WO2019052562A1 - Protéine de fusion d'une il-4r et son utilisation - Google Patents

Protéine de fusion d'une il-4r et son utilisation 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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Chinese (zh)
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李宗海
王益
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464419—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—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
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    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/53—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).

Abstract

La présente invention concerne une cellule effectrice immunitaire exprimant une protéine de fusion. La protéine de fusion contient un domaine extracellulaire de liaison aux cytokines et un domaine intracellulaire de transduction de signal, le domaine extracellulaire étant le domaine extracellulaire d'un récepteur IL4 et le domaine intracellulaire étant le domaine intracellulaire d'un récepteur IL-21. La cellule effectrice immunitaire présente une capacité anti-tumorale significative dans le micro-environnement tumoral des tumeurs solides, et de là n'est pas seulement efficace contre les cellules tumorales solides in vitro, mais également présente d'excellents effets d'élimination concernant les cellules tumorales solides in vivo. La présente invention concerne également des compositions pharmaceutiques contenant la cellule effectrice immunitaire et son utilisation.
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