WO2020143631A1 - 细胞免疫治疗的组合 - Google Patents

细胞免疫治疗的组合 Download PDF

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WO2020143631A1
WO2020143631A1 PCT/CN2020/070717 CN2020070717W WO2020143631A1 WO 2020143631 A1 WO2020143631 A1 WO 2020143631A1 CN 2020070717 W CN2020070717 W CN 2020070717W WO 2020143631 A1 WO2020143631 A1 WO 2020143631A1
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cancer
cells
tumor
immune effector
gemcitabine
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PCT/CN2020/070717
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English (en)
French (fr)
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李宗海
吴秀奇
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Shanghai Cancer Institute
Carsgen Therapeutics Ltd
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Shanghai Cancer Institute
Carsgen Therapeutics Ltd
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Priority to CN202080007968.0A priority Critical patent/CN113271953B/zh
Priority to JP2021539884A priority patent/JP2022524906A/ja
Priority to KR1020217024837A priority patent/KR20210126008A/ko
Priority to EP20738673.1A priority patent/EP3909590A4/en
Priority to US17/421,133 priority patent/US20220152101A1/en
Publication of WO2020143631A1 publication Critical patent/WO2020143631A1/zh
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    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
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    • 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
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
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    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/54—Pancreas
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
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    • 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 invention belongs to the field of cell immunotherapy, and specifically relates to the combination of immune effector cells and gemcitabine for anti-tumor therapy.
  • CAR-T cells can be more easily contacted with tumor cells to achieve killing through intravenous infusion, and CAR-T cells have greater difficulty in homing tumor tissue of solid tumors.
  • solid tumors usually have a complex and dynamic tumor microenvironment, which enables tumor cells, benign cells, stromal cells, and vascular cells to interact.
  • cytokines and growth factors there is a network of cytokines and growth factors in the tumor microenvironment.
  • CAR-T treatment is usually not effective when used in solid tumor treatment.
  • the object of the present invention is to provide a tumor treatment method to improve the application effect of immune cell therapy, especially CAR-T cell therapy in solid tumors.
  • a method of treating a tumor which comprises administering an immune effector cell and gemcitabine to an individual with a tumor, the immune effector cell expressing a receptor that recognizes a tumor antigen.
  • a method for reducing the growth, survival, or vitality of cancer cells or all of them which comprises administering immune effector cells and gemcitabine to individuals with tumors, the immune effector cells expressing tumor antigens Receptor.
  • the administration time of immune effector cells and gemcitabine are in no particular order; gemcitabine can be administered first and then immune effector cells can be administered; they can also be administered simultaneously; and immune effector cells can be administered first and then gemcitabine. That is, the way to administer immune effector cells and gemcitabine to an individual with a tumor is selected from any of the following: (1) give gemcitabine first and then give immune effector cells, (2) give both immune effector cells and gemcitabine, and (3) first Gemcitabine was given to immune effector cells.
  • the above-mentioned receptors are selected from the group consisting of: Chimeric Antigen Receptor (CAR), T Cell Receptor (TCR), T Cell Fusion Protein (TFP), T cell antigen coupler (Tcell antigen coupler, TAC) or a combination thereof.
  • CAR Chimeric Antigen Receptor
  • TCR T Cell Receptor
  • TFP T Cell Fusion Protein
  • TAC T cell antigen coupler
  • the tumor antigen is a solid tumor antigen.
  • the tumor antigen is selected from the epidermal growth factor receptor family and its mutants (ie EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin 18.2, Claudin 18.1, Claudin 6, Phosphatidyl muscle Any of proteoglycan-3 (GPC3) and vascular endothelial growth factor receptor.
  • epidermal growth factor receptor family and its mutants ie EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII
  • Claudin 18.2 Claudin 18.1, Claudin 6, Phosphatidyl muscle Any of proteoglycan-3 (GPC3) and vascular endothelial growth factor receptor.
  • the tumor antigen is selected from: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20 ; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit ⁇ (IL- 13R ⁇ ); interleukin 11 receptor ⁇ (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
  • the chimeric antigen receptor has:
  • the amino acid sequence of the antibody that recognizes the tumor antigen has at least 90% identity with the amino acid sequence shown in any one of SEQ ID NO: 4 and SEQ ID NO: 14-22;
  • the amino acid sequence of the antibody that recognizes the tumor antigen is the amino acid sequence shown in any one of SEQ ID NO: 4 and SEQ ID NO: 14-22.
  • the amino acid sequence of the chimeric antigen receptor has at least 90% identity with the amino acid sequence shown in any one of SEQ ID NO: 23-44; preferably the chimeric antigen receptor Is the amino acid sequence shown in any one of SEQ ID NO: 23-44.
  • the tumor includes breast cancer, colon cancer, rectal cancer, renal cell cancer, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer , Uterine cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis Cancer, bladder cancer, ureteral cancer, renal pelvis cancer, spinal tumors, gliomas, pituitary adenoma, Kaposi's sarcoma, a combination of the cancers, and metastatic lesions of the cancers.
  • the tumors include breast cancer, blood cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, bone cancer, Pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, Vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, children's solid tumors, bladder cancer, Renal or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem gliom
  • CNS
  • the immune effector cells include: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytic cells or a combination thereof; preferably The immune effector cells are selected from autologous T cells, allogeneic T cells or allogeneic NK cells. More preferably, the T cells are autologous T cells.
  • the gemcitabine is administered orally, intraperitoneally and/or by injection.
  • the individual does not undergo lymphocyte clearance. .
  • the third aspect of the present invention provides an application of immune effector cells expressing receptors that recognize tumor antigens in the preparation of a medicament, characterized in that the medicament contains the cells and gemcitabine for treatment in human patients Tumor or reduce tumor cell growth, survival or viability, wherein the cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the effects of the cells and gemcitabine when used alone.
  • the fourth aspect of the present invention provides the use of immune effector cells and gemcitabine expressing receptors that recognize tumor antigens in the preparation of medicines, characterized in that the medicines are used to treat tumors or reduce tumor cells in human patients Growth, survival, or viability, wherein the cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the effects of the cells and gemcitabine when used alone.
  • the immune effector cells are CAR T cells.
  • the CAR T cells recognize the epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin 18.2 , Claudin 18.1, Claudin 6, Glypican-3 (GPC3), BCMA and/or vascular endothelial growth factor receptor.
  • kits for treating tumors comprising:
  • the immune effector cells and gemcitabine are formulated to provide a greater therapeutic effect than the sum of the respective effects of the agents; preferably, the immune effector cells are CAR T cells, more preferably, the CAR T cells Identify epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin 18.2, Claudin 18.1, Claudin 6, Glypican-3 (GPC3), BCMA and/ Or vascular endothelial growth factor receptor.
  • the immune effector cells are CAR T cells, more preferably, the CAR T cells Identify epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII), Claudin 18.2, Claudin 18.1, Claudin 6, Glypican-3 (GPC3), BCMA and/ Or vascular endothelial growth factor receptor.
  • the combination of gemcitabine and immune effector cells provided by the present invention can significantly improve the ability to kill tumor cells.
  • the treatment scheme of the present invention can resist immunosuppression in the cancer microenvironment, thereby significantly enhancing the effect on solid tumors, and also having better effects on refractory and progressive cancers.
  • Figure 1 is a diagram of the recombinant vector MSCV-8E5-2I-mBBZ plasmid.
  • Figure 2 shows in vivo experiments to observe the inhibitory effect of gemcitabine combined with CART cells on pancreatic cancer in situ in mice.
  • Figure 3 shows in vivo imaging data of mice treated with gemcitabine in combination with CART cells.
  • Figure 4 shows the survival data of mice treated with gemcitabine in combination with CART cells.
  • the present invention relates to the combined application of immune effector cells and gemcitabine for the treatment of tumors, it should be understood that the present invention is not limited to the methods and experimental conditions described. Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics, molecular biology, and medicinal chemistry.
  • the invention derives, at least in part, from the recognition that a combination treatment regimen of one or more cycles and/or doses of gemcitabine and immune effector cells is administered continuously, in any order, or substantially simultaneously, during treatment of some subjects
  • the cancer of the person may be more effective in increasing, enhancing or prolonging the activity and/or number of immune cells to achieve the anti-tumor effect.
  • Gemcitabine has the chemical name 2'-deoxy-2',2'-difluorocytidine hydrochloride; its molecular formula is C 9 H 11 F 2 N 3 O 4 .HCl and its molecular weight is 299.66.
  • Gemcitabine is a pyrimidine antimetabolite tumor drug that can interfere with the synthesis and repair of tumor cell DNA by inhibiting ribonucleotide reductase and cell replication, and is effective for a variety of solid tumors.
  • the combination of gemcitabine and tumor cell-targeted immune cell therapy can significantly improve the anti-tumor effect and even achieve a complete remission treatment effect.
  • the present invention can not only improve the anti-cancer effect of refractory cancer, when CAR-T cells are used, there is no need for lymphocyte clearance, thus greatly reducing the low anti-cancer treatment effect caused by clearing Toxic side effects caused by damage to normal tissues, especially severe suppression of bone marrow.
  • an individual with a tumor has a dose of approximately 1250 mg/m 2 of intravenous infusion of gemcitabine, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800, 750, 700, 650 , 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2 , or 1 mg/m 2 .
  • the individual suffering from the tumor is infused with gemcitabine approximately once every 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, or 1 week.
  • the administration time of the immune effector cells and gemcitabine is in no particular order; the gemcitabine can be administered first and then the immune effector cells can be administered; or they can be administered at the same time; the immune effector cells can be administered first and then the gemcitabine.
  • the immune effector cell therapy is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours before gemcitabine administration , 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days Days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month or any combination thereof.
  • the immune effector cell therapy is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours after gemcitabine administration , 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days Days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month or any combination thereof.
  • immune effector cell refers to a cell that exerts an effector function during an immune response.
  • immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages (or bone marrow-derived Phagocytic cells) and dendritic cells.
  • T cells include autologous T cells, heterologous T cells, and allogeneic T cells, and the natural killer cells are allogeneic NK cells.
  • immune effector function or immune effector response refers to immune effector cells, such as a function or response that enhances or promotes immune attack of target cells.
  • immune effector function or response refers to the properties of T cells or NK cells that promote the killing of target cells or inhibit growth or proliferation.
  • immune effect function includes any function mediated by the composition of the immune system, which can lead to inhibition of tumor growth and/or inhibition of tumorigenesis, including inhibition of tumor spread and metastasis.
  • the immune effector function kills tumor cells.
  • the immune effect function in the present invention is antibody-mediated, including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis ( ADCP), induction of apoptosis in cells carrying tumor-associated antigens (eg, through the binding of antibodies to surface antigens), inhibition of CD40L-mediated signal transduction (eg, through the interaction of antibodies with CD40 receptors or CD40 ligands (CD40L) Combined), and/or inhibits the proliferation of cells carrying tumor-associated antigens, preferably ADCC and/or CDC.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • induction of apoptosis in cells carrying tumor-associated antigens eg, through the binding of antibodies to surface antigens
  • CD40L-mediated signal transduction eg, through the interaction of antibodies with CD40 receptors or CD40 ligand
  • antibodies capable of mediating one or more immune effector functions are preferably capable of inducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypic adhesion, and/or phagocytosis (preferably by inducing CDC-mediated Lysis and/or ADCC-mediated lysis) to mediate the killing of tumor cells.
  • Antibodies can also function simply by binding to tumor-associated antigens on the surface of cancer cells. For example, antibodies can block the function of tumor-associated antigens or induce apoptosis by binding to tumor-associated antigens on the surface of tumor cells.
  • terapéuticaally effective amount refers to the amount of a compound, preparation, substance or composition that is effective to achieve a specific biological result, such as but not Limited to an amount or dose sufficient to promote a T cell response.
  • therapeutically effective amount refers to the amount of a compound, preparation, substance or composition that is effective to achieve a specific biological result, such as but not Limited to an amount or dose sufficient to promote a T cell response.
  • the effective amount of immune effector cells refers to, but not limited to, increase, enhance or prolong the antitumor activity of immune effector cells; increase the number of antitumor immune effector cells or activated immune effector cells; promote the secretion of IFN- ⁇ and TNF ⁇ ; tumor regression, The number of immune effector cells with tumor shrinkage and tumor necrosis.
  • lymphocytes in the subject are not cleared. Including but not limited to not giving lymphocyte scavengers, systemic radiation therapy or a combination thereof or other means to cause lymphocyte count clearance; however, when lymphocyte scavengers, systemic radiation therapy or a combination thereof or other means to cause lymphocyte count clearance are administered After the method, when the lymphocyte clearance rate of the subject is less than 60%, it will also fall into the category of "not clear" in this application.
  • peptide refers to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • the protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide.
  • Polypeptides include any peptide or protein that contains two or more amino acids bonded to each other by peptide bonds.
  • chimeric receptor refers to a fusion molecule formed by linking corresponding cDNAs of DNA fragments or proteins from different sources using gene recombination technology, including extracellular domain, transmembrane domain and intracellular domain. Chimeric receptors include but are not limited to: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TCR modified T cell
  • T cell fusion protein T cell fusion protein
  • TAC T cell antigen coupler
  • chimeric antigen receptor refers to a group of polypeptides that, when they are in immune effector cells, provide the cells with specificity against target cells (usually cancer cells) and have Intracellular signal generation.
  • CAR generally includes at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain”), which includes stimulatory molecules derived from and/or Or functional signaling domains of costimulatory molecules.
  • the groups of polypeptides are contiguous with each other.
  • the polypeptide group includes dimerization switches that can couple polypeptides to each other in the presence of a dimerization molecule, for example, an antigen-binding domain can be coupled to an intracellular signaling domain.
  • the stimulatory molecule is a delta chain that binds to the T cell receptor complex.
  • the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (ie, CD137), CD27, and/or CD28.
  • CAR includes a chimeric fusion protein that includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain that includes a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and a functional signaling domain derived from a costimulatory molecule and a functionality derived from a stimulatory molecule The intracellular signaling domain of the signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and comprising two functional signaling derived from one or more costimulatory molecules.
  • the present invention contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, scFv) that produces functionally equivalent molecules.
  • the VH or VL of the antigen-binding domain of the cancer-associated antigen described herein, such as the scFv contained in the CAR can be modified to retain the initial VH or VL framework of the antigen-binding domain of the cancer-associated antigen described herein At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity .
  • the present invention contemplates modification of the entire CAR construct, such as modification of one or more amino acid sequences of multiple domains of the CAR construct, to produce functionally equivalent molecules.
  • the CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 of the starting CAR construct %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the amino acid sequence of the antibody that recognizes tumor antigens herein is shown in SEQ ID NO: 4, or shown in SEQ ID NO: 14, or shown in SEQ ID NO: 15, or shown in SEQ ID NO: 16, as shown in SEQ ID NO: 17, or as SEQ ID NO: 18, or as SEQ ID NO: 19, or as SEQ ID NO: 20, or as SEQ ID NO: 21, or the amino acid sequence shown in SEQ ID NO: 22 has at least 90% identity; preferably SEQ ID NO: 4, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or SEQ ID NO: 19, or SEQ The amino acid sequence shown in ID NO: 20, or shown in SEQ ID NO: 21, or shown in SEQ ID NO: 22.
  • transmembrane domain refers to a region of a protein sequence that spans a cell membrane, and may include one or more additional amino acids adjacent to the transmembrane region, such as one or more proteins that are derived from the transmembrane Amino acids associated with the extracellular region (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or derived from the transmembrane protein One or more additional amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region) associated with the extracellular region of the protein.
  • the transmembrane domain is a domain related to one of the other domains of the chimeric receptor.
  • the transmembrane domain may be from a signaling domain, costimulatory The same protein from which the domain or hinge domain is derived.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, for example, to allow interaction with other members of the receptor complex Interaction is minimized.
  • the transmembrane domain is capable of homodimerizing with another chimeric receptor on the cell surface expressing the chimeric receptor.
  • the transmembrane domain can be derived from natural or recombinant sources. When the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein.
  • the transmembrane domain is capable of signaling to the intracellular domain as long as the chimeric receptor binds to the target antigen.
  • the transmembrane domains particularly used in the present invention may include at least the following transmembrane domains: for example, ⁇ , ⁇ , or ⁇ chains of T-cell receptors, CD28, CD27, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain may include at least the following transmembrane regions: for example KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , 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, 2B
  • the transmembrane domain may be connected to the extracellular region of the CAR via a hinge (eg, a hinge from a human protein), such as the antigen binding domain of the CAR.
  • a hinge eg, a hinge from a human protein
  • the hinge may be a human Ig (immunoglobulin) hinge (eg, IgG4 hinge, IgD hinge), GS linker (eg, GS linker described herein), KIR2DS2 hinge, or CD8a hinge.
  • the transmembrane domain may be recombinant, in which case it will mainly contain hydrophobic residues, such as leucine and valine.
  • a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.
  • a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length can form a bond between the transmembrane domain of the CAR and the cytoplasmic region.
  • the glycine-serine doublet provides a particularly suitable linker.
  • intracellular domain or "cytoplasmic domain” includes intracellular signaling domains.
  • the intracellular signaling domain is generally responsible for the activation of at least one of the normal immune effector functions of immune cells into which the chimeric receptor has been introduced.
  • effector function refers to the specialized function of a cell.
  • the immune effector function of T cells may be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein that transduces immune effector function signals and directs cells to perform specific functions. Although generally all intracellular signaling domains can be used, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain In the case of using truncated portions of intracellular signaling domains, such truncated portions can be used instead of complete chains, as long as they transduce immune effector function signals. Therefore, the term intracellular signaling domain is meant to include truncated portions of the intracellular signaling domain that are sufficient to transduce immune effector function signals.
  • T cell activation can be said to be mediated by two different kinds of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation by TCR (primary intracellular signaling domain) and in an antigen-independent manner Those that function to provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains).
  • the term "stimulatory molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that regulates the signaling pathway used for immune cells in a stimulatory manner Activation of immune cells in at least some aspects.
  • the signal is a primary signal initiated by, for example, the binding of the TCR/CD3 complex to the peptide-loaded MHC-antigen peptide complex, and it results in mediating T cell responses, including, but not limited to proliferation, activation, differentiation Wait.
  • Primary cytoplasmic signaling sequences that function in a stimulating manner may contain signaling motifs called immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM-containing cytoplasmic signaling sequences specifically used in the present invention include, but are not limited to those derived from: CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (FcEpsilon R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10 and DAP12.
  • the intracellular signaling domain in any CAR of the present invention includes intracellular signaling sequences, such as the primary signaling sequence of CD3- ⁇ .
  • the primary signaling sequence of CD3- ⁇ is the equivalent residue from human or non-human species such as mouse, rodent, monkey, ape, etc.
  • costimulatory molecule refers to a homologous binding partner on T cells that specifically binds to the costimulatory ligand, thereby mediating the T cell costimulatory response, such as but not limited to proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which promote an effective immune response.
  • Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4- 1BB (CD137).
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD22), CD19
  • the costimulatory intracellular signaling domain may be the intracellular part of the costimulatory molecule.
  • Costimulatory molecules can be represented by the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), and NK cell receptor.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind CD83, etc.
  • the intracellular signaling domain may include all or part of the natural intracellular signaling domain of the molecule, or a functional fragment or derivative thereof.
  • 4-1BB refers to a member of the TNFR superfamily having the amino acid sequence provided by GenBank Accession No. AAA62478.2, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc.;
  • the "4-1BB costimulatory domain” is defined as the amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • the "4-1BB costimulatory domain” is an equivalent residue from human or from a non-human species such as mouse, rodent, monkey, ape, etc.
  • the chimeric antigen receptor herein has: (i) an antibody or fragment thereof that recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signal domain of CD28, and CD3 ⁇ ; or (ii) an antibody or a antibody that recognizes a tumor antigen Fragment, CD28 or CD8 transmembrane region, CD137 costimulatory signal domain and CD3 ⁇ ; or (iii) an antibody or fragment thereof that recognizes a tumor antigen, CD28 or CD8 transmembrane region, CD28 costimulatory signal domain, CD137 Costimulatory signal domain and CD3 ⁇ .
  • amino acid sequence of the chimeric antigen receptor and SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43 or 44 have at least 90% identity; preferably the amino acid sequence of the chimeric antigen receptor is SEQ ID NO: 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 amino acid sequences.
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen.
  • the antibody may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulin, and may be derived from natural sources or recombinant sources.
  • the antibody may be a tetramer of immunoglobulin molecules.
  • antibody fragment refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution).
  • antibody fragments include, but are not limited to Fab, Fab', F(ab') 2 , Fv fragments, scFv, disulfide bond-linked Fvs (sdFv), Fd fragments composed of VH and CH1 domains, linear antibodies , A single-domain antibody (such as sdAb), camelid VHH domain, a multispecific antibody formed by an antibody fragment (for example, a bivalent fragment including two Fab fragments connected by a disulfide bond in a hinge region) and an isolated CDR of the antibody Or other epitope binding fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed in the form of 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
  • the 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 the VL-linker-VH or VH-linker-VL may be included.
  • antibody heavy chain refers to the larger of the two polypeptide chains that exist in the antibody molecule in their naturally occurring configuration and generally determine the type of antibody.
  • antibody light chain refers to the smaller of the two polypeptide chains present in the antibody molecule in its naturally occurring configuration.
  • ⁇ (k) and ⁇ (l) light chains refer to the two major antibody light chain isotypes.
  • recombinant antibody refers to antibodies produced using recombinant DNA technology, such as, for example, antibodies expressed by phage or yeast expression systems.
  • the term should also be interpreted as referring to an antibody that has been produced by synthesizing an antibody-encoding DNA molecule (and in which the DNA molecule expresses an antibody protein) or an amino acid sequence of a specified antibody, where the DNA or amino acid sequence has been obtained using recombinant DNA or available in the art And well-known amino acid sequence technology.
  • antigen refers to a molecule that causes an immune response.
  • the immune response may involve antibody production or activation of cells with specific immunity or both.
  • antigens can be derived from recombinant or genomic DNA.
  • DNA including a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response.
  • the antigen need not be encoded only by the full-length nucleotide sequence of the gene.
  • the invention includes but is not limited to the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode polypeptides that elicit the desired immune response.
  • the antigen need not be encoded by a "gene” at all.
  • Antigens can be produced synthetically, or can be derived from biological samples, or can be large molecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.
  • tumor-associated antigen refers to a protein that is specifically expressed under normal conditions in a limited number of tissues and/or organs or at a specific developmental stage.
  • tumor-associated The antigen is specifically expressed in gastric tissues under normal conditions, preferably in the pancreas, gastric mucosa, in the reproductive organs (eg testis), in the trophoblastic tissue (eg placenta) or in germline cells, and Expressed or abnormally expressed in one or more tumor tissues or cancer tissues.
  • the tumor-associated antigen refers to a differentiation antigen, preferably a cell type-specific differentiation antigen, ie, a specific stage of differentiation under normal conditions Proteins specifically expressed in cell types.
  • tumor-associated antigens are located in tumor cells, but are not expressed in normal tissues or only in small amounts.
  • tumor-associated antigens or abnormal expression of tumor-associated antigens It can be used to identify tumor cells.
  • tumor-associated antigens expressed by tumor cells in an individual are autologous proteins of the individual.
  • tumor-associated antigens are under normal conditions.
  • tumor-associated antigen is the same as the amino acid sequence of the tumor-associated antigen expressed in tumor tissue.
  • the tumor antigens of the present invention include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD70; CD123; CD138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); interleukin 11 receptor ⁇ (IL-11R ⁇ ); Prostate Stem Cell Antigen (PSCA); Prostate Specific Membrane Antigen (PSMA); Carcinoembryonic Antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Tyrosine Enzymes; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endothelial Growth
  • tumor refers to swelling or lesions (called neoplastic cells or tumor cells) caused by abnormal growth of cells.
  • Tumor cells refers to cells that can proliferate rapidly and uncontrollably and continue to grow after the stimulation that caused the proliferation ceases. Tumors show partial or complete lack of structural tissue and lack of functional coordination with normal tissues, usually forming unique tissue clumps, which can be benign, premalignant or malignant.
  • cancer (medical term: malignant tumor) is a type of disease in which a group of cells exhibit uncontrolled growth (divide beyond normal limits), invade (invade and destroy neighboring tissues), and sometimes metastasize (through the lymph Or blood spread to other places in the body). These three malignant characteristics of cancer distinguish them from benign tumors, which are self-limiting and do not invade or metastasize. Most cancers form tumors, but some (such as leukemia) are not.
  • cancer and “tumor” or “cancer disease” and “tumor disease” are interchangeable and refer to a disease in which cells exhibit uncontrolled growth, and/or invasion, and/or metastasis.
  • leukemia seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, renal cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer , Brain cancer, cervical cancer, bowel cancer, liver cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ENT cancer, breast cancer, Prostate cancer, uterine cancer, ovarian cancer and lung cancer, and their metastases.
  • lung cancer breast cancer, prostate cancer, colon cancer, renal cell carcinoma, cervical cancer, or the metastasis of the above-mentioned various types of cancer or tumor.
  • Transfection or “transduction” refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • Transfected or “transduced” cells are cells that have been transfected, transformed or transduced with exogenous nucleic acid. The cells include primary subject cells and their progeny.
  • refractory refers to a disease, such as cancer or tumor, which does not respond to treatment.
  • the refractory tumor may be resistant to treatment before or at the beginning of treatment.
  • refractory tumors can become resistant during treatment.
  • Refractory cancer is also called resistant tumor.
  • refractory tumors include but are not limited to tumors that are insensitive to radiotherapy, relapse after radiotherapy, insensitive to chemotherapy, relapse after chemotherapy, insensitive to CAR-T treatment, or relapse after treatment.
  • Refractory or recurrent malignant tumors can use the treatment protocol described herein.
  • relapsed refers to a period of improvement, for example, returning to a disease (eg, cancer) or signs and symptoms of a disease such as cancer after a previous treatment of the therapy, such as cancer therapy.
  • a disease eg, cancer
  • signs and symptoms of a disease such as cancer after a previous treatment of the therapy, such as cancer therapy.
  • subject means “subject”, “individual”, “organism” or “patient” are used interchangeably and include vertebrates, preferably mammals.
  • mammals are humans, non-human primates, domestic animals (eg, dogs, cats, sheep, cows, goats, pigs, horses, etc.), experimental animals (eg, mice, rats, rabbits, guinea pigs) Etc.) and captive animals (eg zoo animals).
  • animal as used herein also includes humans.
  • subject and “individual” include patients, ie, diseased animals, preferably humans, and the diseases are preferably those described herein.
  • the term “enhancement” refers to allowing a subject or tumor cell to improve its ability to respond to the treatment disclosed herein.
  • the enhanced response may include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 in response %, 75%, 80%, 85%, 90%, 95%, or 98% or more increase.
  • “enhancement” may also refer to increasing the number of subjects that respond to treatments such as immune effector cell therapy.
  • an enhanced response may refer to the total percentage of subjects responding to treatment, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
  • the treatment results from the clinical; the increase, enhancement or prolongation of the anti-tumor activity of T cells; compared with the number before treatment, the increase in the number of anti-tumor T cells or activated T cells promotes the secretion of IFN- ⁇ and TNFa, Or a combination of them.
  • the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response through the immune system; tumor expansion, recurrence or spread, or a combination thereof.
  • the therapeutic effect is predicted by the presence of T cells, the presence of genetic markers indicative of T cell inflammation, promoting IFN- ⁇ , TNFa secretion, or a combination thereof.
  • the immune effector cells as disclosed herein can be administered to an individual by various routes including, for example, oral or parenteral, such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral , Intranasally (intravasally), intradermally, or passively or through the skin to promote absorption, for example using skin patches or transdermal iontophoresis.
  • oral or parenteral such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral , Intranasally (intravasally), intradermally, or passively or through the skin to promote absorption, for example using skin patches or transdermal iontophoresis.
  • the total amount of agent to be administered in practicing the method of the present invention may be administered as a single dose as a bolus or by infusion over a relatively short period of time to the subject, or may be administered using a graded treatment regimen, where for an extended period of time Multiple doses are administered in stages.
  • a graded treatment regimen where for an extended period of time Multiple doses are administered in stages.
  • the amount of the composition that treats the pathological condition in the subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking into account these factors, the technician will adjust the specific dosage as needed.
  • phase I and phase II clinical trials were used to determine the formulation of the composition and the route and frequency of administration.
  • a range such as 95-99% identity includes a range with 95%, 96%, 97%, 98%, or 99% identity, and includes sub-ranges such as 96-99%, 96-98%, 96 to 97%, 97 to 99%, 97 to 98% and 98 to 99% identity. This is applicable regardless of the width of the range.
  • the present application relates to adoptive cells or immune effector cells for the treatment of solid tumors, including the administration of multiple doses or repeated doses of cells, and the methods, compositions, and products used therefor.
  • Cells generally express chimeric antigen receptors, for example, chimeric antigen receptors (CAR) or other transgenic receptors such as T cell receptors (TCR).
  • CAR chimeric antigen receptors
  • TCR T cell receptors
  • Human claudin 18 (Claudin 18, CLD18) molecule (Genbank accession number: splice variant 1 (CLD18A1 or Claudin 18.1): NP_057453, NM016369, and splice variant 2 (CLD18A2 or Claudin 18.2): NM_001002026, NP_001002026) are about molecular weight It is an intrinsic transmembrane protein of 27,9/27,72kD. Mouse claudin Claudin 18.2 (NP_001181850.1).
  • CLD18 refers to claudin 18 and includes any variant of CLD18 (including CLD18A1 and CLD18A2), conformation, isoform, and interspecies homology expressed by cells naturally expressed or transfected by cells transfected with the CLD18 gene Sources (specieshomologs).
  • Cleavin 18 is an intrinsic membrane protein located in the tight junction of epithelium and endothelium. A network of tightly interconnected chains of particles in the tissue membrane between adjacent cells. Among tight junctions, occludin and claudin are the most important transmembrane protein components.
  • CLD18 refers to human CLD18, in particular CLD18A2 (amino acid sequence shown in SEQ ID NO: 2) and/or CLD18A1 (amino acid sequence shown in SEQ ID NO: 3), more preferably CLD18A2.
  • CLD18A1 includes any post-translationally modified variants, isoforms, and interspecies homologs of any human CLD18A1 that are naturally expressed by cells or expressed by cells transfected with the CLD18A1 gene.
  • CLD18A2 includes any post-translationally modified variants, isoforms, and interspecies homologs of any human CLD18A2 that are naturally expressed by cells or expressed by cells transfected with the CLD18A2 gene.
  • CLD18 variants shall include (i) CLD18 splice variants, (ii) CLD18 post-translational modification variants, especially including variants with different N-glycosylation status, (iii) CLD18 conformational variants, especially including CLD18-conformation-1, CLD18-conformation-2 and CLD18-conformation-3, (iv) free CLD18 and homotypic/heteromorphic variants located at the tight junctions between cells, (v) CLD18 cancer-associated variants and CLD18 non- Cancer-related variants.
  • the claudin 18A2 is a protein/peptide comprising a peptide of the amino acid sequence of SEQ ID NO: 2 or a variant of the amino acid sequence.
  • variant refers to mutants, splice variants, conformations, isotypes, allelic variants, species variants and species homologs, especially naturally occurring variants .
  • CLDN shall cover any post-translationally modified variants and conformational variants.
  • CLD18A1 is selectively expressed in the epithelium of normal lungs and stomach, while CLD18A2 is only expressed in gastric cells. Moreover, CLD18A2 is restricted to differentiated gastric epithelial short-lived cells, but does not exist in the gastric stem cell area.
  • the primary tumor refers to gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer and gallbladder cancer
  • the metastasis particularly refers to gastric cancer metastasis such as Krukenberg Tumor, peritoneal metastasis and lymph node metastasis.
  • the cell expressing CLDN18A2 refers to a tumor cell, and is specifically selected from tumorigenic gastric cancer cells, esophageal cancer cells, pancreatic cancer cells, lung cancer cells, ovarian cancer cells, colon cancer cells, liver cancer cells, head and neck cancers Cells and gallbladder cancer cells.
  • the tumor includes breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterus Cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Bladder cancer, ureteral cancer, renal pelvis cancer, spine tumors, gliomas, pituitary adenoma, Kaposi's sarcoma, a combination of the above cancers, and metastatic lesions of the above cancers.
  • the present invention provides therapeutic methods and compositions for treating diseases (such as tumors) with CLD18 expression.
  • the present invention provides a method for adopting cells or immune effector cells to treat solid tumors in which the subject expresses genetically engineered (recombinant) immune effector cell chimeric receptors for treating the subject's tumor.
  • the method generally includes a single course of reinfusion of such cells, or multiple courses of reinfusion.
  • the dose is increased during the course of treatment or repeated dose infusion during the course of treatment or the dose is reduced during the course of treatment.
  • the first dose is the lower dose and/or the adjusted or reduced dose and/or the subsequent dose is the consolidation dose and/or the adjusted or reduced dose.
  • Cells, compositions and preparations that can be used in such methods are also provided.
  • the chimeric receptor is a genetically engineered antigen receptor, such as a functional non-TCR antigen receptor, for example, a chimeric antigen receptor (CAR) and other recombinant antigen receptors such as transgenic T cell receptors Body (TCR).
  • Receptors also include other recombinant chimeric receptors, such as those containing extracellular and intracellular portions that specifically bind to ligands or receptors or other binding partners, such as the intracellular signal transduction portion of CAR.
  • the dose includes a lower first dose.
  • the method includes (a) administering a single course of cells expressing a chimeric antigen receptor (eg, CAR) to a subject with a tumor; and (b) administering a multiple course of expression to a subject Antigen receptor (eg, CAR) cells.
  • a single course of cells expressing a chimeric antigen receptor eg, CAR
  • a multiple course of expression eg, CAR
  • one or more subsequent doses may be administered.
  • Exemplary antigen receptors of the present invention including CAR, and methods for engineering and introducing receptors into cells, refer to, for example, Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A , CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication Nos. WO2017186121A1, WO2018006882A1, WO2015172339A8, those disclosed in WO2018/018958A1.
  • Example 1 Panc02/Luc-GFP-Claudin 18.2, 8E5-2I-mBBZ CAR T cell establishment
  • the Panc02 pancreatic cancer cell model overexpressing mouse Claudin 18.2 (SEQ ID NO: 1) (Panc02 pancreatic cancer cell model was purchased from Banna Bio) was established using conventional methods of molecular biology. Using pWPT as a vector, the mouse Claudin 18.2 (SEQ ID NO: 1) was inserted to construct the plasmid pWPT-mClaudin 18.2. Then the pancreatic cancer cell Panc02/Luc-GFP-Claudin 18.2 overexpressing mouse Claudin 18.2 was established by lentivirus packaging infection method.
  • the scFv used in this example is a murine claudin 18.2 antibody, and the nucleic acid sequence is shown in SEQ ID NO: 5.
  • the 8E5-2I-mBBZ sequence consists of the murine CD8 ⁇ signal peptide (its amino acid sequence is shown in SEQ ID NO: 6, its nucleotide sequence is shown in SEQ ID NO: 7), and scFv targeting claudin 18.2 (the The amino acid sequence is shown in SEQ ID NO: 4, the nucleotide sequence is shown in SEQ ID NO: 5), murine CD8 hinge and transmembrane region (the amino acid sequence is shown in SEQ ID NO: 8, the nucleotide sequence is shown in SEQ ID NO: 9) and murine 4-1BB intracellular signaling domain (the amino acid sequence is shown in SEQ ID NO: 10, the nucleotide sequence is shown in SEQ ID NO: 11) and the mouse CD8 ⁇ signal peptide (its amino acid sequence is shown in SEQ ID NO: 6, its nucleotide sequence is shown in SEQ ID NO: 7), and scFv targeting claudin 18.2 (the The amino acid sequence is shown in SEQ ID NO: 4, the
  • the MSCV-8E5-2I-mBBZ constructed above was transfected into 293T (derived from ATCC) packaging retrovirus to obtain retrovirus.
  • the infection method is a conventional infection method in the preparation process of T cells expressing chimeric antigen receptors in the art.
  • CAR T cell construction take splenic T lymphocytes of C57BL/6 mice (from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.), and purify mouse CD3+ T lymphocytes in 1:1 volume Than add Dynabeads Mouse T-activator CD3/CD28, wash once with PBS, activate, put in incubator culture, the medium is RPMI 1640 complete medium (purchased from invitrogen company), add 10% FBS serum.
  • the activated mouse spleen T lymphocytes for 24h were inoculated into recombinant human fibrin fragment-coated 12-well plates, and retroviral infection was added for 12 hours, and then cultured and amplified to the required number to obtain 8E5-2I- mBBZ CAR T cells.
  • pancreas of 6-week-old C57BL/6 mice was inoculated with 2 ⁇ 10 5 Panc02/Luc-GFP-Claudin 18.2 cells in situ, and the tumor cell inoculation diary was Day 0 (that is, Day 0).
  • mice On the 6th day after tumor inoculation (that is, Day 6), mouse T cells were taken and the 8E5-2I-mBBZ CAR T cell line was constructed as in Example 1. At the same time, the mice were imaged in vivo, and the mice were randomly grouped according to the tumor burden of the mice. The main groups were:
  • Group 1 UTD group, that is, the group that was given T-cell therapy only to untransfected mice;
  • Group 2 The CART group, that is, the group given only 8E5-2I-mBBZ CAR T cell therapy;
  • Group 3 The gemcitabine group (or, the gemcitabine group), that is, the group given only gemcitabine alone;
  • Group 4 Gemcitabine+CART group (or called gemcitabine+CART group), that is, 8E5-2I-mBBZ CAR T cells combined with gemcitabine treatment group.
  • mice in the Gemcitabine group and Gemcitabine+CART group were treated with 30 mg/kg gemcitabine for intraperitoneal administration once daily.
  • mice in the CART group and Gemcitabine+CART group were given a dose of 3 ⁇ 10 6 8E5-2I-mBBZ CAR-T cell tail vein injection, and the UTD group was given a 3 ⁇ 10 injection 6 Untransfected mouse T cells, and then weekly live imaging to observe the therapeutic effect.

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Abstract

一种治疗肿瘤的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和吉西他滨，所述免疫效应细胞表达有识别肿瘤抗原的受体。一种用于治疗肿瘤的试剂盒，其特征在于，所述试剂盒包含：1)表达有识别肿瘤抗原的受体的免疫效应细胞；2)吉西他滨；3)用于包含以上1)和2)所述物质的容器；和4)利用所述试剂盒治疗肿瘤的给药说明书。

Description

细胞免疫治疗的组合 技术领域
本发明属于细胞免疫治疗领域，具体涉及免疫效应细胞和吉西他滨联合用于抗肿瘤治疗。
背景技术
近年来，细胞免疫治疗如CAR-T细胞治疗在血液瘤治疗中显示出惊人的治疗效果，目前已经超过200项CAR-T细胞用于血液瘤治疗的临床实验(Clinical development of CAR T cells-challenges and opportunities in translating innovative treatment concepts,Jessica Hartmann et al.,EMBO Molecule Medicine,Published on line,August 1,2017)。然而对于实体瘤的治疗却难以达到血液瘤治疗的效果。
这是因为对于血液瘤而言，CAR-T细胞通过静脉输入能够更容易的接触到肿瘤细胞实现杀伤，而CAR-T细胞想要归巢到实体瘤的肿瘤组织则具有更大的难度。并且，实体瘤通常具有复杂的动态的肿瘤微环境，能够使肿瘤细胞、良性细胞、基质细胞、血管细胞等相互作用，另外，肿瘤微环境中还存在由细胞因子和生长因子相互作用的网络因此，CAR-T治疗在用于实体瘤治疗时，通常疗效不佳。
发明内容
本发明的目的在于提供一种肿瘤治疗方法，以提高免疫细胞治疗特别是CAR-T细胞治疗在实体瘤中应用效果。
本发明的第一方面，提供一种治疗肿瘤的方法，其包括对患有肿瘤的个体施用免疫效应细胞和吉西他滨，所述免疫效应细胞表达有识别肿瘤抗原的受体。
本发明的第二方面，还提供一种降低癌细胞生长、存活或活力或全部的方法，其包括对患有肿瘤的个体施用免疫效应细胞和吉西他滨，所述免疫效应细胞表达有识别肿瘤抗原的受体。
在一优选方案中，免疫效应细胞和吉西他滨给予时间不分先后；可以先给予吉西他滨再给予免疫效应细胞；也可以同时给药；还可以先给予免疫效应细胞再给予吉西他滨。即对患有肿瘤的个体给予免疫效应细胞和吉西他滨的方式选自以下中的任意：(1)先给予吉西他滨再给予免疫效应细胞，(2)同时给予免疫效应细胞和吉西他滨，以及(3)先给予免疫效应细胞再给予吉西他滨。
在另一优选方案中，上述受体选自：嵌合抗原受体(Chimeric Antigen Receptor，CAR)、T细胞受体(T cell receptor，TCR)、T细胞融合蛋白(T cell fusionprotein，TFP)、T细胞抗原耦合器(T cell antigen coupler，TAC)或其组合。
在另一优选方案中，所述肿瘤抗原为实体瘤抗原。
在另一优选方案中，所述肿瘤抗原选自表皮生长因子受体家族及其突变体(即EGFR、EGFR2、ERBB3、ERBB4、EGFRvIII)、Claudin18.2、Claudin18.1、Claudin 6、磷脂酰肌醇蛋白聚糖-3(GPC3)、血管内皮生长因子受体中的任意。
在另一优选方案中，所述肿瘤抗原选自：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1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员 D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
在另一优选方案中，所述的嵌合抗原受体具有：
(i)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3δ；或
(ii)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3δ；或
(iii)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3δ。
在另一优选方案中，所述识别肿瘤抗原的抗体的氨基酸序列与SEQ ID NO：4以及SEQ ID NO：14-22中任一项所示的氨基酸序列具有至少90％的同一性；优选所述识别肿瘤抗原的抗体的氨基酸序列为SEQ ID NO：4以及SEQ ID NO： 14-22中任一项所示的氨基酸序列。
在另一优选方案中，所述嵌合抗原受体的氨基酸序列与SEQ ID NO：23-44中任一项所示的氨基酸序列具有至少90％的同一性；优选所述嵌合抗原受体的氨基酸序列为SEQ ID NO：23-44中任一项所示的氨基酸序列。
在另一优选方案中，其中所述肿瘤包括：乳腺癌，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，子宫癌，卵巢癌，直肠癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，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，输尿管癌，肾盂癌，脊椎肿瘤，脑胶质瘤，垂体腺瘤，卡波西肉瘤，所述癌症的组合和所述癌症的转移性病灶。
在另一优选方案中，所述肿瘤包括：乳腺癌，血液癌症，结肠癌，直肠癌，肾细胞癌，肝癌，肺的非小细胞癌，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，霍奇金氏病，非霍奇金淋巴瘤，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，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，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，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶。
在另一优选方案中，所述的免疫效应细胞包括：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；优选地，所述免疫效应细胞选自自体T细胞、同种异体T细胞或同种异体NK细胞，更优选地，所述T细胞为自体T细胞。
在另一优选方案中，所述的吉西他滨为口服给药、腹腔给药和/或注射给药。
在另一优选方案中，不对所述的个体进行淋巴细胞清除。。
本发明的第三方面，提供一种表达有识别肿瘤抗原的受体的免疫效应细胞在制备药物中的应用，其特征在于，所述药物含有所述细胞和吉西他滨，用于在人类患者中治疗肿瘤或降低肿瘤细胞生长、存活或活力，其中将所述细胞和吉西他滨配制成能提供比所述细胞和吉西他滨各自单独使用时的效果之和更大的治疗效果。
本发明的第四方面，提供一种表达有识别肿瘤抗原的受体的免疫效应细胞和吉西他滨在制备药物中的应用，其特征在于，所述药物用于在人类患者中治 疗肿瘤或降低肿瘤细胞生长、存活或活力，其中将所述细胞和吉西他滨配制成能提供比所述细胞和吉西他滨各自单独使用时的效果之和更大的治疗效果。
在一优选方案中，所述免疫效应细胞为CAR T细胞，优选地，所述CAR T细胞识别表皮生长因子受体家族及其突变体(EGFR、EGFR2、ERBB3、ERBB4、EGFRvIII)、Claudin18.2、Claudin18.1、Claudin 6、磷脂酰肌醇蛋白聚糖-3(GPC3)、BCMA和/或血管内皮生长因子受体。
本发明的第五方面，提供一种用于治疗肿瘤的试剂盒，所述试剂盒包含：
1)表达有识别肿瘤抗原的受体的免疫效应细胞；
2)吉西他滨；
3)用于包含以上1)和2)所述物质的容器；和
4)利用所述试剂盒治疗肿瘤的给药说明书；
其中，所述免疫效应细胞和吉西他滨配制成能提供比所述试剂各自的效果之和更大的治疗效果；优选地，所述免疫效应细胞为CAR T细胞，更优选地，所述CAR T细胞识别表皮生长因子受体家族及其突变体(EGFR、EGFR2、ERBB3、ERBB4、EGFRvIII)、Claudin18.2、Claudin18.1、Claudin 6、磷脂酰肌醇蛋白聚糖-3(GPC3)、BCMA和/或血管内皮生长因子受体。
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。
本发明的有益效果：
1、本发明所提供的吉西他滨和免疫效应细胞联用，能够显著提高杀伤肿瘤细胞的能力。
2、采用本发明的治疗方案能够对抗癌症微环境中的免疫抑制，从而对于实体瘤的作用显著增强，对于难治性和进行性的癌症也具有较好的效果。
附图说明
图1为重组载体MSCV-8E5-2I-mBBZ质粒图。
图2显示了体内实验观察吉西他滨与CART细胞联用对原位小鼠胰腺癌的抑制效果。
图3显示了吉西他滨与CART细胞联用治疗小鼠的活体成像数据。
图4显示了吉西他滨与CART细胞联用治疗小鼠的生存期数据。
具体实施方式
本发明涉及免疫效应细胞和吉西他滨联合应用于治疗肿瘤，应理解本发明并不限于所述的方法和实验条件。除非本文中专门定义，所使用的所有技术和科学术语具有在基因治疗、生物化学、遗传学、分子生物学、以及药物化学领域内的技术人员通常理解的相同含义。
类似或等效于本文中描述的方法和材料都可以在本发明的实践或测试中使用。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书包括定义为准。此外，除非另有说明，材料、方法和实施例仅是说明性的，而并非进行限制。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
本发明至少部分源于下述认知：连续地、以任一次序地或基本上同时地给 予吉西他滨和免疫效应细胞的一个或多个周期和/或剂量的组合治疗方案，在治疗一些受试者的癌症时可以是更有效的增加、增强或延长免疫细胞的活性和/或数目，从而达到抗肿瘤效果。
术语“吉西他滨(Gemcitabine)”,化学名称为2’-脱氧-2’,2’-二氟胞苷盐酸盐；分子式为C 9H 11F 2N 3O 4.HCl,分子量为299.66。吉西他滨是嘧啶类抗代谢肿瘤药物,能通过抑制核糖核苷酸还原酶及细胞复制干扰肿瘤细胞DNA的合成修复，对多种实体肿瘤有效。在具体实施例中吉西他滨与靶向肿瘤抗原的免疫细胞治疗联用可以显著提高抗肿瘤作用，甚至达到完全缓解的治疗效果。
申请人还发现，本发明不仅可以提高难治型癌症的抗癌效果，在使用CAR-T细胞时，还不需要进行淋巴细胞清除，从而大大减轻清淋造成的抗癌治疗效果低以及减轻对正常组织的损伤所造成的毒副作用，尤其是对骨髓的严重抑制。
在某些实施方案中，患有肿瘤的个体每次静脉输注吉西他滨的剂量约为1250mg/m 2、1250、1150、1100、1050、1000、950、900、850、800、750、700、650、600、550、500、450、400、350、300、250、200、150、100、50、40、30、20、10、5、4、3、2或1mg/m 2。在某些实施方案中，患有肿瘤的个体约每6周、5周、4周、3周、2周或1周静脉输注一次吉西他滨。
本发明中免疫效应细胞和吉西他滨给予时间不分先后；可以先给予吉西他滨再给予免疫效应细胞；也可以同时给药；还可以先给予免疫效应细胞再给予吉西他滨。在某些实施方案中，免疫效应细胞治疗在吉西他滨给予之前1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、1个月或其任何组合施用。在某些实施方案中，免疫效应细胞治疗在吉西他滨给予之后1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、1个月或其任何组合施用。
术语“免疫效应细胞”，是指在免疫反应期间发挥效应功能的细胞。例如，包括分泌细胞因子和/或趋化因子、杀伤微生物、分泌抗体、识别或清除肿瘤细胞的免疫细胞。在一些实施例中，免疫效应细胞包括T细胞(细胞毒性T细胞、 辅助T细胞、肿瘤浸润性T细胞)、B细胞、自然杀伤细胞、嗜中性粒细胞、巨噬细胞(或骨髓源性吞噬细胞)和树突细胞。优选地，所述T细胞包括自体T细胞、异种T细胞、同种异体T细胞，所述的自然杀伤细胞是同种异体NK细胞。
正如本文中使用的那样，术语“免疫效应功能或免疫效应应答”是指免疫效应细胞，例如增强或促进靶细胞的免疫攻击的功能或反应。例如，免疫效应功能或应答是指促进靶细胞的杀伤或者抑制生长或增殖的T细胞或NK细胞的属性。
术语“免疫效应功能”包括免疫系统的组成所介导的任何功能，可导致抑制肿瘤生长和/或抑制肿瘤发生，包括抑制肿瘤的扩散和转移。优选地，免疫效应功能杀伤肿瘤细胞。优选地，本发明中的免疫效应功能是抗体介导，包括补体依赖的细胞毒性作用(CDC)、抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞介导的吞噬作用(ADCP)、在携带肿瘤相关抗原的细胞中诱导凋亡(例如，通过抗体与表面抗原的结合)、抑制CD40L介导的信号转导(例如通过抗体与CD40受体或CD40配体(CD40L)相结合)，和/或抑制携带肿瘤相关抗原的细胞的增殖，优选ADCC和/或CDC。因此，能够介导一种或更多种免疫效应功能的抗体优选地能够通过诱导CDC介导的裂解、ADCC介导的裂解、凋亡、同型黏着和/或吞噬作用(优选通过诱导CDC介导的裂解和/或ADCC介导的裂解)来介导对肿瘤细胞的杀伤。抗体还可简单地通过与癌细胞表面上的肿瘤相关抗原结合而发挥作用。例如，抗体可通过与肿瘤细胞表面上的肿瘤相关抗原结合来阻断肿瘤相关抗原的功能或诱导凋亡。
术语“治疗有效量”、“治疗有效的”、“有效量”在本文中可互换地使用，是指有效地实现特定生物学结果的化合物、制剂、物质或组合物的量，例如但不限于足以促进T细胞应答的量或剂量。当指示“免疫学上有效量”、“抗肿瘤有效量”、“抑制肿瘤有效量”或“治疗有效量”时，本发明的免疫效应细胞、或治疗剂的精确给药数量可以由医师在考虑个体在年龄、体重、肿瘤大小、感染或转移的程度以及患者(受试者)的状况的情况下确定。有效量的免疫效应细胞是指但不限于能使免疫效应细胞抗肿瘤活性增加、增强或延长；抗肿瘤免疫效应细胞或活化免疫效应细胞数目的增加；促进IFN-γ、TNFα分泌；肿瘤消退、肿瘤缩小、肿瘤坏死的免疫效应细胞的数量。
术语“不清淋”或“不进行淋巴细胞清除”，即不清除受试者体内的淋巴细胞。包括但不限于不给予淋巴细胞清除剂、全身辐射治疗或其组合或其他引起淋巴细胞数量清除的手段；但是，在给予了淋巴细胞清除剂、全身辐射治疗或其组合或其他引起淋巴细胞数量清除的手段后，当受试者体内淋巴细胞清除率低于 60％，也将落入本申请“不清淋”的范畴。
术语“肽”、“多肽”和“蛋白质”可互换地使用，并且是指由肽键共价连接的氨基酸残基组成的化合物。蛋白质或肽必须含有至少两个氨基酸，并且对于可以包括蛋白质或肽的序列的氨基酸的最大数量没限制。多肽包括含有彼此通过肽键结合的两个或多个氨基酸的任何肽或蛋白质。本文所用的“嵌合受体”,即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：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)。
本文所用的“嵌合抗原受体”或“CAR”是指一组多肽，当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域、跨膜结构域和细胞质信号传导结构域(本文中也称为“胞内信号传导结构域”)，其包括来源于如下定义的刺激性分子和/或共刺激性分子的功能信号传导结构域。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。在一个方面，刺激性分子为与T细胞受体复合体结合的δ链。在一个方面，细胞质信号传导结构域进一步包括一种或多种来源于至少一个如下定义的共刺激性分子的功能性信号传导结构域。在一个方面，共刺激性分子选自本文所述共刺激性分子，例如4-1BB(即CD137)、CD27和/或CD28。在一个方面，CAR包括嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于共刺激性分子的功能性信号传导结构域和来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面中，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于一个或更多个共刺激性分子的两个功能性信号传导。
在一个方面，本发明设想产生功能等同分子的起始抗体或片段(例如scFv)氨基酸序列的修饰。例如，本文所述的癌症相关抗原的抗原结合结构域的VH或VL，例如CAR中包含的scFv，可以被修饰以保留本文所述的癌症相关抗原的抗原结合结构域的起始VH或VL构架区(例如scFv)的至少约70％、71％、72％.73％、74％、75％、76％、77％、78％、79％、80％,81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95 ％、96％、97％、98％、99％的同一性。本发明设想整个CAR构建体的修饰，例如CAR构建体的多个结构域的一个或更多个氨基酸序列的修饰，以产生功能等同分子。CAR构建体可以被修饰以保留起始CAR构建体的至少约70％、71％、72％.73％、74％、75％、76％、77％、78％、79％、80％,81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。
在一个具体的实施方式中，本文的识别肿瘤抗原的抗体的氨基酸序列与SEQ ID NO：4所示，或如SEQ ID NO：14所示，或如SEQ ID NO：15所示，或如SEQ ID NO：16所示，或如SEQ ID NO：17所示，或如SEQ ID NO：18所示，或如SEQ ID NO：19所示，或如SEQ ID NO：20所示，或如SEQ ID NO：21所示，或如SEQ ID NO：22所示的氨基酸序列具有至少90％的同一性；优选为SEQ ID NO：4所示，或如SEQ ID NO：14所示，或如SEQ ID NO：15所示，或如SEQ ID NO：16所示，或如SEQ ID NO：17所示，或如SEQ ID NO：18所示，或如SEQ ID NO：19所示，或如SEQ ID NO：20所示，或如SEQ ID NO：21所示，或如SEQ ID NO：22所示的氨基酸序列。
本文所用的“跨膜结构域”，指蛋白质序列中跨越细胞膜的区域，可以包括一个或更多个邻接跨膜区域的另外氨基酸，例如一个或更多个与所述跨膜所源自的蛋白质的胞外区域相关联的氨基酸(例如，胞外区域的1、2、3、4、5、6、7、8、9、10直至15个氨基酸)和/或与所述跨膜蛋白所源自的蛋白质的胞外区域相关联的一个或更多个另外的氨基酸(例如，胞内区域的1、2、3、4、5、6、7、8、9、10直至15个氨基酸)。在一个方面，跨膜结构域是与嵌合受体的其它结构域中的一个有关的结构域，例如，在一种实施方式中，所述跨膜结构域可以来自信号传导结构域、共刺激结构域或铰链结构域所源自的相同蛋白质。在某些情况下，跨膜结构域可以被选择或通过氨基酸取代修饰以避免这样的结构域与相同或不同表面膜蛋白的跨膜结构域结合，例如以使与受体复合体的其它成员的相互作用最小化。在一个方面，跨膜结构域能够与表达嵌合受体的细胞表面上的另一个嵌合受体同型二聚化。跨膜结构域可以来源于天然或重组来源。当所述来源是天然的时，所述结构域可以来源于任何膜结合的蛋白质或跨膜蛋白质。在一个方面，只要所述嵌合受体与所述靶抗原结合时，跨膜结构域能够向胞内结构域传导信号。在本发明中特别使用的跨膜结构域可以包括至少以下的跨膜结构域：例如，T-细胞受体的α、β或δ链、CD28、CD27、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、 CD137、CD154。在某些实施方式中，跨膜结构域可以包括至少下述跨膜区域:例如KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、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、NKG2D、NKG2C。
在某些情况下，跨膜结构域可以经由铰链(例如，来自人蛋白质的铰链)连接至CAR的胞外区域，例如CAR的抗原结合结构域。例如，在一种实施方式中，铰链可以是人Ig(免疫球蛋白)铰链(例如，IgG4铰链、IgD铰链)、GS接头(例如，本文所述的GS接头)、KIR2DS2铰链或CD8a铰链。在一个方面，跨膜结构域可以是重组的，在这样情况下，其将会主要包含疏水性残基，比如亮氨酸和缬氨酸。在一个方面，在重组跨膜结构域的每个末端可以发现苯丙氨酸、色氨酸和缬氨酸的三联体。任选地，长度在2至10个氨基酸之间的短的寡肽或多肽接头可以在CAR的跨膜结构域与细胞质区之间形成键。甘氨酸-丝氨酸二联体提供一种特别合适的接头。
本文所用的“胞内结构域”或“细胞质结构域”，是包括胞内信号传导结构域。胞内信号传导结构域通常负责其中已经引入嵌合受体的免疫细胞的正常免疫效应子功能中至少一个的活化。术语“效应子功能”是指细胞的特化功能。T细胞的免疫效应子功能例如可以是细胞溶解活性或辅助活性，包括分泌细胞因子。因此，术语“胞内信号传导结构域”是指转导免疫效应子功能信号且引导细胞执行特定功能的蛋白质的部分。虽然通常可以应用全部胞内信号传导结构域，但是在许多情况下不必使用整个链。就使用胞内信号传导结构域的截短部分来说，可以使用这样的截短部分代替完整的链，只要其转导免疫效应子功能信号。因此，术语胞内信号传导结构域意味着包括足以转导免疫效应子功能信号的胞内信号传导结构域的截短部分。
众所周知通过单独的TCR产生的信号不足以完全活化T细胞，并且也需要二级和/或共刺激信号。因此，T细胞活化可以被称为是由两个不同种类的细胞 质信号传导序列介导的:通过TCR引发抗原依赖性一级活化的那些(一级胞内信号传导结构域)以及以抗原独立方式起作用以提供二级或共刺激信号的那些(二级细胞质结构域，例如共刺激结构域)。
术语“刺激性分子”是指由免疫细胞(例如，T细胞、NK细胞、B细胞)表达的提供细胞质信号传导序列的分子，该信号传导序列以刺激性方式调节用于免疫细胞信号传导途径的至少一些方面的免疫细胞的活化。在一个方面，信号是通过例如TCR/CD3复合体与负载有肽的MHC-抗原肽复合物的结合启动的初级信号，并且其导致介导T细胞应答，包括，但不限于增殖、活化、分化等。以刺激方式起作用的一级细胞质信号传导序列(也称为“一级信号传导结构域”)可以含有被称为基于免疫受体酪氨酸的活化基序或ITAM的信号传导基序。特别地用于本发明的含有ITAM的细胞质信号传导序列的实例包括，但不限于来源于下述的那些：CD3δ、常见的FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。在本发明的任一个CAR中的胞内信号传导结构域包括细胞内信号传导序列，例如CD3-δ的初级信号传导序列。在本发明的CAR中，CD3-δ的初级信号传导序列是来自人或非人类种类例如小鼠、啮齿类动物、猴、猿等的等同残基。
术语“共刺激性分子”是指T细胞上的同源结合配偶体，其特异性地结合共刺激配体，从而介导T细胞的共刺激反应，比如但不限于增殖。共刺激性分子是除了抗原受体或其配体之外的细胞表面分子，其促进有效的免疫应答。共刺激性分子包括但不限于MHC I类分子，BTLA和Toll配体受体，以及OX40、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)。这样的共刺激性分子的进一步实例包括CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、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、NKG2D、NKG2C、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、CD19a，以及特异性地结合CD83的配体。
共刺激性胞内信号传导结构域可以为共刺激性分子的细胞内部分。共刺激性分子可以以下述蛋白质家族代表：TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导淋巴细胞性活化分子(SLAM蛋白质)、和NK细胞受体。这样的分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、GITR、CD30、CD40、ICOS、BAFFR、HVEM、ICAM-1、与淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CDS、CD7、CD287、LIGHT、NKG2C、NKG2D、SLAMF7、NKp80、NKp30、NKp44、NKp46、CD160、B7-H3、以及特异性地结合CD83的配体等。
胞内信号传导结构域可以包括分子的全部细胞内部分或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“4-1BB”是指具有如GenBank Accession No.AAA62478.2提供的氨基酸序列的TNFR超家族的成员，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基；并且“4-1BB共刺激结构域”被定义为GenBank Accession No.AAA62478.2的氨基酸残基214～255，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。在一个方面，“4-1BB共刺激结构域”为来自人或者来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。
本文的嵌合抗原受体具有：(i)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3δ；或(ii)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3δ；或(iii)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3δ。
本文的嵌合抗原受体的氨基酸序列与SEQ ID NO：23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43或44所示的氨基酸序列具有至少90％的同一性；优选所述嵌合抗原受体的氨基酸序列为SEQ ID NO：23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43或44所以的氨基酸序列。
术语“抗体”是指源于特异性地结合抗原的免疫球蛋白分子的蛋白质或多肽序列。抗体可以为多克隆的或单克隆的、多链或单链、或完整的免疫球蛋白，并且可以来源于天然来源或重组来源。抗体可以为免疫球蛋白分子的四聚体。
术语“抗体片段”是指保留与抗原的表位特异性地相互作用(例如，通过结合、空间位阻、稳定化/去稳定化、空间分布)的能力的抗体的至少一部分。抗体片段的 实例包括，但不限于Fab，Fab'、F(ab') 2、Fv片段、scFv、二硫键-连接的Fvs(sdFv)、由VH和CH1结构域组成的Fd片段、线性抗体、单域抗体(如sdAb)、camelid VHH结构域、由抗体片段(例如包括在铰链区通过二硫键连接的两个Fab片段的二价片段)形成的多特异性抗体和抗体的分离的CDR或其它表位结合片段。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。
术语“抗体重链”是指以其天然存在的构型存在于抗体分子中且通常决定抗体所属类型的两种多肽链中较大者。
术语“抗体轻链”是指以其天然存在构型存在于抗体分子中的两种多肽链的较小者。κ(k)和λ(l)轻链是指两种主要的抗体轻链的同种型。
术语“重组抗体”是指使用重组DNA技术产生的抗体，比如例如由噬菌体或酵母菌表达系统表达的抗体。该术语也应当解释为指已经通过合成编码抗体的DNA分子(且其中DNA分子表达抗体蛋白质)或指定抗体的氨基酸序列产生的抗体，其中所述DNA或氨基酸序列已经使用重组DNA或本领域可获得且熟知的氨基酸序列技术获得。
术语“抗原”或“Ag”是指引起免疫应答的分子。该免疫应答可以涉及抗体产生或有特异性免疫能力的细胞的活化或两者都包括。本领域技术人员应当理解并且实际上所有蛋白质或肽的任何大分子都可以充当抗原。此外，抗原可以来源于重组或基因组DNA。当在本文中使用该术语时，本领域技术人员应当理解包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA。此外，本领域技术人员应当理解抗原无需仅通过基因的全长核苷酸序列编码。本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列以不同组合排列以编码引发期望免疫应答的多肽。本领域技术人员应当理解抗原根本无需由“基因”编码。抗原可以合成产生，或者可以来源于生物样品，或者可以是除了多肽之外的大分子。这样的生物样品可以包括，但不限于组织样品、肿瘤样品、具有其它生物组分的细胞或液体。
术语“肿瘤相关抗原(TAA)或“肿瘤抗原”,是指在正常条件下在有限数目的组织和/或器官中或者在特定发育阶段中特异性表达的蛋白质。在一些实施方案 中，肿瘤相关抗原是在正常条件下胃组织中特异性表达，优选地在胰腺、胃粘膜中、在生殖器官(例如，睾丸)中、在滋养层组织(例如，胎盘)中或者在生殖系细胞中，以及在一种或更多种肿瘤组织或癌组织中表达或异常表达。在一些实施方案中，肿瘤相关抗原是指分化抗原，优选细胞类型特异性分化抗原，即，正常条件下特定分化阶段的特定细胞类型中特异性表达的蛋白质。在一些实施方案中，肿瘤相关抗原位于肿瘤细胞，而在正常组织中不表达或仅少量表达。在一些实施方案中，肿瘤相关抗原或肿瘤相关抗原的异常表达可用于鉴定肿瘤细胞。在一些实施方案中，个体(例如，肿瘤患者)中的肿瘤细胞表达的肿瘤相关抗原为所述个体的自体蛋白。在一些实施方案中，肿瘤相关抗原在正常条件下在个体受到免疫系统损伤时不致死的组织或器官中特异性表达；或者在机体的免疫系统无法接近或很难接近的器官或结构中特异性表达。在一些实施方案中，在正常组织中表达的肿瘤相关抗原的氨基酸序列与在肿瘤组织中表达的肿瘤相关抗原的氨基酸序列相同。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD70；CD123；CD138；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,血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6，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)；uroplakin2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
术语“肿瘤”，是指由细胞异常生长造成的膨胀或病变(称为赘生性细胞或肿瘤细胞)。“肿瘤细胞”是指能迅速的、不受控增殖的细胞，且在引起该增殖的刺激停止之后继续生长的异常细胞。肿瘤表现为部分或完全缺乏结构性组织，也缺乏与正常组织的功能性协调，通常形成独特的组织团块，其可以是良性的、恶变前的或恶性的。
术语“癌症”，(医学术语：恶性瘤)是一类疾病，其中一组细胞展现出不受控制的生长(超过正常限制的分裂)、侵袭(侵入并且破坏邻近组织)并且有时转移(通过淋巴或血液扩散至身体中的其他位置)。癌症的这三种恶性特性将它们与 良性肿瘤区分开，良性肿瘤有自限性，并且不会侵袭或转移。大部分癌症形成肿瘤，但是有一些(如白血病)不是。
术语“癌症”和“肿瘤”或“癌疾病”和“肿瘤疾病”可互换，指其中细胞表现出不受控生长、和/或侵袭、和/或转移的疾病。包括白血病、精原细胞瘤、黑色素瘤、畸胎瘤、淋巴瘤、成神经细胞瘤、神经胶质瘤、直肠癌、子宫内膜癌、肾癌、肾上腺癌、甲状腺癌、血液癌症、皮肤癌、脑癌、宫颈癌、肠癌、肝癌、结肠癌、胃癌、肠癌、头颈癌、胃肠癌、淋巴结癌、食管癌、结肠直肠癌、胰腺癌、耳鼻喉(ENT)癌、乳腺癌、前列腺癌、子宫癌、卵巢癌和肺癌，及其转移。其实例有肺癌、乳癌、前列腺癌、结肠癌、肾细胞癌、宫颈癌或上述各类癌或肿瘤的转移。
术语“转染”或“转导”是指外源性核酸转移或引入到宿主细胞中的过程。“转染的”或“转导的”细胞是已经用外源性核酸转染、转化或转导的细胞。所述细胞包括原发性受试者细胞及其后代。
术语“特异性地结合”、“特异性地识别”在本文中具有相同的含义，是指识别并且结合存在于样品中的抗原(例如肿瘤抗原)的抗体或配体，但是该抗体或配体基本上不会识别或结合样品中的其它分子。
术语“难治”指的是一种疾病，例如，癌症或肿瘤，其不应答治疗。在实施方案中，难治性肿瘤可以是对治疗开始前或开始时的治疗有抗性。在其他实施方案中，难治性肿瘤可以成为治疗期间抗性的。难治性癌症也称为抗性肿瘤。在本发明中，难治性肿瘤包括但不限于放疗不敏感、放疗后复发、化疗不敏感、化疗后复发、对CAR-T治疗不敏感或治疗后复发的肿瘤。难治性或复发性恶性肿瘤可以使用本文中描述的治疗方案。
如本文所用“复发的”是指在一段改进期，例如，在疗法，例如癌症疗法的先前治疗后，返回疾病(例如癌症)或疾病如癌症的体征和症状。
可互换地使用术语“对象”、“个体”、“生物体”或“患者”，包括脊椎动物，优选哺乳动物。本发明中，哺乳动物是人、非人灵长类、家养动物(例如，犬、猫、绵羊、牛、山羊、猪、马等)、实验动物(例如，小鼠、大鼠、兔、豚鼠等)以及圈养的动物(例如，动物园的动物)。本文使用的术语“动物”还包括人。术语“对象”、“个体”包括患者，即，患病的动物，优选人，所述疾病优选本文所述的疾病。
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强的应答可以包含应答性中5％、10％、15％、20％、25％、30％、 35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫效应细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％更多。
在一个方面，治疗由临床结果；通过T细胞的抗肿瘤活性增加、增强或延长；与治疗前的数目相比较，抗肿瘤T细胞或活化T细胞数目的增加，促进IFN-γ、TNFa分泌,或其组合决定。在另一个方面，临床结果是肿瘤消退；肿瘤缩小；肿瘤坏死；通过免疫系统的抗肿瘤应答；肿瘤扩大，复发或扩散或其组合。在一个另外方面，治疗效应通过T细胞的存在、指示T细胞炎症的基因标记的存在，促进IFN-γ、TNFa分泌,或其组合预测。
如本文公开的免疫效应细胞可以通过各种途径施用于个体，包括例如经口或肠胃外，例如静脉内、肌内、皮下、眶内、囊内、腹膜内、直肠内、脑池内、瘤内、鼻内(intravasally)、皮内或者分别使用例如皮肤贴片或透皮离子电渗疗法通过皮肤的被动或促进吸收。
在实践本发明的方法中待施用的试剂总量可以作为单一剂量以推注或通过在相对短时间段的输注，施用于受试者，或可以使用分级治疗方案进行施用，其中在延长时间段施用多个剂量。本领域技术人员将知道治疗受试者中的病理状况的组合物的量取决于许多因素，包括受试者的年龄和一般健康、以及施用途径和待施用的治疗次数。考虑到这些因素，技术人员将根据需要调整具体剂量。一般而言，最初，使用I期和II期临床试验测定组合物的配制以及施用途径和频率。
范围：在整个公开中，本发明的各个方面都可以以范围形式存在。应当理解，范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本发明的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。例如，范围的描述比如从1至6就应当被认为具体地公开了子范围比如1至3、1至4、1至5、2至4、2至6、3至6等，以及该范围内的单独数值，例如1、2、2.7、3、4、5、5.3、和6。作为另一个实例，范围比如95-99％的同一性包括具有95％、96％、97％、98％或99％同一性的范围，并且包括子范围比如96～99％、96～98％、96～97％、97～99％、97～98％和98～99％的同一性。不考虑范围的宽度，这均适用。
根据本公开内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等同的方法和组分在本发明的范围内。
本申请涉及过继细胞或免疫效应细胞治疗实体肿瘤，包括给予多剂量或重复剂量的细胞，及其使用的方法、组合物和制品。细胞一般表达嵌合抗原受体例如，嵌合抗原受体(CAR)或其他转基因受体如T细胞受体(TCR)。
人密蛋白18(Claudin 18，CLD18)分子(Genbank登记号：剪接变体1(CLD18A1或Claudin 18.1)：NP_057453、NM016369，以及剪接变体2(CLD18A2或Claudin 18.2)：NM_001002026、NP_001002026)是分子量约为27,9/27,72kD的内在跨膜蛋白。小鼠密蛋白Claudin 18.2(NP_001181850.1)。
术语“CLD18”指密蛋白18，并包括细胞天然表达的或转染了CLD18基因的细胞所表达的任何CLD18的变体(包括CLD18A1和CLD18A2)、构象、同工型(isoform)和种间同源物(specieshomologs)。密蛋白18是位于上皮和内皮的紧密连接中的内在膜蛋白。紧密连接在相邻细胞之间组织膜内颗粒互联链的网。在紧密连接中，闭合蛋白(occludin)和密蛋白是最主要的跨膜蛋白组分。由于其强胞间粘附特性，它们产生了防止和控制溶质的细胞旁转运并限制膜脂和蛋白质侧向扩散以维持细胞极性的一级屏障。形成紧密连接的蛋白质关键性地参与了组织上皮组织结构。优选地，“CLD18”指人CLD18，特别是CLD18A2(氨基酸序列如SEQ ID NO:2所示)和/或CLD18A1(氨基酸序列如SEQ ID NO:3所示)，更优选CLD18A2。
术语“CLD18A1”包括细胞天然表达的或转染了CLD18A1基因的细胞所表达的任何人CLD18A1的翻译后修饰变体、同工型和种间同源物。
术语“CLD18A2”包括细胞天然表达的或转染了CLD18A2基因的细胞所表达的任何人CLD18A2的翻译后修饰变体、同工型和种间同源物。
术语“CLD18变体”应包括(i)CLD18剪接变体，(ii)CLD18翻译后修饰变体，特别是包括N糖基化状态不同的变体，(iii)CLD18构象变体，特别是包括CLD18-构象-1、CLD18-构象-2和CLD18-构象-3，(iv)位于胞间紧密连接处的游离CLD18和同型/异型相关联变体，(v)CLD18癌症相关变体和CLD18非癌症相关变体。
在一些实施方案中，所述密蛋白18A2是包含SEQ ID NO:2的氨基酸序列的肽或所述氨基酸序列变体的蛋白质/肽。术语“变体”是指突变体、剪接变体、构象、同种型、等位基因变体、种变体(species variant)和种同源物(species  homolog)，特别是天然存在的变体。术语“CLDN”、“CLDN18”、“CLDN18A1”和“CLDN18A2”应涵盖任何翻译后修饰的变体和构象变体。
CLD18A1在正常肺和胃的上皮中选择性表达，而CLD18A2仅在胃细胞中表达。并且，CLD18A2局限在已分化的胃上皮短寿细胞中，但在胃干细胞区中不存在。
在一些实施例中，原发肿瘤是指胃癌、食管癌、胰腺癌、肺癌例如非小细胞肺癌、卵巢癌、结肠癌、肝癌、头颈癌和胆囊癌，所述转移特别是指胃癌转移例如Krukenberg肿瘤、腹膜转移和淋巴结转移。在一些实施例中，表达CLDN18A2的细胞是指肿瘤细胞，并且特别地选自致瘤性胃癌细胞、食管癌细胞、胰腺癌细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、肝癌细胞、头颈癌细胞和胆囊癌细胞。
在一些实施例中，所述肿瘤包括：乳腺癌，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，子宫癌，卵巢癌，直肠癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，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，输尿管癌，肾盂癌，脊椎肿瘤，脑胶质瘤，垂体腺瘤，卡波西肉瘤，上述癌症的组合和上述癌症的转移性病灶。
本发明的提供用于治疗有CLD18表达的疾病(例如肿瘤)的治疗方法及组合物。
本发明提供了过继细胞或免疫效应细胞治疗实体肿瘤中，受试者表达遗传工程改造(重组)的免疫效应细胞嵌合受体，用于治疗受试者的肿瘤的方法。该方法一般包括这类细胞的单疗程回输、或多疗程回输。在一些实施方式中，为疗程内剂量递增或者疗程内重复剂量输注或者疗程内剂量递减。在一些实施方式中，首剂量是较低剂量和/或是调节或减量剂量和/或后续剂量是巩固剂量和/或是调节或减量剂量。还提供可用于这类方法的细胞、组合物和制品。在一些实施方式中，嵌合受体是遗传工程改造的抗原受体，如功能性非-TCR抗原受体，例如，嵌合抗原受体(CAR)和其他重组抗原受体如转基因T细胞受体(TCR)。受体还包括其他重组嵌合受体，如含特异性结合至配体或受体或其他结合伴侣的胞外部分和胞内部分，如CAR的胞内信号转导部分的那些受体。在一些实施方式中，剂量包括较低的首剂量。
在一些实施方式中，该方法包括(a)给予患有肿瘤的受试者单疗程的表达嵌合抗原受体(例如，CAR)的细胞；和(b)给予受试者多疗程的表达嵌合抗原受体(例如，CAR)的细胞。在其他实施方式中，可给予一个或多个后续剂量。
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。
本发明的示例性的抗原受体，包括CAR，以及用于工程改造和将受体导入细胞中的方法，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1中公开的那些。
实施例一、Panc02/Luc-GFP-Claudin 18.2、8E5-2I-mBBZ CAR T细胞建立
1.Panc02/Luc-GFP-Claudin 18.2细胞的构建
利用分子生物学常规手段建立过表达小鼠Claudin 18.2(SEQ ID NO:1)的Panc02胰腺癌细胞模型(Panc02胰腺癌细胞模型购自北纳生物)。以pWPT为载体，插入小鼠Claudin 18.2(SEQ ID NO:1)，构建了质粒pWPT-mClaudin 18.2。然后采用慢病毒包装感染方法建立过表达小鼠Claudin 18.2的胰腺癌细胞Panc02/Luc-GFP-Claudin 18.2。
2.表达嵌合抗原受体的T细胞的构建
(1)质粒构建
采用本领域常规分子生物学方法，本实施例采用的scFv为靶向鼠claudin18.2抗体，核酸序列如SEQ ID NO：5所示。
以MSCV-IRES-GFP(购自Addgene)为载体，构建了表达二代嵌合抗原受体的逆转录病毒质粒MSCV-8E5-2I-mBBZ(如图1所示)。其中，8E5-2I-mBBZ序列由鼠CD8α信号肽(其氨基酸序列如SEQ ID NO：6所示，其核苷酸序列如SEQ ID NO：7所示)、靶向claudin18.2的scFv(其氨基酸序列如SEQ ID NO：4所示，其核苷酸序列如SEQ ID NO：5)、鼠CD8 hinge和跨膜区(其氨基酸序列如SEQ ID NO：8所示，其核苷酸序列如SEQ ID NO：9所示)和鼠4-1BB胞内信号传导结构域(其氨基酸序列如SEQ ID NO：10所示，其核苷酸序列如SEQ ID NO：11所示)以及鼠CD3的胞内段CD3δ(其氨基酸序列如SEQ ID NO：12所示，其核苷酸序列如SEQ ID NO：13所示)组成。
将上述构建的MSCV-8E5-2I-mBBZ转染293T(来源于ATCC)包装逆转录病毒，得到逆转录病毒。感染方法为本领域表达嵌合抗原受体的T细胞制备过程 中常规的感染方法。
(2)CAR T细胞构建：取C57BL/6小鼠(来源于上海西普尔－必凯实验动物有限公司)的脾脏T淋巴细胞，将纯化的小鼠CD3+T淋巴细胞按1：1的体积比加入Dynabeads Mouse T-activator CD3/CD28，PBS清洗一次，活化，放培养箱培养，培养基为RPMI 1640完全培养基(购买自invitrogen公司)，添加10％FBS血清。
将活化24h的小鼠脾脏T淋巴细胞接种于重组人纤维蛋白片段包被的12孔板中，加入逆转录病毒感染12小时，随后培养扩增至需要的数量，得到小鼠的8E5-2I-mBBZ CAR T细胞。
实施例二、体内实验观察吉西他滨(Gemcitabine)与CAR T细胞联用对小鼠胰腺癌的抑制效果
6周龄的C57BL/6小鼠胰腺原位接种2×10 5 Panc02/Luc-GFP-Claudin 18.2细胞，肿瘤细胞接种日记为第0天(即Day 0)。
接种肿瘤后第6天(即Day6)，取小鼠T细胞并按实施例一来构建8E5-2I-mBBZ CAR T细胞系。同时将小鼠进行活体成像，根据小鼠肿瘤负荷将小鼠随机分组，主要组别为：
第1组：UTD组，即只给予未转染的小鼠T细胞治疗的组别；
第2组：CART组，即只给予8E5-2I-mBBZ CAR T细胞治疗的组别；
第3组：Gemcitabine组(或称为，吉西他滨组)，即只给予吉西他滨单独治疗的组别；以及
第4组：Gemcitabine+CART组(或称为，吉西他滨+CART组)，即给予8E5-2I-mBBZ CAR T细胞联合吉西他滨治疗组。
在第6、7、8天(即Day6、7、8)，针对Gemcitabine组及Gemcitabine+CART组的小鼠分别给予30mg/kg吉西他滨进行治疗，腹腔给药，每天给药一次。
接种肿瘤后第9天(即Day9)，针对CART组、Gemcitabine+CART组的小鼠分别给予剂量为3×10 6 8E5-2I-mBBZ CAR-T细胞尾静脉注射，UTD组给予注射3×10 6未转染的小鼠T细胞，此后每周一次活体成像观察治疗效果。
对原位小鼠胰腺癌的抑制效果的结果如图2所示，在接受治疗后第13天(day13)，相比较UTD组、CART组、Gemcitabine组，Gemcitabine+CART组的小鼠体内的肿瘤负荷(白色高亮部位)有明显的下降，并且该Gemcitabine+CART组中的两只小鼠出现了完全缓解。第21天(Day21)，在Gemcitabine+CART组中，有四只小鼠完全缓解。而针对其他三组，在第21天均没有出现完全缓解的小鼠。 活体成像数据显示，UTD组、CART组和Gemcitabine组的小鼠的肿瘤均持续增长；与UTD组相比较，Gemcitabine+CART组小鼠的肿瘤增长得到了有效的控制(P<0.05，图3)。小鼠存活数据显示，Gemcitabine+CART组小鼠的生存期最长，明显超过UTD组小鼠的生存期(P<0.01，图4)及Gemcitabine组小鼠的生存期(P<0.05，图4)。
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。
本申请中涉及的序列如下：
Figure PCTCN2020070717-appb-000001
Figure PCTCN2020070717-appb-000002
Figure PCTCN2020070717-appb-000003
Figure PCTCN2020070717-appb-000004
Figure PCTCN2020070717-appb-000005
Figure PCTCN2020070717-appb-000006

Claims (19)

  1. 治疗肿瘤的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和吉西他滨，所述免疫效应细胞表达有识别肿瘤抗原的受体。
  2. 降低癌细胞生长、存活或活力或全部的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和吉西他滨，所述免疫效应细胞表达有识别肿瘤抗原的受体。
  3. 如权利要求1或2所述的方法，其特征在于，对患有肿瘤的个体给予免疫效应细胞和吉西他滨的方式选自以下中的任意：
    (1)先给予吉西他滨再给予免疫效应细胞，
    (2)同时给予免疫效应细胞和吉西他滨，以及
    (3)先给予免疫效应细胞再给予吉西他滨。
  4. 如权利要求1-3中任一项所述的方法，其特征在于，所述受体选自：嵌合抗原受体(Chimeric Antigen Receptor，CAR)、T细胞受体(T cell receptor，TCR)、T细胞融合蛋白(T cell fusionprotein，TFP)、T细胞抗原耦合器(T cell antigen coupler，TAC)或其组合。
  5. 如权利要求1-4中任一项所述的方法，其特征在于：
    所述肿瘤抗原为实体瘤抗原；优选所述肿瘤抗原为表皮生长因子受体家族及其突变体(即EGFR、EGFR2、ERBB3、ERBB4、EGFRvIII)、Claudin18.2、Claudin18.1、Claudin 6、磷脂酰肌醇蛋白聚糖-3(GPC3)和/或血管内皮生长因子受体。
  6. 如权利要求4或5所述的方法，其特征在于，所述嵌合抗原受体具有：
    (i)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；或
    (ii)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；或
    (iii)识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
  7. 如权利要求5或6所述的方法，其特征在于，
    所述识别肿瘤抗原的抗体的氨基酸序列与SEQ ID NO：4以及SEQ ID NO：14-22中任一项所示的氨基酸序列具有至少90％的同一性；
    优选所述识别肿瘤抗原的抗体的氨基酸序列为SEQ ID NO：4以及SEQ ID NO：14-22中任一项所示的氨基酸序列。
  8. 权利要求6或7所述的方法，其特征在于，所述嵌合抗原受体的氨基酸序列与SEQ ID NO：23-44中任一项所示的氨基酸序列具有至少90％的同一性；
    优选所述嵌合抗原受体的氨基酸序列为SEQ ID NO：23-44中任一项所示的氨基酸序列。
  9. 如权利要求1-8中任一所述的方法，其特征在于，其中所述肿瘤包括：乳腺癌，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，子宫癌，卵巢癌，直肠癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，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，输尿管癌，肾盂癌，脊椎肿瘤，脑胶质瘤，垂体腺瘤，卡波西肉瘤，所述癌症的组合和所述癌症的转移性病灶。
  10. 如权利要求1-9中任一所述的方法，其特征在于，所述的免疫效应细胞包括：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、嗜中性粒细胞、树突细胞或骨髓源性吞噬细胞或其组合；优选地，所述免疫效应细胞选自自体T细胞、同种异体T细胞或同种异体NK细胞，更优选地，所述T细胞为自体T细胞。
  11. 如权利要求1-10中任一所述的方法，其特征在于，所述的吉西他滨为口服给药、腹腔给药和/或注射给药。
  12. 如权利要求1-11中任一所述的方法，其特征在于，不对所述的个体进 行淋巴细胞清除。
  13. 表达有识别肿瘤抗原的受体的免疫效应细胞在制备药物中的应用，其特征在于，所述药物含有所述细胞和吉西他滨，用于在人类患者中治疗肿瘤或降低肿瘤细胞生长、存活或活力，其中将所述细胞和吉西他滨配制成能提供比所述细胞和吉西他滨各自单独使用时的效果之和更大的治疗效果。
  14. 表达有识别肿瘤抗原的受体的免疫效应细胞和吉西他滨在制备药物中的应用，其特征在于，所述药物用于在人类患者中治疗肿瘤或降低肿瘤细胞生长、存活或活力，其中将所述细胞和吉西他滨配制成能提供比所述细胞和吉西他滨各自单独使用时的效果之和更大的治疗效果。
  15. 如权利要求13或14所述的应用，其特征在于，所述免疫效应细胞为CAR T细胞，优选地，所述CAR T细胞识别表皮生长因子受体家族及其突变体(EGFR、EGFR2、ERBB3、ERBB4、EGFRvIII)、Claudin18.2、Claudin18.1、Claudin 6、磷脂酰肌醇蛋白聚糖-3(GPC3)、BCMA和/或血管内皮生长因子受体。
  16. 一种用于治疗肿瘤的试剂盒，其特征在于，所述试剂盒包含：
    1)表达有识别肿瘤抗原的受体的免疫效应细胞；
    2)吉西他滨；
    3)用于包含以上1)和2)所述物质的容器；和
    4)利用所述试剂盒治疗肿瘤的给药说明书；
    其中，所述免疫效应细胞和吉西他滨配制成能提供比所述试剂各自的效果之和更大的治疗效果；优选地，所述免疫效应细胞为CAR T细胞，更优选地，所述CAR T细胞识别表皮生长因子受体家族及其突变体(EGFR、EGFR2、ERBB3、ERBB4、EGFRvIII)、Claudin18.2、Claudin18.1、Claudin 6、磷脂酰肌醇蛋白聚糖-3(GPC3)、BCMA和/或血管内皮生长因子受体。
  17. 如权利要求13-15中任一项所述的应用或者如权利要求16所述的试剂盒，其特征在于，
    所述识别肿瘤抗原的抗体的氨基酸序列与SEQ ID NO：4以及SEQ ID NO：14-22中任一项所示的氨基酸序列具有至少90％的同一性；优选所述识别肿瘤抗 原的抗体的氨基酸序列为SEQ ID NO：4以及SEQ ID NO：14-22中任一项所示的氨基酸序列；
    所述嵌合抗原受体的氨基酸序列与SEQ ID NO：23-44中任一项所示的氨基酸序列具有至少90％的同一性；
    优选所述嵌合抗原受体的氨基酸序列为SEQ ID NO：23-44中任一项所示的氨基酸序列。
  18. 如权利要求13-15中任一项所述的应用或者如权利要求16或17所述的试剂盒，其特征在于，其中所述肿瘤包括：乳腺癌，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，子宫癌，卵巢癌，直肠癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，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，输尿管癌，肾盂癌，脊椎肿瘤，脑胶质瘤，垂体腺瘤，卡波西肉瘤，所述肿瘤的组合和所述肿瘤的转移性病灶。
  19. 如权利要求13-15中任一项所述的应用或者如权利要求16-18中任一项所述的试剂盒，其特征在于，所述的免疫效应细胞包括：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、嗜中性粒细胞、树突细胞或骨髓源性吞噬细胞或其组合；优选地，所述免疫效应细胞选自自体T细胞、同种异体T细胞或同种异体NK细胞，更优选地，所述T细胞为自体T细胞。
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