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WO2019052562A1 - Il-4r的融合蛋白及其应用 - Google Patents

Il-4r的融合蛋白及其应用 Download PDF

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WO2019052562A1
WO2019052562A1 PCT/CN2018/106034 CN2018106034W WO2019052562A1 WO 2019052562 A1 WO2019052562 A1 WO 2019052562A1 CN 2018106034 W CN2018106034 W CN 2018106034W WO 2019052562 A1 WO2019052562 A1 WO 2019052562A1
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cancer
receptor
antigen
cells
cell
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French (fr)
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李宗海
王益
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Shanghai Cancer Institute
Carsgen Therapeutics Ltd
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Shanghai Cancer Institute
Carsgen Therapeutics Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4217—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4261—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 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 A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/53—Liver
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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

Definitions

  • the present invention is in the field of adoptive cell therapy; in particular, the present invention relates to an improved immune cell which can significantly enhance the anti-tumor ability of immune cells.
  • Cancer cells in solid tumors are capable of forming a tumor microenvironment around them to support the growth and metastasis of cancer cells.
  • the tumor microenvironment is the cell environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix and can promote tumor transformation, support tumor growth and invasion, and protect tumors from immunity.
  • the tumor and its surrounding microenvironment are closely related and constantly interacting. Tumors can affect their microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance. See Warts et al, "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy" Cancer Res, Vol. 72, pp. 2473-2480, 2012. Therefore, the treatment of solid tumors is often difficult to work.
  • the present invention provides a fusion protein comprising an IL-4 receptor (IL-4R) extracellular domain or a variant thereof and an IL-21 receptor (IL-21R) intracellular domain or Variants.
  • IL-4R IL-4 receptor
  • IL-21R IL-21 receptor
  • the fusion protein comprises:
  • IL-4 receptor IL-4R
  • transmembrane domain preferably a transmembrane region of IL-4R or a transmembrane region of IL-21R;
  • Iii IL-21 receptor (IL-21R) intracellular domain.
  • the nucleotide sequence encoding the extracellular domain of IL-4R has at least 90%, at least 91%, at least 92%, at least 93%, at least 94 of the sequence set forth in SEQ ID NO:2. %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • nucleotide sequence encoding the extracellular domain of IL-4R is the sequence set forth in SEQ ID NO:2.
  • the nucleotide sequence encoding the intracellular domain of IL-21R has at least 90%, at least 91%, at least 92%, at least 93%, at least the sequence set forth in SEQ ID NO:4. 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • nucleotide sequence encoding the intracellular domain of IL-21R is the sequence set forth in SEQ ID NO:4.
  • the fusion protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, and the sequence set forth in SEQ ID NO. At least 97%, at least 98%, or at least 99% identity.
  • the fusion protein has the sequence set forth in SEQ ID NO.
  • the IL-4R is selected from the group consisting of IL-4R ⁇ , IL-4R ⁇ c.
  • the IL-4R may bind to IL-4 or mutant IL-4 or IL-13 or mutant IL-13; preferably, the IL-4R may bind to IL-4 or a mutant IL-4.
  • the mutant IL-4 comprises a KFR variant, a KF variant, or an RGA variant.
  • each domain is joined together directly or through a linker molecule.
  • the invention provides a nucleic acid molecule encoding the fusion protein of the first aspect of the invention.
  • the present invention provides a vector comprising the nucleic acid molecule of the second aspect of the invention.
  • the invention provides a host cell comprising the vector of the third aspect of the invention.
  • the present invention provides an immune effector cell which expresses the fusion protein of the first aspect of the invention.
  • the immune effector cell is a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell or a bone marrow-derived phagocytic cell or a combination thereof; more preferably, The immune effector cells are T cells, natural killer (NK) cells, or natural killer T (NKT) cells; more preferably, the immune effector cells are T cells.
  • the immune effector cells are autologous cells, such as autologous T cells, autologous NK cells.
  • the immune effector cells are autologous T cells.
  • the immune effector cells are allogeneic cells, such as allogeneic T cells, allogeneic NK cells, or NK cell lines (eg, NK-92 cells).
  • the immune effector cell further expresses an exogenous receptor having a second extracellular binding domain that specifically binds to a tumor antigen, a second transmembrane domain, and a second Intracellular domain.
  • the immune effector cell further expresses an exogenous receptor having a second extracellular binding domain that specifically binds to a tumor antigen, a second transmembrane domain, and a second Intracellular domain; preferably, the tumor antigen is different from the binding antigen of the IL-4 receptor.
  • the exogenous receptor is selected from the group consisting of a chimeric antigen receptor (CAR), a modified T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), and a T cell antigen. Coupler (TAC) or a combination thereof.
  • the exogenous receptor is a chimeric antigen receptor.
  • the fusion protein is constitutively expressed.
  • the fusion protein is inducible.
  • the exogenous receptor is a chimeric antigen receptor
  • the second extracellular domain, the second transmembrane domain, and the second intracellular domain of the chimeric antigen receptor have the following Features:
  • the second extracellular binding domain comprises: an antibody, an antibody fragment, an scFv, an Fv, a Fab, a (Fab') 2, a single domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain , or a natural ligand of the corresponding antigen, or a combination thereof; and/or
  • the second transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of alpha, beta or ⁇ chain of T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR , CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1
  • the second intracellular domain comprises: a primary signaling domain and/or a costimulatory signaling domain, wherein: (1) the primary signaling domain comprises a selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , a functional signaling domain of a protein of CD3 ⁇ , CD3 ⁇ , common FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10, and DAP12, or a combination thereof; and/or (2) said costimulatory signaling The domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) ), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVE
  • the chimeric antigen receptor comprises:
  • the tumor antigen comprises:
  • Thyroid stimulating hormone receptor 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); interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); interleukin 11 receptor alpha (IL-11R ⁇ ); Prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; Protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR- ⁇ ); stage-
  • the tumor antigen is a solid tumor antigen
  • the solid tumor antigen is selected from the group consisting of prostate specific membrane antigen, carcinoembryonic antigen, IL13Ralpha, HER-2, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3 (GPC3), EphA2, HER3, EpCAM, MUC16, MUC1, claudin 18.2, folate receptor, claudin 6, CD138, MAGE3, ASGPR1 or CDH16, more preferably, the solid tumor antigen is GPC3.
  • the solid tumor is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, and skin.
  • CNS central nervous system
  • the second extracellular binding domain has at least 90% (eg, at least 91%, 92%, 93%, 94) of the sequence set forth in SEQ ID NO: 7, 20, 21, 22, or 23. %, 95%, 96%, 97%, 98% or 99%) sequences of identity.
  • the second extracellular binding domain of the immune effector cell has at least 90% of the sequence set forth in SEQ ID NO: 7 (eg, at least 91%, 92%, 93%, 94%, 95%) , 96%, 97%, 98%, or 99%) sequences of identity.
  • the coding nucleotide sequence of the second transmembrane domain of the chimeric antigen receptor has at least 90% of the sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 29 or 30 (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity.
  • the nucleotide sequence encoding the second intracellular domain of the chimeric antigen receptor comprises the nucleotide sequence set forth in SEQ ID NOs: 31 and 33, or contains SEQ ID NO: 31 and
  • the nucleotide sequence shown at 33 has a nucleotide sequence of at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity.
  • the nucleotide sequence encoding the second intracellular domain of the chimeric antigen receptor comprises the nucleotide sequence set forth in SEQ ID NOs: 32 and 33, or contains SEQ ID NO: 32 and The nucleotide sequence shown at 33 has a nucleotide sequence of at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity.
  • the nucleotide sequence encoding the second intracellular domain of the chimeric antigen receptor comprises the nucleotide sequence set forth in SEQ ID NOs: 31, 32, and 33, or contains SEQ ID NO: Nucleotides having a nucleotide sequence of 31, 32 and 33 having at least 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity Acid sequence.
  • the nucleotide sequence encoding the second intracellular domain of the chimeric antigen receptor comprises the nucleotide sequence set forth in SEQ ID NO: 33 or comprises the core set forth in SEQ ID NO:
  • the nucleotide sequence has a nucleotide sequence that is at least 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical.
  • the chimeric antigen receptor has the sequence set forth in SEQ ID NO: 9, 10, 11, 12 or at least 90% of the sequence set forth in SEQ ID NO: 9, 10, 11, and 12. At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • the chimeric antigen receptor and the fusion protein are encoded by the nucleotide sequence set forth in SEQ ID NO: 16, 17, 18, or 19 or by SEQ ID NO: 17, 18, or 19 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • the nucleotide sequence is encoded.
  • the antigen is a tumor antigen or a pathogenic microorganism antigen.
  • the pathogenic microorganism comprises a virus, a bacterium, a fungus, a protozoa or a parasite; more preferably, the pathogenic microorganism is a virus; or more preferably, the pathogenic microorganism is selected from the group consisting of a cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and influenza virus.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the third aspect of the invention
  • the present invention provides a method of inducing cell death, the method comprising administering to a subject in need thereof: the fusion protein of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, The vector of the third aspect of the invention, the host cell of the fourth aspect of the invention, the immune effector cell of the fifth aspect of the invention, or the pharmaceutical composition of the sixth aspect of the invention.
  • the present invention provides a method of treating cancer, comprising administering to a subject in need thereof: the fusion protein of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the present invention
  • the present invention provides a method of treating cancer, the method comprising fusing a malignant cell expressing IL-4 in a tumor microenvironment of a subject in need thereof with the fusion of the first aspect of the invention
  • the protein, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, the host cell of the fourth aspect of the invention, the immune effector cell of the fifth aspect of the invention or the sixth aspect of the invention The pharmaceutical composition of the aspect is in contact.
  • the malignant cells are contacted prior to the subject starting treatment.
  • the malignant cell is a malignant cell.
  • the present invention provides a method of treating a hyperproliferative or differentiation disorder, the method comprising administering to a subject in need thereof: the fusion protein of the first aspect of the invention, the second of the present invention
  • the hyperproliferative or differentiation disorder is fibrosis or hyperplasia, an inflammatory disease, or an autoimmune disease.
  • the present invention provides the fusion protein of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, the host of the fourth aspect of the invention.
  • the cell, the immune effector cell of the fifth aspect of the invention or the pharmaceutical composition of the sixth aspect of the invention is for inducing cell death, or treating cancer, or treating hyperproliferative or differentiation in a patient in need thereof Disordered application.
  • the subject is a human.
  • the invention provides the fusion protein of the first aspect, the nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, or the fifth aspect Use of an immune effector cell for the preparation of a medicament for inducing cell death, or treating cancer, or treating a hyperproliferative or differentiation disorder in a patient in need thereof.
  • Figure 1A is a schematic diagram of the plasmid of chIL4-21R-CAR
  • Figure 1B is a plasmid map constructed by chIL4-21R-CAR
  • Figures 1C and 1D show the effect of different cells on the phosphorylation level of STAT3/5;
  • 2A, 2B show the ability of CAR-T cells to proliferate or survive under IL-4 stimulation
  • Figures 3A, 3B show in vitro cytotoxicity of different CAR-T cells
  • Figure 4A shows the expression level of Bcl-6 of chIL4-21R-CAR T cells stimulated by IL-4;
  • Figure 4B shows the expression level of T-bet of chIL4-21R-CAR T cells stimulated by IL-4;
  • Figure 4C shows the expression level of Blimp-1 of chIL4-21R-CAR T cells stimulated by IL-4;
  • Figure 4D shows the expression level of granzyme B of chIL4-21R-CAR T cells stimulated by IL-4;
  • Figure 4E shows the expression level of CD26 of chIL4-21R-CAR T cells stimulated by IL-4;
  • Figure 4F shows the expression level of ROR ⁇ t of chIL4-21R-CAR T cells stimulated by IL-4;
  • the expression level of GATA3 of chIL4-21R-CAR T cells under IL-4 stimulation is shown.
  • Figure 5A shows the cell population ratio of CD25+ in the GPC3-CAR-T cell group after IL-4 or IL-2 stimulation
  • Figure 5B shows the CD25+ in the chIL4-21R-CAR T cell group after IL-4 or IL-2 stimulation. Ratio of cell population
  • Figure 6 shows the results of detection of cell depletion markers
  • Figure 7A shows the persistence of IL-4 secreted tumor cells by chIL4-21R-CAR T cells
  • Figure 7B shows the chIL4-21R-CAR T cell depletion marker after persistent killing of IL-4 secreted tumor cells. Detection of matter;
  • Figure 8 shows the tumor killing effect of chIL4-21R-CAR T cells on the GPC-3-SMMC-7721 xenograft model
  • Figure 9A shows the survival of chIL4-21R-CAR T cells in experimental animals
  • Figure 9B shows the CD4/8 population ratio of chIL4-21R-CAR T cells surviving in experimental animals;
  • Figure 10 shows the tumor killing of the chIL4-21R-CAR T cells to the PLC/PRF/5 liver cancer xenograft model.
  • a fusion protein composed of IL4R and IL21R is co-expressed with a chimeric antigen receptor (CAR) on T cells, and the obtained T cells can be in the tumor microenvironment of solid tumors. It has significant anti-tumor ability.
  • CAR chimeric antigen receptor
  • Embodiments of the present invention provide a novel protocol to render tumor-reactive T cells resistant to immunosuppressive/inhibitory cytokines present in the tumor microenvironment.
  • the present invention relates to a fusion protein using a cytokine receptor to improve amplification and antitumor activity of tumor-specific immune effector cells.
  • Such a scheme includes natural or genetically modified tumor-specific T cells with a fusion protein of a cytokine receptor that binds to the inhibitory/repressor cytokine IL4 and makes their intracellular results Converted to IL21 immunostimulatory/activating signal, thus improving the efficacy of tumor-specific T cells.
  • the invention includes a vector, such as an exemplary bicistronic retroviral vector, which encodes the extracellular domain of an IL4 cytokine receptor fused to the signal transduction intracellular domain of the IL21 cytokine receptor.
  • a vector such as an exemplary bicistronic retroviral vector, which encodes the extracellular domain of an IL4 cytokine receptor fused to the signal transduction intracellular domain of the IL21 cytokine receptor.
  • the cancer in which the IL4 is present in the microenvironment comprises substantially all solid tumors.
  • Specific exemplary cancers include: fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphatic endothelial sarcoma, mesothelioma, Ewing's tumor, leiomyosarcoma , rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, carcinoma, bronchial carcinoma , medullary carcinoma, renal cell carcinoma, liver cancer, Nile tube cancer, choriocarcino
  • Embodiments of the invention can be used to modulate, for example, primary T cells, naturally occurring tumor antigen-specific cytotoxic T lymphocytes, and NK cells.
  • T/NK cells modified with the present invention can be used in an autologous or allogeneic environment.
  • chimeric molecules that can convert a negative immunomodulatory signal into a forward signal.
  • this approach involves fusing the IL-4 extracellular domain to the signal transduction intracellular domain of the IL-21 receptor. This approach can be used to make immune effector cells resistant to negative cytokine signaling that is often present in the tumor microenvironment.
  • the invention provides a fusion protein using a cytokine receptor, an extracellular domain of an IL4 cytokine receptor fused to IL-21, the intracellular domain of a receptor, to reverse the effects of the tumor microenvironment .
  • the present invention provides a fusion protein using a cytokine receptor, an extracellular domain of an IL4 cytokine receptor fused to the intracellular domain of the IL-21 receptor, which reverses the effects of the tumor microenvironment:
  • CD25 acts as a marker of T cell activation. After IL-4 stimulation, the ratio of CD25+ cell population in chIL4-21R-CAR T cell group was not significantly different from that in IL-2 stimulation group, indicating that the fusion protein chIL4-21R-CAR blocked the inhibition of T cell activation by IL-4. The effect, that is to say the induction of downstream signals of IL-4, was significantly inhibited in chIL4-21R-CAR-T cells.
  • IL-4 stimulation can induce high expression of T cells as PD-1, and TIM3 levels as important markers of T cell depletion.
  • the expression of the chIL4-21R-CAR fusion protein significantly inhibited the expression levels of PD-1 and TIM3, which are important markers of T cell depletion caused by IL-4 stimulation. This indicates that the IL-4 signal is converted into the IL-21 signal by the fusion protein chIL4-21R and functions to inhibit cell depletion and maintain cell survival, thereby improving the effect of killing tumor cells.
  • Bcl-6, T-bet and Blimp-1 are target genes of IL-21 signaling.
  • the expression levels of Bcl-6, T-bet and Blimp-1 in chIL4-21R-CAR-T cells were significantly higher than those in GPC3-CAR T cells, indicating that the chIL4-21R-CAR fusion protein can stimulate extracellular IL-4.
  • Transduction into IL-21 signaling, and by up-regulating the level of STAT3/5 phosphorylation in cells upregulates the level of granzyme B that acts on T cell activation to release cytoplasmic granules, suggesting that stimulation of extracellular IL-4 has been It is converted to activation of the IL-21 signaling pathway and exerts immunostimulatory/activating effects.
  • IL-4 stimulation can promote the expansion of chIL4-21R-CAR-T cells, indicating that the growth of chIL4-21R-CAR T cells in IL-4 environment can reverse the influence of tumor microenvironment, inhibition or resistance
  • the inhibitory cytokine signal is converted to promote immunostimulatory/activation signals with greater proliferation or viability.
  • chIL4-21R-CAR-T can significantly retain or even enhance cytotoxicity/killing ability; while for IL-4-expressing tumor cells, chIL4-21R-CAR-T also has Significant long-lasting ability to kill tumor cells; and chIL4-21R-CAR T can significantly inhibit tumor growth in mice, and the subpopulations of cells that survive in vivo are mainly CD4+T, CD8+ T cells; these results indicate that chIL4 -21R-CAR T cells can reverse the effects of tumor microenvironment after IL-4 stimulation or in the sustained secretion of IL-4, converting inhibitory or repressible cytokine signals into immune stimulatory/activation signals, Stronger cell killing ability.
  • tumor refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues.
  • tumor microenvironment refers to any and all elements of the tumor environment, including elements that create a structural and/or functional environment for a malignant process to survive and/or expand and/or spread.
  • IL-4R or "IL-4R” includes the native IL-4R protein as well as the variant IL-4R protein.
  • sequence of "native” or “wild-type” IL-4R refers to human IL-4R sequences, whether purified from natural sources or using recombinant techniques.
  • IL-4R functional portion of IL-4R
  • IL-4R refers to the full length of IL-4R that retains IL-4R function or a partial fragment of IL-4R; for example, the extracellular portion of IL-4R (see SEQ ID) NO: 14), the extracellular portion of IL-4R and the transmembrane portion. It can come from natural or from artificial recombination.
  • IL-4" refers to interleukin 4, NCBI Gene ID: 3565, an anti-inflammatory cytokine that induces T cell differentiation into a Th2 type, is a pleiotropic cytokine produced by activated T cells, and is an IL4 receptor (IL4R).
  • Ligand. GATA3 is a downstream signal after IL-4 activation.
  • a variant IL-4 protein with high selectivity for IL-13R ⁇ 1 is a human IL-4 protein containing the following mutations relative to native IL-4 (numbering excludes methyl sulfide at the N-terminus) Amino acid): R121K/Y124F/S125R ("KFR" or "KFR4" variant) or R121K/Y124F ("KF" variant).
  • a variant IL-4 protein having a higher selectivity for ⁇ c (type I receptor) than IL-13R ⁇ 1 (type II receptor) is an IL-4 having the following mutation relative to the sequence of native human IL-4. Protein (numbering excludes methionine at the N-terminus): R121Q/Y124W/S125F ("RGA” or "super-4" or "S4" variant), as in, for example, Junttila et al (Nature Chemical Biology 8:990) -998, 2012).
  • IL-21R or "IL-21R” includes the native IL-21R protein as well as the variant IL-21R protein.
  • sequence of "native” or “wild-type” IL-21R refers to human IL-21R sequences, whether purified from natural sources or using recombinant techniques.
  • IL-21R functional portion of IL-21R
  • IL-21R refers to the full length of IL-21R that retains IL-21R function or a partial fragment of IL-21R; for example, the intracellular signal portion of IL-21R (SEQ ID NO: 15), intracellular signal portion and transmembrane portion of IL-21R. It can come from natural or from artificial recombination.
  • the intracellular signal portion of IL-21R has the same meaning as "the intracellular domain of IL-21R”.
  • Bcl-6, T-bet and Blimp-1 are the target genes of IL-21 signaling
  • Bcl-6 is a transcription factor that maintains the survival of memory T cells
  • T-bet and Blimp-1 promote the differentiation of CD8+ T cells into effects.
  • the transcription factor of the cell is the target genes of IL-21 signaling.
  • IL-2 is a type of cell growth factor in the immune system, NCBI Gene ID: 3558, which regulates the cellular activity of leukocytes in the immune system and promotes the proliferation of Th0 and CTL. In this application, it can be used for T cells. Cultivation.
  • the IL-4R protein according to the present disclosure includes a fragment that can be shorter than the native IL-4R protein as long as the IL-4R protein fragment retains the ability to bind IL-4. It is also to be understood that the disclosure encompasses nucleic acid molecules encoding the IL-4R proteins described herein or known in the art, including, but not limited to, RNA sequences corresponding to the DNA sequences described herein.
  • fusion protein includes IL-4R proteins that bind to IL-21R using alternative additional sequences or portions (eg, linkers), as described herein, as well as nucleic acid molecules encoding such fusion proteins. Also contemplated are recombinant nucleic acid molecules in which a nucleic acid sequence encoding a fusion protein is operably linked to a promoter, a vector comprising the molecule, and a transgenic cell comprising such a molecule. Methods of producing these fusion proteins are routine methods in the art, for example using recombinant molecular biology methods.
  • transmembrane domain may include the transmembrane domain of a variety of natural receptor proteins that function to link the extracellular, intracellular regions of the receptor and anchor to the cell membrane, including but not limited to IL-4R, IL- The transmembrane region of 21R.
  • antigen refers to a molecule that can provoke an immune response.
  • the immune response can involve the production of antibodies, or activation of specific immunocompetent cells, or both.
  • any macromolecule including substantially all proteins or peptides, can serve as an antigen.
  • the antigen may be derived from recombinant DNA or genomic DNA. It will be apparent to those skilled in the art that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that can elicit an immune response, thus encoding an "antigen" (as the term is used herein).
  • the antigen need not be encoded only by the full length nucleotide sequence of the gene. It will be readily apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, which may be arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, it will be apparent to those skilled in the art that the antigen need not be encoded by a "gene”. It will be readily apparent that the antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide.
  • the biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell or a liquid, and other biological components.
  • antibody as used herein includes intact antibodies and any antigen-binding fragments (ie, "antigen-binding portions") or single chains thereof.
  • a naturally occurring "antibody” is a glycoprotein comprising at least 2 heavy (H) chains and 2 light (L) chains joined by a disulfide bond.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • VH and VL regions can be further subdivided into regions of high variability called complementarity determining regions (CDRs) separated by a more conserved region called the framework region (ER).
  • CDRs complementarity determining regions
  • ER framework region
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • scFv refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein said light and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • an scFv can have the VL and VH variable regions in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv can include a VL-linker-VH or A VH-linker-VL can be included.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in the light chain variable region.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • Lentivirus refers to the genus of the family Lentiviridae. Lentiviruses are unique in retroviruses and are capable of infecting non-dividing cells; they are capable of delivering a significant amount of genetic information into the DNA of a host cell, whereby they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses.
  • homologous or identical refers to between two polymer molecules, for example between two nucleic acid molecules, for example between two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • Unit sequence identity When a subunit position is occupied by the same monomer subunit in two molecules, for example when two DNA molecules are occupied by adenosine at one position, they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matched or homologous positions; for example, when half of the positions in the two sequences (for example, 5 positions in a polymer of 10 subunits) are homologous
  • the two sequences are 50% homologous; if 90% of the positions (eg, 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.
  • transfected or “transformed” or “transduced” refers to the process of transferring or introducing an exogenous nucleic acid into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid.
  • the cells include primary test cells and their progeny.
  • CAR Chimeric Antigen Receptor
  • Chimeric antigen receptors typically comprise a (fine) extracellular antigen binding region.
  • the extracellular antigen binding region can be fully human. In other cases, the extracellular antigen binding region can be humanized. In other instances, the extracellular antigen binding region can be murine or the chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region can be non-human.
  • the extracellular antigen binding region comprises a hinge or spacer.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be considered as part of a CAR for providing flexibility to the extracellular antigen binding region.
  • the hinge can be used to detect CAR on the cell surface of a cell, particularly when detecting antibodies to the extracellular antigen binding region are ineffective or available.
  • the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the location of the extracellular antigen binding region that targets the epitope on the target.
  • the transmembrane domain of CAR can anchor the CAR to the plasma membrane of the cell, such as the transmembrane domain of CD8, the transmembrane domain of CD28, and the like.
  • the skilled person can replace it according to the known transmembrane domain.
  • the intracellular signal domain of CAR may be responsible for activating at least one of the effector functions of the immune response cells into which the CAR has been placed.
  • CAR can induce effector functions of T cells, for example, the effector function is cytolytic activity or helper activity, including secretion of cytokines.
  • the term "intracellular signal domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function.
  • the entire intracellular signaling region can generally be used, in many cases it is not necessary to use the entire chain of the signal domain. In some cases, a truncated portion of the intracellular signaling region is used. In some instances, the term intracellular signal domain is therefore intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
  • the intracellular signaling domain of CAR can be selected from any of the domains of Table 1.
  • the intracellular signaling region of CAR may further comprise one or more costimulatory domains.
  • the intracellular signaling region may comprise a single costimulatory domain, such as an ⁇ chain (first generation CAR) or it is with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling region can comprise two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • signals generated by the CAR may be combined with an auxiliary or costimulatory signal.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains.
  • T cell activation Several receptors have been reported to provide co-stimulation for T cell activation including, but not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signaling pathways used by these costimulatory molecules work synergistically with the primary T cell receptor activation signal.
  • the signals provided by these costimulatory signaling regions can act synergistically with primary effect activation signals derived from one or more ITAM motifs (eg, the CD3zeta signal transduction domain) and can fulfill the requirements for T cell activation.
  • ITAM motifs eg, the CD3zeta signal transduction domain
  • the functional portion of IL-4R can be directly linked to the functional portion of IL-21R.
  • a functional portion of IL-4R can be linked to a functional portion of IL-21R by a linker.
  • a linker that functionalizes the functional portion of IL-4R and the functional portion of IL-21R can be designed to: (1) allow the two molecules to fold and function independently of each other; (2) have no formation potential interference The tendency of the ordered secondary structure of the functions of these two parts; (3) the smallest hydrophobic or charged character that may interact with the functional protein domain; and/or (4) the space providing two regions Separation.
  • Linkers suitable for use in fusion proteins according to the present disclosure include peptides.
  • the linker can be conjugated to a functional portion of IL-4R and/or a functional portion of IL-21R using recombinant DNA techniques. These methods are known in the art, and details of such techniques can be found, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 or Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons , 1994) or found in its upgraded version.
  • the fusion protein can include one or more linkers as well as other moieties as needed and/or as discussed herein. They may include binding regions, such as avidin or an epitope, or a tag such as a polyhistidine tag that can be used to purify and process the fusion protein, as well as other linkers described herein.
  • a detectable label can be bound to the fusion protein such that transport of the fusion protein through the body or cells can be conveniently monitored. These markers include radionuclides, enzymes, fluorophores, chromophores, and the like.
  • DNA can be altered in a wide variety of ways without affecting the biological activity of the encoded protein.
  • PCR can be used to generate a change in the DNA sequence encoding the fusion protein.
  • These changes in the DNA encoding the fusion protein can be used to optimize codon preferences in the host used to express the protein, or can include other sequence changes that facilitate expression.
  • Fusion proteins, or immune effector cells expressing the fusion protein can be analyzed using standard methods known in the art or described herein.
  • the fusion protein comprising IL-4R/IL-21R as described herein can be used in a variety of therapeutic applications.
  • the fusion proteins described herein can be used in the treatment or prevention of cells, cells that express IL-4, and any disease, disorder, or condition that would benefit from inhibition of cell proliferation or promotion of cell death. In some embodiments, it can be used to induce apoptosis or cell death, or to treat disorders associated with abnormal apoptosis or cell proliferation, such as cancer.
  • cancer refers to a cell that has the ability to grow automatically (eg, an abnormal state or condition characterized by a proliferation of cells that are proliferating). Hyperproliferative or neoplastic disease states can be classified as pathological types (eg, because they deviate from normal but are not associated with disease states). Thus, “cancer” or “tumor” refers to any unwanted growth of a cell that has no physiological function.
  • cancer includes cell growth that is technically benign but may present a risk of becoming malignant.
  • Malignant refers to the abnormal growth of any cell type or tissue.
  • malignancy includes cell growth that is technically benign but at risk of becoming malignant.
  • the term also includes any cancer, cancer, neoplasm, tumor formation or tumor. Thus, these terms are meant to include all types of cancer growth or tumorigenesis processes, metastatic tissues or malignant transformed cells, tissues or organs, whether of histopathological type or invasive stage.
  • cancer which is the majority of cancers and epidermal cells or covers organs, glands, or other body structures (eg, skin, uterus, lung cancer, breast cancer, prostate cancer). , cancer of the outer or inner surface of the stomach, intestines, and often metastasis; sarcoma, which is derived from connective tissue or supporting tissue (eg, bone, cartilage, tendons, ligaments, fat, muscle); and blood Tumors, which are derived from bone marrow and lymphoid tissues.
  • connective tissue or supporting tissue eg, bone, cartilage, tendons, ligaments, fat, muscle
  • Tumors which are derived from bone marrow and lymphoid tissues.
  • Examples of cancer include, but are not limited to, cancer, sarcoma, and hematological tumor formation disorders such as leukemia.
  • the cancer can be an adenocarcinoma (which is typically in an organ or gland that can be secreted, such as the breast, lung, colon, prostate, or bladder), or can be a squamous cell carcinoma (which is derived from the squamous epithelium and is generally large in the body). Part of the area is formed).
  • adenocarcinoma which is typically in an organ or gland that can be secreted, such as the breast, lung, colon, prostate, or bladder
  • a squamous cell carcinoma which is derived from the squamous epithelium and is generally large in the body. Part of the area is formed).
  • Sarcoma can be osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), striated muscle (skeletal muscle), mesothelioma or mesothelioma (membranous lining of body cavity), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelial blood vessels), liposarcoma (fat), glioma or astrocytoma (found in the brain's neurogenic connective tissue), mucinous sarcoma (primary embryonic connective tissue) or between Leaf cell tumor or mesodermal mixed tumor (mixed connective tissue type).
  • Hematopoietic tumor-forming disorders include proliferative/neoplastic cells involved in the origin of hematopoiesis, for example, derived from the myeloid, lymphoid or erythroid cell lines or their precursor cells.
  • the disease is derived from poorly differentiated acute leukemia (eg, erythroblastic leukemia and acute megakaryoblastic leukemia).
  • Additional exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myelogenous leukemia (CML); lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-line acute lymphoblastic leukemia and T-line acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia, and Waldens Waldenstrom's macroglobulinemia.
  • ALL acute lymphoblastic leukemia
  • ALL including B-line acute lymphoblastic leukemia and T-line acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • hairy cell leukemia and Waldens Waldenstrom's macroglobulinemia.
  • malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymph Cellular leukemia (LGF), Hodgkin's disease, and Ris-Sick disease.
  • non-Hodgkin's lymphoma and its variants include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymph Cellular leukemia (LGF), Hodgkin's disease, and Ris-Sick disease.
  • a pharmaceutical composition according to the present disclosure may comprise a fusion protein provided herein or an immune effector cell provided herein and one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients, and adjuvants. These compositions may be suitable for use in the treatment of the therapeutic indications described herein.
  • the fusion protein or immune effector cells can be administered in a therapeutically effective amount to treat one or more cancers or in combination with other therapies.
  • the fusion protein or immune effector cells can be administered before, during or after treatment with an anti-tumor or other therapy.
  • the fusion protein or immune effector cells can also be used in combination with a radiation sensitizer such as a radiotherapy sensitizer (see, eg, Diehn et al, J. Natl. Cancer Inst. 98: 1755-7, 2006).
  • a radiation sensitizer such as a radiotherapy sensitizer (see, eg, Diehn et al, J. Natl. Cancer Inst. 98: 1755-7, 2006).
  • a sensitizer is any agent capable of increasing the activity of the fusion protein.
  • a sensitizer will increase the ability of the fusion protein to inhibit cancer cell growth or kill cancer cells.
  • exemplary sensitizers include antibodies against IL-10, bone morphogenetic proteins, and HDAC inhibitors (see, eg, Sakariassen et al, Neoplasia 9(11): 882-92, 2007).
  • the fusion protein or immune effector cell may be used as part of a neoadjuvant therapy (to primary therapy) as part of an adjuvant therapy regimen in which the goal is to cure cancer in the subject.
  • the fusion protein can also be administered at different stages of tumorigenesis and progression, including in advanced and/or invasive neoplasms (eg, in a subject by topical treatment (eg, a dominant disease that cannot be cured by surgery or radiotherapy), Metastatic disease. Administration of various stages in the treatment of locally advanced disease and/or refractory tumors (eg, cancer or tumors that do not respond to treatment).
  • Primary therapy refers to the initial diagnosis of cancer in a subject. First-line treatment.
  • Exemplary primary therapy may involve surgery, a wide range of chemotherapy and radiation therapy.
  • “Auxiliary therapy” refers to a therapy that is administered to a subject following a primary therapy and at the risk of recurrence.
  • Adjuvant systemic therapy begins shortly after primary therapy, such as at 2, 3, 4, 5, or 6 weeks after the last primary therapy treatment to delay relapse, prolong survival, or cure the subject.
  • the fusion protein or the immune effector cells can be used alone or as part of an adjuvant therapy.
  • Use or more other chemotherapeutic agents in combination The combination of fusion proteins or immune effector cells and standard chemotherapy agents can serve to improve the effect of chemotherapy, and therefore, it can be used to improve the standard cancer therapy.
  • a "subject” can be a mammal in need of treatment, such as a human or veterinary patient (e.g., a rodent such as a mouse or rat, a cat, a dog, a cow, a horse, a sheep, a goat, or other animal).
  • a "subject” can be a clinical patient, a clinical trial volunteer, an experimental animal, and the like.
  • the subject may be suspected of having a disease characterized by cell proliferation or having a disease characterized by cell proliferation, being diagnosed as having a disease characterized by cell proliferation, or being confirmed not to have a cell Control subjects for proliferative diseases, as described herein, diagnostic methods for diseases characterized by cell proliferation and clinical division of such diagnosis are known to those skilled in the art.
  • the composition may be a liquid solution, suspension, emulsion, sustained release formulation or powder, and may be formulated with a pharmaceutically acceptable carrier.
  • the composition can be formulated as a suppository using conventional binders and carriers such as triglycerides.
  • “Pharmaceutically acceptable carrier” refers to a carrier matrix or vehicle that does not interfere with the effectiveness of the biological activity of the active ingredient and which does not confer toxicity to the host or subject.
  • the fusion protein or immune effector cells can be delivered with a pharmaceutically acceptable vehicle.
  • the vehicle can enhance stability and/or delivery properties.
  • Vehicles such as artificial membrane vesicles (including liposomes, nonionic surfactant noisome, nanolipid vesicles, etc.), microparticles or microcapsules, or colloidal formulations comprising pharmaceutically acceptable polymers.
  • a pharmaceutical composition comprising one or more fusion proteins or immune effector cells can be formulated aseptically injectable according to methods known in the art and using one or more suitable dispersing or wetting agents and/or suspending agents.
  • the sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent.
  • the human IL-4R signal peptide (SEQ ID NO: 1), the IL-4R extracellular domain DNA coding sequence (SEQ ID NO: 2) and the transmembrane domain of human IL-21R are used in routine molecular biology methods in the art ( SEQ ID NO: 3), the intracellular domain of human IL-21R (SEQ ID NO: 4) is ligated in turn, and then self-cleaved peptide with F2A with GPC3-28z-CAR (SEQ ID NO: 8) (SEQ ID NO: 13) Ligation, insertion of pRRLSIN lentiviral expression vector, lentiviral plasmid chIL4-21R-CAR expressing GPC3-28z-CAR (SEQ ID NO: 8) and fusion protein (SEQ ID NO: 5), nucleotide sequence such as SEQ ID NO: 16 (Figs. 1A and 1B).
  • Human IL-4R signal peptide (SEQ ID NO: 1), IL-4R extracellular domain DNA coding sequence (SEQ ID NO: 2) and human IL-7R transmembrane domain (SEQ ID NO: 24), human IL
  • the intracellular domain of -7R (SEQ ID NO: 25) was ligated in turn, and ligated with GPC3-28z-CAR (SEQ ID NO: 8) with F2A self-shearing peptide (SEQ ID NO: 13), inserted into pRRLSIN lentiviral expression.
  • the vector gave the lentiviral plasmid chIL4-7R-CAR expressing GPC3-28z-CAR (SEQ ID NO: 8) and the fusion protein (SEQ ID NO: 26), and the nucleotide sequence is shown in SEQ ID NO: 27.
  • T cell activation Human PBMC were cultured in AIM-V medium, 2% human AB type serum was added, 500 U/mL recombinant human IL-2 was added, and CD3/CD28 antibody was added to activate magnetic beads for 48 h.
  • the activated T cells were infected with the lentiviral plasmids chIL4-21R-CAR or chIL4-7R-CAR, and the serum was allowed to rest for 24 h to obtain the expression fusion protein chIL4-21R (amino acid sequence is SEQ ID NO: 6) or chIL4-7R ( The amino acid sequences are SEQ ID NO: 28) and CAR (amino acid sequence is SEQ ID NO: 10) chIL4-21R-CAR T cells.
  • UTX cells untreated T cells, no transfection
  • chIL4-21R-CAR T cells were stimulated with recombinant human IL-4 for 30 min, and the extracted proteins were collected for Western blot analysis to analyze STAT3/5 phosphorylation. The level changes and the result is shown in Figure 1C.
  • the STAT3/5 phosphorylation level of the chIL4-21R-CAR T cell group was significantly up-regulated, indicating that the fusion protein can convert IL-4 stimulation into IL-21. signal of.
  • FIG. 1D shows that chIL4-21R-CAR T cells stimulated STAT3 levels significantly higher than chIL4-7R-CAR T cells stimulated by rIL-4, whereas chIL4-7R-CAR T cells tended to phosphorylate STAT5, indicating that chIL4 -21R-CAR T cells and chIL4-7R-CAR T cells produce different downstream signals under rIL-4 stimulation.
  • Example 2 Determination of the ability of chIL4-21R-CAR T cells to proliferate or survive under stimulation with IL-4
  • GPC3-28z-CAR (SEQ ID NO: 8) was inserted into the RRLSIN lentiviral expression vector, and a lentiviral vector expressing GPC3-28z-CAR was constructed, and then transfected into 293T-packaged lentivirus, which was slow.
  • Virus 2 T cells were infected with lentivirus 2 to obtain GPC3-CAR T cells.
  • chIL4-21R-CAR T cells and GPC3-CAR T cells prepared in Example 1 were induced to culture for 4 days with recombinant human IL-2 (concentration: 500 U/mL), and the cells were collected for antibodies against human Fab fragments. Mark CAR, flow detection cell positive rate.
  • b ChIL4-21R-CAR T cells and GPC3-CAR T cells prepared in Example 1 were induced to culture for 20 days with 20 ng/mL recombinant human IL-4 (Peprotech), and cells were collected for antibodies against human Fab fragments. Mark CAR, flow detection cell positive rate.
  • Example 3 Killing ability of chIL4-21R-CAR T cells to tumor cells stimulated by IL-4 or IL-2
  • T cells were cultured with recombinant human IL-2 or IL-4 for 6 days, and Huh7 liver cancer cells (GPC-3 positive) were used as target cells for cytotoxicity experiments.
  • Huh7 liver cancer cells GPC-3 positive
  • 1 ⁇ 10 4 Huh7 hepatoma cells were per well in a 96-well plate, and the corresponding number of GPC3-CAR T cells and chIL4-7R obtained in the above examples were added at a specific target ratio of 3:1, 1:1, 1:3.
  • chIL4-21R-CAR T cells were co-cultured in 100 ⁇ L RPMI-1640 medium + 10% FBS, and after 50 h, 50 ⁇ L of culture supernatant was collected, and the LDH level in the supernatant was detected by Promega CytoTox 96 kit. The calculation of efficiency is carried out in accordance with the manufacturer's instructions. Results As shown in Figures 3A and 3B, the cytotoxicity of GPC3-CAR T cells and chIL4-7R-CAR T cells in the control group was significantly reduced under IL-4 treatment, and the cells were significantly retained or even enhanced by chIL4-21R-CAR T cells. toxicity. The in vitro toxicity assay shown in Figure 3B further demonstrates that chIL4-21R-CAR T cells maintain stronger cell killing toxicity under rIL-4 induction than chIL4-7R-CAR T cells.
  • GPC3-CAR T cells, chIL4-7R-CAR T cells and chIL4-21R-CAR T cells prepared in the above Example 1 were co-incubated with rIL-4 (20 ng/mL) or IL-2 (500 U/mL), respectively. After 72 h, cells were harvested, RNA was extracted, and Bcl-6, T-bet, Blimp-1, Granzyme B, and GATA3 were detected by RT-qPCR.
  • Bcl-6, T-bet, and Blimp-1 are target genes for IL-21 signaling.
  • Bcl-6 is a transcription factor that maintains the survival of memory T cells
  • T-bet and Blimp-1 are transcription factors that promote the differentiation of CD8 + T cells into effector cells.
  • Granzyme B is a cytoplasmic granule released by T cell activation
  • GATA3 is a downstream signal after IL-4 activation.
  • Figures 4A-4E One-way ANOVA statistic* indicates p ⁇ 0.05, ** indicates p ⁇ 0.01)
  • Figure 4A shows Bcl-6 of chIL4-21R-CAR T cells stimulated by IL-4.
  • the expression level is much higher than GPC3-CAR T cells and chIL4-7R-CAR T cells;
  • Figure 4B shows that the expression level of T-bet in chIL4-21R-CAR T cells is higher than that of GPC3-CAR T cells under IL-4 stimulation;
  • Figure 4C shows that under IL-4 stimulation, the expression level of Blimp-1 of chIL4-21R-CAR T cells is higher than that of GPC3-CAR T cells;
  • Figure 4D shows that granzyme B expression levels of chIL4-21R-CAR T cells were significantly higher than GPC3-CAR T cells and chIL4-7R-CAR-T cells under IL-4 stimulation. This indicates that for chIL4-21R-CAR T cells, the presence of IL-4 not only induces the expression of Bcl-6 in chIL4-21R-CAR T cells, but also the expression of T-bet, Blimp-1 and granzyme B by IL-4. Inhibition was also attenuated or even abolished in chIL4-21R-CAR T cells, and high expression of granzyme B may be one of the reasons why chIL4-21R-CAR T cells maintain high cytotoxicity in Example 3.
  • Figure 4E shows that under IL-4 stimulation, the expression level of CD26 of chIL4-21R-CAR T cells is much higher than that of GPC3-CAR T cells and chIL4-7R-CAR T cells. Since CD26 + CAR T cells expressing CD26 have strong anti-tumor ability, they are even stronger than CD8 + CART cells, which are generally considered to kill tumor cells. This indicates that the chIL4-21R fusion protein can improve the efficacy of tumor-specific T cells and produce CAR-T cells with stronger anti-tumor function.
  • Figure 4F shows that IL-4 induces a significant increase in RORyt expression levels in chIL4-21R-CAR T cells, improving the efficacy of tumor-specific T cells, resulting in CAR-T cells with enhanced anti-tumor function.
  • Figure 4G shows that GATA3 of GPC3-CAR T cells that do not express chIL4-21R is significantly upregulated under IL-4 induction, but the degree of GATA3 upregulation by chIL4-21R-CAR T cells is significantly attenuated.
  • Figure 4G illustrates that the action of IL-4 downstream signaling molecules is significantly inhibited in chIL4-21R-CAR T cells.
  • chIL4-21R fusion protein can not only change the immunosuppressive environment induced by IL-4, but also enhance the expression of T cells and survival and effect-related genes through IL-21 signaling pathway, resulting in stronger anti-tumor function. CAR-T cells.
  • chIL4-21R-CAR T cells expressed high levels of Bcl-6 compared to chIL4-7R-CAR T cells under rIL-4 induction, suggesting that they have greater viability and Memory T cell phenotype.
  • chIL4-21R-CAR T cells maintained a higher level of granzyme B expression, which is consistent with the enhancement of granzyme B expression by native IL-21 signaling, suggesting that chIL4-21R-CAR T cells are induced in rIL-4 The cell killing toxicity is maintained or even enhanced, while the chIL4-7R-CAR T cell has no similar ability.
  • GPC3-CAR-T cells, chIL4-7R-CAR-T cells and chIL4-21R-CAR T cells prepared in Example 1 above were combined with rIL-4 (20 ng/mL) and IL-2 (500 U/mL), respectively.
  • Cells were harvested after 6 days of incubation, and PD-1 and TIM3 were detected by flow.
  • PD-1, and TIM3 were important markers of T cell depletion. The results are shown in Figure 6. Under IL-4 treatment, the level of PD-1 expressed by the chIL4-21R-CAR T cell group was lower than that of the GPC3-CAR T cell group, and the level of TIM3 was lower than that of the GPC3-CAR T cell group and chIL4.
  • the IL-4 signal is converted into the IL-21 signal by the fusion protein chIL4-21R and functions to inhibit cell depletion.
  • the IL-21 signal has the function of inhibiting cell depletion and maintaining cell survival, thereby improving The effect of killing tumor cells.
  • Example 6 Killing of IL-4 secreting tumor cells by chIL4-21R-CAR T cells
  • the pWPT lentiviral expression plasmid of GFP-F2A-IL-4 was constructed, and the virus was infected and infected with Huh-7 cells. After 3 days, the GFP was positive by flow cytometry, and IL-4-Huh-7 cells capable of secreting IL-4 were obtained. .
  • IL-4-Huh-7 cells As tumor cells, the killing of IL-4-Huh-7 cells by chIL4-21R-CAR T cells and GPC3-CAR T cells was compared, and on the other hand, CAR T cells were exposed to tumors in vivo. The state of the cells, on the other hand, shows changes in the depletion state of CAR T cells after killing tumor cells.
  • the chIL4-7R-CAR T cells, the chIL4-21R-CAR T cells and the GPC3-CAR T cells prepared in the previous examples were taken as the target cells with IL-4-Huh-7, and the target ratio was 1:1.
  • the round was killed and observed under the microscope after 48 hours: except for the UTD group, there were no adherent cells in each group.
  • the suspended T cells were collected for flow detection, and the CAR positive rate and depletion marker expression were analyzed.
  • the remaining T cells were co-cultured with the target cells at a target ratio of 1:5 for a second round of killing. After 48 hours, the PBS was washed and suspended. T cells, adherent target cells were observed by crystal violet staining.
  • the first round of killing was performed on the day when chIL4-21R-CAR T cells and GPC3-CAR T cells were co-incubated with IL-4-Huh-7 cells (reported as the first round of pre-killing R0) and the effective target ratio was 1:1.
  • 48h (reported as the first round of post-killing R1)
  • the effect of the target than 1:5 for a second round of killing 48h (reported as the second round of post-killing R2).
  • T cells were collected and T cell depleted PD-1 and TIM3 were detected by flow. The results are shown in Fig. 7B. As the number of kills increased, the expression of markers depleted by CAR T cells was significantly enhanced. UTD was used as a control because it had no killing effect.
  • the expression of PDIL and TIM3 in the chIL4-21R-CAR T cell group was significantly lower than that in the control group, and the chIL4-21R-CAR T cells were further decreased.
  • the cells can kill the target cells of IL-4 + more persistently, and have the effect of killing tumor cells more persistently.
  • the chIL4-21R-CAR T cells were continuously killed by IL-4-secreting target cells compared to chIL4-7R-CAR T cells, and the T cell depletion marker PD-1 and The lower expression level of TIM3 suggests that chIL4-21R-CAR T cells are more resistant to depletion in the tumor microenvironment of IL-4+, and the killing of target cells further demonstrates that chIL4-21R-CAR T cells have more Long-lasting target cell killing ability.
  • B-NDG mice (Bai Osei), randomly divided into 4 groups at 6-8 weeks old, 4 in each group, respectively Untransduced (UTD), GPC3-CAR T cell group, chIL4-7R - CAR T cell group, chIL4-21R-CAR T cell group.
  • Inoculation of subcutaneous xenografts 7721 cells in a logarithmic growth phase and in good growth state were collected by trypsin digestion, and each mouse was inoculated with 3 ⁇ 10 6 tumor cells, and the inoculation diary was the 0th day.
  • CAR-T cell reinfusion When the average tumor volume is about 85 mm 3 , that is, on the 11th day after tumor inoculation, 2 ⁇ 10 6 /CAR-T cells or UTD cell control are injected. The experimental results are shown in Figure 8.
  • Figure 8 (One-way ANOVA *** indicates p ⁇ 0.001). Compared with the UTD control group, the anti-tumor effect was observed in each group 26 days after CAR T injection. The inhibition rates were: GPC3-CAR T Group: 66.5% ⁇ 17.2%, chIL4-7R-CAR T group: 96.7% ⁇ 3.6%, chIL4-21R-CAR T group: 100% ⁇ 0.
  • the tumor volume of the infused UTD-T cell group continued to increase, and the tumor volume growth of the GPC3-CAR T cell group was relatively slowed and showed an inhibition trend, while the tumor growth of the chIL4-21R-CAR T cell group was significantly inhibited.
  • the tumor volume was significantly smaller than that of the GPC3-CAR T cell group, indicating that chIL4-21R-CAR T cells have stronger anti-tumor function than GPC3-CAR T cells.
  • chIL4-21R-CAR T cells have more CD4+ cell subsets in IL-4+ tumor-bearing mice, due to CD4+ CAR T cells in vivo than CD8+CAR T The cells are not easily depleted and have a longer persistence, suggesting that chIL4-21R-CAR T cells may have more sustained immune killing and memory functions than chIL4-7R-CAR T cells in vivo.
  • mice were randomly divided into 4 groups at 6-8 weeks old, 6-7 in each group, which were Untransduced (UTD), GPC3-CAR T cell group and chIL4-7R-CAR T cell group. , chIL4-21R-CAR T cell group.
  • CAR-T cell reinfusion When the average tumor volume is about 150 mm 3 , that is, on the 13th day after tumor inoculation, 3.0 ⁇ 10 6 /CAR-T cells or untransduced T cell controls are injected. The experimental results are shown in Figures 10A and 10B.
  • CAR T cells targeting GPC3 are exemplarily employed, and those skilled in the art can employ CAR-T cells targeting other targets, such as CAR T cells targeting EGFR, according to the teachings of the present application.
  • the sequence of the scFv of the EGFR-targeting CAR-T cell is as set forth in SEQ ID NO: 20), such as a CAR T cell targeting CLD18A2 (exemplary, scFv of a CAR-T cell targeting CLD18A2)
  • the sequence is as shown in SEQ ID NO: 21), such as CAR T cells targeting CD19 (exemplary, the sequence of the scFv targeting CD19-secreting CAR T cells is shown in SEQ ID NO: 22), such as targeting BCMA CAR T cells (exemplary, the sequence of the scFv of CAR T cells targeting BCMA is shown in SEQ ID NO: 23).

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Abstract

提供表达一种融合蛋白的免疫效应细胞，所述融合蛋白包含细胞因子结合胞外结构域和信号转导胞内结构域，其中所述胞外结构域为IL4受体胞外结构域，所述胞内结构域为IL-21受体胞内结构域。所述免疫效应细胞在实体瘤的肿瘤微环境中具备显著的抗肿瘤能力，从而不仅在体外对实体瘤细胞有效，在体内对实体瘤细胞也具备优异的杀伤效果。还提供包含所述免疫效应细胞的药物组合物以及它们的应用。

Description

IL-4R的融合蛋白及其应用 技术领域
本发明属于过继性细胞治疗领域；具体地说，本发明涉及一种改进的免疫细胞，改进的免疫细胞可以显著增强免疫细胞的抗肿瘤能力。
背景技术
实体瘤中的癌细胞能够在其周围形成肿瘤微环境以支持癌细胞的生长和转移。肿瘤微环境是肿瘤存在的细胞环境，包括周围的血管、免疫细胞、成纤维细胞、其他细胞、可溶性因子、信号传导分子、细胞外基质和可促进肿瘤转化、支持肿瘤生长和侵袭、保护肿瘤免受宿主免疫、培养治疗抗性并为休眠转移茁壮成长提供微环境的机械因子(mechanical cue)。肿瘤及其周围的微环境密切相关，不断交互，肿瘤可以通过释放细胞外信号、促进肿瘤血管生成和诱导外周免疫耐受来影响它们的微环境。参见warts等，“Tumor Microenvironment Complexity:Emerging Roles in Cancer Therapy”Cancer Res,72卷,第2473-2480页,2012。因此实体瘤的治疗通常难以奏效。
发明内容
本发明的目的在于提供一种融合蛋白，这种融合蛋白能够使得表达它的免疫效应细胞在实体瘤的肿瘤微环境中具备显著的抗肿瘤能力；本发明还提供表达所述融合蛋白的免疫效应细胞、包含所述免疫效应细胞的药物组合物以及它们在诱导细胞死亡、或者治疗癌症、或者治疗过度增生性或者分化性紊乱中的应用。
在第一方面，本发明提供一种融合蛋白，所述融合蛋白包含IL-4受体(IL-4R)胞外域或其变体和IL-21受体(IL-21R)胞内域或其变体。
在具体的实施方式中，所述融合蛋白包含：
i)IL-4受体(IL-4R)胞外域；
ii)跨膜域，优选IL-4R的跨膜区或者IL-21R的跨膜区；和
iii)IL-21受体(IL-21R)胞内域。
在具体的实施方式中，所述IL-4R的胞外域的编码核苷酸序列与SEQ ID NO:2所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
在具体的实施方式中，所述IL-4R的胞外域的编码核苷酸序列是SEQ ID NO:2所示的序列。
在具体的实施方式中，所述IL-21R的胞内域的编码核苷酸序列与SEQ ID NO:4所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
在具体的实施方式中，所述IL-21R的胞内域的编码核苷酸序列是SEQ ID NO:4所示的序列。
在具体的实施方式中，所述融合蛋白与SEQ ID NO.6所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
在具体的实施方式中，所述融合蛋白具有SEQ ID NO.6所示的序列。
在具体的实施方式中，所述IL-4R选自IL-4Rα、IL-4Rγc。
在具体的实施方式中，所述IL-4R可以结合IL-4或者突变型IL-4或者IL-13或者突变型IL-13；优选地，所述IL-4R可以结合IL-4或者突变型IL-4。
在具体的实施方式中，所述突变型IL-4包含KFR变体、KF变体、或RGA变体。
在具体的实施方式中，各结构域直接或通过接头分子连接在一起。
在第二方面，本发明提供一种核酸分子，所述核酸分子编码本发明第一方面所述的融合蛋白。
在第三方面，本发明提供一种载体，所述载体包含本发明第二方面所述的核酸分子。
在第四方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明第三方面所述的载体。
在第五方面，本发明提供一种免疫效应细胞，所述免疫效应细胞表达本发明第一方面所述的融合蛋白。
在优选的实施方式中，所述的免疫效应细胞为T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞或其组合；更优选地，所述的免疫效应细胞为T细胞、自然杀伤(NK)细胞、或自然杀伤T(NKT)细胞；更优选地，所述的免疫效应细胞为T细胞。
在具体的实施方式中，所述的免疫效应细胞为自体细胞，如自体T细胞、自体NK细胞。
在一优选例中，所述的免疫效应细胞为自体T细胞。
在具体的实施方式中，所述的免疫效应细胞为同种异体细胞，如同种异体的T细胞、同种异体的NK细胞、或者NK细胞系(如NK-92细胞)。
在具体的实施方式中，所述免疫效应细胞还表达外源性的受体，该外源性的受体具有特异结合肿瘤抗原的第二胞外结合域、第二跨膜域、及第二胞内域。
在一具体实施方案中，所述免疫效应细胞还表达外源性的受体，该外源性的受体具有特异结合肿瘤抗原的第二胞外结合域、第二跨膜域、及第二胞内域；优选地，所述的肿瘤抗原与IL-4受体的结合抗原不同。
在具体的实施方式中，所述外源性受体选自：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)或其组合。
在具体的实施方式中，所述外源性受体为嵌合抗原受体。
在具体的实施方式中，所述的融合蛋白为组成性表达。
在具体的实施方式中，所述的融合蛋白为诱导性表达。
在具体的实施方式中，所述外源性受体为嵌合抗原受体，该嵌合抗原受体的第二胞外结合域、第二跨膜域、及第二胞内域分别具有以下特点：
(i)所述第二胞外结合域包含：抗体，抗体片段，scFv，Fv，Fab，(Fab')2，单结构域抗体(SDAB)，VH或VL结构域，或骆驼科VHH结构域，或相应抗原的天然配体，或其组合；和/或
(ii)所述第二跨膜结构域包含选自下组的蛋白质的跨膜结构域：T细胞受体的α、β或ζ链、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NKG2C；和/或
(iii)所述的第二胞内域包括：一级信号传导结构域和/或共刺激信号传导结构域，其中：(1)所述一级信号传导结构域包含选自：CD3ζ、CD3γ、CD3δ、CD3ε、常见FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10、和DAP12的蛋白质的功能信号传导结构域，或其组合；和/或(2)所述共刺激信号传导结构域包含选自如下的蛋白质的功能信号传导结构域：CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关的抗原-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和NKG2D，或其组合。
在具体的实施方式中，所述的嵌合抗原受体包括：
(i)特异性结合抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域和CD3ζ；或
(ii)特异性结合抗原的抗体或其片段、CD28或CD8的跨膜域、CD137的共刺激信号结构域和CD3ζ；或
(iii)特异性结合抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
在具体的实施方式中，所述的肿瘤抗原包括：
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；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251(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5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10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)。
在具体的实施方式中，所述的肿瘤抗原为实体瘤抗原；
在具体的实施方式中，所述实体瘤抗原选自前列腺特异性膜抗原、癌胚抗原、IL13Ralpha，HER-2，间皮素，EGFR、EGFRvIII，磷脂酰肌醇蛋白聚糖3(GPC3)、EphA2，HER3，EpCAM，MUC16，MUC1，claudin 18.2，叶酸受体，claudin 6、CD138、MAGE3、ASGPR1或CDH16，更优选的，所述实体瘤抗原为GPC3。
在具体的实施方式中，所述实体瘤选自结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌；优选地，所述实体瘤选自肝癌，肺癌，鳞状细胞癌。
在具体的实施方式中，所述第二胞外结合域具有与SEQ ID NO:7、20、21、22、或23所示序列至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的序列。
在具体的实施方式中，所述的免疫效应细胞的第二胞外结合域具有与SEQ ID NO:7所示序列至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的序列。
在具体的实施方式中，所述嵌合抗原受体的第二跨膜域的编码核苷酸序列与SEQ ID NO:29或30所示核苷酸序列编码的序列具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性。
在具体的实施方式中，所述嵌合抗原受体的第二胞内域的编码核苷酸序列含有SEQ ID NO:31和33所示核苷酸序列，或含有与SEQ ID NO:31和33所示核苷酸序列具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的核苷酸序列。
在具体的实施方式中，所述嵌合抗原受体的第二胞内域的编码核苷酸序列含有SEQ ID NO:32和33所示核苷酸序列，或者含有与SEQ ID NO:32和33所示核苷酸序列具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的核苷酸序列。
在具体的实施方式中，所述嵌合抗原受体的第二胞内域的编码核苷酸序列含有SEQ ID NO:31、32和33所示核苷酸序列，或者含有与SEQ ID NO:31、32和33所示核苷酸序列具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的核苷酸序列。
在具体的实施方式中，所述嵌合抗原受体的第二胞内域的编码核苷酸序列含有SEQ ID NO:33所示核苷酸序列，或者含有与SEQ ID NO:33所示核苷酸序列具有至少90％(例如至 少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的核苷酸序列。
在具体的实施方式中，所述的嵌合抗原受体具有SEQ ID NO:9、10、11、12所示序列或与SEQ ID NO:9、10、11、12所示序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的同一性。
在具体的实施方式中，所述的嵌合抗原受体及所述的融合蛋白由SEQ ID NO：16、17、18、或19所示核苷酸序列编码或由与SEQ ID NO：16、17、18、或19具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的核苷酸序列编码。
在具体的实施方式中，所述的抗原为肿瘤抗原或病原微生物抗原。
在具体的实施方式中，所述病原微生物包括病毒、细菌、真菌、原生动物或寄生虫；更佳地，所述病原微生物为病毒；或者更佳地，所述病原体微生物选自巨细胞病毒、爱泼斯坦-巴尔病毒、人类免疫缺陷病毒及流感病毒。
在第六方面，本发明提供一种药物组合物，所述药物组合物包含本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、或者本发明第五方面所述的免疫效应细胞。
在第七方面，本发明提供一种诱导细胞死亡的方法，所述方法包括向需要的受试者施用：本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、本发明第五方面所述的免疫效应细胞或者本发明第六方面所述的药物组合物。
在第八方面，本发明提供一种治疗癌症的方法，所述方法包括向需要的受试者施用：本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、本发明第五方面所述的免疫效应细胞或者本发明第六方面所述的药物组合物。
在第九方面，本发明提供一种治疗癌症的方法，所述方法包括使在有此需要的受试者的肿瘤微环境中表达IL-4的恶性细胞与本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、本发明第五方面所述的免疫效应细胞或者本发明第六方面所述的药物组合物接触。
在具体的实施方式中，所述恶性细胞在所述受试者开始治疗之前接触。
在具体的实施方式中，所述恶性细胞是恶性肿瘤细胞。
在第十方面，本发明提供一种治疗过度增生性或者分化性紊乱的方法，所述方法包括向有此需要的受试者施用：本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分 子、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、或者本发明第五方面所述的免疫效应细胞。
在具体的实施方式中，所述过度增生性或者分化性紊乱是纤维化症或增生症、炎性疾病或自身免疫疾病。
在第十一方面，本发明提供本发明第一方面所述的融合蛋白、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、本发明第四方面所述的宿主细胞、本发明第五方面所述的免疫效应细胞或者本发明第六方面所述的药物组合物用于在有此需要的患者中诱导细胞死亡、或者治疗癌症、或者治疗过度增生性或者分化性紊乱的应用。
在具体的实施方式中，所述受试者是人类。
在第十二方面，本发明提供第一方面所述的融合蛋白、第二方面所述的核酸分子、第三方面所述的载体、第四方面所述的宿主细胞或者第五方面所述的免疫效应细胞在制备药物中的用途，所述药物用于在有此需要的患者中诱导细胞死亡、或者治疗癌症、或者治疗过度增生性或者分化性紊乱。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1A为chIL4-21R-CAR的的质粒示意图；图1B为chIL4-21R-CAR构建的质粒图；图1C、1D显示了不同细胞对STAT3/5磷酸化水平的影响；
图2A、2B显示了CAR-T细胞在IL-4刺激下增殖或存活的能力；
图3A、3B显示了不同CAR-T细胞的体外细胞毒性；
图4A显示了在IL-4刺激下chIL4-21R-CAR T细胞的Bcl-6的表达水平；图4B显示了在IL-4刺激下chIL4-21R-CAR T细胞的T-bet的表达水平；图4C显示了在IL-4刺激下chIL4-21R-CAR T细胞的Blimp-1的表达水平；图4D显示了在IL-4刺激下chIL4-21R-CAR T细胞的颗粒酶B的表达水平；图4E显示了在IL-4刺激下，chIL4-21R-CAR T细胞的CD26的表达水平；图4F显示了在IL-4刺激下，chIL4-21R-CAR T细胞的RORγt的表达水平；图4G显示了在IL-4刺激下，chIL4-21R-CAR T细胞的GATA3的表达水平。
图5A显示了GPC3-CAR-T细胞组在IL-4或IL-2刺激后CD25+的细胞群体比例；图5B显示了chIL4-21R-CAR T细胞组在IL-4或IL-2刺激后CD25+的细胞群体比例；
图6显示了细胞耗竭标志物的检测结果；
图7A显示了chIL4-21R-CAR T细胞对IL-4分泌型的肿瘤细胞持续性杀伤情况；图7B显示了持续性杀伤IL-4分泌型的肿瘤细胞后chIL4-21R-CAR T细胞耗竭标志物的检测；
图8显示了chIL4-21R-CAR T细胞对GPC-3-SMMC-7721移植瘤模型的肿瘤杀伤情况；
图9A显示了chIL4-21R-CAR T细胞在实验动物体内的存活情况；图9B显示了在实验动物体内存活的chIL4-21R-CAR T细胞的CD4/8群体比例；
图10显示了chIL4-21R-CAR T细胞对PLC/PRF/5肝癌移植瘤模型的肿瘤杀伤情况。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现将IL4R和IL21R构成的融合蛋白与嵌合抗原受体(CAR)共表达在T细胞上，得到的T细胞能够在实体瘤的肿瘤微环境中具备显著的抗肿瘤能力。在此基础上完成了本发明。
本发明的实施方案提供一项新方案以使肿瘤反应性T细胞抵抗肿瘤微环境存在的免疫阻抑性/抑制性细胞因子。本发明涉及使用细胞因子受体的融合蛋白改善肿瘤特异性免疫效应细胞的扩增和抗肿瘤活性。
这类方案包括具有细胞因子受体的融合蛋白的天然或基因修饰的肿瘤特异性T细胞，所述细胞因子受体的融合蛋白结合抑制性/阻抑性细胞因子IL4并且使得它们的胞内结果转换成IL21免疫刺激性/活化信号，因此改善肿瘤特异性T细胞的功效。
本发明包括载体，如示例性双顺反子逆转录病毒载体，所述载体编码与IL21细胞因子受体的信号转导胞内结构域融合的IL4细胞因子受体的胞外结构域。
在特定的实施方案中，其中IL4存在微环境中的癌包括基本上全部实体瘤。具体的示例性癌包括：纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、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，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤、食管癌、胆囊癌、肾癌、多发性骨髓瘤。本发明的实施方案可用来调节例如原代T细胞、天然存在的肿瘤抗原特异性细胞毒T淋巴细胞和NK细胞。利用本发明修饰的T/NK细胞可以在自体或同种异型环境下使用。
在本发明的一些实施方案中，存在可以使负向免疫调节信号转化成正向信号的嵌合分子。仅以举例方式，这种方案涉及使IL-4胞外结构域与IL-21受体的信号转导胞内结构域融合。这种方案可以用来使得免疫效应细胞抵抗肿瘤微环境中经常存在的负向细胞因子信号。
在一些实施方案中，本发明提供了使用细胞因子受体的融合蛋白，与IL-21、受体的胞内结构域融合的IL4细胞因子受体的胞外结构域，逆转肿瘤微环境的影响。
本发明提供了使用细胞因子受体的融合蛋白，与IL-21受体的胞内结构域融合的IL4 细胞因子受体的胞外结构域，逆转肿瘤微环境的影响：
1.IL-4活化后的下游信号GATA3在chIL4-21R-CAR-T细胞中被显著抑制，说明IL-4的下游信号的诱导作用在chIL4-21R-CAR-T细胞中被显著抑制；
2.CD25作为T细胞激活的标志物。chIL4-21R-CAR T细胞组在IL-4刺激后，CD25+的细胞群体比例与IL-2刺激组基本没有差异，说明融合蛋白chIL4-21R-CAR阻断了IL-4对T细胞激活的抑制作用，也就是说IL-4的下游信号的诱导作用在chIL4-21R-CAR-T细胞中被显著抑制。
3.IL-4的刺激能诱导T细胞高表达作为T细胞耗竭的重要标志物的PD-1、和TIM3水平。而在IL-4存在的条件下，chIL4-21R-CAR融合蛋白的表达能显著抑制由IL-4刺激引起的T细胞耗竭重要标志物PD-1、TIM3的表达水平。说明IL-4信号由融合蛋白chIL4-21R转化为IL-21信号并发挥抑制细胞耗竭、维持细胞存活的功能，从而提高杀伤肿瘤细胞的效果。
4.Bcl-6、T-bet、Blimp-1是IL-21信号的靶基因。chIL4-21R-CAR-T细胞中，Bcl-6、T-bet、Blimp-1的表达水平大大高于GPC3-CAR T细胞，表明chIL4-21R-CAR融合蛋白能够将胞外IL-4的刺激转变成IL-21的信号传递，并且通过显著上调细胞中STAT3/5磷酸化水平，上调作用于T细胞活化释放细胞浆颗粒的颗粒酶B的水平，这都提示胞外IL-4的刺激已经转变成IL-21信号通路的激活，并且发挥免疫刺激性/活化作用。
5.IL-4的刺激能促使chIL4-21R-CAR-T细胞扩增，表明chIL4-21R-CAR T细胞在IL-4环境下的生长，能逆转肿瘤微环境的影响，将抑制性或阻抑性细胞因子信号转化成促进免疫刺激性/活化信号，具有更强的增殖或存活能力。
6.在IL-4的刺激后，chIL4-21R-CAR-T能显著地保留甚至增强了细胞毒性/杀伤能力；而对持续分泌IL-4的肿瘤细胞，chIL4-21R-CAR-T也具有显著的持久的杀伤肿瘤细胞的能力；并且chIL4-21R-CAR T能明显抑制小鼠体内肿瘤的生长，并且体内存活的细胞亚群主要为CD4+T、CD8+T细胞；这些结果都表明chIL4-21R-CAR T细胞能在IL-4刺激后或在IL-4持续分泌环境下逆转肿瘤微环境的影响，将抑制性或阻抑性细胞因子信号转化成促进免疫刺激性/活化信号，具有更强的细胞杀伤能力。
为了进一步理解本文提供的技术方案，下面对方法和/或术语在本文中的定义作进一步说明。
术语“肿瘤”是指所有赘生细胞生长和增殖，无论是恶性的还是良性的，以及所有癌前和癌性细胞和组织。
术语“肿瘤微环境”是指肿瘤环境的任何和所有元素，包括为恶性过程产生结构和/或功能环境以存活和/或扩张和/或扩散的元素。
术语“IL-4R”或“IL-4R”包括天然的IL-4R蛋白以及变体型IL-4R蛋白。本文使用的“天然的”或“野生型”IL-4R的序列是指人类IL-4R序列，不管是从天然来源纯化还是使用重组技术制得。
术语“IL-4R(IL-4R)的功能性部分”是指保留了IL-4R功能的IL-4R的全长或者IL-4R的 部分片段；例如IL-4R的胞外部分(见SEQ ID NO：14)、IL-4R的胞外部分及跨膜部分。其可以来自天然的，也可以来自人工重组。
术语“IL-4”指白介素4，NCBI Gene ID:3565,诱导T细胞分化为Th2型的抗炎细胞因子，是由激活T细胞产生的多效性细胞因子，是IL4受体(IL4R)的配体。GATA3是IL-4活化后的下游信号。对IL-13Rα1(II型受体)具有高的选择性的变体型IL-4蛋白是相对于天然IL-4而言包含如下突变的人类IL-4蛋白(编号排除了在N末端的甲硫氨酸)：R121K/Y124F/S125R(“KFR”或者“KFR4”变体)或者R121K/Y124F(“KF”变体)。
对γc(I型受体)具有比对IL-13Rαl(II型受体)的选择性高的变体型IL-4蛋白是相对于天然人类IL-4的序列而言包含如下突变的IL-4蛋白(编号排除了在N末端的甲硫氨酸)：R121Q/Y124W/S125F(“RGA”或者“super-4”或者“S4”变体)，如在例如Junttila等(Nature Chemical Biology 8:990-998,2012)中所述。
术语“IL-21R”或“IL-21R”包括天然的IL-21R蛋白以及变体型IL-21R蛋白。本文使用的“天然的”或“野生型”IL-21R的序列是指人类IL-21R序列，不管是从天然来源纯化还是使用重组技术制得。
术语“IL-21R(IL-21R)的功能性部分”是指保留了IL-21R功能的IL-21R的全长或者IL-21R的部分片段；例如IL-21R的胞内信号部分(SEQ ID NO：15)、IL-21R的胞内信号部分及跨膜部分。其可以来自天然的，也可以来自人工重组。在本文中，“IL-21R的胞内信号部分”与“IL-21R的胞内域”具有相同的含义。Bcl-6、T-bet、Blimp-1是IL-21信号的靶基因，Bcl-6是维持记忆性T细胞存活的转录因子，T-bet和Blimp-1是促进CD8+T细胞分化为效应细胞的转录因子。
术语“IL-2”是免疫系统中的一类细胞生长因子，NCBI Gene ID：3558，能调控免疫系统中白血球的细胞活性，促进Th0和CTL的增殖，在本申请中，可以用于T细胞的培养。
应当理解的是，根据本公开内容的IL-4R蛋白包括可以比天然的IL-4R蛋白短的片段，只要IL-4R蛋白片段保持结合IL-4的能力。还应当理解的是，本公开内容涵盖编码本文所述的或者本领域已知的IL-4R蛋白的核酸分子，包括但不限于与本文所述的DNA序列对应的RNA序列。
术语“融合蛋白”包括利用可选的额外的序列或者部分(例如接头)结合至IL-21R的IL-4R蛋白，如本文所述，以及编码这些融合蛋白的核酸分子。还涵盖的是其中编码融合蛋白的核酸序列被可操作地连接至启动子的重组核酸分子、包含所述分子的载体、和包含这种分子的转基因细胞。生成这些融合蛋白的方法是本领域中的常规方法，例如使用重组分子生物学方法。
术语“跨膜域”可包括多种天然受体蛋白的跨膜结构域，起到连接受体胞外、胞内区并锚定于细胞膜上的作用，包括但不限于IL-4R、IL-21R的跨膜区。
术语“抗原”或“Ag”指可以激起免疫反应的分子。免疫反应可以涉及抗体的产生、或特定的免疫活性细胞的激活、或两者。本领域技术人员明了，任何大分子，包括基本上所有的蛋白或肽，均可以充当抗原。此外，抗原可以源自重组DNA或基因组DNA。本领域技术 人员明了，包含编码可以引起免疫反应的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA，因此编码“抗原”(正如该术语在本文所用的)。此外，本领域技术人员明了，抗原不必仅由基因的全长核苷酸序列编码。容易明了的，本发明包括，但不限于，使用一个以上基因的部分核苷酸序列，这些核苷酸序列可以以各种组合方式排列以编码可以引起期望免疫反应的多肽。此外，本领域技术人员明了，抗原无需由“基因”编码。容易明了的，抗原可以合成产生、或可以源自生物样品、或可以是除多肽之外的大分子。所述生物样品可以包括，但不限于，组织样品、肿瘤样品、细胞或液体以及其它生物学成分。
术语“抗体”在本文中包括完整的抗体和任何抗原结合片段(即，“抗原结合部分”)或其单链。天然存在的“抗体”是包含通过二硫键链间连接的至少2个重(H)链和2个轻(L)链的糖蛋白。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为称为互补决定区(CDR)的具有高可变性的区域，其间隔以更保守的称为框架区(ER)的区域。每一个VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q))的结合。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
如本文中所用，术语“互补决定区”和“CDR”是指抗体可变区内赋予抗原特异性和结合亲和力的氨基酸的序列。一般而言，在每一个重链可变区中存在3个CDR(HCDR1、HCDR2、HCDR3)并且在轻链可变区中存在3个CDR(LCDR1、LCDR2、LCDR3)。
术语“表达”指启动子驱动的特定核苷酸序列的转录和/或翻译。
术语“慢病毒”指慢病毒科的属。慢病毒在逆转录病毒中是独特的，能够感染非分裂细胞；它们能将显著量的遗传信息递送到宿主细胞的DNA中，由此它们是最为有效的基因递送载体方法之一。HIV、SIV和FIV均是慢病毒的例子。
术语“同源性”或“同一性”指两个聚合物分子之间，例如两个核酸分子之间、例如两个DNA分子或两个RNA分子之间、或两个多肽分子之间的亚单位序列同一性。当一个亚单位位置在两个分子中被相同单体亚单位占据时，例如当两个DNA分子在一个位置上都被腺苷占据时，则它们在该位置是同源的或同一的。两个序列之间的同源性是匹配的或同源的位置数的直接函数；例如，当两个序列中半数位置(例如在长10个亚单位的聚合物中5个位置)是同源的，则这两个序列50％同源；如果90％的位置(例如，10个中9个)是匹配的或同源 的，则这两个序列90％同源。
术语“转染的”或“转化的”或“转导的”指，将外源核酸转移或引入宿主细胞中的过程。“转染的”或“转化的”或“转导的”细胞是已经被转染了、转化了或转导了外源核酸的细胞。该细胞包括原代受试细胞和其后代。
本文所用的术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”指一种融合到细胞内信号转导域的肿瘤抗原结合结构域，能激活T细胞。常见地，CAR的胞外结合结构域来源于小鼠或人源化或人的单克隆抗体。
嵌合抗原受体通常包含(细)胞外抗原结合区。在一些实施方案中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区域可以被人源化。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施方案中，所述胞外抗原结合区可以是非人的。
在一些情况下，细胞外抗原结合区域包括铰链或间隔区。术语铰链和间隔区可以互换使用。铰链可以被认为是用于向细胞外抗原结合区提供柔性的CAR的一部分。在一些情况下，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测细胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于细胞外抗原结合区域靶向靶上的表位的位置。
CAR的跨膜域可以将CAR锚定在细胞的质膜上，如CD8的跨膜域、CD28的跨膜域等均可使用。技术人员可以根据已知的跨膜域进行替换。
CAR的胞内信号域可以负责活化CAR已经置于其中的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌。因此，术语“细胞内信号域”是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区域，但是在许多情况下，不必使用信号结构域的整个链。在一些情况下，使用细胞内信号传导区的截短部分。在一些情况下，术语细胞内信号域因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
CAR的细胞内信号传导结构域可以选自表1的任何一个结构域。CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如ζ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区域提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
表1.共刺激结构域
接头
在一些实施方式中，可以直接将IL-4R的功能性部分与IL-21R的功能性部分相连。
在一些实施方式中，可以通过接头将IL-4R的功能性部分与IL-21R的功能性部分相连。将IL-4R的功能性部分和IL-21R的功能性部分的接头可以被设计成用于：(1)允许所述两个分子折叠并且独立于彼此发挥作用；(2)不具有形成可能干扰这两个部分的功能的有序二级结构的倾向性；(3)具有最小的可能与功能性蛋白域相互作用的疏水性或者带电荷特性；和/或(4)提供两个区域的空间分离。
适合于在根据本公开内容的融合蛋白中使用的接头包括肽。可以使用重组DNA技术将所述接头结合至IL-4R的功能性部分和/或IL-21R的功能性部分。这些方法在本领域中是已知的，并且这种技术的细节可以在例如Sambrook等，MolecularCloning：A LaboratoryManual.第二版，ColdSpringHarborLaboratory，ColdSpringHarbor LaboratoryPress，ColdSpringHarbor，N.Y.，1989或者Ausubel等，CurrentProtocols inMolecularBiology，JohnWiley&Sons，1994)或者其升级版中找到。
所述融合蛋白可以根据需要和/或如本文所讨论的那样包括一个或者多个接头以及其他部分。它们可以包括结合区域，例如抗生物素或者表位、或者可以用于纯化和加工所述融合蛋白的多组氨酸标签等标签以及本文所述的其他接头。另外，可以将可检测的标记物结合至 所述融合蛋白，使得可以方便地监视所述融合蛋白通过身体或者细胞的运输。这些标记物包括放射性核素、酶、荧光团、发色团以及类似的标记物。
本领域技术人员应当理解的是，可以以很多种方式改变所述DNA而不影响编码蛋白的生物学活性。例如，可以使用PCR来在编码所述融合蛋白的DNA序列中产生变化。编码融合蛋白的DNA中的这些变化可以被用来对用于表达所述蛋白的宿主中的密码子偏好进行优化，或者可以包含能够便于表达的其他序列变化。
分析
可以使用本领域已知的或者本文描述的标准方法对融合蛋白、或者表达融合蛋白的免疫效应细胞进行分析。
应用
包含如本文所述的IL-4R/IL-21R的所述融合蛋白可以被用于各种治疗用途。一般来说，本文所述的融合蛋白可以被用于涉及表达IL-4的细胞以及将受益于对细胞增殖的抑制或者细胞死亡的促进的任意疾病、紊乱或者病症的治疗或者预防。在一些实施方式中，可以被用来诱导凋亡或者细胞死亡、或者用来治疗与异常凋亡或者细胞增殖相关联的紊乱例如癌症。
本文使用的术语“癌症”、“癌”、“过度增生”或者“肿瘤”是指具有自动生长的能力的细胞(例如，以正在增殖的细胞生长激增为特征的异常状态或者情况)。过度增生性或者赘生性疾病状态可以被分类为病理性类型(例如，因为偏离正常状态但是与疾病状态不相关)。因此，“癌症”或者“肿瘤”是指细胞的没有生理学功能的任何不需要的生长。术语癌症包括从技术上来说是良性的但是可能存在变成恶性的风险的细胞生长。“恶性”是指任何细胞类型或者组织的异常生长。术语恶性包括从技术上来说是良性的但是存在变成恶性的风险的细胞生长。该术语还包括任何癌症、癌、赘生物、肿瘤形成或者肿瘤。因此，这些术语是指包括所有类型的癌生长或者肿瘤发生过程、转移性组织或者恶性转化细胞、组织或者器官，不管是组织病理学类型或者侵袭阶段。
大多数癌症分为三个大的组织学类别：癌，其是占多数的癌症以及表皮细胞或者覆盖在器官、腺体、或者其他身体结构(例如，皮肤、子宫、肺癌、乳腺癌、前列腺癌、胃、肠)的外部或者内部表面上的细胞的癌症，并且往往会发生转移；肉瘤，其衍生于结缔组织或者支撑组织(例如，骨、软骨、肌腱、韧带、脂肪，肌肉)；和血液肿瘤，其衍生自骨髓和淋巴组织。癌症的示例包括但不限于：癌、肉瘤和血液肿瘤形成紊乱例如白血病。
癌可以是腺癌(其一般在能够分泌的器官或腺体中，如乳腺、肺、结肠、前列腺或膀胱)，或者可以是鳞状细胞癌(其衍生自鳞状上皮并且一般在身体的大部分区域形成)。
肉瘤可以是骨肉瘤或骨源性肉瘤(骨)、软骨肉瘤(软骨)、平滑肌肉瘤(平滑肌)、横纹肌(骨骼肌)，间皮肉瘤或间皮瘤(体腔的膜质内层)、纤维肉瘤(纤维组织)、血管肉瘤或血管内皮瘤血管)、脂肪肉瘤(脂肪)、神经胶质瘤或星形细胞瘤(见于大脑的神经源性结缔组织)、粘液肉瘤(原始胚胎结缔组织)或间叶细胞肿瘤或中胚叶混合瘤(混合结缔组织类型)。
造血肿瘤形成性紊乱包括涉及造血起源的增生性/赘生性细胞，例如源自于髓系、淋巴系或红细胞系或其前体细胞。优选的是，所述疾病源自于分化不良的急性白血病(例如，成红细胞性白血病和急性成巨核细胞性白血病)。另外的例示性髓性紊乱包括但不是限于急性前髓细胞性白血病(APML)、急性骨髓性白血病(AML)和慢性骨髓性白血病(CML)；淋巴恶性疾病包括但不是限于急性成淋巴细胞性白血病(ALL)，其包括B系急性成淋巴细胞性白血病和T系急性成淋巴细胞性白血病，慢性淋巴细胞性白血病(CLL)、前淋巴细胞性白血病(PLL)、毛细胞白血病和瓦尔登斯特伦巨球蛋白血症(Waldenstrom's macroglobulinemia)。
其他形式的恶性淋巴瘤包括但不是限于非霍奇金淋巴瘤及其变体，外周T细胞淋巴瘤，成人T细胞白血病/淋巴瘤(ATL)、皮肤T细胞淋巴瘤(CTCL)、大颗粒淋巴细胞白血病(LGF)、霍奇金病和里-斯疾病。
药物组合物
根据本公开内容的药物组合物可以包含本申请提供的融合蛋白或者本申请提供的免疫效应细胞和一种或多种非毒性的药学上可接受的载体、稀释剂、赋形剂和佐剂。这些组合物可以适合在本文所述的治疗性适应症的治疗中使用。
如果需要，可以将其它活性成分包含在所述组合物中。因此，在一些实施方式中，所述的融合蛋白或免疫效应细胞可以以治疗有效量治疗一种或多种癌症或与其他疗法联合施用。可以在使用抗肿瘤或者其他疗法进行治疗之前、期间或之后施用所述融合蛋白或免疫效应细胞。还可以与辐射敏化剂如放射性治疗敏化剂结合使用所述融合蛋白或免疫效应细胞(参见例如Diehn等，J.Natl.CancerInst.98：1755-7，2006)。一般而言，敏化剂是能够增加所述融合蛋白的活性的任何试剂。例如，敏化剂将增加融合蛋白抑制癌细胞生长或杀死癌细胞的能力。例示性的敏化剂包括抗IL-10的抗体、骨形态发生蛋白和HDAC抑制剂(参见例如Sakariassen等，Neoplasia 9(11)：882-92，2007)。
所述融合蛋白或免疫效应细胞可能用作新辅助治疗(到初级治疗)的一部分、作为辅助治疗方案的一部分，其中目的是为了治愈受试者中的癌症。所述融合蛋白也可以在肿瘤发生和发展的不同阶段施用，包括在晚期和/或侵袭性赘生物(例如受试者中通过局部治疗形式(例如手术或者放射性治疗不能治愈的显性疾病)、转移性疾病。局部晚期疾病和/或难治性肿瘤(例如，对治疗不反应的癌症或肿瘤)的治疗中的各个阶段施用。“初级疗法”是指受试者中癌症的初次诊断后的一线治疗。例示性的初级疗法可能涉及手术、大范围的化学治疗和放射性治疗。“辅助治疗”是指这样的一种疗法，其跟随一种初级疗法之后以及在存在复发风险时施用于受试者。辅助系统性治疗在初级疗法之后很快开始，例如在最后一次的初级疗法治疗之后2、3、4、5、或6周开始以延迟复发，延长存活时间或治愈所述受试者。如此处讨论的那样，考虑到了所述融合蛋白或所述免疫效应细胞可以单独使用或作为辅助疗法的一部分与一种或多种其他化疗药物结合使用。所述融合蛋白或免疫效应细胞与标准化疗试剂的组合可以起到提高化疗疗效的作用，因此，可以用来改善标准癌症疗法。
“受试者”可以是需要治疗的哺乳动物，如人类或兽医学患者(例如，啮齿类动物，如 小鼠或者大鼠、猫、狗、奶牛、马、绵羊、山羊，或其他牲畜)。在一些实施方式中，“受试者”可以是临床患者、临床试验志愿者、实验动物等等。所述受试者可能被怀疑患有以细胞增殖为特征的疾病或者具有患上以细胞增殖为特征的疾病、被诊断为患有以细胞增殖为特征的疾病、或者是被证实不患有以细胞增殖为特征的疾病的对照受试者，如本文所述，用于以细胞增殖为特征的疾病的诊断方法和这种诊断的临床划分对于本领域技术人员是已知的。
所述组合物可以是液体溶液、悬浮液、乳液、缓释制剂或粉末，并且可以和药学上可接受的载体配制。所述组合物可以使用传统的粘合剂和载体例如甘油三酯配制成栓剂。“药学上可接受的载体”是指不会干扰活性成分的生物活性的有效性并且不对所述宿主或者受试者产生毒性的载体基质或者媒介物(vehicle)。
融合蛋白或免疫效应细胞可以和药学上可接受的媒介物一起输送。在一个实施例中，媒介物可以增强稳定性和/或输送性质。媒介物如人造膜囊泡(包括脂质体、非离子表面活性剂泡囊(noisome)、纳米微脂囊等)、微粒或者微胶囊、或者包含药学可接受的聚合物的胶体制剂。
包含一种或者多种融合蛋白或免疫效应细胞的药物组合物可以根据本领域已知的方法并且使用适当的一种或者多种分散试剂或润湿试剂和/或悬浮试剂配制成无菌可注射水性或者油质悬浮液。所述无菌可注射制剂可以是在非毒性的亲本可接受的稀释剂或者溶剂中的无菌可注射溶液或者悬浮液。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。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。
实施例1.表达嵌合抗原受体的T细胞的构建
采用本领域常规分子生物学方法，将人IL-4R信号肽(SEQ ID NO:1)、IL-4R胞外区DNA编码序列(SEQ ID NO:2)与人IL-21R的跨膜域(SEQ ID NO:3)、人IL-21R的胞内域(SEQ ID NO:4)依次连接，再与GPC3-28z-CAR(SEQ ID NO:8)以F2A自剪切肽(SEQ ID NO:13)连接，插入pRRLSIN慢病毒表达载体，得到表达GPC3-28z-CAR(SEQ ID NO:8)和融合蛋白(SEQ ID NO:5)的慢病毒质粒chIL4-21R-CAR，核苷酸序列如SEQ ID NO:16所示(图1A和1B)。
将人IL-4R信号肽(SEQ ID NO:1)、IL-4R胞外区DNA编码序列(SEQ ID NO:2)与人IL-7R的跨膜域(SEQ ID NO:24)、人IL-7R的胞内域(SEQ ID NO:25)依次连接，再与GPC3-28z-CAR(SEQ ID NO:8)以F2A自剪切肽(SEQ ID NO:13)连接，插入pRRLSIN慢病毒表达载体，得到表达GPC3-28z-CAR(SEQ ID NO:8)和融合蛋白(SEQ ID NO:26)的慢病毒质粒chIL4-7R-CAR，核苷酸序列如SEQ ID NO:27所示。
T细胞活化：取人PBMC培养于AIM-V培养基，添加2％人AB型血清，500U/mL重组人IL-2，并加入CD3/CD28抗体结合磁珠活化48h。
采用慢病毒质粒chIL4-21R-CAR或chIL4-7R-CAR感染活化后的T细胞，去血清静息 24h，得到表达融合蛋白chIL4-21R(氨基酸序列为SEQ ID NO:6)或chIL4-7R(氨基酸序列为SEQ ID NO:28)和CAR(氨基酸序列为SEQ ID NO:10)的chIL4-21R-CAR T细胞。
以重组人IL-4分别刺激UTD细胞组(未做处理的T细胞，没有转染空载)和chIL4-21R-CAR T细胞30min，收集细胞提取蛋白进行Western blot检测，分析STAT3/5磷酸化水平变化，结果如图1C所示。相比于未感染组(UTD)，chIL4-21R-CAR T细胞组(chIL4-21R)的STAT3/5磷酸化水平发生显著上调，说明该融合蛋白能够将IL-4的刺激转变为IL-21的信号。
而图1D显示chIL4-21R-CAR T细胞在rIL-4刺激下，磷酸化STAT3水平显著高于chIL4-7R-CAR T细胞，而chIL4-7R-CAR T细胞则倾向于STAT5磷酸化，说明chIL4-21R-CAR T细胞和chIL4-7R-CAR T细胞在rIL-4刺激下，产生不同的下游信号。
实施例2.chIL4-21R-CAR T细胞与IL-4刺激下增殖或存活的能力测定
参照实施例1的操作，将GPC3-28z-CAR(SEQ ID NO:8)插入RRLSIN慢病毒表达载体，构建了表达GPC3-28z-CAR的慢病毒载体，再转染293T包装慢病毒，得到慢病毒2。采用慢病毒2感染T细胞，得到GPC3-CAR T细胞。
采用如下述a或者b所述的操作，进行不同的细胞因子诱导培养：
a：分别将实施例1制备的chIL4-21R-CAR T细胞和GPC3-CAR T细胞，以重组人IL-2(浓度是500U/mL)诱导培养4天，收集细胞以抗人Fab段的抗体标记CAR，流式检测细胞阳性率。b：分别将实施例1制备的chIL4-21R-CAR T细胞和GPC3-CAR T细胞，以20ng/mL重组人IL-4(Peprotech公司)诱导培养4天，收集细胞以抗人Fab段的抗体标记CAR，流式检测细胞阳性率。
结果如图2A、2B所示，结果说明：对照组GPC3-CAR T细胞在IL-2或IL-4处理下的阳性率无显著差异，而chIL4-21R-CAR T细胞组在IL-4处理下阳性率显著上升，说明IL-4可选择性地使融合蛋白阳性的细胞扩增，提示表达融合蛋白的细胞受到IL-4刺激后比普通T细胞具有更强的增殖或存活能力，提示我们chIL4-21R-CAR T细胞在IL-4环境下的生长，能逆转肿瘤微环境的影响，将抑制性或阻抑性细胞因子信号转化成促进免疫刺激性/活化信号，比常规的CAR-T细胞更有优势。
实施例3.chIL4-21R-CAR T细胞在IL-4或IL-2刺激下对肿瘤细胞的杀伤能力
参照实施例2的操作，对T细胞分别以重组人IL-2或IL-4诱导培养6天，以Huh7肝癌细胞(GPC-3阳性)为靶细胞进行细胞毒性实验。于96孔板每孔接1×10 4个Huh7肝癌细胞，以效靶比3:1、1:1、1:3分别加入相应数量的上述实施例所得的GPC3-CAR T细胞、chIL4-7R-CAR T细胞、chIL4-21R-CAR T细胞共培养于100μL RPMI-1640培养液+10％FBS，18h后收取50μL培养液上清，用Promega公司CytoTox 96试剂盒检测上清中LDH水平，杀伤效率的计算按照厂家说明书建议进行。结果如图3A、3B所示，对照组GPC3-CAR T细胞以及chIL4-7R-CAR T细胞在IL-4处理下细胞毒性明显降低，chIL4-21R-CAR T细胞则 显著地保留甚至增强了细胞毒性。其中图3B所示的体外毒性实验进一步证明了，相比chIL4-7R-CAR T细胞，chIL4-21R-CAR T细胞在rIL-4诱导下维持更强的细胞杀伤毒性。
实施例4.T细胞活化和抑制信号基因的测定
取上述实施例1制备的GPC3-CAR T细胞、chIL4-7R-CAR T细胞和chIL4-21R-CAR T细胞，分别与rIL-4(20ng/mL)或IL-2(500U/mL)共孵育72h后，收取细胞，提取RNA，RT-qPCR检测Bcl-6、T-bet、Blimp-1、颗粒酶B、及GATA3。
Bcl-6、T-bet、Blimp-1是IL-21信号的靶基因。其中，Bcl-6是维持记忆性T细胞存活的转录因子，T-bet和Blimp-1是促进CD8 +T细胞分化为效应细胞的转录因子。颗粒酶B为T细胞活化释放的细胞浆颗粒，GATA3是为IL-4活化后的下游信号。
结果如图4A-4E(One-way ANOVA统计*表示p〈0.05，**表示p<0.01)所示，图4A显示了在IL-4刺激下，chIL4-21R-CAR T细胞的Bcl-6的表达水平大大高于GPC3-CAR T细胞和chIL4-7R-CAR T细胞；
图4B显示了在IL-4刺激下，chIL4-21R-CAR T细胞的T-bet的表达水平高于GPC3-CAR T细胞；
图4C显示了在IL-4刺激下，chIL4-21R-CAR T细胞的Blimp-1的表达水平高于GPC3-CAR T细胞；
图4D显示了在IL-4刺激下，chIL4-21R-CAR T细胞的颗粒酶B的表达水平大大高于GPC3-CAR T细胞和chIL4-7R-CAR-T细胞。说明对于chIL4-21R-CAR T细胞，IL-4的存在不仅能够诱导chIL4-21R-CAR T细胞大量表达Bcl-6，且IL-4对T-bet、Blimp-1和颗粒酶B的表达的抑制作用也在chIL4-21R-CAR T细胞中减弱甚至消除，而颗粒酶B的高表达可能是chIL4-21R-CAR T细胞在实施例3中维持高细胞毒性的原因之一。
图4E显示了在IL-4刺激下，chIL4-21R-CAR T细胞的CD26的表达水平大大高于GPC3-CAR T细胞和chIL4-7R-CAR T细胞。由于表达CD26的CD4 +CAR T细胞具有极强的抗肿瘤能力，甚至比一般认为的主要杀伤肿瘤细胞的CD8 +CART细胞更强。说明chIL4-21R融合蛋白能改善肿瘤特异性T细胞的功效,产生具有更强抗肿瘤功能的CAR-T细胞。
图4F显示了在chIL4-21R-CAR T细胞中，IL-4诱导下RORγt表达水平显著增强，改善肿瘤特异性T细胞的功效,产生具有更强抗肿瘤功能的CAR-T细胞。
图4G显示了不表达chIL4-21R的GPC3-CAR T细胞的GATA3在IL-4诱导下显著上调，但chIL4-21R-CAR T细胞的GATA3上调程度显著减弱。图4G说明IL-4下游信号分子的作用在chIL4-21R-CAR T细胞中被显著抑制。
以上结果显示了chIL4-21R融合蛋白不仅能够改变IL-4诱导的免疫抑制性环境，反而能够通过IL-21信号通路增强了T细胞与存活、效应相关基因的表达，产生具有更强抗肿瘤功能的CAR-T细胞。
综合图4A/D可以看出，chIL4-21R-CAR T细胞在rIL-4诱导下，相比chIL4-7R-CAR T细胞，表达高水平的Bcl-6，提示了其具有更强存活能力和记忆性T细胞表型。此外， chIL4-21R-CAR T细胞维持更高水平的颗粒酶B表达，这与天然的IL-21信号可增强颗粒酶B的表达相符，提示了chIL4-21R-CAR T细胞在rIL-4诱导下可保持甚至增强细胞杀伤毒性，而chIL4-7R-CAR T细胞则没有类似能力。
取上述在IL-2或IL-4刺激下，得到的GPC3-CAR T细胞和chIL4-21R-CAR T细胞，流式检测T细胞激活的标志物CD25，结果如图5A和5B所示：GPC3-CAR T细胞组在IL-4刺激后，CD25+的细胞群体比例显著低于IL-2刺激组(图5A)，chIL4-21R-CAR T细胞组在在IL-4刺激后，CD25+的细胞群体比例与IL-2刺激组基本没有差异(图5B)，说明IL-4显著抑制GPC3-CAR T细胞的CD25表达，而chIL4-21R-CAR T细胞的CD25表达基本不受抑制，提示融合蛋白阻断了IL-4对T细胞激活的抑制作用。
实施例5.CAR-T细胞耗竭标志物的检测
取上述实施例1制备的GPC3-CAR-T细胞、chIL4-7R-CAR-T细胞和chIL4-21R-CAR T细胞，分别与rIL-4(20ng/mL)、IL-2(500U/mL)共孵育6天后收取细胞，流式检测PD-1和TIM3,PD-1、和TIM3是T细胞耗竭的重要标志物。结果如图6所示：在IL-4处理下，chIL4-21R-CAR T细胞组所表达的PD-1水平低于GPC3-CAR T细胞组，TIM3水平低于GPC3-CAR T细胞组和chIL4-7R-CAR T细胞组，说明IL-4信号由融合蛋白chIL4-21R转化为IL-21信号并发挥抑制细胞耗竭的功能，IL-21信号具有抑制细胞耗竭、维持细胞存活的功能，从而提高杀伤肿瘤细胞的效果。
实施例6.chIL4-21R-CAR T细胞对IL-4分泌型的肿瘤细胞的杀伤
构建GFP-F2A-IL-4的pWPT慢病毒表达质粒，包装病毒并感染Huh-7细胞，3天后流式检测GFP呈阳性，得到能够分泌IL-4的靶细胞IL-4-Huh-7细胞。
采用IL-4-Huh-7细胞作为肿瘤细胞，比较chIL4-21R-CAR T细胞和GPC3-CAR T细胞对IL-4-Huh-7细胞的杀伤,一方面更模拟CAR T细胞在体内遭遇肿瘤细胞的状态，另一方面可显示CAR T细胞在杀伤肿瘤细胞后耗竭状态的改变。
取前述实施例制备的chIL4-7R-CAR T细胞、chIL4-21R-CAR T细胞和GPC3-CAR T细胞，以IL-4-Huh-7为靶细胞，效靶比＝1:1进行第一轮杀伤，48h后显微镜下观察：除UTD组外，各组已无贴壁细胞。收集悬浮T细胞进行流式检测，分析CAR阳性率和耗竭标志物表达，将剩余T细胞再次以效靶比1:5与靶细胞共培养，进行第二轮杀伤，48h后PBS洗去悬浮的T细胞，贴壁的靶细胞以结晶紫染色观察。结晶紫染色结果如图7A所示，相比于GPC3-CAR T细胞、chIL4-7R-CAR T细胞，chIL4-21R-CAR T细胞在第二轮刺激后维持更好的靶细胞杀伤能力。UTD指未感染的T细胞。
分别在chIL4-21R-CAR T细胞和GPC3-CAR T细胞与IL-4-Huh-7细胞共孵育的当天(记为第一轮杀伤前R0)、效靶比1:1进行第一轮杀伤48h(记为第一轮杀伤后R1)、效靶比1:5进行第二轮杀伤48h(记为第二轮杀伤后R2)。收集T细胞，流式检测T细胞耗竭物PD-1和TIM3，结果如图7B所示，随着杀伤次数增多，CAR T细胞耗竭的标志物表达显著增强，UTD作为对照 由于没有杀伤作用，因此表达情况基本不变，chIL4-21R-CAR T细胞组的PD-1和TIM3表达水平较对照组GPC3-CAR T细胞、chIL4-7R-CAR T细胞均明显降低，进一步说明chIL4-21R-CAR T细胞可更持久地对IL-4 +的靶细胞进行杀伤，具有更持久杀伤肿瘤细胞的效果。
而综合图7A/B可以看出，相比chIL4-7R-CAR T细胞，chIL4-21R-CAR T细胞在对分泌IL-4的靶细胞的持续杀伤后，T细胞耗竭标志物PD-1和TIM3表达水平更低，提示了chIL4-21R-CAR T细胞在IL-4+的肿瘤微环境中抵抗耗竭的能力更强，对靶细胞杀伤的结果进一步证明了chIL4-21R-CAR T细胞具有更持久的靶细胞杀伤能力。
实施例7小鼠体内实验
一.7721肝癌细胞小负荷皮下移植瘤模型
1)实验分组：B-NDG小鼠(百奥赛图公司)，6-8周龄随机分为4组，每组4只，分别为Untransduced(UTD)、GPC3-CAR T细胞组、chIL4-7R-CAR T细胞组、chIL4-21R-CAR T细胞组。
2)皮下移植瘤的接种：胰酶消化法收集处于对数生长期且生长状态良好的7721细胞，每只小鼠接种3×10 6个肿瘤细胞，接种日记为第0天。
3)CAR-T细胞回输：当肿瘤平均体积约为85mm 3时，即接种肿瘤后第11天，注射2×10 6/只CAR-T细胞或UTD细胞对照。实验结果如图8所示。
图8(One-way ANOVA统计***表示p<0.001)显示，CAR T注射后26天，与UTD对照组相比，各组均见明显抑瘤效果，抑制率分别为：GPC3-CAR T组：66.5％±17.2％，chIL4-7R-CAR T组：96.7％±3.6％，chIL4-21R-CAR T组：100％±0。
图8中可见，输注UTD-T细胞组的肿瘤体积持续增长，GPC3-CAR T细胞组的肿瘤体积生长相对减缓并呈现抑制趋势，而chIL4-21R-CAR T细胞组肿瘤生长明显受抑制，肿瘤体积显著小于GPC3-CAR T细胞组，说明chIL4-21R-CAR T细胞相比GPC3-CAR T细胞具有更强的抗肿瘤功能。
在给予CAR T细胞第14天时，小鼠颌下取外周血进行流式检测和细胞计数，以CD3/4/8作为标志物，检测CAR-T细胞的体内存活情况。结果如图9A和图9B(One-way ANOVA统计*表示p<0.05，**表示p<0.01)所示，表达融合蛋白chIL4-21R的CAR T细胞在外周血中的数量相比GPC3-CAR T细胞更多，提示具有更持久的抗肿瘤功能。图9B显示了chIL4-21R-CAR T细胞在体内存活的细胞亚群为CD4 +T细胞占多，而chIL4-7R-CAR T细胞则为CD8 +T细胞占多。与chIL4-7R-CAR T细胞不同，chIL4-21R-CAR T细胞在IL-4+荷瘤小鼠体内有更多CD4+细胞亚群的存活，由于CD4+CAR T细胞在体内比CD8+CAR T细胞不易耗竭，持久性更好，提示chIL4-21R-CAR T细胞可能比chIL4-7R-CAR T细胞在体内具有更持久的免疫杀伤和记忆功能。
有文献(Yang et al.,TCR engagement negatively affects CD8but not CD4CAR T cell expansion and leukemic clearance,Sci.Transl.Med.9,eaag 1209(2017)22November 2017)报道，与CD8 +T细胞相比，CD4 +T细胞在体内不易耗竭，具有更强的存活能力，这提示 chIL4-21R-CAR T细胞可能比chIL4-7R-CAR T细胞具有更持久的抗肿瘤作用。
二.PLC/PRF/5肝癌细胞大负荷皮下移植瘤模型
1)实验分组：B-NDG小鼠6-8周龄随机分为4组，每组6-7只，分别为Untransduced(UTD)、GPC3-CAR T细胞组、chIL4-7R-CAR T细胞组、chIL4-21R-CAR T细胞组。
2)皮下移植瘤的接种：胰酶消化法收集处于对数生长期且生长状态良好的PLC/PRF/5细胞，每只小鼠接种3×10 6的肿瘤细胞，接种日记为第0天。
3)CAR-T细胞回输：当肿瘤平均体积约为150mm 3时，即接种肿瘤后第13天，注射3.0×10 6/只CAR-T细胞或未经转导的T细胞对照。实验结果如图10A、10B所示。
图10A可见，CAR T注射后14天，与UTD对照组相比，各组均见明显抑瘤效果，抑制率分别为：GPC3-CAR T组：53.3％±12.0％，chIL4-7R-CAR T组：62.3％±13.8％，chIL4-21R-CAR T组：89.1％±10.1％。
在动物模型中，小鼠的体重变化是研究CAR-T细胞疗法毒副作用的重要指标之一。在本实施例中，输注CAR T细胞后第7天，chIL4-7R-CAR T组小鼠体重下降率明显高于其他CAR T组，达7.5％±2.8％，而GPC3-CAR T组和chIL4-21R-CAR T组分别为-0.1％±1.8％和-0.1％±1.5％，第9天chIL4-7R-CAR组有两只小鼠死亡。以上数据提示GPC3-CAR T细胞在表达chIL4-7R后，可能会增强CAR T细胞的毒副作用，而表达chIL4-21R后则未发现类似的不良效应，具有更好的安全性。
在上述实施例中，示例性地采用了靶向GPC3的CAR T细胞，本领域技术人员可以依据本申请的教导，采用靶向其他靶点的CAR-T细胞，如靶向EGFR的CAR T细胞(示例性的，靶向EGFR的CAR-T细胞的scFv的序列如SEQ ID NO：20所示)，如靶向CLD18A2的CAR T细胞(示例性的，靶向CLD18A2的CAR-T细胞的scFv的序列如SEQ ID NO：21所示)，如靶向CD19的CAR T细胞(示例性的，靶向CD19的CAR T细胞的scFv的序列如SEQ ID NO：22所示)，如靶向BCMA的CAR T细胞(示例性的，靶向BCMA的CAR T细胞的scFv的序列如SEQ ID NO：23所示)。
本发明所用的序列总结于下表：
Figure PCTCN2018106034-appb-000002
Figure PCTCN2018106034-appb-000003
Figure PCTCN2018106034-appb-000004
Figure PCTCN2018106034-appb-000005
Figure PCTCN2018106034-appb-000006
Figure PCTCN2018106034-appb-000007
Figure PCTCN2018106034-appb-000008
Figure PCTCN2018106034-appb-000009
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，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。

Claims (33)

  1. 一种融合蛋白，包含IL-4受体(IL-4R)胞外域或其变体和IL-21受体(IL-21R)胞内域或其变体。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述融合蛋白包含：
    i)IL-4受体(IL-4R)胞外域；
    ii)跨膜域，优选IL-4R的跨膜区或者IL-21R的跨膜区；和
    iii)IL-21受体(IL-21R)胞内域。
  3. 根据权利要求1所述的融合蛋白，其特征在于，所述IL-4R的胞外域的编码核苷酸序列与SEQ ID NO:2所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
  4. 根据权利要求3所述的融合蛋白，其特征在于，所述IL-4R的胞外域的编码核苷酸序列是SEQ ID NO:2所示的序列。
  5. 根据权利要求1所述的融合蛋白，其特征在于，所述IL-21R的胞内域的编码核苷酸序列与SEQ ID NO:4所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
  6. 根据权利要求5所述的融合蛋白，其特征在于，所述IL-21R的胞内域的编码核苷酸序列是SEQ ID NO:4所示的序列。
  7. 根据权利要求2所述的融合蛋白，其特征在于，所述融合蛋白与SEQ ID NO.6所示的序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％的同一性。
  8. 根据权利要求7所述的融合蛋白，其特征在于，所述融合蛋白具有SEQ ID NO:6所示的序列。
  9. 根据权利要求1所述的融合蛋白，其特征在于，所述IL-4R选自IL-4Rα、IL-4Rγc。
  10. 根据权利要求1所述的融合蛋白，其特征在于，所述IL-4R可以结合IL-4或者突变型IL-4或者IL-13或者突变型IL-13；优选地，所述IL-4R结合IL-4或者突变型IL-4。
  11. 根据权利要求10所述的融合蛋白，其特征在于，所述突变型IL-4包含KFR变体、KF变体、或RGA变体。
  12. 根据权利要求1-11任一所述的融合蛋白，其特征在于，各结构域直接或通过接头分子连接在一起。
  13. 一种核酸分子，所述核酸分子编码权利要求1-12中任一项所述的融合蛋白。
  14. 一种载体，所述载体包含权利要求13所述的核酸分子。
  15. 一种宿主细胞，所述宿主细胞包含权利要求14所述的载体。
  16. 一种免疫效应细胞，其特征在于，所述免疫效应细胞表达权利要求1-12任一所述的融合蛋白，或包含权利要求13所述的核酸分子或权利要求14所述的载体。
  17. 根据权利要求16所述的免疫效应细胞，其特征在于，所述的免疫效应细胞为自体 细胞或者同种异体细胞；
  18. 根据权利要求16所述的免疫效应细胞，其特征在于，所述免疫效应细胞还表达外源性的受体，该外源性的受体具有特异结合肿瘤抗原的第二胞外结合域、第二跨膜域、及第二胞内域。
  19. 根据权利要求18所述的免疫效应细胞，其特征在于，所述的融合蛋白为组成性表达或诱导性表达。
  20. 如权利要求18或19所述的免疫效应细胞，其特征在于：所述外源性受体选自：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)或其组合。
  21. 根据权利要求18-20任一所述的免疫效应细胞，其特征在于，所述的嵌合抗原受体包括：
    (i)特异性结合抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域和CD3ζ；或
    (ii)特异性结合抗原的抗体或其片段、CD28或CD8的跨膜域、CD137的共刺激信号结构域和CD3ζ；或
    (iii)特异性结合抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
  22. 根据权利要求18-21任一所述的免疫效应细胞，其特征在于，所述的肿瘤抗原包括：
    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-1 Gag；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；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25 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5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10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)。
  23. 根据权利要求18-21任一所述的免疫效应细胞，其特征在于，所述的肿瘤抗原为实体瘤抗原。
  24. 根据权利要求23所述的免疫效应细胞，所述实体瘤选自结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌；优选地，所述实体瘤选自肝癌，肺癌，鳞状细胞癌。
  25. 如权利要求18-24任一所述的免疫效应细胞，其特征在于，所述第二胞外结合域具有与SEQ ID NO:7、20、21、22、或23所示序列至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)同一性的序列。
  26. 根据权利要求20-25任一所述的免疫效应细胞，其特征在于，所述的嵌合抗原受体具有SEQ ID NO:9、10、11、12所示序列或与SEQ ID NO:9、10、11、12所示序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的同一性。
  27. 根据权利要求20-26任一所述的免疫效应细胞，其特征在于，所述的嵌合抗原受体及所述的融合蛋白由SEQ ID NO：16、17、18、或19所示核苷酸序列编码或由与SEQ ID NO： 16、17、18、或19具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同一性的核苷酸序列编码。
  28. 一种药物组合物，所述药物组合物包含权利要求1-12中任一项所述的融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞、或者权利要求16-27任一所述的免疫效应细胞。
  29. 一种诱导细胞死亡的方法，所述方法包括向需要的受试者施用权利要求1-12中任一项所述的融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞、或者权利要求16-27任一所述的免疫效应细胞。
  30. 一种治疗癌症的方法，所述方法包括向需要的受试者施用：权利要求1-12中任一项所述的融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞、权利要求16-27任一所述的免疫效应细胞、或者权利要求28所述的药物组合物。
  31. 权利要求1-12中任一项所述的融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞、或者权利要求16-27任一所述的免疫效应细胞、或权利要求28所述的药物组合物用于在有此需要的患者中诱导细胞死亡、或者治疗癌症、或者治疗过度增生性或者分化性紊乱的应用。
  32. 根据权利要求29或30所述的方法或者权利要求31所述的应用，其中，所述受试者是人类。
  33. 权利要求1-12中任一项所述的融合蛋白、权利要求13所述的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞或者权利要求16-27任一所述的免疫效应细胞在制备药物中的用途，所述药物用于在有此需要的患者中诱导细胞死亡、或者治疗癌症、或者治疗过度增生性或者分化性紊乱。
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