric receptor can effectively kill spike protein-positive A549- Spike cells, target cell killing rates were as high as 84%.
  • Example 15 T cells expressing hrCD16 chimeric receptor combined with highly lethal CD47 targeting antibody Internal tumor suppressor effect
  • NCI-H292-Luc lung cancer cells were inoculated into the left lower abdomen of B-NDG immunodeficient mice, 2 ⁇ 10 6 cells per mouse. After the tumor grew for 4 days, the tumor-bearing mice were randomly divided into the following 3 groups, expressing hrCD16 embedded cells. There were 3 mice in the T cell injection group with receptors; CD47 targeting antibody combined with untransduced T cell injection group, 3 mice; CD47 targeting antibody combined with T cell injection group expressing hrCD16 chimeric receptor, 3 mice. Tumor growth was monitored using a small animal in vivo imaging system.
  • Antibody administration 50 ⁇ g (125 ⁇ L) of each mouse was injected intraperitoneally each time, and injected on the 4th, 7th and 18th days after tumor inoculation, respectively.
  • Cell administration each mouse was intraperitoneally injected with untransduced T cells or T cells expressing hrCD16 chimeric receptor 2 ⁇ 10 6 /125 ⁇ L, and re-infused one day after antibody administration, a total of 3 times.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

一种嵌合受体，所述嵌合受体包含：(1)Fc片段的胞外识别域；(2)胞外间隔区；(3)跨膜区；和(4)胞内信号传导结构域；任选地，所述嵌合受体还包含(5)一个或多个共刺激信号传导结构域和/或(6)一个或多个细胞因子受体信号传导结构域。表达所述嵌合受体的免疫细胞，包含所述免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体的组合，以及一种Fc片段高亲和性嵌合受体，其具备更高的抗体Fc片段亲和力，联合肿瘤抗原靶向抗体或病毒抗原靶向抗体使用，可显著增强ADCC作用。

Description

一种嵌合受体及其应用 技术领域
本发明属于生物医药技术领域。具体地，本发明涉及一种嵌合受体。本发明还涉及所述嵌合受体及其应用。
背景技术
2013年是癌症治疗的一个重要转折点，基于逆转机体肿瘤免疫抑制性微环境的癌症免疫疗法获得了《科学》公布的2013年度十大科学突破之首。随后，嵌合受体T细胞免疫疗法(Chimeric antigen receptor T-cell immunotherapy，CAR-T)在血液肿瘤的治疗中呈现显著的疗效。CAR-T细胞疗法通过病毒或非病毒的外源基因递送技术，将识别肿瘤特异/相关抗原的识别域和T细胞活化相关序列的融合蛋白表达在T细胞表面，使其以抗原依赖但非MHC限制的方式特异结合肿瘤抗原，启动活化并特异杀伤肿瘤细胞。但在实体瘤中，由于缺乏特异性的肿瘤抗原，使得该疗法的应用受到一定的限制。
目前使用的肿瘤抗原多为肿瘤相关抗原，在肿瘤细胞高表达，但在正常组织细胞亦有少量表达，导致CAR-T细胞疗法出现“靶向非肿瘤细胞的毒副效应”(On-target off-tumor)，限制了其在实体瘤治疗的临床应用。一项靶向碳酸酐酶IX(CAIX)的CAR-T临床研究结果显示，CAIX CAR-T细胞治疗不仅不能控制患者肿瘤生长，反而导致患者肝功能异常和胆管炎的发生(Lamers CH,Sleijfer S,Vulto AG,et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX:first clinical experience[J].J Clin Oncol.2006,24(13):e20-e22)。另外一项靶向酪氨酸激酶受体2(ERBB2)的CAR-T细胞治疗更是引起致死性的炎症因子风暴导致患者死亡(Morgan RA,Yang JC,Kitano M,et al.Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2[J].Mol Ther.2010,18:843-851)。因此，有必要开发一种更为安全的免疫细胞治疗技术。
此外，临床研究发现CAR-T细胞疗法治疗血液瘤后复发率高达50％，特别是CD19/CD22CAR-T细胞治疗后易发抗原逃逸(Shah NN,Fry TJ.Mechanisms of resistance to CAR T cell therapy[j].Nat Rev Clin Oncol.2019, 16(6):372-385)。由于目标抗原的突变或丢失，导致针对该靶点的原有CAR-T细胞无法控制肿瘤的生长，需切换至靶向新抗原的CAR-T细胞治疗方可抑制肿瘤的生长。新靶点CAR-T细胞制备与质控极大地增加了患者治疗的时间成本与经济负担。因此，有必要开发一种通用型免疫细胞治疗技术，可灵活切换不同的肿瘤抗原靶向抗体，达到有效治疗一种或多种肿瘤的目的。
CD16分子，又名FcγRIII，主要表达于自然杀伤细胞(Natural killer cells,NK)、中性粒细胞和巨噬细胞。具有杀伤功能细胞的CD16分子可结合抗体的Fc片段，从而启动抗体依赖的细胞介导的细胞毒作用(Antibody-dependent cell-mediated cytotoxicity,ADCC)，其中，发挥作用的主要为NK细胞。愈来愈多的研究报道，将用于识别肿瘤抗原的嵌合受体的胞外单链抗体(Single chain antibody fragment，scFv)更换为野生型CD16分子，通过联合使用多种肿瘤抗原靶向抗体，可实现不同肿瘤细胞的杀伤(Clémenceau B,Congy-Jolivet N,Gallot G,et al.Antibody-dependent cellular cytotoxicity(ADCC)is mediated by genetically modified antigen-specific human T lymphocytes[J].Blood.2006,107(12):4669-4677；Ochi F,Fujiwara H,Tanimoto K,et al.Gene-modified humanα/β-T cells expressing a chimeric CD16-CD3ζreceptor as adoptively transferable effector cells for anticancer monoclonal antibody therapy[J].Cancer Immunol Res.2014,2(3):249-262)。
研究发现CD16分子存在基因多态性，野生型CD16分子的亲和力通常较低，减弱了抗体依赖的细胞介导的细胞毒作用。另有研究表明，免疫细胞激活后表达的或外环境存在的解整合素金属蛋白酶17(ADAM17)可切割细胞膜表面的CD16分子，下调细胞膜表面CD16的表达而导致免疫细胞特别是NK细胞功能受损(Romee R,Foley B,Lenvik T,et al.NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17(ADAM17)[J].Blood.2013,121(18):3599-3608)。
综上所述，有必要构建一种特异结合Fc片段的嵌合受体，特别是Fc高亲和性、细胞蛋白酶耐受型CD16(High-affinity,protease-resistant CD16,hrCD16)嵌合受体。
发明简述
针对现有技术的不足，本发明的目的是提供一种嵌合受体。本发明提供的嵌合受体，特别是hrCD16嵌合受体，其具有Fc高亲和性和细胞蛋白酶耐受性，可通过病毒/非病毒递送方式导入多种杀伤性免疫细胞，如T细胞、NK细胞或NKT细胞等免疫细胞，并稳定表达于其上。使用本发明的嵌合受体，特别是hrCD16嵌合受体修饰的免疫细胞，联合多种肿瘤抗原靶向抗体 或病毒抗原靶向抗体，能够广谱杀伤不同类型的肿瘤细胞和病毒。由于本申请的嵌合受体只有在联合肿瘤抗原靶向抗体或病毒抗原靶向抗体时方可发挥ADCC作用进而杀伤肿瘤细胞或经病毒感染的细胞，因此本申请提供的方法具有能够提升治疗安全性、增强疗效、避免肿瘤抗原逃逸或病毒逃逸以及快速灵活切换治疗靶点抗体等诸多优点。
本发明通过如下技术方案以实现所述目的：
一方面，本发明提供了一种嵌合受体，所述嵌合受体包含：
(1)Fc片段的胞外识别域；
(2)胞外间隔区；
(3)跨膜区；和
(4)胞内信号传导结构域；
任选地，所述嵌合受体还包含：
(5)一个或多个共刺激信号传导结构域；和/或
(6)一个或多个细胞因子受体信号传导结构域；
其中，所述Fc片段的胞外识别域为特异性结合抗体Fc片段的CD16胞外域、CD32胞外域、CD64胞外域、CD89胞外域、CD23胞外域、FcεRI胞外域、FcRn胞外域、Fc结合抗体、Protein A、Protein G或其突变体或多个重复串联胞外域；
优选地，所述Fc片段的胞外识别域为CD16胞外域；
优选地，所述Fc片段的胞外识别域为野生型CD16胞外域、F176V突变型CD16胞外域、S197P突变型CD16胞外域或F176V和S197P双突变型CD16胞外域，其包含如SEQ ID NO:1-4中任一项所示的氨基酸序列；更优选地，所述Fc片段的胞外识别域为F176V和S197P双突变型CD16胞外域，其包含如SEQ ID NO:4所示的氨基酸序列。
根据本发明所述的嵌合受体，其特征在于，所述胞外间隔区包括但不限于以下任何一种或多种：
(1)抗体IgG4的铰链区及其突变体；
(2)抗体IgG4的铰链区及其突变体和CH2区；
(3)抗体IgG4的铰链区及其突变体、CH2区和CH3区；
(4)抗体IgG1的铰链区及其突变体；
(5)抗体IgG1的铰链区及其突变体和CH2区；
(6)抗体IgG1的铰链区及其突变体、CH2区和CH3区；
(7)免疫球蛋白Fc受体的铰链区：CD64、CD32、CD16、CD89、FcεRI、FcεRII(CD23)和FcRn；
(8)CD28铰链区、CD137铰链区、CD8α铰链区、CD4铰链区、PD-1 铰链区和CTLA-4铰链区；和
(9)以上的任何一种组合。
根据本发明所述的hrCD16嵌合受体，其特征在于，所述胞外间隔区为CD8铰链区；优选地，所述胞外间隔区包含如SEQ ID NO:5所示的氨基酸序列。
根据本发明所述的hrCD16嵌合受体，其特征在于，所述跨膜区包括但不限于以下任何一种或多种：T细胞受体复合物的CD3ξ链的跨膜区、CD28跨膜区、CD137跨膜区、CD8α跨膜区、CD4跨膜区、PD-1跨膜区、CTLA-4跨膜区、、免疫球蛋白Fc受体跨膜区及其组合。
优选地，所述跨膜区为CD8跨膜区；更优选地，所述跨膜区包含如SEQ ID NO:6所示的氨基酸序列。
根据本发明所述的hrCD16嵌合受体，其特征在于，所述共刺激信号传导结构域包括但不限于以下任何一种或多种的信号传导结构域：CD2、CD27、CD28、CD30、CD40、CD40L、CD137(4-1BB)、CD134(OX40)、CD278(ICOS)、GITR、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、Dap10、ICAM-1、LFA-1、Lck、TNFRI、TNFRII、TIM-1、TIM-2、TIM-3、TIM-4及其组合。
优选地，所述共刺激信号传导结构域为CD137信号传导结构域；更优选地，所述共刺激信号传导结构域包含如SEQ ID NO:7所示的氨基酸序列。
根据本发明所述的嵌合受体，其特征在于，所述细胞因子受体信号传导结构域包括但不限于以下任何一种或多种信号传导结构域：IL-2R、IL-7R、IL-15R、IL-18R、IL-21R、IL-23R及其组合。
根据本发明所述的嵌合受体，其特征在于，所述胞内信号传导结构域包括但不限于以下任何一种或多种信号传导结构域：T细胞受体复合物的CD3ξ链、FcγRIII、FcεRI、Fc受体胞内信号传导域、携带免疫受体酪氨酸活化基序(ITAM)的信号传导结构域及其组合。
优选地，所述胞内信号传导结构域为CD3ξ链信号传导结构域；更优选地，所述胞内信号转导结构域如SEQ ID NO:8所示。
优选地，所述受体由包含如SEQ ID NO:1-4中任一项所示的氨基酸序列的野生型CD16胞外域、F176V突变型CD16胞外域、S197P突变型CD16胞外域和F176V和S197P双突变型CD16胞外域中任一CD16胞外域、人CD8铰链区、人CD8跨膜区、人CD137共刺激信号传导结构域和CD3ξ链信号传导结构域组成；
优选地，所述嵌合受体的氨基酸序列如SEQ ID NO:9-12中任一项所示。
更优选地，根据本发明所述的嵌合受体，其特征在于，所述受体由包含如SEQ ID NO:4所示的氨基酸序列的F176V和S197P双突变型CD16胞外域、人CD8铰链区、人CD8跨膜区、人CD137共刺激信号传导结构域和CD3ξ链信号传导结构域组成。
更优选地，所述双突变型hrCD16嵌合受体的氨基酸序列如SEQ ID NO:12所示。
本发明还提供了一种多核苷酸，其编码所述嵌合受体。
优选地，所述多核苷酸序列如SEQ ID NO:13-16中任一项所示；
更优选地，所述多核苷酸序列如SEQ ID NO:16所示
本发明还提供了一种载体，所述载体包含所述多核苷酸。
优选地，所述载体共表达细胞因子、趋化因子、趋化因子受体、免疫检查点阻断抗体或其组合；
更优选地，所述细胞因子包括但不限于IL-2、IL-7、IL-15、IL-21、IL-12、IL-18、IL-23及其组合；所述趋化因子包括但不限于CXCL9、CXCL10、CXCL11、CCL19、CCL20和CCL21；所述趋化因子受体包括但不限于CCR1、CCR3、CCR9、CXCR1和CXCR2；所述免疫检查点阻断抗体包括但不限于CTLA-4阻断抗体、PD-1阻断抗体、PD-L1阻断抗体、LAG-3阻断抗体、Tim-3阻断抗体、TIGIT阻断抗体、VISTA阻断抗体、Siglec-15阻断抗体及其组合。
本发明还提供了一种病毒，所述病毒包含所述的多核苷酸；
优选地，所述病毒包括但不限于逆转录病毒、慢病毒、腺病毒、腺相关病毒、痘病毒和疱疹病毒。
另一方面，本发明提供了一种表达所述嵌合受体的免疫细胞。
优选地，所述免疫细胞包括但不限于T细胞、天然杀伤细胞(Natural killer cells，NK)、固有淋巴细胞((Innate lymphoid cells,ILC)、造血干细胞、胚胎干细胞和多能干细胞等；
更优选地，所述T细胞包括但不限于未经分选纯化的T细胞、分选纯化的T细胞、分选纯化的PD-1 +T细胞、分选纯化的CD137 +T细胞、分选纯化的CD160 +T细胞、分选纯化的纯真T细胞(T naive)、分选纯化的中央记忆型T细胞(T CM)、分选纯化的效应记忆型T细胞(T EM)、分选纯化的效应T细胞(T EMRA)、分选纯化的过渡记忆型T细胞(Transitional Memory T cells，T TM)、分选纯化的组织记忆型T细胞(Tissue residential memory T cells，T RM)和天然杀伤T细胞(Natural killer T cells，NKT)等。
再一方面，本发明提供了包含所述免疫细胞和肿瘤抗原靶向抗体的组合。
优选地，所述肿瘤抗原包括但不限于以下一种或多种：CD19、BCMA、CD20、CD22、CD30、CD33、CD38、CD47、CD70、CD117、CD123、CD133、CD138、CD147、CD171、NKG2DL、HER2、MUC1、MUC16、CEA、EpCAM、IL-13Rα2、EGFR、EGFRvIII、GD2、DR5、EphA2、FRα、PSCA、PSMA、TARP、cMet、VEGFR2、BCMA、CTLA-4、PD-L1、AFP、GPC3、AXL、ROR1、ROR2、FAP、Mesothelin、DLL3和CLDN18。
更优选地，所述肿瘤抗原包括但不限于以下一种或多种：HER2、EGFR、CD47、AXL和FAP。
再一方面，本发明提供了包含所述免疫细胞和病毒抗原靶向抗体的组合。
优选地，所述病毒抗原选自以下的一种或多种：人类获得性免疫缺陷病毒HIV-1的gp120、乙型肝炎病毒HBV的表面抗原、流感病毒的血凝素或神经氨酸酶，埃博拉病毒的刺突蛋白、严重急性呼吸系统综合征冠状病毒SARS-CoV的表面刺突蛋白，中东呼吸综合征冠状病毒MERS-CoV的表面刺突蛋白和新型冠状病毒SARS-CoV-2的表面刺突蛋白；
更优选地，所述病毒抗原选自新型冠状病毒SARS-CoV-2的表面刺突蛋白。
再另一方面，本发明提供了本发明所述的嵌合受体、免疫细胞、免疫细胞和肿瘤抗原靶向抗体的组合以及免疫细胞和病毒抗原靶向抗体的组合在制备用于治疗肿瘤或病毒感染性疾病的药物中的用途；
优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤和平滑肌瘤；
优选地，所述病毒感染性疾病选自以下的一种或多种：人类获得性免疫缺陷综合征、乙型肝炎、流感、埃博拉病毒病、严重急性呼吸系统综合征(SARS)、中东呼吸综合征(MERS)和新型冠状病毒肺炎。
再另一方面，本发明提供了治疗肿瘤的方法，所述方法包括给予受试者治疗有效量的嵌合受体、免疫细胞、免疫细胞和肿瘤抗原靶向抗体的组合；
优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、 小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤、平滑肌瘤。
在优选的实施方案中，本发明提供了治疗FAP+肿瘤的方法，所述方法包括给予患者本发明所述的免疫细胞和FAP靶向抗体的组合。
在优选的实施方案中，本发明提供了治疗AXL+肿瘤的方法，所述方法包括给予患者本发明所述的免疫细胞和AXL靶向抗体的组合。
在优选的实施方案中，本发明提供了治疗CD47+肿瘤的方法，所述方法包括给予患者本发明所述的免疫细胞和CD47靶向抗体的组合。在优选的实施方案中，本发明提供了治疗HER2+肿瘤的方法，所述方法包括给予患者本发明所述的免疫细胞和HER2靶向抗体的组合。
在优选的实施方案中，本发明提供了治疗EGFR+肿瘤的方法，所述方法包括给予患者本发明所述的免疫细胞和EGFR靶向抗体的组合。
再另一方面，本发明提供了治疗病毒感染性疾病的方法，所述方法包括给予受试者治疗有效量的免疫细胞，免疫细胞和病毒抗原靶向抗体的组合；
优选地，所述病毒选自以下的一种或多种：人类获得性免疫缺陷病毒HIV-1，乙型肝炎病毒HBV、流感病毒，埃博拉病毒、严重急性呼吸系统综合征冠状病毒SARS-CoV，中东呼吸综合征冠状病毒MERS-CoV和新型冠状病毒SARS-CoV-2。
在优选的实施方案中，本发明提供了治疗新型冠状病毒SARS-CoV-2感染性疾病的方法，所述方法包括给予患者本发明所述的免疫细胞和SARS-CoV-2表面刺突蛋白靶向抗体的组合。
再另一方面，本发明提供了本发明的免疫细胞的制备方法，其包括如下步骤：
1)获取嵌合受体的核酸序列；
2)将嵌合受体的核酸序列克隆至慢病毒表达载体中，获得编码嵌合受体的慢病毒表达质粒；
3)将慢病毒表达质粒、骨架质粒和包膜质粒共转染至HEK293T细胞，包装并获得慢病毒颗粒，经离心浓缩后获得慢病毒浓缩液；
4)将慢病毒转导免疫细胞，从而获得表达嵌合受体的免疫细胞；
优选地，所述免疫细胞为T细胞。
本发明还提供了一种用于扩增大量表达嵌合受体的T细胞的方法，所述方法包括：用所述的载体转染或用如所述的病毒感染T细胞；及额外添加抗人CD3刺激抗体和抗人CD28刺激抗体、表达肿瘤抗原的细胞或重组肿瘤抗 原以及肿瘤抗原靶向抗体以刺激所述T细胞，大量增殖以产生大量的嵌合受体工程化的T细胞。
与现有技术相比，本发明具有以下优点：
1)本发明提供了一种全新的Fc高亲和性、细胞蛋白酶耐受的嵌合受体，其具备更高的抗体Fc片段亲和力，联合肿瘤抗原靶向抗体使用，可显著增强ADCC作用。
2)本发明的嵌合受体同时具备抵抗金属蛋白酶的切割，保证嵌合受体高效表达于杀伤细胞上，避免因嵌合受体下调而导致的功能受损。
3)本发明的嵌合受体区别于传统的单靶点CAR策略，可灵活切换不同肿瘤抗原的靶向抗体，从而实现多癌种的广谱免疫治疗的功效。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为重组慢病毒表达质粒图谱
图2为根据本发明的嵌合受体结合抗体Fc段的效果图。
图3为根据本发明的嵌合受体在原代T细胞膜表面的表达情况图。
图4为根据本发明的表达嵌合受体的T细胞联合FAP靶向抗体杀伤肿瘤细胞曲线图。
图5为根据本发明的表达嵌合受体的T细胞联合AXL靶向抗体杀伤肿瘤细胞曲线图。
图6为根据本发明的表达嵌合受体的T细胞联合CD47靶向抗体杀伤肿瘤细胞曲线图。
图7为根据本发明的表达野生型/单点突变/双突变嵌合受体的T细胞联合CD47靶向抗体杀伤肿瘤细胞效率图和存活肿瘤细胞的荧光素酶表达水平统计图；
图8为根据本发明的表达嵌合受体的T细胞联合HER2靶向抗体杀伤肿瘤细胞效率图。
图9为本发明的表达野生型/单点突变/双突变嵌合受体的T细胞联合HER2靶向抗体杀伤肿瘤细胞效率图和存活肿瘤细胞的荧光素酶表达水平统计图。
图10为根据本发明的表达嵌合受体的T细胞联合EGFR靶向抗体杀伤肿瘤细胞效率图。
图11为根据本发明的表达嵌合受体的T细胞联合SARS-CoV-2表面刺突蛋白靶向抗体杀伤刺突蛋白阳性A549-Spike细胞效率图。
图12根据本发明的表达嵌合受体的T细胞联合CD47靶向抗体抑制肿瘤生长图。
实施发明的最佳方式
以下实施例仅用于说明本发明，但不用来限制本发明的范围。
以下实施例的实验方法，如无特殊说明，均为本领域的常规实验方法。以下实施例中所使用的实验材料，若无特殊说明，均为自常规生化试剂销售公司购买所得，其中：
DMEM培养基、RPMI1640培养基均购自Corning公司，淋巴细胞培养基X-VIVO 15购自Lonza公司。
T细胞生长培养基由基础培养基和细胞因子组成，基础培养基为淋巴细胞培养基X-VIVO 15，细胞因子为终浓度5ng/mL的IL-7，10ng/mL的IL-15和30ng/mL的IL-21。其中，细胞因子IL-7和IL-15购自R&D公司，IL-21购自近岸蛋白质科技有限公司。
胎牛血清购自BI公司。
TurboFect转染试剂盒购自Thermo Fisher Scientific公司。
Lenti-X慢病毒浓缩试剂购自Takara公司。
肿瘤靶向抗体和新型冠状病毒SARS-CoV-2表面刺突蛋白靶向抗体由上海鑫湾生物科技有限公司制备并提供，包括HER2靶向抗体(XW-HER2-02)、EGFR靶向抗体(XW-EGFR-02)、CD47(XW-CD47-02)、GPC3靶向抗体(XW-GPCR-02)、AXL靶向抗体(XW-AXL-02)，FAP靶向抗体(XW-FAP-02)和SARS-CoV-2表面刺突蛋白靶向抗体(XW-SARS-CoV-2)。
合成基因购自上海捷瑞生物工程有限公司。
慢病毒表达质粒pXW-EF1α-MCS-P2A-EGFP由上海鑫湾生物科技有限公司提供，包装质粒psPAX2和包膜质粒PMD2.G购自Addgene公司。
Stable 3化学感受态细胞购自上海唯地生物技术有限公司。
无内毒素质粒小提试剂盒和无内毒素质粒中提试剂盒分别购自OMEGA公司和Macherey Nagel公司。
荧光素酶底物购自普洛麦格生物技术有限公司。
HEK293T细胞、A549肺癌细胞、NCI-H292高转移肺癌细胞、U251胶质瘤细胞、MDA-MB-231乳腺癌细胞、HepG2肝癌细胞购自美国ATCC。A549-luc肺癌细胞、NCI-H292-luc高转移肺癌细胞、U25-luc胶质瘤细胞、MDA-MB-231-luc乳腺癌细胞、HepG2-luc肝癌细胞、A549-FAP细胞购自上海鑫湾生物科技有限公司。
实时无标记细胞功能分析仪(RTCA)购自上海优者生物科技有限公司。
Figure PCTCN2021104764-appb-000001
微孔板发光检测仪购自普洛麦格生物技术有限公司。
B-NDG免疫缺陷小鼠购自北京百奥赛图基因生物技术有限公司。
除非另有规定，以下实施例中，hrCD16嵌合受体表示根据本发明的F176V和S197P双突变性CD16嵌合受体；CD16嵌合受体表示含有野生型CD16的嵌合受体，CD16F176V嵌合受体表示含有F176V突变的CD16的嵌合受体，CD16S197P嵌合受体表示含有S197P突变的CD16的嵌合受体。
实施例1慢病毒表达质粒的构建
由上海捷瑞生物工程有限公司合成野生型CD16.BBz、F176V突变CD16.BBz、S197P突变CD16.BBz和hrCD16.BBz基因(分别如SEQ ID NO:13-16所示)，并克隆至空白慢病毒表达质粒(pXW-EF1α-MCS-P2A-EGFP)分别获得pXW-EF1α-CD16.BBz-P2A-EGFP、pXW-EF1α-CD16F176V.BBz-P2A-EGFP、pXW-EF1α-CD16S197P.BBz-P2A-EGFP和pXW-EF1α-hrCD16.BBz-P2A-EGFP重组慢病毒表达质粒，质粒图谱如图1所示。
实施例2 HEK 293T细胞的慢病毒表达质粒转染
实验前一天，在12孔平底细胞培养板上接种HEK293T细胞，4×10 5个细胞/2mL/孔。第二天，通过TurboFect转染试剂分别转染pXW-CD16.BBz和XW-hrCD16.BBz慢病毒表达质粒，质粒总量合计2μg/孔。以质粒量(μg)：转染试剂(μL)＝1：2的比例加入TurboFect转染试剂4μL，新鲜配置的质粒转染复合物于室温孵育15～20min后加至上述细胞培养板中。置于37℃，5％CO 2条件下继续培养48小时，500×g，室温离心5min后，弃除上清，收集细胞备用。
实施例3 hrCD16嵌合受体高效地结合抗体Fc段
采用实施例2所述的方法制备分别表达野生型CD16嵌合受体(CD16.BBz)和表达hrCD16嵌合受体(hrCD16.BBz)的HEK293T细胞。
分别取1×10 5个野生型CD16嵌合受体和hrCD16嵌合受体修饰的HEK293T细胞置于1.5mL EP管中，加入荧光染料AF647标记的EGFR靶向抗体，调整抗体的工作浓度为0.5、1和5μg/mL，4℃避光孵育30min，结束后用FACS缓冲液(含有2％FBS的1×PBS)洗脱2遍，200～300μL FACS缓冲液重悬后使用流式细胞仪检测。
结果如下：图2A为表达野生型CD16嵌合受体和hrCD16嵌合受体的 HEK293T细胞分别结合EGFR靶向抗体Fc片段的流式图。在5μg/mL抗体的作用浓度下，表达hrCD16嵌合受体的HEK293T细胞结合EGFR靶向抗体Fc片段的效率远远高于表达野生型CD16嵌合受体的HEK293T细胞(百分比：85.8％vs 40.4％；荧光强度：3720vs 1033)。图2B为表达野生型CD16嵌合受体的HEK293T细胞和表达hrCD16嵌合抗体的HEK293T细胞结合EGFR靶向抗体Fc片段的效率统计图。在Fc片段结合率和Fc片段强度上，表达hrCD16嵌合受体的HEK293T细胞结合抗体Fc片段的能力优于表达野生型CD16嵌合抗体的HEK293T细胞，特别是在较高抗体作用浓度下，差异尤为明显。
实施例4慢病毒的包装、浓缩和滴度测定
1.1慢病毒的包装
HEK293T细胞处理：转染前24小时，收集处于对数生长期的HEK293T细胞，将其接种于10cm细胞培养皿中(6～8×10 6个细胞)，细胞在含有10mL的完全DMEM培养基中生长，置于37℃，5％CO 2条件下培养18～24小时，细胞密度达到70～90％以上即可进行转染。
HEK293T细胞转染：在15mL离心管中加入1mL基础DMEM培养基，按照质量比为慢病毒表达质粒:包装质粒psPAX2:包膜质粒PMD2.G＝1:3:1配制转染混合液，质粒总量合计15μg/皿。以质粒量(μg)：转染试剂(μL)＝1：2的比例加入TurboFect转染试剂30μL，室温孵育15～20min后加至细胞培养皿中，置于37℃，5％CO 2细胞培养箱中培养48小时并收集病毒上清，1000×g，4℃离心10min，收集上清病毒。
1.2慢病毒的浓缩
将离心收集的病毒上清采用0.45μm滤器过滤，加入1/3病毒上清体积的Lenti-X慢病毒浓缩试剂，颠倒混匀数次，4℃孵育过夜，2000×g，4℃离心45min，离心管底部可见白色沉淀，即为病毒。小心弃除上清，以原病毒上清的1/50～1/100体积的空白RPMI1640培养基重悬白色沉淀，分装并于-80℃冻存备用。
1.3慢病毒滴度测定将Jurkat T细胞按照1×10 5个/孔接种于96孔U底板上，将所收集的慢病毒浓缩液按10倍递增稀释。将100μL的病毒稀释液加入到相应孔中，加入促感染试剂硫酸鱼精蛋白并调整浓度至10μg/mL，
1000×g，32℃离心感染90min，过夜培养后，更换培养液，继续培养48小时，流式细胞仪检测荧光阳性细胞比例，采用下面的公式计算病毒滴度：病毒滴度(TU/mL)＝1×10 5×荧光阳性细胞比例/100×1000×相应的稀释倍数。
实施例5 T细胞的感染和扩增
在48孔平底细胞培养板中(含有1×10 6个预先活化的外周血单个核细胞)，加入实施例4包装、浓缩的慢病毒载体(LV-CD16BBz、LV-CD16F176V、LV-CD16S197P或LV-hrCD16.BBz)(MOI＝5～10)，添加促感染试剂硫酸鱼精蛋白10μg/mL，1000×g，32℃离心感染90min后过夜培养。第二天，更换培养液为新鲜的T细胞生长培养基继续培养。每2～3天添加新鲜的T细胞生长培养基，并调整细胞密度至0.5～2×10 6个细胞。自感染后6～7天，移除活化T细胞的免疫磁珠，继续培养扩增hrCD16嵌合受体修饰的T细胞，待细胞静息(移除免疫磁珠6～7天)后方可进行后续的功能实验。
实施例6 hrCD16嵌合受体有效地表达在原代T细胞表面
采用实施例4所述的方法制备表达hrCD16嵌合受体(hrCD16.BBz)修饰的原代T细胞。
取1×10 5个未转导T细胞/hrCD16修饰的T细胞置于1.5mL EP管中，加入0.5μL流式抗体PerCP/Cy5.5-anti-human CD16，室温避光孵育20min，结束后用FACS buffer(含有2％FBS的1×PBS)洗脱2遍，200～300μL FACS buffer重悬后使用流式细胞仪检测。
结果显示：图3A为hrCD16在原代T细胞上的表达流式图。三人份的hrCD16嵌合受体修饰的T细胞均正常表达hrCD16，阳性率在33.9～50.6％，荧光强度在3021～4987(图3B)。
实施例7表达hrCD16嵌合受体的T细胞联合高杀伤性FAP靶向抗体杀伤 FAP+肿瘤细胞
肿瘤细胞杀伤效率采用实时无标记细胞分析(RTCA，Real Time Cellular Analysis)技术进行检测。首先，将1×10 4个A549-FAP(FAP修饰的人肺癌细胞)接种于16孔E-Plate电极板上，每孔100μL培养基。使用RTCA系统动态监测细胞生长8～9小时。以效应细胞：靶细胞＝3：1的比例加入表达hrCD16嵌合受体的T细胞至含有靶细胞的孔中，并分别加入FAP靶向抗体和高杀伤性FAP靶向抗体，抗体工作浓度调整为1μg/mL，每隔15分钟记录一次测定结果，连续记录24小时。
结果如图4所示：图4实线为表达hrCD16嵌合受体的T细胞联合FAP靶向抗体杀伤肿瘤细胞的靶细胞生长曲线，虚线为表达hrCD16嵌合受体的T细胞杀伤肿瘤细胞的靶细胞生长曲线，结果显示表达hrCD16嵌合受体的 T细胞联合FAP靶向抗体组的靶细胞生长曲线与表达hrCD16嵌合受体的T细胞的靶细胞生长曲线差异明显，表明表达hrCD16嵌合受体的T细胞联合FAP靶向抗体能够高效杀伤肿瘤细胞，肿瘤细胞杀伤率高达100％，有效抑制肿瘤细胞的生长，从而使得肿瘤细胞生长曲线快速下降。
实施例8表达hrCD16嵌合受体的T细胞联合高杀伤性AXL靶向抗体杀伤 AXL+肿瘤细胞
肿瘤细胞杀伤效率由实时无标记细胞分析(RTCA，Real Time Cellular Analysis)技术进行检测。首先，将1×10 4个U251(人脑胶质瘤细胞)或MDA-MB-231(人乳腺癌细胞)接种于16孔E-Plate电极板上，每孔100μL培养基。使用RTCA系统动态监测细胞生长18～20小时。以效应细胞：靶细胞＝3：1的比例加入表达hrCD16嵌合受体的T细胞至含有靶细胞的孔中，并加入高杀伤性AXL靶向抗体，抗体工作浓度调整为1μg/mL，每隔15分钟记录一次测定结果，连续记录24小时。
结果如图5所示：图5A实线为表达hrCD16嵌合受体的T细胞联合AXL靶向抗体杀伤U251肿瘤细胞的靶细胞生长曲线，虚线为表达hrCD16嵌合受体的T细胞联合无关抗体杀伤U251肿瘤细胞的靶细胞生长曲线，其余两条曲线分别为U251肿瘤细胞的生长曲线和表达hrCD16嵌合受体的T细胞杀伤肿瘤细胞的靶细胞生长曲线，结果显示只有hrCD16嵌合受体的T细胞联合AXL靶向抗体才能使得U251肿瘤细胞生长曲线快速下降，有效杀伤肿瘤细胞，杀伤率高达94.5％；图5B实线为表达hrCD16嵌合受体的T细胞联合AXL靶向抗体杀伤MDA-MB-231肿瘤细胞的靶细胞生长曲线，虚线为表达hrCD16嵌合受体的T细胞联合无关抗体杀伤MDA-MB-231肿瘤细胞的靶细胞生长曲线，其余两条曲线分别为MDA-MB-231肿瘤细胞的生长曲线和表达hrCD16嵌合受体的T细胞杀伤肿瘤细胞的靶细胞生长曲线，结果显示只有hrCD16嵌合受体的T细胞联合MDA-MB-231靶向抗体才能使得MDA-MB-231肿瘤细胞生长曲线快速下降，有效杀伤肿瘤细胞，杀伤率高达91.8％。
实施例9表达hrCD16嵌合受体的T细胞联合高杀伤性CD47靶向抗体杀 伤CD47+肿瘤细胞
肿瘤细胞杀伤效率由采用实时无标记细胞分析(RTCA，Real Time Cellular Analysis)技术进行检测。首先，将1×10 4个U251(人脑胶质瘤细胞)或MDA-MB-231(人乳腺癌细胞)接种于16孔E-Plate电极板上，每孔100μL培养基。使用RTCA系统动态监测细胞生长18～20小时。以效应细胞： 靶细胞＝3：1的比例加入表达hrCD16嵌合受体的T细胞至含有靶细胞的孔中，并加入高杀伤性CD47靶向抗体，抗体工作浓度调整为1μg/mL，每隔15分钟记录一次测定结果，连续记录24小时。
结果如图6所示，图6A实线为表达hrCD16嵌合受体的T细胞联合CD47靶向抗体杀伤U251肿瘤细胞的靶细胞生长曲线，虚线为表达hrCD16嵌合受体的T细胞联合无关抗体杀伤U251肿瘤细胞的靶细胞生长曲线，其余两条曲线分别为U251肿瘤细胞的生长曲线和表达hrCD16嵌合受体的T细胞杀伤肿瘤细胞的靶细胞生长曲线，结果显示只有hrCD16嵌合受体的T细胞联合CD47靶向抗体才能使得U251肿瘤细胞生长曲线快速下降，有效杀伤肿瘤细胞，杀伤率高达94.2％；图6B实线为表达hrCD16嵌合受体的T细胞联合CD47靶向抗体杀伤MDA-MB-231肿瘤细胞的靶细胞生长曲线，虚线为表达hrCD16嵌合受体的T细胞联合无关抗体杀伤MDA-MB-231肿瘤细胞的靶细胞生长曲线，其余两条曲线分别为MDA-MB-231肿瘤细胞的生长曲线和表达hrCD16嵌合受体的T细胞杀伤肿瘤细胞的靶细胞生长曲线，结果显示只有hrCD16嵌合受体的T细胞联合MDA-MB-231靶向抗体才能使得MDA-MB-231肿瘤细胞生长曲线快速下降，有效杀伤肿瘤细胞。
实施例10表达不同嵌合受体的T细胞联合高杀伤性CD47靶向抗体杀伤 CD47+肿瘤细胞
肿瘤细胞杀伤效率由采用基于荧光素酶的细胞杀伤检测方法(Luciferase-based cytotoxicity assay)进行评估。首先，将1×10 4个SKOV3-Luc(荧光素酶基因修饰的人卵巢癌细胞)接种于96孔平底板上，每孔100μL培养基，置于37℃，5％CO 2细胞培养箱中培养18～20小时。第二天，以效应细胞：靶细胞＝1：1的比例加入表达不同嵌合受体的T细胞至含有靶细胞的孔中，包括CD16.BBz、CD16F176V.BBz、CD16S197P.BBz和hrCD16.BBz，并加入高杀伤性CD47靶向抗体，抗体工作浓度调整为2μg/mL，置于37℃，5％CO 2细胞培养箱中继续培养18～20小时，共培养结束后使用
Figure PCTCN2021104764-appb-000002
微孔板发光检测仪检测靶细胞的荧光素酶活力值。细胞杀伤率的计算如下：
细胞杀伤率(％)＝(靶细胞组荧光素酶活力值-实验组荧光素酶活力值)/靶细胞组荧光素酶活力值×100
结果如图7所示，CD47靶向抗体联合表达不同嵌合受体的T细胞可以杀伤CD47+肿瘤细胞，hrCD16修饰T细胞处理组具有最少的肿瘤存活(图7A)和最高的肿瘤细胞杀伤效率(图7B)，优于野生型CD16、单一突变型CD16F176V和CD16S197P。
实施例11表达hrCD16嵌合受体的T细胞联合高杀伤性HER2靶向抗体杀 伤HER2+肿瘤细胞
肿瘤细胞杀伤效率由采用基于荧光素酶的细胞杀伤检测方法(Luciferase-based cytotoxicity assay)进行评估。首先，将1×10 4个NCI-H292-Luc(荧光素酶基因修饰的人肺癌细胞)或SKOV3-Luc(荧光素酶基因修饰的人卵巢癌细胞)接种于96孔平底板上，每孔100μL培养基，置于37℃，5％CO 2细胞培养箱中培养18～20小时。第二天，以效应细胞：靶细胞＝3：1的比例加入表达hrCD16嵌合受体的T细胞至含有靶细胞的孔中，并加入高杀伤性HER2靶向抗体，抗体工作浓度调整为1μg/mL，置于37℃，5％CO 2细胞培养箱中继续培养18～20小时，共培养结束后使用
Figure PCTCN2021104764-appb-000003
微孔板发光检测仪检测靶细胞的荧光素酶活力值。细胞杀伤率的计算如下：
细胞杀伤率(％)＝(靶细胞组荧光素酶活力值-实验组荧光素酶活力值)/靶细胞组荧光素酶活力值×100
结果如图8所示，表达hrCD16嵌合受体的T细胞针对肿瘤细胞具有一定的非特异杀伤作用，而联合无关对照抗体组的杀伤效率与单独加入表达hrCD16嵌合受体的T细胞组相当，提示无关对照抗体的ADCC作用极弱。HER2靶向抗体联合表达hrCD16嵌合受体的T细胞可以有效地杀伤肿瘤细胞，肺癌细胞(NCI-H292)和卵巢癌细胞(SKOV3)杀伤率分别高达92.2％和89.4％，从而有效清除肿瘤细胞。
实施例12表达不同嵌合受体的T细胞联合高杀伤性HER2靶向抗体杀伤 HER2+肿瘤细胞
肿瘤细胞杀伤效率由采用基于荧光素酶的细胞杀伤检测方法(Luciferase-based cytotoxicity assay)进行评估。首先，将1×10 4个SKOV3-Luc(荧光素酶基因修饰的人卵巢癌细胞)接种于96孔平底板上，每孔100μL培养基，置于37℃，5％CO 2细胞培养箱中培养18～20小时。第二天，以效应细胞：靶细胞＝1：1的比例加入表达不同嵌合受体的T细胞至含有靶细胞的孔中，包括CD16.BBz、CD16F176V.BBz、CD16S197P.BBz和hrCD16.BBz，并加入高杀伤性CD47靶向抗体，抗体工作浓度调整为2μg/mL，置于37℃，5％CO 2细胞培养箱中继续培养18～20小时，共培养结束后使用
Figure PCTCN2021104764-appb-000004
微孔板发光检测仪检测靶细胞的荧光素酶活力值。细胞杀伤率的计算如下：
细胞杀伤率(％)＝(靶细胞组荧光素酶活力值-实验组荧光素酶活力值)/靶细胞组荧光素酶活力值×100
结果如图9所示，HER2靶向抗体联合表达不同嵌合受体的T细胞可以杀伤HER2+肿瘤细胞，hrCD16修饰T细胞处理组具有最少的肿瘤存活(图8A)和最高的肿瘤细胞杀伤效率(图8B)，优于野生型CD16、单一突变型CD16F176V和CD16S197P。
实施例13表达hrCD16嵌合受体的T细胞联合高杀伤性EGFR靶向抗体杀 伤肿瘤细胞
肿瘤细胞杀伤效率由采用基于荧光素酶的细胞杀伤检测方法(Luciferase-based cytotoxicity assay)进行评估。首先，将1×10 4个NCI-H292-Luc(荧光素酶基因修饰的人肺癌细胞)或SKOV3-Luc(荧光素酶基因修饰的人卵巢癌细胞，EGFR+)接种于96孔平底板上，每孔100μL培养基，置于37℃，5％CO 2细胞培养箱中培养18～20小时。第二天，以效应细胞：靶细胞＝3：1的比例加入表达hrCD16嵌合受体的T细胞至含有靶细胞的孔中，并加入高杀伤性EGFR靶向抗体，抗体工作浓度调整为1μg/mL，置于37℃，5％CO 2细胞培养箱中继续培养18～20小时，共培养结束后使用
Figure PCTCN2021104764-appb-000005
微孔板发光检测仪检测靶细胞的荧光素酶活力值。细胞杀伤率的计算如下：
细胞杀伤率(％)＝(靶细胞组荧光素酶活力值-实验组荧光素酶活力值)/靶细胞组荧光素酶活力值×100
结果如图10所示，表达hrCD16嵌合受体的T细胞针对肿瘤细胞具有一定的非特异杀伤作用，而联合无关对照抗体组的杀伤效率与单独加入表达hrCD16嵌合受体的T细胞组相当，提示无关对照抗体的ADCC作用极弱。改造后的高杀伤性EGFR靶向抗体联合表达hrCD16嵌合受体的T细胞可以有效地杀伤肿瘤细胞，肺癌细胞和卵巢癌细胞杀伤率分别高达92.2％和86.1％，从而有效清除肿瘤细胞。
实施例14表达hrCD16嵌合受体的T细胞联合SARS-CoV-2刺突蛋白靶向 抗体杀伤Spike+细胞
细胞杀伤效率由实时无标记细胞分析(RTCA，Real Time Cellular Analysis)技术进行检测。首先，将1×10 4个刺突蛋白阳性A549-Spike细胞接种于16孔E-Plate电极板上，每孔100μL培养基。使用RTCA系统动态监测细胞生长18～20小时。以效应细胞：靶细胞＝2：1的比例加入表达hrCD16嵌合受体的T细胞至含有靶细胞的孔中，并加入SARS-CoV-2刺突 蛋白靶向抗体SARS-CoV-2-505-5和SARS-CoV-2-553-20，抗体工作浓度调整为1μg/mL，每隔15分钟记录一次测定结果，连续记录24小时。
结果如图11所示：表达hrCD16嵌合受体的T细胞针对刺突蛋白阳性A549-Spike具有一定的非特异杀伤作用(40％)。SARS-CoV-2刺突蛋白靶向抗体SARS-CoV-2-505-5和SARS-CoV-2-553-20联合表达hrCD16嵌合受体的T细胞可以有效地杀伤刺突蛋白阳性A549-Spike细胞，靶细胞杀伤率分别高达84％。
实施例15表达hrCD16嵌合受体的T细胞联合高杀伤性CD47靶向抗体体 内抑瘤效果
将NCI-H292-Luc肺癌细胞接种于B-NDG免疫缺陷小鼠的左下腹，2×10 6个细胞/只，待肿瘤生长4天，将荷瘤小鼠随机分成以下3组，表达hrCD16嵌合受体的T细胞注射组，3只；CD47靶向抗体联合未转导T细胞注射组，3只；CD47靶向抗体联合表达hrCD16嵌合受体的T细胞注射组，3只。采用小动物活体成像系统监测肿瘤生长情况。
给药方法：抗体给药：每次每只小鼠腹腔注射50μg(125μL)，分别于肿瘤接种后第4天、第7天和第18天注射。细胞给药：每次每只小鼠腹腔注射未转导T细胞或表达hrCD16嵌合受体的T细胞2×10 6个/125μL，分别于抗体给药后一天回输，合计3次。
结果如图12显示，CD47靶向抗体联合表达hrCD16嵌合受体的T细胞注射组的腹腔肿瘤生长明显抑制，有一只荷瘤小鼠治愈。其余两组的腹腔肿瘤随时间延长而不断增长(图12A)。CD47靶向抗体联合表达hrCD16嵌合受体的T细胞注射组的小鼠全部存活，而其余两组小鼠于肿瘤接种第40天全部死亡(图12B)。
虽然以上仅描述了本发明的具体实施方式的范例，但本领域的技术人员应当理解，以上这些仅为举例说明，本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改，但这些变更或修改均应落入本发明的保护范围。

Claims (10)

  1. 一种嵌合受体，其特征在于，所述嵌合受体包含：
    (1)Fc片段的胞外识别域；
    (2)胞外间隔区；
    (3)跨膜区；和
    (4)胞内信号传导结构域；
    任选地，所述嵌合受体还包含：
    (5)一个或多个共刺激信号传导结构域；和/或
    (6)一个或多个细胞因子受体信号传导结构域；
    其中，所述Fc片段的胞外识别域为特异性结合抗体Fc片段的CD16胞外域、CD32胞外域、CD64胞外域、CD89胞外域、CD23胞外域、FcεRI胞外域、FcRn胞外域、Fc结合抗体、Protein A、Protein G或其突变体或多个重复串联胞外域；
    优选地，所述Fc片段的胞外识别域为CD16胞外域；
    优选地，所述Fc片段的胞外识别域为野生型CD16胞外域、F176V突变型CD16胞外域、S197P突变型CD16胞外域或F176V和S197P双突变型CD16胞外域，其包含如SEQ ID NO:1-4中任一项所示的氨基酸序列；
    更优选地，所述Fc片段的胞外识别域为F176V和S197P双突变型CD16胞外域，其包含如SEQ ID NO:4所示的氨基酸序列。
  2. 根据权利要求1所述的嵌合受体，其特征在于，所述胞外间隔区包含选自以下的一种或多种：
    (1)抗体IgG4的铰链区及其突变体；
    (2)抗体IgG4的铰链区及其突变体和CH2区；
    (3)抗体IgG4的铰链区及其突变体、CH2区和CH3区；
    (4)抗体IgG1的铰链区及其突变体；
    (5)抗体IgG1的铰链区及其突变体和CH2区；
    (6)抗体IgG1的铰链区及其突变体、CH2区和CH3区；
    (7)免疫球蛋白Fc受体的铰链区：CD64、CD32、CD16、CD89、FcεRI、FcεRII(CD23)和FcRn；
    (8)CD28铰链区、CD137铰链区、CD8铰链区、CD4铰链区、PD-1铰链区和CTLA-4铰链区；和
    (9)以上的任何一种组合；
    优选地，所述胞外间隔区为CD8铰链区；更优选地，所述胞外间隔区包含如SEQ ID NO:5所示的氨基酸序列；
    优选地，所述跨膜区选自以下一种或多种：T细胞受体复合物的CD3ξ链的跨膜区、CD28跨膜区、CD137跨膜区、CD8跨膜区、CD4跨膜区、PD-1跨膜区、CTLA-4跨膜区、免疫球蛋白Fc受体跨膜区及其组合；更优选地，所述跨膜区为CD8跨膜区；进一步优选地，所述跨膜区包含如SEQ ID NO:6所示的氨基酸序列；
    优选地，所述共刺激信号传导结构域选自以下一种或多种的信号传导结构域：CD2、CD27、CD28、CD30、CD40、CD40L、CD137、CD134、CD278、GITR、TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR11、Dap10、ICAM-1、LFA-1、Lck、TNFRI、TNFRII、TIM-1、TIM-2、TIM-3、TIM-4及其组合；更优选地，所述共刺激信号传导结构域为CD137信号传导结构域；进一步优选地，所述共刺激信号传导结构域包含如SEQ ID NO:7所示的氨基酸序列；
    优选地，所述细胞因子受体信号传导结构域选自以下一种或多种的信号传导结构域：IL-2R、IL-7R、IL-15R、IL-18R、IL-21R、IL-23R及其组合；
    优选地，所述胞内信号传导结构域选自以下的一种或多种信号传导结构域：T细胞受体复合物的CD3ξ链、FcγRIII、FcεRI、Fc受体胞内信号传导域、携带免疫受体酪氨酸活化基序(ITAM)的信号传导结构域及其组合；更优选地，所述胞内信号传导结构域为CD3ξ链信号传导结构域；进一步优选地，所述胞内信号转导结构域如SEQ ID NO:8所示；
    优选地，所述受体由包含如SEQ ID NO:1-4中任一项所示的氨基酸序列的CD16胞外域、人CD8铰链区、人CD8跨膜区、人CD137共刺激信号传导结构域和CD3ξ链信号传导结构域组成；
    优选地，所述嵌合受体的氨基酸序列如SEQ ID NO:9-12中任一项所示。
    更优选地，所述受体由包含如SEQ ID NO:4所示的氨基酸序列的CD16胞外域、人CD8铰链区、人CD8跨膜区、人CD137共刺激信号传导结构域和CD3ξ链信号传导结构域组成；更进一步优选地，所述嵌合受体的氨基酸序列如SEQ ID NO:12所示。
  3. 一种多核苷酸，其特征在于，所述多核苷酸编码如权利要求1或2所述的嵌合受体；
    优选地，所述多核苷酸包含如SEQ ID NO:13-16中任一项所示的核苷酸 序列；
    更优选地，所述多核苷酸包含如SEQ ID NO:16所示的核苷酸序列
  4. 一种载体，所述载体包含如权利要求3所述的多核苷酸；
    优选地，所述载体共表达细胞因子、趋化因子、趋化因子受体、免疫检查点阻断抗体或其组合；
    更优选地，所述细胞因子选自IL-2、IL-7、IL-15、IL-21、IL-12、IL-18、IL-23及其组合；所述趋化因子选自CXCL9、CXCL10、CXCL11、CCL19、CCL20和CCL21；所述趋化因子受体选自CCR1、CCR3、CCR9、CXCR1和CXCR2；所述免疫检查点阻断抗体选自CTLA-4阻断抗体、PD-1阻断抗体、PD-L1阻断抗体、LAG-3阻断抗体、Tim-3阻断抗体、TIGIT阻断抗体、VISTA阻断抗体、Siglec-15阻断抗体及其组合；
    优选地，所述载体为病毒；
    更优选地，所述病毒选自逆转录病毒、慢病毒、腺病毒、腺相关病毒、痘病毒和疱疹病毒。
  5. 一种表达如权利要求1或2所述的嵌合受体的免疫细胞；
    优选地，所述免疫细胞选自T细胞、天然杀伤细胞(Natural killer cells，NK)、固有淋巴细胞((Innate lymphoid cells,ILC)、造血干细胞、胚胎干细胞和多能干细胞；
    更优选地，所述T细胞选自未经分选纯化的T细胞、分选纯化的T细胞和天然杀伤T细胞(Natural killer T cells，NKT)；
    进一步优选地，所述分选纯化的T细胞选自分选纯化的PD-1 +T细胞、分选纯化的CD137 +T细胞、分选纯化的CD160 +T细胞、分选纯化的纯真T细胞(T naive)、分选纯化的中央记忆型T细胞(T CM)、分选纯化的效应记忆型T细胞(T EM)、分选纯化的效应T细胞(T EMRA)、分选纯化的过渡记忆型T细胞(Transitional Memory T cells，T TM)和分选纯化的组织记忆型T细胞(Tissue residential memory T cells，T RM)。
  6. 一种组合，所述组合包含权利要求5所述的免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体；
    优选地，所述肿瘤抗原选自以下的一种或多种：CD19、BCMA、CD20、CD22、CD30、CD33、CD38、CD47、CD70、CD117、CD123、CD133、CD138、 CD147、CD171、NKG2DL、HER2、MUC1、MUC16、CEA、EpCAM、IL-13Rα2、EGFR、EGFRvIII、GD2、DR5、EphA2、FRα、PSCA、PSMA、TARP、cMet、VEGFR2、BCMA、CTLA-4、PD-L1、AFP、GPC3、AXL、ROR1、ROR2、FAP、Mesothelin、DLL3和CLDN18；
    更优选地，所述肿瘤抗原选自以下的一种或多种：HER2、EGFR、CD47、AXL和FAP；
    优选地，所述病毒抗原选自以下的一种或多种：人类获得性免疫缺陷病毒HIV-1的gp120、乙型肝炎病毒HBV的表面抗原、流感病毒的血凝素或神经氨酸酶、埃博拉病毒的刺突蛋白、严重急性呼吸系统综合征冠状病毒SARS-CoV的表面刺突蛋白，中东呼吸综合征冠状病毒MERS-CoV的表面刺突蛋白和新型冠状病毒SARS-CoV-2的表面刺突蛋白；
    更优选地，所述病毒抗原选自新型冠状病毒SARS-CoV-2的表面刺突蛋白。
  7. 如权利要求1或2所述的嵌合受体、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞和肿瘤抗原靶向抗体或病毒抗原靶向抗体的组合在制备用于治疗肿瘤或病毒感染性疾病的药物中的用途；
    优选地，所述肿瘤选自以下的一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤和平滑肌瘤；
    优选地，所述病毒感染性疾病选自以下的一种或多种：人类获得性免疫缺陷综合征、乙型肝炎、流感、埃博拉病毒病、严重急性呼吸系统综合征(SARS)、中东呼吸综合征(MERS)和新型冠状病毒肺炎。
  8. 一种治疗肿瘤的方法，所述方法包括给予受试者治疗有效量的如权利要求1或2所述的嵌合受体、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞和肿瘤抗原靶向抗体的组合；
    优选地，所述肿瘤选自以下一种或多种：淋巴瘤、神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌、脑癌、横纹肌瘤、平滑肌瘤；
    优选地，所述肿瘤为FAP+肿瘤，所述方法包括给予患者如权利要求5 所述的免疫细胞和FAP靶向抗体的组合；
    优选地，所述肿瘤为AXL+肿瘤，所述方法包括给予患者如权利要求5所述的免疫细胞和AXL靶向抗体的组合；
    优选地，所述肿瘤为CD47+肿瘤，所述方法包括给予患者如权利要求5所述的免疫细胞和CD47靶向抗体的组合；
    优选地，所述肿瘤为HER2+肿瘤，所述方法包括给予患者如权利要求5所述的免疫细胞和HER2靶向抗体的组合；
    优选地，所述肿瘤为EGFR+肿瘤，所述方法包括给予患者如权利要求5所述的免疫细胞和EGFR靶向抗体的组合。
  9. 一种治疗病毒感染性疾病的方法，所述方法包括给予受试者治疗有效量的如权利要求1或2所述的嵌合受体、权利要求5所述的免疫细胞、权利要求6所述的免疫细胞和病毒抗原靶向抗体的组合；
    优选地，所述病毒选自以下的一种或多种：人类获得性免疫缺陷病毒HIV-1，乙型肝炎病毒HBV、流感病毒，埃博拉病毒、严重急性呼吸系统综合征冠状病毒SARS-CoV，中东呼吸综合征冠状病毒MERS-CoV和新型冠状病毒SARS-CoV-2；
    优选地，所述疾病为新型冠状病毒SARS-CoV-2感染性疾病，所述方法包括给予患者如权利要求5所述的免疫细胞和SARS-CoV-2表面刺突蛋白靶向抗体的组合。
  10. 如权利要求5所述的免疫细胞的制备方法，其特征在于，所述方法包括如下步骤：
    1)获取如权利要求1或2所述的嵌合受体的核酸序列；
    2)将嵌合受体的核酸序列克隆至慢病毒表达载体中，获得编码嵌合受体的慢病毒表达质粒；
    3)将慢病毒表达质粒、骨架质粒和包膜质粒共转染至HEK293T细胞，包装并获得慢病毒颗粒，经离心浓缩后获得慢病毒浓缩液；
    4)将慢病毒转导免疫细胞，从而获得表达嵌合受体的免疫细胞；
    优选地，所述免疫细胞为T细胞。
PCT/CN2021/104764 2020-07-06 2021-07-06 一种嵌合受体及其应用 WO2022007795A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010640526.1 2020-07-06
CN202010640526 2020-07-06

Publications (1)

Publication Number Publication Date
WO2022007795A1 true WO2022007795A1 (zh) 2022-01-13

Family

ID=78246083

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/104764 WO2022007795A1 (zh) 2020-07-06 2021-07-06 一种嵌合受体及其应用

Country Status (2)

Country Link
CN (1) CN113583139A (zh)
WO (1) WO2022007795A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838439A (zh) * 2022-12-06 2023-03-24 上海恩凯细胞技术有限公司 嵌合转换受体基因修饰的nk细胞制备方法及应用
CN116574748A (zh) * 2023-07-10 2023-08-11 昆明医科大学 一种用于靶向KRAS高频突变肿瘤的嵌合型nTCR-T构建方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114250201A (zh) * 2021-11-16 2022-03-29 上海市第十人民医院 一种car-nk细胞及其制备方法与应用
CN114574447B (zh) * 2022-03-10 2023-10-20 中国海洋大学 Her2靶向的增强型抗肿瘤nk细胞、其制备方法及其应用
CN116948012A (zh) * 2022-04-13 2023-10-27 星奕昂(上海)生物科技有限公司 增强细胞功能的cd16抗剪切突变体
CN114478806B (zh) * 2022-04-14 2022-07-01 呈诺再生医学科技(北京)有限公司 一种提升免疫细胞杀伤活性的嵌合受体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105683214A (zh) * 2013-10-17 2016-06-15 新加坡国立大学 触发针对多种肿瘤的抗体依赖细胞毒性的嵌合受体
CN107074969A (zh) * 2014-09-09 2017-08-18 优努姆治疗公司 嵌合受体及其在免疫治疗中的应用
WO2019126748A1 (en) * 2017-12-22 2019-06-27 Fate Therapeutics, Inc. Enhanced immune effector cells and use thereof
CN111944062A (zh) * 2019-12-09 2020-11-17 深圳市体内生物医药科技有限公司 一种识别Fc片段的嵌合抗原受体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105683214A (zh) * 2013-10-17 2016-06-15 新加坡国立大学 触发针对多种肿瘤的抗体依赖细胞毒性的嵌合受体
CN107074969A (zh) * 2014-09-09 2017-08-18 优努姆治疗公司 嵌合受体及其在免疫治疗中的应用
WO2019126748A1 (en) * 2017-12-22 2019-06-27 Fate Therapeutics, Inc. Enhanced immune effector cells and use thereof
CN111944062A (zh) * 2019-12-09 2020-11-17 深圳市体内生物医药科技有限公司 一种识别Fc片段的嵌合抗原受体及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN 17 January 2022 (2022-01-17), ANONYMOUS : "low affinity immunoglobulin gamma Fc region receptor III-A isoform c precursor [Homo sapiens]", XP055885917, retrieved from NCBI Database accession no. NP_000560 *
DATABASE PROTEIN 2 September 2016 (2016-09-02), ANONYMOUS : "FCGR3A protein [synthetic construct]", XP055885921, retrieved from NCBI Database accession no. AAH17865 *
F. OCHI, H. FUJIWARA, K. TANIMOTO, H. ASAI, Y. MIYAZAKI, S. OKAMOTO, J. MINENO, K. KUZUSHIMA, H. SHIKU, J. BARRETT, E. ISHII, M. Y: "Gene-Modified Human / -T Cells Expressing a Chimeric CD16-CD3 Receptor as Adoptively Transferable Effector Cells for Anticancer Monoclonal Antibody Therapy", CANCER IMMUNOLOGY RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 2, no. 3, 1 March 2014 (2014-03-01), US , pages 249 - 262, XP055223149, ISSN: 2326-6066, DOI: 10.1158/2326-6066.CIR-13-0099-T *
YAWU JING, ZHENYA NI, JIANMING WU, LEEANN HIGGINS, TODD W. MARKOWSKI, DAN S. KAUFMAN, BRUCE WALCHECK: "Identification of an ADAM17 Cleavage Region in Human CD16 (FcγRIII) and the Engineering of a Non-Cleavable Version of the Receptor in NK Cells", PLOS ONE, vol. 10, no. 3, pages e0121788, XP055218471, DOI: 10.1371/journal.pone.0121788 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115838439A (zh) * 2022-12-06 2023-03-24 上海恩凯细胞技术有限公司 嵌合转换受体基因修饰的nk细胞制备方法及应用
CN115838439B (zh) * 2022-12-06 2023-10-31 上海恩凯细胞技术有限公司 嵌合转换受体基因修饰的nk细胞制备方法及应用
CN116574748A (zh) * 2023-07-10 2023-08-11 昆明医科大学 一种用于靶向KRAS高频突变肿瘤的嵌合型nTCR-T构建方法
CN116574748B (zh) * 2023-07-10 2023-09-12 昆明医科大学 一种用于靶向KRAS高频突变肿瘤的嵌合型nTCR-T构建方法

Also Published As

Publication number Publication date
CN113583139A (zh) 2021-11-02

Similar Documents

Publication Publication Date Title
WO2022007795A1 (zh) 一种嵌合受体及其应用
US11026975B2 (en) Chimeric antigen receptor (CAR) binding to BCMA, and uses thereof
JP6989634B2 (ja) Ｂ細胞成熟抗原を標的指向するキメラ抗原受容体
CN112142854B (zh) 免疫调节特异性嵌合抗原受体细胞及制备方法和应用
CN108384760B (zh) 携带cd20/cd19双特异性嵌合抗原受体的人t淋巴细胞及制备方法和应用
KR20180021137A (ko) 키메라 항원 수용체 (car), 조성물 및 이의 사용 방법
US11739297B2 (en) Method of increasing tumor killing activity of macrophages or monocytes comprising chimeric antigen receptor
WO2020108645A1 (en) Cd19-and bcma-based combined car-t immunotherapy
KR20240046645A (ko) 고형암을 위한 키메라 항원 수용체 및 키메라 항원 수용체가 발현된 t 세포
BR112021001694A2 (pt) polipeptídeos do receptor de antígeno quimérico em combinação com moléculas de metabolismo trans que modulam o ciclo de krebs e seus usos terapêuticos
CN115074331A (zh) 靶向psca的嵌合抗原受体
JP2021525101A (ja) 改変された抗ｃｄ１９ ｃａｒ−ｔ細胞
CN107365798A (zh) 一种携带iCasp9自杀基因的CD19‑CAR‑T细胞及其应用
WO2019129174A1 (zh) 靶向cd19且高水平稳定表达cd40抗体的car-t细胞及其用途
WO2020019983A1 (zh) 一种用于治疗肿瘤的基因工程细胞
WO2021259334A1 (zh) 自我调节型嵌合抗原受体及其在肿瘤免疫中的应用
WO2022007796A1 (zh) 共表达IL-21和hrCD16嵌合受体的免疫细胞及其应用
WO2024055339A1 (zh) 用于制备和扩增通用型人源化抗cd19 car-nk细胞的方法及其用途
US20220267420A1 (en) Foxp3 targeting agent compositions and methods of use for adoptive cell therapy
WO2024131966A1 (zh) 工程化的免疫细胞及其应用
KR20240034205A (ko) 항EGFRviii 항체, 폴리펩타이드, 상기 폴리펩타이드를 발현하는 세포, 상기 세포를 포함하는 의약 조성물, 상기 세포의 제조 방법 및 상기 폴리펩타이드를 코딩하는 염기서열을 포함하는 폴리뉴클레오티드 또는 벡터
KR20240035506A (ko) 키메라 항원 수용체, 상기 수용체를 발현하는 세포, 상기 세포를 포함하는 의약 조성물, 상기 세포의 제조 방법, 및 상기 키메라 항원 수용체를 코딩하는 염기서열을 포함하는 폴리뉴클레오티드 또는 벡터
CN114644716A (zh) 一种抗bxmas1嵌合抗原受体及其修饰的免疫细胞及应用
CN103483453A (zh) 结合egfr家族蛋白的嵌合抗原受体、其组合物及用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21838661

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21838661

Country of ref document: EP

Kind code of ref document: A1