WO2019114762A1 - 免疫效应细胞和辐射联用治疗肿瘤 - Google Patents

免疫效应细胞和辐射联用治疗肿瘤 Download PDF

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WO2019114762A1
WO2019114762A1 PCT/CN2018/120679 CN2018120679W WO2019114762A1 WO 2019114762 A1 WO2019114762 A1 WO 2019114762A1 CN 2018120679 W CN2018120679 W CN 2018120679W WO 2019114762 A1 WO2019114762 A1 WO 2019114762A1
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cancer
tumor
cells
antigen
cell
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PCT/CN2018/120679
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French (fr)
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李宗海
周敏
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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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    • 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
    • A61K39/461—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
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/10—X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast
    • 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
    • A61K2239/50—Colon

Definitions

  • the invention belongs to the field of immunotherapy, and particularly relates to an immune effector cell having a receptor for recognizing a tumor antigen and triggering cell activation, and a local tumor radiation therapy combined for tumor treatment.
  • Adoptive immune cell therapy such as chimeric antigen receptor-modified T lymphocytes (CAR-T) has been expected to become an important treatment for tumors, especially malignant tumors, but the immune effector cells still face how to expand and improve in vivo. Anti-tumor activity and other issues.
  • CAR-T chimeric antigen receptor-modified T lymphocytes
  • lymphocyte clearance (clearing) pretreatment can increase the in vivo expansion of immune effector cells such as CAR-T cells or TIL and its antitumor activity, and recently by treating blood on large scale CD19-CAR-T cells.
  • Analysis of clinical data from malignant tumors found that the response to the CD19-CAR-T cell treatment was 88% in the clear-pretreatment group, which was much higher than that in the untreated group (32%) (Am J Cancer Res) 2016); 6(2): 403-424).
  • the instructions for two CD19-specific CAR-T cell therapy products (Kymriah of Knock Pharmaceuticals and Yescarta (KTE-C19) of Kate Pharmaceuticals), which were marketed in 2017, also clearly stated that before administration of CD19-CAR-T cells
  • the lymphocyte removal process (including the lymphocyte removal protocol of fludarabine and cyclophosphamide lymphocytes) was performed first.
  • clearing pretreatment may also affect the therapeutic effect of adoptive immune cells, and may be involved in the formation of cytokine storms, but also produce serious toxic reactions such as myelosuppression, especially in the treatment of solid tumors, after clearing pretreatment
  • the efficacy of CAR-T cells is not significant (Zhang et al., "Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA + Metastatic Colorectal Cancers", Molecular Therapy (2017), 25(5): 1248-1258 ), and there have been reports of cytokine storms in the treatment of colon metastasis using clear leaching pretreatment plus ERBB2-CAR-T, leading to death (Molecular Therapy vol. 18no. 4, 843–851apr. 2010).
  • One of the objects of the present invention is to provide a method for treating a tumor, which comprises administering a combination of immune effector cells and tumor local radiation to an individual having cancer, and not performing lymphocyte clearance on said individual, said immune effector cell A receptor comprising a tumor antigen that recognizes the tumor and triggering activation of the immune effector cell.
  • the immune effector cell administration and the local tumor radiation treatment are administered in no time; the local radiation therapy of the tumor may be administered first and then administered to the immune effector cells; or may be administered simultaneously; or the immune effector cells may be administered first. Administration is followed by localized radiation therapy of the tumor.
  • the receptor is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a T cell receptor (TCR), a T cell fusion protein (TFP), and a T cell. T cell antigen coupler (TAC) or a combination thereof.
  • the local radiation therapy of the tumor is to irradiate the tumor with a radiation therapy device.
  • the radiation therapy device irradiates the tumor by generating any of the following rays: X-ray, alpha-ray , beta rays, gamma rays, neutrons.
  • the radiation therapy apparatus generates X-rays that perform at least one radiation on the tumor or multiple doses of radiation.
  • the radiation dose of the radiation treatment is between not more than 100 Gy, preferably not more than 80 Gy, and more preferably not more than 70 Gy.
  • the energy source of the radiation therapy is located in the body or outside of the individual.
  • the chimeric antigen receptor comprises:
  • the tumor antigen is selected from the group consisting of: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; ; CD30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); 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; Lysin; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y
  • the immune effector cells are selected from CAR-T cells which specifically recognize EGFR, EGFRvIII, GPC3, Claudin 18.2.
  • the antibody which specifically recognizes a tumor antigen has the amino acid sequence shown in SEQ ID NO: 2.
  • the chimeric antigen receptor has SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 Amino acid sequence.
  • the immune effector cells are administered on the day after the local radiation treatment of the tumor, or after 1 day, 2 days, or 3 days.
  • the method of the present invention further comprising administering to the individual an immune checkpoint inhibitor; preferably, the immune checkpoint inhibitor is a biological therapeutic agent or a small molecule; more preferably, the The immunological checkpoint inhibitor is selected from the group consisting of a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.
  • the immunological checkpoint targeted by the immunological checkpoint inhibitor is selected from the following immunological checkpoint proteins: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3 , VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR and B-7 family ligands or combinations thereof; preferably, the immunological checkpoint inhibitor is a PD-1 or PDL-1 inhibitor .
  • the immunological checkpoint inhibitor interacts with a ligand selected from the following immunological checkpoint proteins: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR and B-7 family ligands or combinations thereof.
  • a ligand selected from the following immunological checkpoint proteins: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR and B-7 family ligands or combinations thereof.
  • the immune effector cells are selected from CAR-T cells which specifically recognize EGFR, EGFRvIII, GPC3, Claudin 18.2, and the immunological checkpoint inhibitor is a monoclonal antibody that specifically recognizes PD-1 or PD-L1.
  • the immune effector cells and the immunological checkpoint inhibitor are administered simultaneously.
  • the therapeutic effect of the method is predicted by the presence of immune effector cells, or the presence of a genetic marker indicative of T cell inflammation, or a combination thereof, preferably by detecting a change in IFN-[gamma] levels.
  • the tumor includes: breast cancer, blood cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell cancer of the lung, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, Skin cancer, glioma, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervix Cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumor of child, bladder Cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor
  • 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, said The immune effector cells are selected from autologous T cells, allogeneic T cells or allogeneic NK cells, and more preferably, the T cells are autologous T cells.
  • a second object of the present invention is to provide a use of immunotherapeutic cells in combination with a radiation source for treating cancer in the absence of clearing.
  • Another object of the present invention is to provide a combined treatment system for tumors, characterized in that the combination therapy system is a combined treatment system consisting of an apparatus for administering immune effector cells and local radiation therapy to a tumor-bearing individual. And the individual is not subjected to lymphocyte clearance, the immune effector cell comprising a receptor that recognizes a tumor antigen of the tumor.
  • the radiation source includes an alpha radiation source, a beta radiation source, a gamma radiation source, a neutron source, and the like.
  • the radiation source is a radiation device, and the radiation device is a linear accelerator; preferably, the linear accelerator generates X-rays and electron lines.
  • the present invention also provides the use of an immune effector cell for the preparation of a medicament for a method for the combined treatment of a tumor, the method comprising the simultaneous or sequential use of a localized radiation treatment of a tumor, the immune effector cell comprising A receptor for a tumor antigen of the tumor is identified, and an individual having the tumor receives the combined treatment of the tumor at a time when the number of lymphocytes in the body is not less than 40% relative to the treatment of the tumor.
  • local tumor radiation therapy can increase the anti-tumor activity of the combination therapy before or after administration of the CAR-T cell; in some embodiments, the local radiation therapy of the tumor combined with the CAR-T administration can reach the post-treatment with the clear rinse.
  • the anti-tumor effect of CAR-T cells in some embodiments, tumor local radiation therapy combined with CAR-T administration is even superior to the anti-tumor effect of CAR-T cells after clear-pretreatment, which is reflected in significant inhibition of tumor growth, Significantly extend the individual's survival and other aspects.
  • prior administration of localized radiation to the CAR-T cells can significantly increase the anti-tumor activity of the combination therapy, which is manifested in more significant inhibition of tumor growth and prolonging survival of the individual.
  • administration of CAR-T cells after a few days of local radiation therapy eg, 1 day apart
  • administration of CAR-T cells within a short period of time eg, the same day
  • Anti-tumor effect is reflected in inhibiting tumor growth, prolonging individual survival and tumor regression.
  • small doses, multiple doses of local radiation therapy to the tumor can also enhance the anti-tumor effect associated with CAR-T cells, which is manifested in inhibiting tumor growth, prolonging individual survival, and the like.
  • tumor local radiation therapy, CAR-T cell administration, and PD-L1 antibody combination have better tumor suppressing effects.
  • Figure 1A is a plasmid map of the recombinant vector MSCV-EGFRvIII-m28Z;
  • Figure 1B is a test for establishing a mouse serum shower model.
  • Figure 2A shows in vivo tumor growth inhibition results of untreated lymphatic tumor local radiation therapy combined with EGFRvIII-m28Z CAR-T cells for colon cancer;
  • Figure 2B shows untreated lymphatic tumor local radiation therapy and EGFRvIII-m28Z CAR-T Comparison of in vivo tumor growth inhibition results between cell combination therapy and clear-leaf pretreatment with CAR-T cells.
  • Figure 3 shows the results of in vivo tumor inhibition rates of tumor local radiation therapy combined with EGFRvIII-m28Z CAR-T cells in the treatment of colon cancer.
  • Figure 4A shows the survival results of untreated lymphatic tumor local radiation therapy combined with EGFRvIII-m28Z CAR-T cells in the treatment of mouse colon cancer
  • Figure 4B shows the localized radiation therapy of untreated lymphoma and EGFRvIII-m28Z CAR-T Comparison of the survival of colon cancer after treatment with cell-based combination therapy and CL-T.
  • Figure 5 shows the detection of plasma IFN- ⁇ concentration in mouse colon cancer in combination with local radiation therapy of tumors and EGFRvIII-m28Z CAR-T cells.
  • Figure 6A shows in vivo tumor growth inhibition assay of tumor local radiation therapy combined with EGFRvIII-m28Z CAR-T cells in C57BL/6 mouse orthotopic breast cancer
  • Figure 6B shows tumor local radiation therapy and EGFRvIII-m28Z CAR-T cells Survival detection of combined treatment of C57BL/6 mouse orthotopic breast cancer.
  • Figure 7A shows in vivo tumor growth inhibition assay of tumor local radiation therapy combined with EGFRvIII-m28Z CAR-T cells in Balb/c mouse orthotopic breast cancer
  • Figure 7B shows tumor local radiation therapy and EGFRvIII-m28Z CAR-T cells Survival detection of combined treatment of Balb/c mice with orthotopic breast cancer.
  • Figure 8 shows the effect of tumor local radiation therapy and EGFRvIII-m28Z CAR-T cell administration interval on the treatment of C57BL/6 mice in situ breast cancer:
  • Figure 8A is the tumor volume assay;
  • Figure 8B is the mouse survival assay.
  • Figure 9 shows the effect of low-dose, multiple-dose local radiation therapy with tumors and EGFRvIII-m28Z CAR-T cell administration on the treatment of Balb/c mice with orthotopic breast cancer:
  • Figure 9A shows tumor volume detection;
  • Figure 9B shows small Mouse survival testing.
  • Figure 10 shows the anti-tumor therapeutic effect of local radiation therapy combined with EGFRvIII-m28Z CAR-T cells and immunological checkpoint inhibitors on subcutaneous xenografts of glioma:
  • Figure 10A is an experimental flow chart;
  • Figure 10B is a tumor growth inhibition experiment.
  • the present invention relates to an immune effector cell having a receptor that recognizes a tumor antigen and triggers cell activation, and a combination of local radiation therapy for treating a tumor, it being understood that the present invention is not limited to the methods and experimental conditions described, because such a method and Conditions can vary.
  • the present disclosure derives, at least in part, from the technological cognition of a combination therapy comprising one or more cycles and/or doses of tumor local radiation therapy and immune effector cell therapy, either continuously, in any order, or substantially simultaneously.
  • the regimen can be more effective in treating cancer in some subjects, and/or can initiate, effect, increase, enhance or prolong the activity and/or number of immune cells, or a medically beneficial response through the tumor.
  • immune effector cell refers to a cell that participates in an immune response, for example, to promote an immune effect.
  • immune effector cells include T cells, for example, ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.
  • the T cells comprise autologous T cells, xenon T cells, allogeneic T cells, and the natural killer cells are allogeneic NK cells.
  • immune effector function or immune effector response refers to an immune effector cell, such as a function or response that enhances or promotes an immune attack by a target cell.
  • an immune effector function or response refers to a property of a T cell or NK cell that promotes killing of a target cell or inhibits growth or proliferation.
  • local radiation therapy or “local radiation treatment” or “local radiation therapy” has the same meaning in the present invention.
  • a radiation radiates a certain part of the body, a reaction causing local cells is called local radiation therapy, including, for example, fractionation.
  • Localized radiation therapy further includes irradiating the tumor with a radiation therapy device that, in a particular embodiment, irradiates the tumor by generating any of the following rays: X-rays, alpha rays, beta rays , gamma rays, neutrons.
  • anti-tumor treatments by ionizing radiation that can be performed according to all available techniques: a non-limiting list of them includes: fractionated radiation therapy, accelerated radiation, intensity modulated radiation therapy, image guided radiation therapy, external beam radiation Treatment, sealed source radiotherapy or brachytherapy, unsealed source radiotherapy, three-dimensional conformal radiotherapy, proton therapy, etc.
  • Localized radiation therapy can also be treated with radiation using an energy source located within the body of the individual.
  • the source of radiation can be external or internal to the subject.
  • the treatment is called external beam radiation therapy (EBRT), also known as external irradiation, often using gamma rays, neutrons, X-rays, etc. Radiation, the biological effect of external irradiation is strong.
  • EBRT external beam radiation therapy
  • BT brachytherapy
  • Its action mainly occurs in the passage of radioactive materials and the tissues and organs at the deposition site, but its effect can be Spread all over the body.
  • the effect of internal irradiation is mainly due to the short range and strong ionization of ⁇ and ⁇ rays.
  • the dose of radiation depends on a number of factors, which are well known in the art. Such factors include the organ to be treated, the healthy organ located in the radiation channel that may be adversely affected inadvertently, the patient's tolerance to radiation therapy, and the area of the body in need of treatment.
  • the dose is typically between no more than 100 Gy, preferably no more than 80 Gy, and even more preferably no more than 70 Gy.
  • the dose used can be administered once, or it can be divided into small doses and administered to the patient multiple times. It should be emphasized that the invention is not limited to any particular dosage. The dosage will be determined by the treating physician based on the particular factors in a given situation, including the factors described above.
  • the distance between the external source and the point of entry into the patient can be any distance that achieves an acceptable balance between killing the target cells and minimizing side effects. Typically, the distance between the external source and the point of entry into the patient is between 70 and 100 centimeters.
  • Brachytherapy is usually performed by placing a sealed radioactive source in the patient.
  • the source is placed approximately 0 to 5 cm from the tissue to be treated.
  • Known methods include interstitial brachytherapy, intraluminal brachytherapy, and surface interstitial brachytherapy.
  • the source of radiation can be permanently or temporarily implanted.
  • Some of the typical radioactive atoms that have been used in permanent implants include iodine-125 and strontium.
  • Some typical radioactive atoms that have been used in temporary implants include technetium-137 and technetium-192.
  • the radiation dose of brachytherapy may be the same as that used in external beam radiation therapy described above.
  • the characteristics of the radioactive atoms used are also taken into account in determining the brachytherapy dose.
  • Immune effector cell administration is administered before, during, and after radiation therapy, and may also be administered in combination, i.e., before, during, and after, during, and after, or before, during, and after radiation therapy.
  • the immune effector cell treatment 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 prior to radiation treatment.
  • 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 after radiation therapy administration.
  • the term "radiation therapy device” is a device that emits radiation. According to the degree to which the radiation device may cause harm to human health and the environment, the radiation device is classified into Class I, Class II, and Class III from high to low.
  • the medical ray device and the non-medical ray device are classified according to the use.
  • the ray device includes: 1) devices for accelerating charge particles, such as a cyclotron, an electromagnetic induction accelerator, various acceleration devices, and the like. 2) A device that emits X-rays, such as an X-ray generating device, an X-ray diffractometer, an X-ray fluorescence analyzer, or the like. 3).
  • a device containing radioactive materials such as an X-ray generating device, an X-ray diffractometer, an X-ray fluorescence analyzer, or the like.
  • Common medical ray devices include: medical accelerators, radiation therapy, X-ray electron beam accelerators, heavy ion therapy accelerators, proton therapy devices, accelerators for the preparation of radioactive drugs for positron emission computed tomography (PET), and other medical accelerators.
  • medical accelerators radiation therapy, X-ray electron beam accelerators, heavy ion therapy accelerators, proton therapy devices, accelerators for the preparation of radioactive drugs for positron emission computed tomography (PET), and other medical accelerators.
  • PET positron emission computed tomography
  • X-ray deep therapy machine digital subtraction angiography device
  • medical X-ray CT machine general X-ray machine for radiology diagnosis
  • X-ray imaging device dental X-ray machine
  • mammography machine mammography machine
  • radiation therapy simulation positioning machine others X-ray machines above the exemption level.
  • terapéuticaally effective amount refers to a compound that effectively achieves a particular biological result as described herein.
  • the amount, formulation, substance or composition for example, but not limited to, an amount or dose sufficient to promote a T cell response.
  • the immunological effector cells, immunological checkpoint inhibitors, therapeutic agents of the present invention to be administered when indicated as “immunologically effective amount”, “antitumor effective amount”, “inhibitory tumor effective amount” or “therapeutically effective amount” The exact number can be determined by the physician in consideration of the individual's age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).
  • An effective amount of localized radiation therapy means, but is not limited to, an increase, increase or prolongation of anti-tumor activity of an immune effector cell when combined with an immune effector cell; an increase in the number of anti-tumor immune effector cells or activated immune effector cells; and promotion of IFN- ⁇ secretion Radiation dose or radiation source for tumor regression, tumor shrinkage, tumor necrosis.
  • An effective amount of an immune effector cell means, but is not limited to, an increase, increase or prolongation of anti-tumor activity of the immune effector cells when combined with local radiation; an increase in the number of anti-tumor immune effector cells or activated immune effector cells; and promotion of IFN- ⁇ secretion; The number of immune effector cells with tumor regression, tumor shrinkage, and tumor necrosis.
  • lymphocyte clearance means that lymphocytes in the subject are not cleared. These include not administering lymphocyte depleting agents, systemic radiation therapy, or a combination thereof, or other means of causing clearance of lymphocytes; and, after administration of lymphocyte depleting agents, systemic radiation therapy, or a combination thereof, or other means of causing clearance of lymphocytes, When the subject's lymphocyte clearance rate is less than 60%.
  • lymphocyte clearance is the removal of lymphocytes from a subject in the body. This includes administering a lymphocyte scavenger, systemic radiation therapy, or a combination thereof.
  • a lymphocyte scavenger e.g, a SELvIII-binding CAR molecule
  • the subject may be administered alone or in combination with one or more agents capable of substantially clearing the lymphocytes of the subject, systemic radiation therapy, or a combination thereof.
  • the clearing treatment can be administered under conditions sufficient to achieve a subject's lymphocyte clearance of 60% to 100%.
  • the number of lymphocytes in the subject is reduced by at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 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% or 100%, the subject's lymphocytes were cleared.
  • Lymphocyte scavengers are anti-tumor chemotherapeutic agents.
  • lymphocyte scavengers include, but are not limited to, fludarabine, cyclophosphamide, or combinations thereof.
  • the treating physician can select a specific lymphocyte scavenger and a suitable dose according to the subject to be treated, such as CAMPATH, anti-CD3 antibody, cyclosporin, FK506, rapamycin, mycophenolic acid, steroid, FR901228, Melphalan, cyclophosphamide, fludarabine, and whole body radiation therapy.
  • Immune effector cell administration is administered before, during, and after the clearing treatment, or in combination, i.e., before, during, and after, during, and after, or before, during, and after the clearing treatment.
  • the clearing treatment is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 before the immune effector cell treatment Hours, 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, Administration is performed on days 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 1 or any combination thereof.
  • the clearing treatment is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, after administration of the immune effector cell therapy, 12 hours, 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, 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.
  • chimeric antigen receptor refers to a group of polypeptides that, when administered in an immune effector cell, provide said cells with specificity for a target cell, typically a cancer cell, and have Intracellular signal production.
  • CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain”), including stimulatory molecules derived from the definitions below and / Or a functional signaling domain of a co-stimulatory molecule.
  • the polypeptide groups are contiguous with each other.
  • a polypeptide group includes a dimerization switch that can couple the 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 an ⁇ chain that binds to a T cell receptor complex.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from a costimulatory molecule described herein, such as 4-1BB (ie, CD137), CD27, and/or CD28.
  • a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising 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 comprising 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 two functional signaling comprising one or more costimulatory molecules.
  • EGFRvIII was selected as a target of CAR-T cells, and in order to more accurately verify the anti-tumor effect in mice, the selected signal peptide, transmembrane region, intracellular region and the like were mouse-derived.
  • the method of preparation is operated according to conventional CAR-T cell preparation methods in the art.
  • the above embodiment selects a CAR of a murine source, but its signal peptide, hinge region, transmembrane region, and the like may be selected from other species depending on the purpose. These include, but are not limited to, human signal peptides, hinge regions, transmembrane regions, intracellular regions.
  • Antibodies can also be selected for murine anti- or humanized antibodies or whole human antibodies against different targets for different purposes.
  • stimulation refers to the binding of a stimulatory molecule (eg, a TCR/CD3 complex or CAR) to its cognate ligand (or a tumor antigen in the case of a CAR), thereby mediating signal transduction events (eg, However, it is not limited to the initial response induced via signal transduction of the TCR/CD3 complex or via signal transduction of a suitable NK receptor or CAR signaling domain. Stimulation can mediate altered expression of certain molecules.
  • a stimulatory molecule eg, a TCR/CD3 complex or CAR
  • the term "irritating molecule” refers to a molecule that provides a cytoplasmic signaling sequence expressed by immune cells (eg, T cells, NK cells, B cells) that modulate the signaling pathway for immune cells in an irritating manner. At least some aspects of activation of immune cells.
  • the signal is a primary signal initiated by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence that functions in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM-containing cytoplasmic signaling sequences specifically for use 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 one or more of the CARs of the invention comprises an intracellular signaling sequence, such as a CD3- ⁇ primary signaling sequence.
  • the primary signaling sequence of CD3- ⁇ is an equivalent residue derived from a human or non-human species such as mouse, rodent, monkey, donkey, and the like.
  • co-stimulatory molecule refers to a homologous binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that promotes an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and 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 can be an intracellular portion of a costimulatory molecule.
  • Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), and NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-related antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.
  • the intracellular signaling domain may comprise part or all of the native intracellular signaling domain, or a functional fragment or derivative thereof, of all cells within the molecule.
  • 4-1BB refers to a member of the TNFR superfamily having an amino acid sequence as provided by GenBank Accession No. AAA62478.2, or an equivalent residue from a non-human species such as a mouse, rodent, monkey, donkey, and the like;
  • the "4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, ticks, and the like.
  • the "4-1BB costimulatory domain” is an equivalent residue from a human or from a non-human species such as a mouse, rodent, monkey, ape or the like.
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain produces a signal that promotes the immune effector function of cells containing CAR, such as CAR-T cells.
  • immune effector functions in, for example, CAR-T cells include cell lytic activity and helper activity, including secretion of cytokines.
  • the intracellular signaling domain can comprise a first order intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen dependent stimulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • the first-level intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of primary cytoplasmic signaling sequences containing ITAM 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.
  • T cell receptor a characteristic marker on the surface of all T cells, binds to CD3 with a non-covalent bond to form a TCR-CD3 complex.
  • the TCR is responsible for identifying antigens that bind to major histocompatibility complex molecules.
  • TCR is a heterodimer composed of two different peptide chains, consisting of two peptide chains, ⁇ and ⁇ . Each peptide chain can be further divided into variable region (V region), constant region (C region), transmembrane. The region and the cytoplasmic region are several parts; it is characterized by a short cytoplasmic region.
  • TCR molecule belonging to the immunoglobulin superfamily that is present in the antigen-specific V regions; V region (V ⁇ , V ⁇ ) and have three hypervariable regions CDR1, CDR2, CDR3, CDR3 maximum variation which directly determines the TCR antigen Binding specificity.
  • CDR1, CDR2 recognizes and binds to the side wall of the MHC molecule antigen binding groove, and CDR3 binds directly to the antigen peptide.
  • TCR is divided into two categories: TCR1 and TCR2; TCR1 consists of two chains of ⁇ and ⁇ , and TCR2 consists of two chains of ⁇ and ⁇ .
  • T cell fusion protein includes various polypeptide-derived recombinant polypeptides constituting a TCR, which are capable of binding to a surface antigen on a target cell, and to other polypeptides of the intact TCR complex. The effect is usually located on the surface of the T cell.
  • TFP consists of an antigen binding domain consisting of a TCR subunit and a human or humanized antibody domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular domain.
  • the TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, and the TFP is integrated TCR expressed on T cells.
  • T cell antigen coupler includes three functional domains: a tumor targeting domain, including a single-chain antibody, designed ankyrin repeat protein (DARPin). Or other targeting group; 2 extracellular domain domain, a single-chain antibody that binds to CD3, such that the TAC receptor is adjacent to the TCR receptor; 3 the transmembrane region and the intracellular region of the CD4 co-receptor, wherein The inner region is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen.
  • Antibodies can be polyclonal or monoclonal, multi-stranded or single-stranded, or intact immunoglobulins, and can be derived from natural or recombinant sources.
  • the antibody can 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 antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, Linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domain, multispecific antibodies formed by antibody fragments (eg, bivalent fragments comprising two Fab fragments joined by a disulfide bond in the hinge region) and An isolated CDR or other epitope binding fragment of an antibody.
  • Antigen-binding fragments can also be incorporated into single domain antibodies, maximal antibodies, minibodies, Nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature Biotechnology (23): 1126-1136, 2005).
  • 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.
  • antibody heavy chain refers to the larger of the two polypeptide chains that are present in the antibody molecule in their naturally occurring configuration and which typically determine the type to which the antibody belongs.
  • antibody light chain refers to the smaller of the two polypeptide chains present in the antibody molecule in their naturally occurring configuration.
  • the ⁇ (k) and ⁇ (l) light chains refer to the isoforms of the two major antibody light chains.
  • recombinant antibody refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be interpreted to mean an antibody that has been produced by synthesizing a DNA molecule encoding an antibody (and wherein the DNA molecule expresses the antibody protein) or an amino acid sequence of a specified antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or is available in the art. And well known amino acid sequence techniques are available.
  • antigen refers to a molecule that elicits an immune response.
  • the immune response can involve activation of the antibody-producing or cells with specific immunity or both.
  • any macromolecule comprising virtually all proteins or peptides can serve as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, thus encoding an "antigen.”
  • the antigen need not be encoded only by the full length nucleotide sequence of the gene. It will be apparent that 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 a polypeptide that elicits a desired immune response.
  • the antigen does not need to be encoded by a "gene” at all. It will be 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. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or liquids with other biological components.
  • Tumor antigen refers to a common antigen of a particular hyperproliferative disease.
  • the hyperproliferative disorder antigen of the invention is derived from cancer.
  • the tumor antigen of the present invention includes, but is not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); 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; Lysin; mesothelin
  • cancer refers to a broad class of disorders characterized by hyperproliferative cell growth in vitro (eg, transformed cells) or in vivo.
  • Conditions which may be treated or prevented by the methods of the invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias and the like.
  • the methods of the invention may achieve inhibition and/or reversal of undesirable hyperproliferative cell growth involved in such conditions.
  • cancer examples include, but are not limited to, breast cancer, blood cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell cancer of the lung, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, Skin cancer, glioma, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervix Cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumor of child, bladder Cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis,
  • transfected or “transformed” or “transduced” refers to the process by which an exogenous nucleic acid is transferred or introduced 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 subject cells and their progeny.
  • the term "specifically binds” refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind other molecules in the sample. .
  • biologically equivalent refers to an agent different from a reference compound, an immune effector cell, or an irradiation agent required to produce an effect equivalent to a reference compound or a reference amount of a reference compound, an immune effector cell, or an irradiation-producing effect. the amount.
  • refractory refers to a disease, such as cancer, which does not respond to treatment.
  • the refractory cancer can be resistant to treatment prior to or at the onset of treatment. In other embodiments, the refractory cancer can be resistant during treatment.
  • Refractory cancer is also known as anti-cancer.
  • refractory cancers include, but are not limited to, cancers that are insensitive to radiotherapy, relapse after radiotherapy, insensitive to chemotherapy, relapse after chemotherapy, insensitive to CAR-T therapy, or relapse after treatment.
  • the treatment regimens described herein can be used for refractory or recurrent malignancies.
  • Relapsed refers to the return of a disease (eg, cancer) or signs and symptoms of a disease, such as cancer, over a period of improvement, for example, after a previous treatment of a therapy, such as a cancer therapy.
  • a disease eg, cancer
  • signs and symptoms of a disease such as cancer
  • a therapy such as a cancer therapy
  • therapeutic agent refers to any medical product that produces a therapeutic response in a subject. These include, but are not limited to, immunostimulatory agents, T cell growth factors, interleukins, antibodies and vaccines, chemotherapeutic agents, or combinations thereof.
  • An immunostimulatory agent is a substance (drug and nutrient) that stimulates the immune system by inducing activation of any one of the components of the immune system or increasing the activity of any of its components.
  • Immunostimulants include bacterial vaccines, colony stimulating factors, interferons, interleukins, other immunostimulants, therapeutic vaccines, vaccine combinations, and viral vaccines.
  • T cell growth factor is a protein that stimulates T cell proliferation.
  • T cell growth factors include Il-2, IL-7, IL-15, IL-17, IL21, and IL-33.
  • Interleukins are a group of cytokines that are first thought to be expressed by white blood cells. The function of the immune system depends to a large extent on interleukins, and many of the rare defects of interleukins have been described, all characterized by autoimmune diseases or immunodeficiencies. Most of the interleukins are synthesized by helper CD4 T lymphocytes, as well as by monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes as well as hematopoietic cells.
  • interleukins examples include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12. IL-13, IL-14, IL-15 and IL-17.
  • Chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, acetaminophen and piperazine; aziridines such as benzotropipine ( Benzodopa), carbofuran, meturedopa and uredopa; ethyleneimine and methylamelamines, including hexamethylene melamine, triethylene melamine, three Ethylenephosphoramide, triethyl thiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, nitrogen Mustard, oxychloride mustard, melphalan, neo-nitrogen mustard, fentanyl sterol, pine benzene mustard, tromethamine, uracil mustard; nitroure
  • an antihormonal agent for regulating or inhibiting the action of a hormone on a tumor
  • an antiestrogenic agent including, for example, tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxytamoxifen, trovaxifene, keoxifene, LY117018, ol's ketone and faremis (Fareston); and antiandrogens such as flutamide, nis Rummet, bicalutamide, leuprolide, and goserelin; and a pharmaceutically acceptable salt, acid or derivative of any of the above.
  • cancer therapeutics include sorafenib and other protein kinase inhibitors such as afatinib, axitinib, bevacizumab, cetuximab, crizotinib, dasatinib, erg Lotitinib, fotininib, gefitinib, imatinib, lapatinib, levabinib, moritinib, nilotinib, panitumumab, pazopanib, pega Tani, ranibizumab, rosobinib, trastuzumab, vandetanib, vemurafenib, and sunitinib; sirolimus (rapamycin), ivimo Division and other mTOR inhibitors.
  • protein kinase inhibitors such as afatinib, axitinib, bevacizumab, cetuximab, crizotinib, dasatinib,
  • chemotherapeutic agents include topoisomerase I inhibitors (eg, irinotecan, topotecan, camptothecin and its analogs or metabolites, and doxorubicin); topoisomerase II inhibition Agents (eg etoposide, teniposide and daunorubicin); alkylating agents (eg melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine) , lomustine, semustine, streptozotocin, azomethamine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (eg, cisplatin, oxaliplatin, and Carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimics (eg 5-fluorouracil, capecitabine, gemcitabine, fludarabine, cytarabine,
  • chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastine, and related analogs; thalidomide, lenalidomide, and related analogs (eg, CC-5013 and CC-4047); protein tyrosine kinase inhibitors (eg imatinib mesylate and gefitinib); proteasome inhibitors (eg bortezomib); NF- ⁇ B inhibitors including I ⁇ B Inhibitors of kinases; antibodies that bind to proteins overexpressed in cancer, and other inhibitors of proteins or enzymes known to be upregulated, overexpressed or activated in cancer, which inhibit downregulation of cells.
  • Local radiation and immune effector cell therapy combined with checkpoint inhibitors and therapeutic agents may be more effective in treating cancer in some subjects, and/or may initiate, achieve, increase, enhance or prolong immune cells (Including the activity and/or number of T cells, B cells, NK cells and/or others, or promoting IFN- ⁇ secretion, or communicating a medically beneficial response (including regression, necrosis or elimination thereof) through the tumor.
  • the immune effector cells are administered concurrently with the checkpoint inhibitor, or the immune effector cell therapy is administered chronologically before or after the checkpoint inhibitor.
  • the immune effector cell treatment is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours prior to administration of the checkpoint inhibitor.
  • 12 hours 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 Administration on days, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 1 or any combination thereof.
  • the immune effector cell treatment is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours after administration of the checkpoint inhibitor. , 12 hours, 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 Administration on days, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 1 or any combination thereof.
  • the therapeutic agent is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 before the administration of the immune effector cells.
  • the therapeutic agent is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 days, 8 days, 9 hours, 10 hours, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, Administration is performed on days 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 1 or any combination thereof.
  • the therapeutic agent is administered 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 administration of the immune effector cells.
  • biological therapeutic agent refers to any medical product that is manufactured in a biological source or extracted from a biological source.
  • Biopharmaceuticals are different from chemically synthesized pharmaceutical products.
  • biological agents include vaccines, blood or blood components, allergens, somatic cells, gene therapy, tissues, recombinant therapeutic proteins, including antibody therapeutics and fusion proteins, and living cells.
  • Biological products can be composed of sugars, proteins or nucleic acids or complex combinations of these substances, or can be living entities such as cells and tissues.
  • Biological products are isolated from a variety of natural sources (human, animal or microbial) and can be produced by biotechnological methods and other techniques.
  • Specific examples of biological therapeutic agents include, but are not limited to, immunostimulatory agents, T cell growth factors, interleukins, antibodies, fusion proteins, and vaccines such as cancer vaccines.
  • treating refers to slowing or ameliorating the progression, severity, and/or progression of a proliferative disorder due to administration of one or more therapies (eg, one or more therapeutic agents such as CAR-T, topical radiation therapy of the invention). Duration, or amelioration of one or more symptoms of a proliferative disorder (preferably, one or more discernible symptoms). In a particular embodiment, the term “treating” refers to ameliorating at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that is not necessarily discernible by a patient.
  • the term “treating” refers to inhibiting the progression of a proliferative disorder physically, by, for example, stabilizing physical parameters, physiologically, or both, by, for example, stabilizing the discernible symptoms. In other embodiments, the term “treating” refers to reducing or stabilizing tumor size, counting cancer cells, or prolonging the survival of an individual.
  • improving survival refers to an increase in the lifespan or quality of life of a subject having cancer or a proliferative disease. For example, improving survival also includes promoting cancer remission, preventing tumor invasion, preventing tumor recurrence, slowing tumor growth, preventing tumor growth, reducing tumor size, and reducing total cancer cell count.
  • treating cancer is not meant to be an absolute term.
  • the methods of the invention seek to reduce tumor size or number of cancer cells, promote cancer into remission, or prevent the growth of cancer cells in size or number of cells. In some cases, treatment results in an improved prognosis.
  • autologous refers to any substance derived from an individual that is subsequently introduced to the same individual.
  • allogeneic refers to any substance derived from a different animal of the same species as the individual into which the substance will be introduced. When the genes of one or more loci are not identical, two or more individuals are considered to be allogeneic to each other. In certain aspects, allogeneic materials from individuals of the same species may be genetically sufficiently different to interact antigenically.
  • subject refers to an individual or subject who has been diagnosed with a cancer or cell proliferative disorder.
  • tumor response refers to a cellular response including, but not limited to, triggering programmed cell death.
  • anti-tumor response refers to an immune system response including, but not limited to, activating T cells to challenge an antigen or antigen presenting cells.
  • an enhanced response refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein.
  • an enhanced response may include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 in responsiveness.
  • “enhanced” may also refer to increasing the number of subjects in response to treatment, such as immune effector cell therapy.
  • an enhanced response can refer to the total percentage of subjects responding to treatment, with percentages being 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
  • small molecule refers to a low molecular weight ( ⁇ 900 Daltons) organic compound that can help regulate biological processes with a size on the order of 10-9 m. Most drugs are small molecules.
  • the immune checkpoint regulates T cell function in the immune system.
  • T cells play a key role in cell-mediated immunity.
  • the checkpoint protein interacts with a specific ligand that signals the T cells and substantially shuts down or inhibits T cell function.
  • Cancer cells utilize this system by driving high levels of checkpoint protein expression on their surface, which results in the control of T cells expressing checkpoint proteins on the surface of T cells entering the tumor microenvironment, thus suppressing the anti-cancer immune response . As such, inhibition of checkpoint proteins will result in restoration of T cell function and immune response against cancer cells.
  • checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (of the CD2 family of molecules, and All NK, ⁇ and memory CD8+ ( ⁇ ) T cells are expressed), CD160 (also known as BY55), CGEN-15049, CHK 1 and CHK2 kinase, A2aR and various B-7 family ligands.
  • PD-1 antibody refers to an antibody that antagonizes the activity and/or proliferation of lymphocytes by agonizing PD-1.
  • antagonistic activity refers to a decrease (or decrease) in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of lymphocyte proliferation or activity.
  • antagonist can be used interchangeably with the terms “inhibiting” and “inhibiting”.
  • PD-1 mediated activity can be quantified using a T cell proliferation assay as described herein.
  • the anti-PD-1 antibody can be a novel antigen binding fragment.
  • the anti-PD-1 antibodies disclosed herein are capable of binding to human PD-1 and antagonizing PD-1, thereby inhibiting the function of immune cells expressing PD-1.
  • the immune cell is an activated lymphocyte that expresses PD-1, such as a T cell, a B cell, and/or a monocyte.
  • PD-1 Programmed cell death protein 1
  • PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family.
  • PD-L1 protein is up-regulated in macrophages and dendritic cells (DC) in response to LPS and GM-CSF treatment, and up-regulated on T and B cells after TCR and B cell receptor signaling, In resting mice, PDL1 mRNA can be detected in the heart, lung, thymus, spleen and kidney. PD-1 negatively regulates T cell responses.
  • CTLA4 cytotoxic T lymphocyte-associated protein
  • CTLA4 is a protein receptor that down-regulates the immune system.
  • CTLA4 is found on the surface of T cells, which results in cellular immune attack on antigen.
  • T cell challenge can be initiated by stimulating the CD28 receptor on T cells.
  • T cell challenge can be turned off by stimulating the CTLA4 receptor.
  • the first class of immunotherapy Yervoy (a monoclonal antibody that targets CTLA-4 on the surface of T cells), is approved for the treatment of melanoma.
  • the checkpoint inhibitor is a biological therapeutic agent or a small molecule.
  • the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.
  • the checkpoint inhibitor inhibits checkpoint proteins, which can be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 , CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or combinations thereof.
  • the checkpoint inhibitor interacts with a ligand that can be a checkpoint protein: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands or combinations thereof.
  • the therapeutic agent is an immunostimulatory agent, a T cell growth factor, an interleukin, an antibody, a vaccine, a chemotherapeutic drug, or a combination thereof.
  • the immunological checkpoint inhibitor is administered prior to, concurrently with, or subsequent to the topical radiation therapy, or the immunological checkpoint inhibitor is administered prior to, concurrently with, or subsequent to the immunotherapeutic cell therapy.
  • Checkpoint inhibitors include any agent that blocks or inhibits the inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies that bind to and block or inhibit the immunological checkpoint receptor or antigen-binding fragments thereof, or antibodies that bind to and block or inhibit the immunological checkpoint receptor ligand.
  • Exemplary checkpoint molecules that can be targeted for blockade or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA , KIR, 2B4 (belonging to the CD2 family of molecules, and expressed on all NK, ⁇ and memory CD8+ ( ⁇ ) T cells), CD160 (also known as BY55), CGEN-15049, CHK 1 and CHK2 kinase, A2aR and various B-7 family ligand.
  • B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7.
  • Checkpoint inhibitors include antibodies or antigen-binding fragments thereof, other binding proteins, biological therapeutics or small molecules that bind to and block or inhibit the activity of one or more of the following: CTLA-4, PDL1, PDL2 , PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049.
  • Illustrative immunological checkpoint inhibitors include tromezumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocking) , Nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (antibiotic) PDL1 antibody) and Yervoy/Ipilimumab (anti-CTLA-4 checkpoint inhibitor).
  • Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.
  • Suitable anti-CTLA-4 antagonists for use in the methods of the invention include, but are not limited to, anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, monoclonal antibodies CTLA4 antibody, polyclonal anti-CTLA4 antibody, chimeric anti-CTLA4 antibody, MDX-010 (Ipilizumab), Trumeimumab, anti-CD28 antibody, anti-CTLA4adnectin, anti-CTLA4 domain antibody, single-stranded anti-CTLA4 fragment , heavy chain anti-CTLA4 fragment, light chain anti-CTLA4 fragment, inhibitor of CTLA4 agonizing the co-stimulatory pathway, antibody disclosed in PCT Publication No.
  • CTLA-4 antibodies are described in U.S. Patent Nos. 5,811,097, 5,855,887, 6,051,227 and 6,984,720; PCT Publication No. WO 01/14424 and WO 00/37504; and U.S. Publication Nos. 2002/0039581 and 2002/086014.
  • Other anti-CTLA-4 antibodies that can be used in the methods of the invention include, for example, those disclosed in WO 98/42752; U.S. Patent Nos.
  • Additional anti-CTLA4 antagonists include, but are not limited to, the ability to disrupt the ability of the CD28 antigen to bind to its cognate ligand, inhibit the ability of CTLA4 to bind its cognate ligand, enhance T cell responses via a costimulatory pathway, and disrupt B7 binding to CD28. And/or the ability of CTLA4 to disrupt B7's ability to activate costimulatory pathways, disrupt CD80's ability to bind to CD28 and/or CTLA4, disrupt CD80's ability to activate costimulatory pathways, disrupt CD86 binding to CD28 and/or CTLA4, and disrupt CD86 activation The ability to co-stimulate pathways, as well as any inhibitor that disrupts the activation of the costimulatory pathway in general.
  • Such requisites include small molecule inhibitors of CD28, CD80, CD86, CTLA4, and other members of the costimulatory pathway; antibodies to CD28, CD80, CD86, CTLA4, and other members of the costimulatory pathway; CD28 for the costimulatory pathway, Antisense molecules of CD80, CD86, CTLA4 and other members; CDnectin, CD80, CD86, CTLA4 and other member of the adnectin for co-stimulatory pathways, CDi, CD80, CD86, CTLA4 and other members of the RNAi inhibitors of the co-stimulatory pathway ( Both single-stranded and double-stranded, as well as other anti-CTLA4 antagonists.
  • the treatment is by clinical outcome; the anti-tumor activity of the T cell is increased, enhanced or prolonged; the increase in the number of anti-tumor T cells or activated T cells, promotes IFN- ⁇ secretion, or Combination decision.
  • the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response through the immune system; tumor enlargement, recurrence or spread, or a combination thereof.
  • the therapeutic effect is predicted by the presence of T cells, the presence of a genetic marker indicative of T cell inflammation, promotion of IFN-[gamma] secretion, or a combination thereof.
  • the methods described herein are used to treat cancer.
  • the methods described herein can be used to reduce the size of a solid tumor or to reduce the number of cancer cells in a cancer.
  • the methods described herein can be used to slow the rate of cancer cell growth.
  • the methods described herein can be used to stop the rate of cancer cell growth.
  • An immune effector cell, therapeutic agent, checkpoint inhibitor, biological therapeutic, or pharmaceutical composition as disclosed herein can be administered to an individual by a variety of routes including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, Intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral, intravasally, intradermal or by passive or accelerated absorption through the skin, for example, by skin patch or transdermal iontophoresis.
  • the therapeutic agent, checkpoint inhibitor, biological therapeutic, or pharmaceutical composition can also be administered to a site of a pathological condition, such as intravenously or intraarterially into a blood vessel that supplies the tumor.
  • the total amount of agent to be administered in practicing the methods of the invention may be administered as a single dose by bolus or by infusion over a relatively short period of time, or may be administered using a fractionated treatment regimen, wherein over extended periods of time Multiple doses are administered in segments.
  • One skilled in the art will recognize that the amount of composition that treats a pathological condition in a subject depends on a number of 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. With these factors in mind, the technician will adjust the specific dose as needed. In general, initially, Phase I and Phase II clinical trials are used to determine the formulation of the composition as well as the route and frequency of administration.
  • a range such as 95-99% identity includes a range having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96 to 97%, 97 to 99%, 97 to 98%, and 98 to 99% identity. This does not apply regardless of the width of the range.
  • Exemplary antigen receptors of the present invention including CAR, and methods for engineering and introducing a receptor into a cell, are described, for example, in Chinese Patent Application Publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A. , CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication No. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018018958A1.
  • this example employs a second generation CAR that targets EGFRvIII, which is used to construct the transmembrane and intracellular domains of CAR using the mouse gene sequence for animal experiments.
  • the coding sequence of the mouse CD8 ⁇ signal peptide (SEQ ID NO: 1), the coding sequence of the monoclonal antibody recognizing human activated EGFR, which also recognizes human EGFRvIII (SEQ ID NO: 2), the murine CD8 alpha hinge region and The coding sequence of the transmembrane region (SEQ ID NO: 3), the coding sequence of the murine CD28 intracellular domain (SEQ ID NO: 4), and the coding sequence of the murine CD3 sputum intracellular domain (SEQ ID NO: 5) were sequentially ligated in vitro.
  • the 293T cells were infected with the recombinant vector MSCV-EGFRvIII-m28Z to obtain a packaged retrovirus. Infection methods are routine infection methods in the preparation of T cells expressing chimeric antigen receptors in the art.
  • mice The spleen T lymphocytes of Balb/c mice were taken, and the purified mouse CD3 + T lymphocytes were added to Dynabeads Mouse T-activator CD3/CD28 (Thermo Fisher) in a volume ratio of 1:1, washed once with PBS, activated, and placed. The incubator was cultured in RPMI 1640 complete medium.
  • Mouse spleen T lymphocytes activated for 24 h were inoculated into a retronectin (Takara T100A)-coated 12-well plate, and retrovirus infection was added overnight to obtain mouse EGFRvIII-m28Z CAR-T cells.
  • Balb/c mice are mice with normal immune systems. Balb/c mice were given 5Gy ⁇ -rays for whole body irradiation. On the 14th day after irradiation, 50 ⁇ l of mouse mandibular blood was collected into anticoagulation tubes, and PerCP Cy5.5-labeled anti-mouse CD3 antibody was added and incubated for 1 hour at room temperature. 450 ⁇ l of red blood cell lysate was added, and after ten minutes, the BD flow analyzer was used to detect the CD3 positive rate.
  • CT26-EGFRvIII a CT26 cell model of mouse EGFR excluding the exon of murine EGFR exon 2-7 was constructed by conventional methods of molecular biology using a mammalian epitope 287-302 amino acid epitope.
  • CT26 cells were purchased from the American Type Culture Collection (ATCC CRL-2638).
  • mice were first divided into two groups: the unclearing group and the Qinglin group.
  • UT (untreated) cell group (UT) Day13 tail vein infusion of 5 ⁇ 10 6 untreated mouse T cells;
  • X-ray local tumor radiation treatment group
  • EGFRvIII-m28Z CAR-T cell group EGFRvIII-m28Z: Day13 tail vein Infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells
  • EGFRvIII-m28Z CAR-T+ tumor local radiation treatment group (EGFRvIII-m28Z+X-ray): Day13 tail vein infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells, one day apart Day15 tumor locally received 10Gy X-ray radiation;
  • Tumor local radiation therapy + EGFRvIII-m28Z CAR-T group (X-ray+EGFRvIII-m28Z): Day13 tumor locally received 10Gy X-ray radiation, interval 1 day Day15 tail 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells were intravenously infused.
  • Qingle group 6 weeks old female Balb/c mice were irradiated with 5Gy ⁇ -rays whole body, and 3 ⁇ 10 5 CT26-EGFRvIII cells were inoculated subcutaneously on the day, which was recorded as Day0 on the day of inoculation; Day 13 after tumor inoculation (Day13), The tumor volume was 150-250 mm 3 , and the mice in the Qinglin group were divided into 3 groups (6 in each group):
  • UT (untreated) cell group (UT (Lymphodeletion): Day13 tail vein infusion of 5 ⁇ 10 6 untreated mouse T cells,
  • Tumor local radiation treatment group (X-ray (Lymphodeletion): Day13 tumor locally receives 10Gy X-ray radiation,
  • EGFRvIII-m28Z CAR-T cell group (EGFRvIII-m28Z (Lymphodeletion): Day13 was intravenously infused with 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells.
  • tumor volume (tumor length ⁇ tumor width 2 )/2.
  • day 21 mouse plasma was taken to examine the concentration of IFN- ⁇ .
  • the tumor volume of the X-ray and CAR-T combination group (EGFRvIII-m28Z+X-ray, X-ray+EGFRvIII-m28Z) was significantly smaller than that of the single CAR-T cell treatment group ( Tumor volume of EGFRvIII-m28Z) (P ⁇ 0.05, Two way ANOVA).
  • the tumor volume of the X-ray and CAR-T combination group (EGFRvIII-m28Z+X-ray, X-ray+EGFRvIII-m28Z) of the uncleared mice was administered with CAR-T cells after pretreatment with clearing.
  • the tumor inhibition rate of the untreated lymphatic CAR-T cell treatment group (EGFRvIII-m28Z) was 35.98% ⁇ 18.15%, X-ray+EGFRvIII-
  • the tumor inhibition rate was 92.44% ⁇ 2.24% in the m28Z combination group and 75.06% ⁇ 13.06% in the EGFRvIII-m28Z+X-ray combination group, and the CAR-T cell treatment group (EGFRvIII-m28Z (after treatment).
  • the tumor inhibition rate of Lymphodeletion) was 77.85% ⁇ 17.92%.
  • the tumor inhibition rate of the unbleached mouse combination group (EGFRvIII-m28Z+X-ray, X-ray+EGFRvIII-m28Z) was higher than that of the untreated mouse CAR-T cell treatment group (EGFRvIII-m28Z) ( P ⁇ 0.001, One way ANOVA), the tumor suppression rate of the unfractionated mouse combination group (EGFRvIII-m28Z+X-ray, X-ray+EGFRvIII-m28Z) and the CAR-T cell treatment group after clearing the pretreatment ( There was no significant difference in the inhibition rate of EGFRvIII-m28Z (Lymphodeletion). The above results indicate that the effect of CAR-T cell therapy combined with local tumor radiation therapy can achieve the anti-tumor effect of the treatment group administered with CAR-T cells after clearing pretreatment.
  • the X-ray+EGFRvIII-m28Z group had the longest survival (P ⁇ 0.05, Log-rank test) in the combination group, which was significantly higher than the EGFRvIII-m28Z CAR-T group.
  • X-ray group The survival time of the X-ray+EGFRvIII-m28Z group in the unbleached mice in Fig. 4B was compared with that of the untreated EGFRvIII-m28Z+X-ray group and the supernatant of the lymphocyte-treated group (EGFRvIII- m28Z (Lymphodeletion)) was longer (P ⁇ 0.05, Log-rank test).
  • CAR-T cell therapy combined with local tumor radiation therapy can achieve the anti-tumor effect of the CAR-T cell treatment group after clearing pretreatment, such as no statistical difference in tumor growth inhibition, and local tumor radiation and CAR-T cells.
  • the order of administration does not affect the tumor growth inhibition effect of the combination therapy.
  • CAR-T cell therapy combined with local tumor radiation treatment was significantly better than the treatment of CAR-T cells after clearing the pre-treatment, including X-ray+EGFRvIII-m28Z group. The impact of survival is particularly significant.
  • CAR-T cell therapy combined with local tumor radiation therapy can promote the secretion of interferon-gamma, and its promotion level can reach the level of the treatment group after treatment with CL-T cells, including X-ray+EGFRvIII-m28Z group.
  • the level of interferon gamma was significantly higher than that of the CAR-T cell treated group after clear pretreatment. The above description of clearing pretreatment is not necessary to improve the therapeutic effect of CAR-T cells.
  • CAR-T cells combined with local tumor radiation treatment can achieve the same anti-tumor effect as the CAR-T treatment group after clearing the pre-treatment, and first perform local tumor irradiation and then re-administer CAR-T cells, ie, X. -ray+EGFRvIII-m28Z was also significantly higher than the anti-tumor treatment effect of the CAR-T cell treatment group after clearing pretreatment.
  • mice breast cancer cell E0771 cell model (E0771-EGFR) overexpressing mouse EGFR expressing the amino acid epitopes 287-302 of human EGFR was established by conventional methods of molecular biology.
  • Mouse breast cancer cell E0771 was presented by Baylor College of Medicine.
  • mice 6-8 weeks old C57BL/6 mice (purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.), 1 ⁇ 10 6 E0771-EGFR cells were inoculated into the right fourth breast fat pad of mice to construct mice.
  • In situ breast cancer model the day of vaccination is Day0. On the 14th day after inoculation (Day 14), the tumor grew to 200-300 mm 3 (), and the tumor-bearing mice were divided into 5 groups (6 in each group):
  • UT (untreated) cell group (UT) Day 14 tail vein infusion of 5 ⁇ 10 6 untreated mouse T cells;
  • Tumor local radiation treatment group (X-ray): Day14 tumor locally received 10Gy X-ray radiation;
  • EGFRvIII-m28Z CAR-T cell group (EGFRvIII-m28Z): Day14 tail vein infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells;
  • EGFRvIII-m28Z CAR-T+ tumor local radiation treatment group (EGFRvIII-m28Z+X-ray): Day14 tail vein infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells, one day apart Day16 tumors locally received 10Gy X-ray radiation;
  • tumor volume (tumor length ⁇ tumor width 2 )/2.
  • survival time of the mice was recorded, and the results are shown in Fig. 6.
  • the tumor inhibition rate of the EGFRvIII-m28Z group was 41.74% ⁇ 13.29%, and the tumor inhibition rate of the EGFRvIII-m28Z+X-ray group was 81.77% ⁇ 12.62%.
  • the tumor inhibition rate of X-ray+EGFRvIII-m28Z group was 80.85% ⁇ 17.82%.
  • both the EGFRvIII-m28Z+X-ray group and the X-ray+EGFRvIII-m28Z group significantly inhibited tumor growth (EGFRvIII-m28Z vs. X-ray+EGFRvIII-m28Z P ⁇ 0.01, One way) ANOVA; EGFRvIII-m28Z vs. EGFRvIII-m28Z+X-rayP ⁇ 0.05, One way ANOVA).
  • a mouse breast cancer cell 4T1 cell model (4T1-EGFR) overexpressing mouse EGFR expressing the amino acid epitopes 287-302 of human EGFR was established by conventional methods in molecular biology. 4T1 cells were purchased from the Chinese Academy of Sciences Cell Bank (TCM32). (1) Establishment of Balb/c mouse orthotopic breast cancer model and group therapy:
  • mice purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.
  • 5 ⁇ 10 5 4T1-EGFR cells were inoculated into the right fourth breast fat pad of mice to construct mice.
  • In situ breast cancer model the day of vaccination is Day0.
  • Day 15 On the 15th day after inoculation (Day 15), the tumor grew to 100-200 mm 3 , and the tumor-bearing mice were divided into 5 groups (6 in each group):
  • UT (untreated) cell group (UT) Day 15 tail vein infusion of 5 ⁇ 10 6 untreated mouse T cells;
  • X-ray local tumor radiation treatment group
  • EGFRvIII-m28Z CAR-T cell group EGFRvIII-m28Z: Day15 tail vein Infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells
  • EGFRvIII-m28Z CAR-T+ tumor local radiation treatment group (EGFRvIII-m28Z+X-ray): Day15 tail vein infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells, one day apart Day17 tumor locally received 10Gy X-ray radiation;
  • Tumor local radiation therapy + EGFRvIII-m28Z CAR-T group (X-ray+EGFRvIII-m28Z): Day15 tumor locally received 10Gy X-ray radiation, one day interval is Day17 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells were infused into the tail vein.
  • tumor volume (tumor length ⁇ tumor width 2 )/2.
  • survival time of the mice was recorded, and the results are shown in Fig. 7.
  • the tumor inhibition rate of the EGFRvIII-m28Z group was 1.05% ⁇ 8.37%, and the tumor inhibition rate of the EGFRvIII-m28Z+X-ray group was 57.17% ⁇ 4.24%.
  • the tumor inhibition rate of X-ray+EGFRvIII-m28Z group was 77.22% ⁇ 11.71%.
  • both the EGFRvIII-m28Z+X-ray group and the X-ray+EGFRvIII-m28Z group significantly inhibited tumor growth (EGFRvIII-m28Z vs. X-ray+EGFRvIII-m28Z, P ⁇ 0.001, One way ANOVA; EGFRvIII-m28Z vs. EGFRvIII-m28Z+X-ray, P ⁇ 0.001, One way ANOVA).
  • mice 6-8 weeks old C57BL/6 mice (purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.), 8 ⁇ 10 5 E0771-EGFR cells were inoculated into the right fourth breast fat pad of mice to construct mice.
  • In situ breast cancer model the day of vaccination is Day0. On the 17th day after inoculation (Day17), the tumor volume was about 100-200 mm 3 , and the tumor-bearing mice were divided into 3 groups (6 in each group);
  • UT (untreated) cell group (UT) Day 17 tail vein infusion of 1 ⁇ 10 7 untreated mouse T cells;
  • X-ray+EGFRvIII-m28Z CAR-T group 1 Day17 tumor locally received 10Gy X-ray radiation, and the day after irradiation, Day17 tail vein was infused with 1 ⁇ 10 7 EGFRvIII-m28Z CAR-T cells;
  • X-ray+EGFRvIII-m28Z CAR-T group 2 Day17 tumors received 10Gy X-ray radiation locally, and 1 ⁇ 10 7 EGFRvIII-m28Z CAR-T cells were infused into the tail vein 1 day.
  • tumor volume (tumor length ⁇ tumor width 2 )/2.
  • survival time of the mice was recorded, and the results are shown in Fig. 8.
  • X-ray+EGFRvIII-m28Z CAR-T group 1 On day 34 after tumor inoculation (Day34), the tumor inhibition rate of X-ray+EGFRvIII-m28Z CAR-T group 1 was 91.16% ⁇ 6.56%, X-ray+EGFRvIII-m28Z CAR-T Group 2 tumor inhibition rate was 50.99% ⁇ 43.27%.
  • X-ray+EGFRvIII-m28Z CAR-T group 1 significantly inhibited tumor growth (X-ray+EGFRvIII-m28Z CAR-T group 1vs.X-ray+ EGFRvIII-m28Z CAR-T group 2P ⁇ 0.05, t-test).
  • mice purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.
  • 5 ⁇ 10 5 4T1-EGFR cells were inoculated into the right fourth breast fat pad of mice to construct mice.
  • In situ breast cancer model the day of vaccination is Day0.
  • Day 14 On the 14th day after inoculation (Day 14), the tumor grew to 100-200 mm 3 , and the tumor-bearing mice were divided into 4 groups (6 in each group):
  • UT (untreated) cell group (UT) Day 14 tail vein infusion of 5 ⁇ 10 6 untreated mouse T cells;
  • Tumor local radiation treatment group Day14 tumor locally received 3Gy X-ray radiation once a day for 3 consecutive days;
  • EGFRvIII-m28Z CAR-T cell group (EGFRvIII-m28Z): Day14 tail vein infusion of 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells;
  • X-ray+EGFRvIII-m28Z Day14 tumor locally received 3Gy X-ray radiation once a day for 3 consecutive days. On the day of the end of the third day of radiation (Day16), 5 ⁇ 10 6 EGFRvIII-m28Z CAR-T was injected into the tail vein. cell.
  • tumor volume (tumor length ⁇ tumor width 2 )/2.
  • survival time of the mice was recorded, and the results are shown in Fig. 9.
  • the tumor inhibition rate of the EGFRvIII-m28Z group was 26.02% ⁇ 7.92%, and the tumor inhibition rate of the X-ray + EGFRvIII-m28Z group was 50.69% ⁇ 4.29%.
  • the X-ray+EGFRvIII-m28Z group significantly inhibited tumor growth relative to the EGFRvIII-m28Z group (EGFRvIII-m28Z vs. X-ray+EGFRvIII-m28Z P ⁇ 0.01, Two way ANOVA).
  • Example 8 Anti-tumor therapeutic effect of local tumor radiation therapy combined with EGFRvIII-m28Z CAR-T cells and immunological checkpoint inhibitors on subcutaneous xenografts of glioma
  • mouse glioma cell line GL261 cells purchased from Shanghai Ruilu Biotechnology Co., Ltd.
  • GL261 cells expressing mouse EGFR expressing chimeric human EGFR epitopes 287-302 were established by routine means of molecular biology.
  • Model, GL261-EGFR cells were obtained.
  • UT (untreated) cell group (UT) Day 7 tail vein infusion of 2 x 10 6 untreated mouse T cells;
  • Treatment group alone (1) tumor local radiation treatment group (X-ray): Day7 tumor locally received 2Gy X-ray radiation; (2) EGFRvIII-m28Z CAR-T cell group (EGFRvIII-m28Z): Day7 tail vein infusion 2 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells; (3) PD-L1 antibody group (PD-L1): Day7, Day9, and Day11 were intraperitoneally injected with anti-PD-L1 antibody (Tecentriq) once daily. 200 ⁇ g;
  • Combination group (1) EGFRvIII-m28Z CAR-T+PD-L1 antibody group (EGFRvIII-m28Z+PD-L1): Day7 tail vein infusion of 2 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells; Day7, Day9, Day11 Intraperitoneal injection of anti-PD-L1 antibody (Tecentriq), once daily, 200 ⁇ g each time; (2) local tumor radiation treatment + EGFRvIII-m28Z CAR-T group (X-ray + EGFRvIII-m28Z): Day7 tumor local acceptance 2Gy X-ray radiation; Day9 tail vein infusion of 2 ⁇ 10 6 EGFRvIII-m28Z CAR-T cells; (3) Tumor local radiation therapy + PD-L1 antibody + EGFRvIII-m28Z CAR-T group (X-ray+PD- L1+EGFRvIII-m28Z): Day7 tumors received 2Gy X-ray radiation locally, and day (Day7) intraperitoneal injection of anti
  • tumor volume (tumor length ⁇ tumor width 2 )/2.
  • the results are shown in Fig. 10B.
  • the tumor inhibition rate of the EGFRvIII-m28Z group was 5.79% ⁇ 6.40%, and the tumor inhibition rate of the X-ray+EGFRvIII-m28Z group was 30.84% ⁇ 3.95%.
  • the tumor inhibition rate of X-ray+PD-L1+EGFRvIII-m28Z group was 43.26% ⁇ 14.51%.
  • the X-ray+EGFRvIII-m28Z group and the X-ray+PD-L1+EGFRvIII-m28Z group had better tumor growth inhibition effects than the EGFRvIII-m28Z group (EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z P) ⁇ 0.01, One way ANOVA; EGFRvIII-m28Z vs. X-ray+PD-L1+EGFRvIII-m28Z P ⁇ 0.0001, One way ANOVA).
  • the X-ray+PD-L1+EGFRvIII-m28Z group had a relatively high tumor inhibition rate relative to the X-ray+EGFRvIII-m28Z group (P ⁇ 0.05, t-test). The above results indicate that the blockade of the PD-1 pathway can further enhance the effect of tumor local radiation and CAR-T cell combination therapy on tumor growth inhibition.
  • a CAR-T prepared by targeting mouse anti-EGFRvIII (SEQ ID NO: 2), murine transmembrane domain and intracellular domain, etc.
  • the coding sequence of the human CD8 ⁇ signal peptide (SEQ ID NO: 6), the coding sequence of the human CD8 ⁇ hinge region and the transmembrane region (SEQ ID NO: 7), and the coding sequence of the human CD28 transmembrane domain can be selected ( SEQ ID NO: 10), the coding sequence of the human CD28 intracellular domain (SEQ ID NO: 8), and the coding sequence of the human CD3 intracellular domain (SEQ ID NO: 9) were prepared.
  • the amino acid sequence of the chimeric antigen receptor targeting EFGRvIII employed is set forth in any one of SEQ ID NOs: 20, 21, 22.
  • the CAR used may be targeted to other antigens, such as GPC3 (exemplary, the amino acid sequence of the chimeric antigen receptor targeting GPC3 is SEQ ID NO: 11, 12, 13, 14, 15 CLD18A2 (exemplary, the amino acid sequence of the chimeric antigen receptor targeting CLD18A2 is as shown in any of SEQ ID NOs: 16, 17, 18, 19); mesothelin (exemplary, targeted) The amino acid sequence of the chimeric antigen receptor of mesothelin is as shown in any of SEQ ID NOs: 23, 24, 25, and 26).

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Abstract

一种治疗肿瘤的方法，具体为对患有肿瘤的个体施用免疫效应细胞和局部辐射联合进行治疗的方法，并且该方法对所述的个体不进行淋巴细胞清除，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原的受体。该治疗肿瘤的方法对实体瘤具有抗肿瘤效果，在没有淋巴细胞清除预处理的情况下，不清淋CAR-T细胞治疗联合局部放疗可以获得比清淋CAR-T治疗更好的治疗效果，提高抗肿瘤疗效。

Description

免疫效应细胞和辐射联用治疗肿瘤 技术领域
本发明属于免疫治疗领域，具体涉及具有靶向识别肿瘤抗原且引发细胞活化的受体的免疫效应细胞和肿瘤局部辐射治疗联合应用于肿瘤治疗。
背景技术
过继免疫细胞治疗如嵌合抗原受体修饰的T淋巴细胞(CAR-T)治疗已经有望成为肿瘤特别是恶性肿瘤的重要治疗手段，但是所采用的免疫效应细胞仍然面临如何在体内扩增、提高抗肿瘤的活性等问题。
现有文献表明淋巴细胞清除(清淋)预处理能增加免疫效应细胞如CAR-T细胞或TIL的体内扩增以及其抗肿瘤活性，并且近来通过对大规模的CD19-CAR-T细胞治疗血液恶性肿瘤的临床实验数据的分析发现，接受清淋预处理组对CD19-CAR-T细胞治疗的应答为88％，远远高于未接受清淋预处理组的32％(Am J Cancer Res(2016)；6(2):403-424)。在2017年上市的2个针对CD19的CAR-T细胞治疗产品(诺华制药的Kymriah和凯特制药的Yescarta(KTE-C19))的说明书上也均明确注明给予CD19-CAR-T细胞治疗前要先进行去除淋巴细胞处理(包括氟达拉滨和环磷酰胺淋巴细胞的去除淋巴细胞方案)。
然而清淋预处理也有可能影响到过继免疫细胞治疗效果，并且有可能参与形成细胞因子风暴，而且还会产生严重的骨髓抑制等毒性反应，特别是在实体肿瘤治疗中，采用清淋预处理后CAR-T细胞的的疗效并不显著(Zhang et al.,“Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+Metastatic Colorectal Cancers”,Molecular Therapy(2017),25(5):1248-1258)，并且还有报道采用清淋预处理加ERBB2-CAR-T治疗结肠转移癌过程中出现细胞因子风暴，导致患者死亡(Molecular Therapy vol.18no.4,843–851apr.2010)。
因此，能否找到不清淋预处理，并且能明显提高过继免疫细胞疗效的手段无疑是非常有价值的。
发明内容
本发明的目的之一在于提供一种肿瘤治疗方法，该方法对有癌症的个体施用免疫效应细胞和肿瘤局部辐射联合进行治疗，并且对所述的个体不进行淋巴细胞清除，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原且引发所述免疫效应细胞活化的受体。
在本发明的治疗方法中，免疫效应细胞施用和肿瘤局部辐射治疗给予时间不分先后；可以先给予肿瘤局部辐射治疗再给予免疫效应细胞施用；也可以同时给与；还可以先给予免疫效应细胞施用再给予肿瘤局部辐射治疗。本发明中，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。
本发明中，所述肿瘤局部辐射治疗为采用辐射治疗设备对所述肿瘤进行辐射，优选的，所述的辐射治疗设备通过产生下述任一射线对所述肿瘤进行辐射：X射线、α射线、β射线、γ射线、中子。
本发明中，所述辐射治疗设备产生X射线，所述X射线对所述的肿瘤进行至少1次辐射，或多次小剂量辐射。
本发明中，所述辐射治疗的射线剂量为介于不高于100Gy之间，优选地不高于80Gy，更为优选地为不高于70Gy。
本发明中，所述的辐射治疗的能量源位于所述个体的体内或体外。
本发明中，所述的嵌合抗原受体包括：
(i)特异性结合所述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)特异性结合所述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)特异性结合所述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。
本发明中，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；CD30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原 (PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体,血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；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)。
本发明中，所述的免疫效应细胞选自特异识别EGFR、EGFRvIII、GPC3、Claudin18.2的CAR-T细胞。
本发明中，所述的特异性识别肿瘤抗原的抗体具有SEQ ID NO:2所示的氨基酸序列。
本发明中，所述的嵌合抗原受体具有SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或26所示的氨基酸序列。
本发明中，所述的免疫效应细胞施用在所述的肿瘤局部辐射治疗之后当天，或1天、2天、或3天后。
本发明所述的方法中，还包括给所述个体施用免疫检查点(immune checkpoint)抑制剂；优选地，所述免疫检查点抑制剂是生物学治疗剂或小分子；更优选地，所述免疫检查点抑制剂选自单克隆抗体、人源化抗体、全人抗体、融合蛋白或其组合。其中所述免疫检查点抑制剂针对的免疫检查点选自下述的免疫检查点蛋白质：CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK 1、CHK2、A2aR和B-7家族配体或其组合；优选地，所述免疫检查点抑制剂是PD-1或PDL-1抑制剂。优选的，所述免疫检查点抑制剂与选自下述的免疫检查点蛋白质的配体相互作用：CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK 1、CHK2、A2aR和B-7家族配体或其组合。
本发明中，所述免疫效应细胞选自特异识别EGFR、EGFRvIII、GPC3、Claudin18.2的CAR-T细胞，所述免疫检查点抑制剂为特异识别PD-1或PD-L1 的单克隆抗体。
本发明所述的方法中，包括在所述肿瘤局部辐射治疗之前、同时或之后，或在所述免疫效应细胞治疗之前、同时或之后，给予所述个体施用所述免疫检查点抑制剂；优选地，所述免疫效应细胞和所述免疫检查点抑制剂同时施用。
本发明中，所述方法的治疗效应通过免疫效应细胞的存在、或指示T细胞炎症的基因标记的存在或其组合预测，优选地，通过检测IFN-γ水平变化来预测。
本发明中，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，脑胶质瘤，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。
本发明中，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。
另外，本发明的目的之二在于，提供了在不清淋的情况下，将免疫效应细胞与辐射源联合应用治疗癌症的用途。
本发明的另一目的在于，提供一种肿瘤的联合治疗系统，其特征在于，所述联合治疗系统为由对患有肿瘤的个体施用免疫效应细胞和肿瘤局部放射治疗的仪器构成的联合治疗系统，并且对所述的个体不进行淋巴细胞清除，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原的受体。
在具体实施方式中，所述的放射源包括α放射源、β放射源、γ放射源和中子源等。
在具体实施方式中，所述的放射源是射线装置，该射线装置为直线加速器；优选地，所述直线加速器产生X射线和电子线。
本发明的再一个目的在于，提供免疫效应细胞在制备治疗肿瘤的药物中的应用，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原的受体，所述药物与肿瘤局部辐射治疗联合施用，包括同时或先后施用，患有所述肿瘤的个体接受所述免疫效应细胞和/或所述肿瘤局部辐射治疗时体内淋巴细胞数目相对于入组所述肿瘤治疗时不低于40％。
此外，本发明还提供免疫效应细胞在制备一种用于联合治疗肿瘤的方法的药物中的应用，所述方法包括肿瘤的局部辐射治疗与所述药物同时或先后使用，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原的受体，患有所述肿瘤的个体接受所述联合治疗肿瘤的方法时体内淋巴细胞数目相对于入组所述肿瘤治疗时不低于40％。
本发明的技术效果
1、提高对放疗、化疗或放化疗联合治疗不敏感的癌症患者或治疗后复发、以及对CAR-T治疗不敏感或治疗后复发的癌症患者的抗癌效果。
2、避免进行清淋预处理，从而避免了清淋预处理所引起的机体免疫系统破坏、正常组织造成的损伤等副作用。
3、不论肿瘤局部辐射治疗在施用CAR-T细胞之前还是之后都能够提高联合治疗的抗肿瘤活性；在一些实施例中，肿瘤局部辐射治疗联合CAR-T施用能到达使用清淋预处理后的CAR-T细胞的抗肿瘤效果，在一些实施例中，肿瘤局部辐射治疗联合CAR-T施用甚至优于使用清淋预处理后的CAR-T细胞的抗肿瘤效果，体现在显著抑制肿瘤生长、显著延长个体生存期等方面。在一些实施例中，先给予肿瘤局部辐射再施用CAR-T细胞能显著提高联合治疗的抗肿瘤活性，体现在能更显著的抑制肿瘤生长、延长个体生存。在一些实施例中，相对于肿瘤局部辐射治疗数天后(如间隔1天)再施用CAR-T细胞，肿瘤局部辐射治疗后短时间内(如当天)施用CAR-T细胞能显著提高联合治疗的抗肿瘤效果，体现在抑制肿瘤生长、延长个体生存期且肿瘤消退等。在一些实施例中，小剂量、多次的给与肿瘤局部辐射治疗也能够提高与CAR-T细胞联用的抗肿瘤作用，体现在抑制肿瘤生长、延长个体生存期等。在一些实 施例中，肿瘤局部辐射疗法、CAR-T细胞施用和PD-L1抗体联用具有更好的抑制肿瘤的效果。
附图说明
图1A为重组载体MSCV-EGFRvIII-m28Z质粒图；图1B为小鼠清淋模型建立的检测。
图2A显示了未清淋的肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗结肠癌的体内肿瘤生长抑制结果；图2B显示了未清淋的肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗与清淋预处理施用CAR-T细胞治疗的体内肿瘤生长抑制结果比较。
图3显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗结肠癌的体内肿瘤抑制率结果。
图4A显示了未清淋的肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗小鼠结肠癌的生存期结果；图4B显示了未清淋的肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗与清淋预处理后施用CAR-T治疗结肠癌的生存期的比较。
图5显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗小鼠结肠癌的血浆IFN-γ浓度检测。
图6A显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗C57BL/6小鼠原位乳腺癌的体内肿瘤生长抑制实验；图6B显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗C57BL/6小鼠原位乳腺癌的生存期检测。
图7A显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗Balb/c小鼠原位乳腺癌的体内肿瘤生长抑制实验；图7B显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合治疗Balb/c小鼠原位乳腺癌的生存期检测。
图8显示了肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞施用间隔时间对治疗C57BL/6小鼠原位乳腺癌影响：图8A为肿瘤体积检测；图8B为小鼠生存期检测。
图9显示了小剂量、多次给与肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T 细胞施用对治疗Balb/c小鼠原位乳腺癌影响：其中，图9A为肿瘤体积检测；图9B为小鼠生存期检测。
图10显示了局部辐射治疗与EGFRvIII-m28Z CAR-T细胞、免疫检查点抑制剂联合应用对脑胶质瘤皮下移植瘤的抗肿瘤治疗作用：图10A为实验流程图；图10B为肿瘤生长抑制实验。
具体实施方式
本发明涉及具有靶向识别肿瘤抗原且引发细胞活化的受体的免疫效应细胞和局部辐射治疗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，因为这样的方法和条件可以改变。
本公开内容至少部分源于下述开创性认知：连续地、以任一次序地或基本上同时地包括肿瘤局部辐射治疗和免疫效应细胞治疗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中的癌症中可以是更有效的，和/或可以起始、使实现、增加、增强或延长免疫细胞的活性和/或数目，或通过肿瘤的医学有益应答。
术语“免疫效应细胞”，是指参与免疫应答，例如，促进免疫效应的细胞。免疫效应细胞的实例包括T细胞，例如，α/β的T细胞和γ/δT细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。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“局部辐射治疗”或“局部辐射处理”或“局部放射治疗”在本发明中具有相同含义，当射线辐射身体某一部位，引起局部细胞的反应者称局部辐射治疗，包括例如，分次辐射治疗、非分次辐射治疗和超分次辐射治疗。局部辐射治疗还包括采用辐射治疗设备对所述肿瘤进行辐射，在具体实施方案中，所述的辐射治疗设备通过产生下述任一射线对所述肿瘤进行辐射：X射线、α射线、β射线、γ射线、中子。还包括根据所有可利用的技术可完成的、通过电离辐射的所有抗肿瘤治疗：其非限制性列表包括：分次放射治疗、加 速辐射、强度调制的放射疗法、图像引导放射治疗、外线束放射治疗、密封源放疗或近距离放射疗法、非密封源放疗、三维适形放射疗法、质子治疗等。局部辐射治疗还可以采用位于所述个体的体内的能量源进行辐射治疗。
术语“个体”和“受试者”在本文中具有同等含义，可以是人和来自其他种属的动物。
放射线的来源可以位于受试者的外部或内部。当来源位于受试者的外部时，治疗就被称为外部射线束放射治疗(external beam radiation therapy，EBRT)也称为外照射，常使用γ射线、中子、X射线等穿透力强的射线，外照射的生物学效应强。当来源位于受试者的内部时，治疗就被称为近距离放射治疗(brachytherapy，BT)，也成为内照射，其作用主要发生在放射性物质通过途径和沉积部位的组织器官，但其效应可波及全身。内照射的效应以射程短、电离强的α、β射线作用为主。
放射线是根据众所周知的标准技术，使用为此目的制造的标准的设备(即辐射治疗设备)进行施用的。放射线的剂量取决于数目众多的因素，这些都是在本工艺中众所周知的。这类因素包括待治疗的器官、位于放射通道中的可能被不注意地不良地影响的健康器官、患者对放射治疗的耐受性、以及需要治疗的身体区域。剂量典型地为介于不高于100Gy之间，优选的，不高于80Gy，更为优选地为不高于70Gy。所用剂量可以一次性给与，也可以分为小剂量、多次给与患者。应该强调的是，本发明并不局限于任何具体的剂量。剂量将由治疗的医师根据在一个给定的情形中的具体的因素，包括上述的因素来确定。
外部放射源和进入患者的点之间的距离可以是任何在杀死目标细胞和将副作用减到最小之间获得可接受平衡的距离。典型地，外部放射源和进入患者的点之间的距离介于70至100厘米之间。
近距离放射疗法通常是通过在患者中放置一个密封放射源来进行的。典型地，放射源放置于距离待治疗组织大约0～5厘米的地方。已知的方法包括间质近距离放射疗法、腔内近距离放射疗法、表面间质近距离放射疗法。放射源可以是永久性或者临时性植入的。已经被用于永久性种植体中的一些典型的放射性原子包括碘-125和氡。已经被用于临时性种植体中的一些典型的放射性原子包括铯-137和铱-192。
近距离放射疗法的放射剂量可以与上述的在外部射线束放射治疗中所用的剂量相同。除了上述的在确定外部射线束放射治疗剂量时所要考虑的因素之外，在确定近距离放射疗法剂量时还考虑了所使用的放射活性原子的特性。
免疫效应细胞施用是在辐射治疗之前、期间、以及之后施用的，也可以结合施用，即在放射治疗之前和期间、之前和之后、期间和之后、或者在之前、期间及之后施用的。在某些实施方案中，免疫效应细胞治疗在辐射治疗之前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辐射治疗施用之后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“辐射治疗设备”就是发出放射线的装置。根据射线装置对人体健康和环境可能造成危害的程度，从高到低将射线装置分为Ⅰ类、Ⅱ类、Ⅲ类。按照使用用途分医用射线装置和非医用射线装置。射线装置包括：1).加速电荷粒子的装置，例如：回旋加速器、电磁感应加速器以及各种加速装置等。2).发射X射线的装置，例如X射线发生装置、X射线衍射仪、X射线荧光分析仪等。3).盛载放射性物质的装置。
常见的医用射线装置包括：医用加速器，放射治疗用，X射线电子束加速器，重离子治疗加速器，质子治疗装置，制备正电子发射计算机断层显像装置(PET)用放射性药物的加速器，其他医用加速器，X射线深部治疗机，数字减影血管造影装置，医用X射线CT机，放射诊断用普通X射线机，X射线摄影装置，牙科X射线机，乳腺X射线机，放射治疗模拟定位机，其它高于豁免水平的X射线机。
术语“治疗有效量”、“治疗有效的”、“有效量”或“以有效的量”在本文中可互换地使用，并且是指如本文中所述有效地实现特定生物学结果的化合物、制剂、物质或组合物的量，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，将要被施用的本发明的免疫效应细胞、免疫检查点抑制剂、治疗剂的精确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。有效量的局部辐射治疗是指但不限于与免疫效应细胞联用时能促进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分泌；肿瘤消退、肿瘤缩小、肿瘤坏死的辐射剂量或辐射源。有效量的免疫效应细胞是指但不限于与局部辐射联用时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；以及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％。
术语“淋巴细胞清除”或“清淋”即清除受试者在体内的淋巴细胞。包括给予淋巴细胞清除剂、全身辐射治疗或其组合。例如，为了增加肿瘤反应的免疫效应细胞(例如，结合EGFRvIII的CAR分子)的一个或多个细胞初始扩增和后期保持，在给予治疗有效量的CAR-T细胞或全身辐射治疗之前、同时、之后、或任一组合，可以单独或组合给予受试者一种或多种能够大幅度地清除受试者的淋巴细胞的药剂、全身辐射治疗或其组合。可以在足以实现受试者淋巴细胞清除率在60％～100％的条件下，给予清淋治疗。受试者中淋巴细胞的数量减少至少60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、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％或100％，则受试者的淋巴细胞被清除了。
淋巴细胞清除剂是抗肿瘤化学治疗剂。淋巴细胞清除剂的例子包括但不限于氟达拉滨、环磷酰胺或它们的组合。治疗医生根据待治疗的受试者能够选择具体的淋巴细胞清除剂及其适合的剂量，例如CAMPATH、抗CD3抗体、 环孢菌素、FK506、雷帕霉素、霉酚酸、类固醇、FR901228、美法仑、环磷酰胺、氟达拉滨和全身辐射治疗。
免疫效应细胞施用是在清淋治疗之前、期间、以及之后施用的，也可以结合施用，即在清淋治疗之前和期间、之前和之后、期间和之后、或者在之前、期间及之后施用的。在某些实施方案中，清淋治疗在免疫效应细胞治疗之前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，清林治疗在免疫效应细胞治疗施用之后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。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ζ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。本实施例选择EGFRvIII作为CAR-T细胞的靶点，为了更准确的验证小鼠体内的抗肿瘤效果，因此，选择的信号肽、跨膜区、胞内区等为鼠源的。制备的方法按照本领域常规的CAR-T细胞制备方法操作。作为示例性的，上述实施例选择鼠源的CAR，但其信号肽、铰链区、跨膜区等可以根据目的的不同选用其他种属。包括但不限于人的信号肽、铰链区、跨膜区、胞内区。抗体也可以根据不同的目的，选择针对不同靶点的鼠抗或者人源化的抗体或者全人的抗体。
术语“刺激”是指由刺激性分子(例如，TCR/CD3复合体或CAR)与其同源配体(或在CAR的情况下为肿瘤抗原)的结合，由此介导信号转导事件(比如但不限于经由TCR/CD3复合体的信号转导或经由适合的NK受体或CAR的信号传导结构域的信号转导)而诱导的初次应答。刺激可以介导某些分子的改变的表达。
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分子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：CD3ζ、常见的FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。在本发明的特异性CAR中，在本发明的任一个或更多个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-ζ的初级信号传导序列。在本发明的特异性CAR中，CD3-ζ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 子为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。
术语“胞内信号传导结构域”是指分子的细胞内部分。胞内信号传导结构域产生促进含有CAR的细胞例如CAR-T细胞的免疫效应子功能的信号。在例如CAR-T细胞中免疫效应子功能的实例包括细胞裂解活性和辅助活性，包括细胞因子的分泌。在一种实施方式中，胞内信号传导结构域可以包括一级胞内信号传导结构域。示例性的一级胞内信号传导结构域包括来源于负责初次刺激或抗原依赖性刺激的分子的那些。在一种实施方式中，胞内信号传导结构域可以包括共刺激细胞内结构域。
一级胞内信号传导结构域可以包括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。含有ITAM的一级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：CD3ζ、常见FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。
术语“T细胞(抗原)受体(T cell receptor，TCR)”，为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。TCR负责识别与主要组织相容性复合体分子结合的抗原。TCR是由两条不同肽链构成的异二聚体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等几部分；其特点是胞质区很短。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区(Vα、Vβ)又各有三个高 变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC- 抗原肽复合体时，CDR1，CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。
术语“T细胞融合蛋白(T cell fusion protein，TFP)”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原,和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。TFP由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该TFP整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1肿瘤靶向结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
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抗体片段、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、单域抗体比如sdAb(VL或VH)、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。抗原结合片段也可以被掺入单域抗体、最大抗体、微小抗体、纳米抗体、胞内抗体、双抗体、三抗体、四抗体、v-NAR和双-scFv(参见，例如Hollinger和Hudson，《Nature Biotechnology》(23)：1126-1136，2005)。
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。本领域技术人员应当理解包括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，因此编码“抗原”。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。显而易见的是，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。而且，本领域技术人员应当理解抗原根本无需由“基因”编码。显而易见的是，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。
“肿瘤抗原”指的是特定过度增生性疾病共同的抗原。在某些方面，本发明的过度增生性病症抗原源自癌症。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；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体,血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经 细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；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“癌症”指特征在于在体外(例如经转化的细胞)或体内的过度增殖性细胞生长的广泛病症类别。可以通过本发明的方法治疗或预防的病况包括例如各种赘生物，包括良性或恶性肿瘤，各种增生等等。本发明的方法可以实现这样的病况中所牵涉的不希望有的过度增殖性细胞生长的抑制和/或逆转。癌症的具体例子包括但不限于：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，脑胶质瘤，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。
术语“转染的”或“转化的”或“转导的”是指外源性核酸通过其转移或引入到宿主细胞中的过程。“转染的”或“转化的”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”是指识别并且结合存在于样品中的结合配偶体(例如肿瘤抗原)蛋白质的抗体或配体，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“生物等同的”是指要产生与参考剂量或参考量的参考化合物、免疫效应细胞、辐照产生的效果等同的效果所需要的不同于参考化合物、免疫效应细胞、辐照的作用剂的量。
这里使用的“难治”指的是一种疾病，例如，癌症，其不应答治疗。在实施方案中，难治性癌症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，难治性癌症可以成为治疗期间抗性的。难治性癌症也称为抗 性癌症。在本发明中，难治性癌症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癌症。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。
术语“治疗剂”，指在受试者中产生治疗应答的任何医学产品。包括但不限于免疫刺激试剂、T细胞生长因子、白介素、抗体和疫苗、化学治疗剂或其组合。
免疫刺激试剂是通过诱导免疫系统组分中任一种的活化或增加其组分中任一种的活性来刺激免疫系统的物质(药物和营养素)。免疫刺激剂包括细菌疫苗、集落刺激因子、干扰素、白介素、其它免疫刺激剂、治疗疫苗、疫苗组合和病毒疫苗。
T细胞生长因子是刺激T细胞增殖的蛋白质。T细胞生长因子的例子包括Il-2、IL-7、IL-15、IL-17、IL21和IL-33。
白介素是首先被视为由白血细胞表达的一组细胞因子。免疫系统的功能在很大程度上取决于白介素，并且许多白介素的罕见缺陷已得到描述，全部表征自身免疫疾病或免疫缺陷。白介素中的大多数通过辅助CD4T淋巴细胞，以及通过单核细胞、巨噬细胞和内皮细胞合成。它们促进T和B淋巴细胞以及造血细胞的发育和分化。白介素的例子包括IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-14、IL-15和IL-17。
化学治疗剂包括烷化剂例如噻替派和环磷酰胺(CYTOXAN)；烷基磺酸盐类例如白消安、英丙舒凡和哌泊舒凡；氮丙啶类例如苯佐替哌(benzodopa)、卡波醌、美妥替哌(meturedopa)和乌瑞替哌(uredopa)；乙撑亚胺类和甲基蜜胺类(methylamelamines)，包括六甲蜜胺、三乙撑蜜胺、三乙撑磷酰胺、三乙撑硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)；氮芥类例如苯丁酸氮芥、萘氮芥、cholophosphamide、磷雌氮芥、异环磷酰胺、氮芥、盐酸氧氮芥、美法仑、新氮芥、苯芥胆甾醇、松龙苯芥、曲磷胺、尿嘧啶氮芥；硝基脲类(nitrosureas)例如卡莫司汀、氯脲菌素、福莫司汀、洛莫司汀、尼莫司汀、雷莫司汀；抗生素例如阿克拉霉素(aclacinomysins)、放射菌素、authramycin、重氮丝氨酸、博来霉素、放线菌素C、卡奇霉素、carabicin、洋红霉素 (caminomycin)、嗜癌霉素、色霉素、放线菌素D、柔红霉素、地托比星、6-重氮基-5-氧代-L-正亮氨酸、阿霉素、表柔比星、依索比星、伊达比星、麻西罗霉素、丝裂霉素、霉酚酸、诺加霉素、橄榄霉素、派来霉素、potfiromycin、嘌呤霉素、三铁阿霉素、罗多比星、链黑霉素、链唑霉素、杀结核菌素、乌苯美司、净司他丁、佐柔比星；抗代谢物例如甲氨蝶呤和5-氟尿嘧啶(5-FU)；叶酸类似物例如二甲叶酸、甲氨蝶呤、蝶罗呤、三甲曲沙；嘌呤类似物例如氟达拉滨、6-巯基嘌呤、硫唑鸟嘌呤、硫鸟嘌呤；嘧啶类似物例如安西他滨、氮杂胞苷、6-氮杂尿苷、卡莫氟、阿糖胞苷、双脱氧尿苷、去氧氟尿苷、依诺他宾、氟尿苷、5-FU；雄激素类例如卡鲁睾酮、屈他雄酮丙酸酯、环硫雄醇、美雄烷、睾内酯；抗肾上腺药(anti-adrenal)例如氨鲁米特、米托坦、曲洛司坦；叶酸补充剂例如亚叶酸(frolinic acid)；醋葡醛内酯；醛磷酰胺糖苷；氨基乙酰丙酸；安吖啶；bestrabucil；比生群；依达曲沙；磷胺氮芥(defofamine)；秋水仙胺；地吖醌；依氟鸟氨酸(elformithine)；依利醋铵；依托格鲁；硝酸镓；羟基脲；香菇多糖；氯尼达明；米托胍腙；米托蒽醌；莫哌达醇；硝氨丙吖啶；喷司他丁；蛋氨氮芥；吡柔比星；鬼臼酸；2－乙基肼；甲基苄肼；PSK.RTM.；丙亚胺；西佐喃；锗螺胺；细交链孢菌酮酸；三亚胺醌；2,2',2"-三氯三乙胺；乌拉坦；长春酰胺；氮烯咪胺；甘露醇氮芥；二溴甘露醇；二溴卫矛醇；哌泊溴烷；gacytosine；阿拉伯糖苷(“Ara-C”)；环磷酰胺；噻替派；紫杉烷类，例如紫杉醇(TAXOLTM，Bristol-Myers Squibb Oncology，Princeton，N.J.)和多西他赛(TAXOTERETM，Rhne-Poulenc Rorer，Antony，法国)；苯丁酸氮芥；吉西他滨；6-硫鸟嘌呤；巯基嘌呤；甲氨蝶呤；铂类似物例如顺铂和卡铂；长春碱；曲妥珠单抗、多西他赛、铂；依托泊苷(VP-16)；异环磷酰胺；丝裂霉素C；米托蒽醌；长春新碱；长春瑞滨；诺维本；诺消灵；替尼泊苷；道诺霉素；氨喋呤；希罗达；伊班膦酸盐；CPT-11；拓扑异构酶抑制剂RFS 2000；二氟甲基鸟氨酸(DMFO)；视黄酸衍生物例如Targretin TM(贝沙罗汀)，PanretinTM(阿利维A酸)；ONTAKTTM(地尼白介素2)；埃斯波霉素；卡培他滨；以及上述任何一种的药物学上可接受的盐、酸或衍生物。这个定义中还包括抗激素剂，所述抗激素剂用于调节或抑制激素对肿瘤的作用，例如抗雌激素剂包括例如他莫昔芬、雷洛昔芬、芳香酶抑制性4(5)-咪唑、4-羟基他莫昔芬、曲沃昔芬、那洛昔芬(keoxifene)、LY117018、奥那司酮和托瑞米芬 (Fareston)；以及抗雄激素剂例如氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林和戈舍瑞林；以及上述任何一种的药物学上可接受的盐、酸或衍生物。进一步的癌症治疗剂包括索拉非尼以及其它蛋白质激酶抑制剂例如阿法替尼、阿西替尼、贝伐珠单抗、西妥昔单抗、克唑替尼、达沙替尼、厄洛替尼、福他替尼、吉非替尼、伊马替尼、拉帕替尼、乐伐替尼、莫立替尼、尼洛替尼、帕尼单抗、帕唑帕尼、哌加他尼、兰尼单抗、鲁索利替尼、曲妥珠单抗、凡德他尼、维罗非尼、和舒尼替尼；西罗莫司(雷帕霉素)、依维莫司以及其它mTOR抑制剂。
另外的化学治疗剂的例子包括拓扑异构酶I抑制剂(例如伊立替康、托泊替康、喜树碱及其类似物或代谢产物、和多柔比星)；拓扑异构酶II抑制剂(例如依托泊苷、替尼泊苷和柔红霉素)；烷化剂(例如美法仑、苯丁酸氮芥、白消安、噻替派、异环磷酰胺、卡莫司汀、洛莫司汀、司莫司汀、链唑霉素、氮烯咪胺、氨甲蝶呤、丝裂霉素C和环磷酰胺)；DNA嵌入剂(例如顺铂、奥沙利铂和卡铂)；DNA嵌入剂和自由基生成剂例如博来霉素；和核苷模拟物(例如5-氟尿嘧啶、卡培他滨、吉西他滨、氟达拉滨、阿糖胞苷、巯嘌呤、硫鸟嘌呤、喷司他丁和羟基脲)。此外，破坏细胞复制的示例性化学治疗剂包括：紫杉醇、多西他赛和相关类似物；长春新碱、长春碱和相关类似物；沙利度胺、来那度胺和相关类似物(例如CC-5013和CC-4047)；蛋白酪氨酸激酶抑制剂(例如甲磺酸伊马替尼和吉非替尼)；蛋白酶体抑制剂(例如硼替佐米)；NF-κB抑制剂包括IκB激酶的抑制剂；结合在癌症中过表达的蛋白质的抗体，以及已知在癌症中上调、过表达或活化的蛋白质或酶的其它抑制剂，所述蛋白质或酶的抑制下调细胞复制。
局部辐射和免疫效应细胞治疗联合检查点抑制剂和治疗剂治疗，在治疗一些受试者中的癌症中可以是更有效的，和/或可以起始、实现、增加、增强或延长免疫细胞(包括T细胞、B细胞、NK细胞和/或其它)的活性和/或数目，或促进IFN-γ分泌，或传达通过肿瘤的医学有益应答(包括其消退、坏死或消除)。
在本发明的某些实施方案中，免疫效应细胞与检查点抑制剂同时给药，或免疫效应细胞治疗在检查点抑制剂前或后按时间顺序施用。在某些实施方案中，免疫效应细胞治疗在检查点抑制剂施用之前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检查点抑制剂施用之后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。在某些实施方案中，治疗剂在免疫效应细胞治疗施用之前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，治疗剂在免疫效应细胞治疗施用之后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“生物学治疗剂”或“生物药剂”指在生物源中制造或由生物源提取的任何医学产品。生物药剂不同于化学合成的药物产品。生物药剂的例子包括疫苗、血液或血液成分、过敏原、体细胞、基因疗法、组织、重组治疗性蛋白质、包括抗体治疗剂和融合蛋白、以及活细胞。生物制品可以由糖、蛋白质或核酸或这些物质的复杂组合组成，或可以是活实体例如细胞和组织。生物制品从各种天然来源(人、动物或微生物)中分离，并且可以通过生物技术方法和其它技术产生。生物学治疗剂的具体例子包括但不限于免疫刺激试剂、T细胞生长因子、白介素、抗体、融合蛋白和疫苗例如癌症疫苗。
术语“治疗”是指由于施用一种或多种疗法(例如一种或多种治疗剂比如本发明的CAR-T、局部辐射治疗)减慢或改善增生性病症的进展、严重程度和/或持续时间，或改善增生性病症的一种或多种症状(优选地，一种或多种可辨 别的症状)。在特定实施方式中，术语“治疗”是指改善增生性病症的至少一种可测量的物理参数比如肿瘤生长，不必是患者可辨别的。在其它实施方案中，术语“治疗”是指通过例如稳定可辨别的症状以物理方式、通过例如稳定物理参数以生理学方式或两者抑制增生性病症的进展。在其它实施方案中，术语“治疗”是指减小或稳定肿瘤尺寸、癌细胞计数或延长个体的生存期。
术语“改善存活”指患有癌症或增殖性疾病的受试者的寿命或生活质量的增加。例如，改善存活还包括促进癌症缓解，预防肿瘤侵入，预防肿瘤复发，减缓肿瘤生长，预防肿瘤生长，减小肿瘤尺寸，并减少总癌细胞计数。
术语“治疗癌症”不意味着是绝对术语。在一些方面，本发明的方法寻求减少肿瘤尺寸或癌细胞数目，促使癌症进入缓解，或阻止癌细胞的尺寸或细胞数目的生长。在一些情况下，治疗导致改善的预后。
术语“自体的”是指来源于个体的任何物质，所述物质随后再被引入到的相同个体。
术语“同种异体的”是指来源于与将导入物质的个体相同物种的不同动物的任何物质。当一个或更多个基因座的基因不相同时，两个或多个个体被认为是彼此同种异体的。在某些方面，来自相同物种的个体的同种异体物质可以在遗传上是足够不同的而以在抗原性上相互作用。
术语“受试者”指已诊断有癌症或细胞增殖性病症的个体或受试者。
术语“肿瘤应答”指细胞应答包括但不限于触发程序性细胞死亡。
术语“抗肿瘤应答”指免疫系统应答，包括但不限于活化T细胞以攻击抗原或抗原呈递细胞。
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。
术语“小分子”指低分子量(<900道尔顿)有机化合物，其可以帮助调节生 物过程，具有在10 -9m级别上的大小。大多数药物是小分子。
免疫检查点调节免疫系统中的T细胞功能。T细胞在细胞介导的免疫中起关键作用。检查点蛋白质与特异性配体相互作用，所述特异性配体将信号发送到T细胞内，并且基本上关闭或抑制T细胞功能。癌细胞通过在其表面上驱动高水平的检查点蛋白质表达来利用这种系统，其导致在进入肿瘤微环境的T细胞的表面上表达检查点蛋白质的T细胞的控制，因此压制抗癌免疫应答。像这样，检查点蛋白质的抑制将导致T细胞功能的恢复和针对癌细胞的免疫应答。检查点蛋白质的例子包括但不限于CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4(属于CD2分子家族，并且在所有NK、γδ和记忆CD8+(αβ)T细胞上表达)、CD160(也称为BY55)、CGEN-15049、CHK 1和CHK2激酶、A2aR和各种B-7家族配体。
术语“PD-1抗体”指通过使PD-1激动来拮抗淋巴细胞的活性和/或增殖的抗体。术语“拮抗活性”指淋巴细胞增殖或活性中至少约10％、20％、30％、40％、50％、60％、70％、80％、90％或更多的下降(或减少)。术语“拮抗”可以与术语“抑制性”和“抑制”互换使用。PD-1介导的活性可以使用如本文描述的T细胞增殖测定法进行定量测定。在一个实施方案中，抗PD-1抗体可以是新型抗原结合片段。本文公开的抗PD-1抗体能够结合包括人PD-1且拮抗PD-1，从而抑制表达PD-1的免疫细胞的功能。在一些实施方案中，免疫细胞是表达PD-1的活化淋巴细胞，例如T细胞、B细胞和/或单核细胞。
程序性细胞死亡蛋白质1(PD-1)是288个氨基酸的细胞表面蛋白质分子，在T细胞和前B细胞上表达，并且在其命运/分化中起作用。PD-1具有两种配体，PD-L1和PD-L2，其为B7家族的成员。PD-L1蛋白质响应LPS和GM-CSF处理在巨噬细胞和树突状细胞(DC)上是上调的，并且在TCR和B细胞受体信号传导后在T细胞和B细胞上是上调的，而在静止小鼠中，PDL1mRNA可以在心脏、肺、胸腺、脾和肾中检测到。PD-1负面调节T细胞应答。
存在目前在临床试验中测试的几种PD-1抑制剂：CT-011、BMS 936558、BMS 936559、MK 3475、MPDL 3280A、AMP 224、Medi 4736。
CTLA4(细胞毒性T淋巴细胞相关蛋白质)是下调免疫系统的蛋白质受体。CTLA4在T细胞的表面上发现，其导致对抗原的细胞免疫攻击。T细胞攻击可以通过刺激T细胞上的CD28受体而开启。T细胞攻击可以通过刺激CTLA4受体 而关闭。第一类免疫疗法伊匹木单抗(Yervoy)(靶向T细胞表面上的CTLA-4的单克隆抗体)被批准用于黑素瘤的治疗。
在一个方面，检查点抑制剂是生物学治疗剂或小分子。在另一个方面，检查点抑制剂是单克隆抗体、人源化抗体、全人抗体、融合蛋白或其组合。在一个进一步方面，检查点抑制剂抑制检查点蛋白质，其可以是CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK 1、CHK2、A2aR、B-7家族配体或其组合。在一个另外方面，检查点抑制剂与可以是下述的检查点蛋白质的配体相互作用：CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK 1、CHK2、A2aR、B-7家族配体或其组合。在一个方面，治疗剂是免疫刺激试剂、T细胞生长因子、白介素、抗体、疫苗、化疗药物或其组合。
在一个进一步方面，在局部辐射治疗之前、同时或之后施用免疫检查点抑制剂，或在免疫效应细胞治疗之前、同时或之后施用免疫检查点抑制剂。
检查点抑制剂包括以统计上显著的方式阻断或抑制免疫系统的抑制途径的任何试剂。这样的抑制剂可以包括小分子抑制剂，或可以包括结合且阻断或抑制免疫检查点受体的抗体或其抗原结合片段，或者结合且阻断或抑制免疫检查点受体配体的抗体。可以被靶向用于阻断或抑制的举例说明性检查点分子包括但不限于CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、GAL9、LAG3、TIM3、VISTA、KIR、2B4(属于CD2分子家族，并且在所有NK、γδ和记忆CD8+(αβ)T细胞上表达)、CD160(也称为BY55)、CGEN-15049、CHK 1和CHK2激酶、A2aR和各种B-7家族配体。B7家族配体包括但不限于B7-1、B7-2、B7-DC、B7-H1、B7-H2、B7-H3、B7-H4、B7-H5、B7-H6和B7-H7。检查点抑制剂包括抗体或其抗原结合片段、其它结合蛋白、生物学治疗剂或小分子，其结合且阻断或抑制下述中的一种或多种的活性：CTLA-4、PDL1、PDL2、PD1、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160和CGEN-15049。举例说明性免疫检查点抑制剂包括曲美木单抗(CTLA-4阻断抗体)、抗OX40、PD-L1单克隆抗体(抗B7-H1；MEDI4736)、MK-3475(PD-1阻断剂)、Nivolumab(抗PD1抗体)、CT-011(抗PD1抗体)、BY55单克隆抗体、AMP224(抗PDL1抗体)、BMS-936559(抗PDL1抗体)、 MPLDL3280A(抗PDL1抗体)、MSB0010718C(抗PDL1抗体)和Yervoy/伊匹木单抗(抗CTLA-4检查点抑制剂)。检查点蛋白质配体包括但不限于PD-L1、PD-L2、B7-H3、B7-H4、CD28、CD86和TIM-3。
用于本发明的方法中的合适抗CTLA-4拮抗试剂包括但不限于抗CTLA4抗体、人抗CTLA4抗体、小鼠抗CTLA4抗体、哺乳动物抗CTLA4抗体、人源化抗CTLA4抗体、单克隆抗CTLA4抗体、多克隆抗CTLA4抗体、嵌合抗CTLA4抗体、MDX-010(伊匹木单抗)、曲美木单抗、抗CD28抗体、抗CTLA4adnectin、抗CTLA4结构域抗体、单链抗CTLA4片段、重链抗CTLA4片段、轻链抗CTLA4片段、激动共刺激途径的CTLA4的抑制剂、在PCT公开号WO2001/014424公开的抗体、在PCT公开号WO2004/035607中公开的抗体、在美国公开号2005/0201994中公开的抗体、以及在授权的欧洲专利号EP 1212422B1中公开的抗体。另外的CTLA-4抗体在美国专利号5,811,097、5,855,887、6,051,227和6,984,720；PCT公开号WO01/14424和WO 00/37504；以及美国公开号2002/0039581和2002/086014中描述。可以用于本发明的方法中的其它抗CTLA-4抗体包括例如公开于下述中的那些：WO98/42752；美国专利号6,682,736和6,207,156；Hurwitz等人，Proc.Natl.Acad.Sci.USA，95(17):10067-10071(1998)；Camacho等人，J.Clin.Oncology，22(145):Abstract No.2505(2004)(抗体CP-675206)；Mokyr等人，Cancer Res.，58:5301-5304(1998)，以及美国专利号5,977,318、6,682,736、7,109,003和7,132,281。
另外的抗CTLA4拮抗剂包括但不限于下述：能够破坏CD28抗原结合其同源配体的能力，抑制CTLA4结合其同源配体的能力，经由共刺激途径加强T细胞应答，破坏B7结合CD28和/或CTLA4的能力，破坏B7活化共刺激途径的能力，破坏CD80结合CD28和/或CTLA4的能力，破坏CD80活化共刺激途径的能力，破坏CD86结合CD28和/或CTLA4的能力，破坏CD86活化共刺激途径的能力，以及破坏一般而言共刺激途径的活化的任何抑制剂。这种必要性包括共刺激途径的CD28、CD80、CD86、CTLA4以及其它成员的小分子抑制剂；针对共刺激途径的CD28、CD80、CD86、CTLA4以及其它成员的抗体；针对共刺激途径的CD28、CD80、CD86、CTLA4以及其它成员的反义分子；针对共刺激途径的CD28、CD80、CD86、CTLA4以及其它成员的adnectin，共刺激 途径的CD28、CD80、CD86、CTLA4以及其它成员的RNAi抑制剂(单链和双链两者)，以及其它抗CTLA4拮抗剂。
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分泌,或其组合预测。
在一个方面，本文描述的方法用于治疗癌症。具体地，本文描述的方法可以用于减小实体瘤的尺寸，或减少癌症的癌细胞数目。本文描述的方法可以用于减慢癌细胞生长的速率。本文描述的方法可以用于停止癌细胞生长的速率。
如本文公开的免疫效应细胞、治疗剂、检查点抑制剂、生物学治疗剂或药物组合物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。治疗剂、检查点抑制剂、生物学治疗剂或药物组合物还可以施用于病理状况的部位，例如静脉内或动脉内进入供应肿瘤的血管内。
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)，并且功能等价的方法和组分在本发明的范围内。事实上，本发明的各种修饰加上本文显示且描述的那些，根据前述描述对于本领域技术人员将变得明显。
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、WO2018018958A1中公开的那些。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，《分子克隆实验指南(第三版)》(科学出版社，2002)中所述的条件，或按照制造厂商所建议的条件。
实施例1 CAR-T细胞的构建和小鼠清淋模型的建立
(1)CAR-T细胞的构建
作为示例性的，本实施例采用靶向EGFRvIII的二代CAR，为了动物实验的需要，本实施例采用鼠的基因序列构建CAR的跨膜域和胞内域。
将小鼠CD8α信号肽的编码序列(SEQ ID NO：1)，即能识别人活化的EGFR、也能识别人EGFRvIII的单抗抗体的编码序列(SEQ ID NO：2)，鼠CD8α铰链区及跨膜区的编码序列(SEQ ID NO：3)，鼠CD28胞内域的编码序列(SEQ ID NO：4)，鼠CD3ζ胞内域的编码序列(SEQ ID NO：5)依次连接，通过体 外基因合成的方法获得EGFRvIII-m28Z基因片段，并用MluⅠ和SalⅠ双酶切位点置换逆转录病毒载体MSCV-IRES-GFP(购自Addgene)中的IRES-GFP片段，获得重组载体MSCV-EGFRvIII-m28Z(或者命名为MSCV-EGFRvIII-mCD28Z)(图1A)。
用重组载体MSCV-EGFRvIII-m28Z感染293T细胞，得到包装后的逆转录病毒。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。
取Balb/c小鼠的脾脏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(Thermo Fisher)，PBS清洗一次，活化，放培养箱培养，培养基为RPMI 1640完全培养基。
将活化24h的小鼠脾脏T淋巴细胞接种于retronectin(Takara T100A)包被的12孔板中，加入逆转录病毒感染过夜，得到小鼠的EGFRvIII-m28Z CAR-T细胞。
(2)小鼠清淋模型的建立
Balb/c小鼠是免疫系统正常的小鼠。给予Balb/c小鼠5Gyγ射线全身辐照，辐照后第14天，小鼠下颌采血50μl至抗凝管，加入PerCP Cy5.5标记的抗小鼠CD3的流式抗体，室温孵育1小时，加入450μl红细胞裂解液，十分钟后，BD流式分析仪检测CD3阳性率。
实验结果：未处理的对照小鼠CD3阳性细胞比例为31.6％，经过辐照后的小鼠CD3阳性细胞比例为5.7％。与未处理的对照小鼠相比较，经过辐照后的小鼠CD3阳性细胞比例明显降低，约82％(P<0.05，t-test)(图1B)。说明5Gyγ射线能有效清除绝大部分CD3阳性细胞。
实施例2肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞联合应用对结肠癌皮下移植瘤的抗肿瘤治疗作用
由于EGFR287-302表位仅在EGFRvIII或过表达EGFR的肿瘤中才暴露，而在正常组织中该表位隐匿(Gan HK et al.,Targeting of a conformationally exposed,tumor-specific epitope of EGFR as a strategy for cancer therapy.Cancer Res,2012,72(12):2924-2930.)。所以利用分子生物学常规手段建立过表达嵌合 有人EGFR第287-302位氨基酸表位的去掉鼠EGFR第2-7号外显子的小鼠EGFR的CT26细胞模型(CT26-EGFRvIII)。CT26细胞购自美国模式培养物保藏所(ATCC CRL-2638)。
(1)小鼠结肠癌模型的建立及分组治疗：
首先将小鼠分为两组：不清淋组及清淋组。
不清淋组：6周龄的雌性Balb/c小鼠(购自上海西普尔-必凯实验动物有限公司)皮下接种3×10 5CT26-EGFRvIII细胞，接种当天记为Day0，肿瘤接种后第13天(Day13),肿瘤体积为150～250mm 3,分为5组(每组6只)，分别为：
UT(未处理)细胞组(UT)：Day13尾静脉输注5×10 6未处理的小鼠T细胞；
2个单独治疗组：(1)肿瘤局部辐射治疗组(X-ray)：Day13肿瘤局部接受10Gy X-ray辐射；(2)EGFRvIII-m28Z CAR-T细胞组(EGFRvIII-m28Z):Day13尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞；
2个联合组：(1)EGFRvIII-m28Z CAR-T+肿瘤局部辐射治疗组(EGFRvIII-m28Z+X-ray):Day13尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞，间隔1天即Day15肿瘤局部接受10Gy X-ray辐射；(2)肿瘤局部辐射治疗+EGFRvIII-m28Z CAR-T组(X-ray+EGFRvIII-m28Z):Day13肿瘤局部接受10Gy X-ray辐射，间隔1天Day15尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞。
清淋组：6周龄的雌性Balb/c小鼠采用5Gyγ射线全身辐照后，当天皮下接种3×10 5CT26-EGFRvIII细胞,接种当天记为Day0；肿瘤接种后第13天(Day13),肿瘤体积为150～250mm 3，将清淋组荷瘤小鼠再各分为3组(每组6只)分别为：
UT(未处理)细胞组(UT(Lymphodeletion)):Day13尾静脉输注5×10 6未处理的小鼠T细胞，
肿瘤局部辐射治疗组(X-ray(Lymphodeletion)):Day13肿瘤局部接受10Gy X-ray辐射，
EGFRvIII-m28Z CAR-T细胞组(EGFRvIII-m28Z(Lymphodeletion)):Day13尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞。
(2)观察记录肿瘤大小，参照对照UT组计算肿瘤抑制率。持续观察并测量小鼠肿瘤体积变化。瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2。 开始治疗后第8天(Day21)取小鼠血浆检查IFN-γ的浓度。
肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T联合在小鼠结肠癌模型中的抗肿瘤作用如图2-4所示。
由图2A可知，未清淋小鼠中，X-ray与CAR-T联合组(EGFRvIII-m28Z+X-ray、X-ray+EGFRvIII-m28Z)肿瘤体积明显小于单CAR-T细胞治疗组(EGFRvIII-m28Z)的肿瘤体积(P<0.05,Two way ANOVA)。由图2B可知，未清淋小鼠的X-ray与CAR-T联合组(EGFRvIII-m28Z+X-ray、X-ray+EGFRvIII-m28Z)肿瘤体积与清淋预处理后施用CAR-T细胞治疗组(EGFRvIII-m28Z(Lymphodeletion))的肿瘤体积无明显差异。上述结果说明CAR-T细胞治疗联合肿瘤局部辐射治疗能达到清淋预处理后施用CAR-T细胞治疗组的抗肿瘤效果。
由图3可知，在肿瘤接种后的第23天(Day23)，未清淋小鼠CAR-T细胞治疗组(EGFRvIII-m28Z)的肿瘤抑制率为35.98％±18.15％，X-ray+EGFRvIII-m28Z联合组肿瘤抑制率为92.44％±2.24％，EGFRvIII-m28Z+X-ray联合组肿瘤抑制率为75.06％±13.06％，而清淋预处理后施用CAR-T细胞治疗组(EGFRvIII-m28Z(Lymphodeletion))的肿瘤抑制率为77.85％±17.92％。未清淋小鼠联合组(EGFRvIII-m28Z+X-ray、X-ray+EGFRvIII-m28Z)肿瘤抑制率高于未清淋小鼠CAR-T细胞治疗组(EGFRvIII-m28Z)的肿瘤抑制率(P<0.001,One way ANOVA)，未清淋小鼠联合组(EGFRvIII-m28Z+X-ray、X-ray+EGFRvIII-m28Z)肿瘤抑制率与清淋预处理后施用CAR-T细胞治疗组(EGFRvIII-m28Z(Lymphodeletion))抑制率无明显差别。上述结果说明CAR-T细胞治疗联合肿瘤局部辐射治疗效果能达到清淋预处理后施用CAR-T细胞治疗组的抗肿瘤效果。
由图4A可知，在未清淋治疗组中，联合治疗组中X-ray+EGFRvIII-m28Z组生存期最长(P<0.05,Log-rank test)，显著高于EGFRvIII-m28Z CAR-T组、X-ray组。图4B中未清淋小鼠中X-ray+EGFRvIII-m28Z组小鼠生存期相对于未清淋EGFRvIII-m28Z+X-ray组以及清淋预处理后施用CAR-T细胞治疗组(EGFRvIII-m28Z(Lymphodeletion))更长(P<0.05,Log-rank test)。
到治疗后的44天为止，未清淋小鼠中X-ray+EGFRvIII-m28Z组6只小鼠中有4只小鼠肿瘤消失。未清淋小鼠中EGFRvIII-m28Z+X-ray的6只小鼠中也有1 只小鼠肿瘤消失，而清淋预处理后施用CAR-T细胞治疗组中没有小鼠肿瘤消失。
上述结果说明清淋预处理并非提高CAR-T细胞治疗效果所必需。CAR-T细胞治疗联合肿瘤局部辐射治疗效果能达到清淋预处理后施用CAR-T细胞治疗组的抗肿瘤效果，如对肿瘤生长的抑制没有统计学差异，并且肿瘤局部辐射与CAR-T细胞施用的先后顺序不影响联合治疗的抑制肿瘤生长作用。但是在对荷瘤小鼠的生存期的影响，CAR-T细胞治疗联合肿瘤局部辐射治疗明显优于清淋预处理后施用CAR-T细胞治疗组，其中以X-ray+EGFRvIII-m28Z组对生存期的影响尤为显著。
实施例3肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T联合治疗小鼠结肠癌模型中的IFN-γ浓度检测
取实施例2中的开始治疗后第8天(Day21)的小鼠血浆50μl，用联科生物公司的ELISA试剂盒测定血液中的干扰素γ的水平，结果如图5所示，未清淋小鼠联合组中EGFRvIII-m28Z+X-ray组与清淋预处理后施用CAR-T治疗组相比没有显著性差异，而X-ray+EGFRvIII-m28Z组干扰素γ的浓度最高，高于清淋预处理后施用CAR-T治疗组(P<0.05,One way ANOVA)。上述结果说明CAR-T细胞治疗联合肿瘤局部辐射治疗能促进干扰素γ的分泌，其促进水平能达到清淋预处理后施用CAR-T细胞治疗组的水平，其中X-ray+EGFRvIII-m28Z组干扰素γ的水平显著高于清淋预处理后施用CAR-T细胞治疗组的水平。上述说明清淋预处理并非提高CAR-T细胞治疗效果所必需。不清淋情况下施用CAR-T细胞联合肿瘤局部辐射治疗可以获得和清淋预处理后施用CAR-T治疗组一样的抗肿瘤效果，并且先进行肿瘤局部辐射再施用CAR-T细胞，即X-ray+EGFRvIII-m28Z还能显著高于清淋预处理后CAR-T细胞治疗组的抗肿瘤治疗效果。
实施例4肿瘤局部辐射治疗和EGFRvIII-m28Z CAR-T细胞联合应用对小鼠原位乳腺癌的抗肿瘤治疗作用
利用分子生物学常规手段建立过表达嵌合有人EGFR第287-302位氨基酸表位的小鼠EGFR的小鼠乳腺癌细胞E0771细胞模型(E0771-EGFR)。小鼠乳腺癌细胞E0771由美国贝勒医学院馈赠。
(1)C57BL/6小鼠原位乳腺癌模型的建立及分组：
取6-8周龄C57BL/6小鼠(购自上海西普尔-必凯实验动物有限公司)，将1×10 6E0771-EGFR细胞接种于小鼠右侧第四乳腺脂肪垫内构建小鼠原位乳腺癌模型,接种当日记为Day0。接种后第14天(Day14),肿瘤长至200～300mm 3()，将荷瘤小鼠分为5组(每组6只)：
UT(未处理)细胞组(UT):Day14尾静脉输注5×10 6未处理的小鼠T细胞；
2个单独治疗组：(1)肿瘤局部辐射治疗组(X-ray):Day14肿瘤局部接受10Gy X-ray辐射；
(2)EGFRvIII-m28Z CAR-T细胞组(EGFRvIII-m28Z):Day14尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞；
2个联合组：(1)EGFRvIII-m28Z CAR-T+肿瘤局部辐射治疗组(EGFRvIII-m28Z+X-ray):Day14尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞，间隔1天即Day16肿瘤局部接受10Gy X-ray辐射；
(2)肿瘤局部辐射治疗+EGFRvIII-m28Z CAR-T组(X-ray+EGFRvIII-m28Z):Day14肿瘤局部接受10Gy X-ray辐射，间隔1天即Day16尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞。
(2)观察记录肿瘤大小，参照对照UT组计算肿瘤抑制率。持续观察并测量小鼠肿瘤体积变化。瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2。记录小鼠生存期，结果如图6所示。
如图6A所示，在肿瘤接种后的第34天(Day34)，EGFRvIII-m28Z组肿瘤抑制率是41.74％±13.29％，EGFRvIII-m28Z+X-ray组肿瘤抑制率是81.77％±12.62％，X-ray+EGFRvIII-m28Z组肿瘤抑制率是80.85％±17.82％。相对于EGFRvIII-m28Z组，不管是EGFRvIII-m28Z+X-ray组还是X-ray+EGFRvIII-m28Z组都显著抑制肿瘤生长(EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z P<0.01，One way ANOVA；EGFRvIII-m28Z vs.EGFRvIII-m28Z+X-rayP<0.05,One way ANOVA)。
如图6B所示，相对于EGFRvIII-m28Z组，不管是EGFRvIII-m28Z+X-ray组还是X-ray+EGFRvIII-m28Z组都显著延小鼠长生存期(EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z P＝0.0409，Log-rank test；EGFRvIII-m28Z vs.EGFRvIII-m28Z+X-rayP＝0.0279，Log-rank test)。
上述结果显示，不论肿瘤局部辐射治疗在CAR-T施用之前还是之后都能够提高联合治疗的抗肿瘤活性，体现在抑制肿瘤生长、延长个体生存期等。而肿瘤局部辐射治疗是在CAR-T细胞施用之前还是之后对联合治疗的效果影响不大。
实施例5肿瘤局部辐射治疗和EGFRvIII-m28Z CAR-T细胞联合应用对小鼠原位乳腺癌的抗肿瘤治疗作用
利用分子生物学常规手段建立过表达嵌合有人EGFR第287-302位氨基酸表位的小鼠EGFR的小鼠乳腺癌细胞4T1细胞模型(4T1-EGFR)。4T1细胞购自中国科学院细胞库(TCM32)。(1)Balb/c小鼠原位乳腺癌模型的建立及分组治疗：
取6-8周龄Balb/c小鼠(购自上海西普尔-必凯实验动物有限公司)，将5×10 54T1-EGFR细胞接种于小鼠右侧第四乳腺脂肪垫内构建小鼠原位乳腺癌模型，接种当日记为Day0。接种后第15天(Day15),肿瘤长至100～200mm 3，将荷瘤小鼠分为5组(每组6只)：
UT(未处理)细胞组(UT):Day15尾静脉输注5×10 6未处理的小鼠T细胞；
2个单独治疗组：(1)肿瘤局部辐射治疗组(X-ray):Day15肿瘤局部接受10Gy X-ray辐射；(2)EGFRvIII-m28Z CAR-T细胞组(EGFRvIII-m28Z):Day15尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞；
2个联合组：(1)EGFRvIII-m28Z CAR-T+肿瘤局部辐射治疗组(EGFRvIII-m28Z+X-ray):Day15尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞，间隔1天即Day17肿瘤局部接受10Gy X-ray辐射；(2)肿瘤局部辐射治疗+EGFRvIII-m28Z CAR-T组(X-ray+EGFRvIII-m28Z):Day15肿瘤局部接受10Gy X-ray辐射，间隔1天即Day17尾静脉输注5×10 6EGFRvIII-m28Z CAR-T 细胞。
(2)观察记录肿瘤大小，参照对照UT组计算肿瘤抑制率。持续观察并测量小鼠肿瘤体积变化。瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2。记录小鼠生存期，结果如图7所示。
如图7A所示，在肿瘤接种后的第29天(Day29)，EGFRvIII-m28Z组肿瘤抑制率是1.05％±8.37％，EGFRvIII-m28Z+X-ray组肿瘤抑制率是57.17％±4.24％，X-ray+EGFRvIII-m28Z组肿瘤抑制率是77.22％±11.71％。相对于EGFRvIII-m28Z细胞组，不管是EGFRvIII-m28Z+X-ray组还是X-ray+EGFRvIII-m28Z组都显著抑制肿瘤生长(EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z,P<0.001,One way ANOVA；EGFRvIII-m28Z vs.EGFRvIII-m28Z+X-ray,P<0.001,One way ANOVA)。
如图7B所示，相对于EGFRvIII-m28Z组，不管是EGFRvIII-m28Z+X-ray组还是X-ray+EGFRvIII-m28Z组都显著延小鼠长生存期(EGFRvIII-m28Z vs.EGFRvIII-m28Z+X-ray,P＝0.0012,Log-rank test；EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z,P＝0.0005,Log-rank test)。
上述结果显示，不论肿瘤局部辐射治疗在CAR-T之前还是之后给与都能够提高联合治疗的抗肿瘤活性，体现在抑制肿瘤生长、延长个体生存期等。而肿瘤局部辐射治疗是在CAR-T细胞施用之前还是之后对联合治疗的效果影响不大。
实施例6肿瘤局部辐射治疗和EGFRvIII-m28Z CAR-T细胞联合应用对小鼠原位乳腺癌的抗肿瘤治疗作用
(1)C57BL/6小鼠原位乳腺癌模型的建立及分组治疗：
取6-8周龄C57BL/6小鼠(购自上海西普尔-必凯实验动物有限公司)，将8×10 5E0771-EGFR细胞接种于小鼠右侧第四乳腺脂肪垫内构建小鼠原位乳腺癌模型，接种当日记为Day0。接种后第17天(Day17),肿瘤体积为约100-200mm 3，将荷瘤小鼠分为3组(每组6只)；
UT(未处理)细胞组(UT):Day17尾静脉输注1×10 7未处理的小鼠T细胞；
X-ray+EGFRvIII-m28Z CAR-T组1:Day17肿瘤局部接受10Gy X-ray辐射，且辐射后当天即Day17尾静脉输注1×10 7EGFRvIII-m28Z CAR-T细胞；
X-ray+EGFRvIII-m28Z CAR-T组2:Day17肿瘤局部接受10Gy X-ray辐射，间隔1天即Day19尾静脉输注1×10 7EGFRvIII-m28Z CAR-T细胞；
(2)观察记录肿瘤大小，参照对照UT组计算肿瘤抑制率。持续观察并测量小鼠肿瘤体积变化。瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2。记录小鼠生存期，结果如图8所示。
如图8A所示，在肿瘤接种后的第34天(Day34)，X-ray+EGFRvIII-m28Z CAR-T组1肿瘤抑制率是91.16％±6.56％，X-ray+EGFRvIII-m28Z CAR-T组2肿瘤抑制率是50.99％±43.27％。相对于X-ray+EGFRvIII-m28Z CAR-T组2，X-ray+EGFRvIII-m28Z CAR-T组1能显著抑制肿瘤生长(X-ray+EGFRvIII-m28Z CAR-T组1vs.X-ray+EGFRvIII-m28Z CAR-T组2P<0.05,t-test)。
如图8B所示，相对于X-ray+EGFRvIII-m28Z CAR-T组2，X-ray+EGFRvIII-m28Z CAR-T组1能显著延小鼠长生存期(X-ray+EGFRvIII-m28Z CAR-T组1vs.X-ray+EGFRvIII-m28Z CAR-T组2P＝0.0494，Log-rank test)。并且，X-ray+EGFRvIII-m28Z CAR-T组1中5只小鼠肿瘤消失，而X-ray+EGFRvIII-m28Z CAR-T组2中仅3只小鼠肿瘤消失。
上述结果显示，相对于肿瘤局部辐射治疗后几天(如间隔1天)给与CAR-T治疗，肿瘤局部辐射治疗后短时间内(如当天)给与CAR-T治疗能显著提高联合治疗的抗肿瘤活性，体现在抑制肿瘤生长、延长个体生存期且肿瘤消退等。
实施例7肿瘤局部辐射治疗和EGFRvIII-m28Z CAR-T细胞联合应用对小鼠原位乳腺癌的抗肿瘤治疗作用
(1)Balb/c小鼠原位乳腺癌模型的建立及分组治疗：
取6-8周龄Balb/c小鼠(购自上海西普尔-必凯实验动物有限公司)，将5×10 54T1-EGFR细胞接种于小鼠右侧第四乳腺脂肪垫内构建小鼠原位乳腺癌模型，接种当日记为Day0。接种后第14天(Day14),肿瘤长至100～200mm 3，将荷瘤小 鼠分为4组(每组6只)：
UT(未处理)细胞组(UT):Day14尾静脉输注5×10 6未处理的小鼠T细胞；
肿瘤局部辐射治疗组(X-ray):Day14肿瘤局部接受3Gy X-ray辐射，每天一次，连续3天；
EGFRvIII-m28Z CAR-T细胞组(EGFRvIII-m28Z):Day14尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞；
X-ray+EGFRvIII-m28Z:Day14肿瘤局部接受3Gy X-ray辐射，每天一次，连续3天，在第3天辐射结束的当天(Day16)尾静脉输注5×10 6EGFRvIII-m28Z CAR-T细胞。
(2)观察记录肿瘤大小，参照对照UT组计算肿瘤抑制率。持续观察并测量小鼠肿瘤体积变化。瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2。记录小鼠生存期，结果如图9所示。
如图9A所示，在肿瘤接种后的第33天(Day33)，EGFRvIII-m28Z组肿瘤抑制率是26.02％±7.92％，X-ray+EGFRvIII-m28Z组肿瘤抑制率是50.69％±4.29％。相对于EGFRvIII-m28Z组，X-ray+EGFRvIII-m28Z组显著抑制肿瘤生长(EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z P<0.01，Two way ANOVA)。
如图9B所示，相对于EGFRvIII-m28Z组，X-ray+EGFRvIII-m28Z CAR-T组显著延小鼠长生存期(EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z P＝0.0386,One way ANOVA)。
上述结果显示，小剂量、多次的给与肿瘤局部辐射治疗也能够提高与CAR-T细胞联合治疗的抗肿瘤活性，体现在抑制肿瘤生长、延长个体生存期等。
实施例8肿瘤局部辐射治疗与EGFRvIII-m28Z CAR-T细胞、免疫检查点抑制剂联合应用对脑胶质瘤皮下移植瘤的抗肿瘤治疗作用
采用小鼠脑胶质瘤细胞系GL261细胞(购自上海瑞鹿生物有限公司)，利用分子生物学常规手段建立过表达嵌合有人EGFR第287-302位氨基酸表位的 小鼠EGFR的GL261细胞模型，得到GL261-EGFR细胞。
(1)C57BL/6小鼠脑胶质瘤皮下移植瘤模型的建立及分组治疗：
将1×10 7GL261-EGFR细胞混合20％的Matrigel皮下注射到6-8周龄C57BL/6小鼠(购自上海西普尔-必凯实验动物有限公司)后腿上,接种当日记为Day0，接种后第7天(Day7),肿瘤长至300-500mm 3，分7组(每组10只)，实验流程图见图10A：
UT(未处理)细胞组(UT):Day7尾静脉输注2×10 6未处理的小鼠T细胞；
单独治疗组：(1)肿瘤局部辐射治疗组(X-ray)：Day7肿瘤局部接受2Gy X-ray辐射；(2)EGFRvIII-m28Z CAR-T细胞组(EGFRvIII-m28Z)：Day7尾静脉输注2×10 6EGFRvIII-m28Z CAR-T细胞；(3)PD-L1抗体组(PD-L1)：Day7、Day9、Day11分别腹腔注射抗PD-L1的抗体(Tecentriq)，每日一次，每次200μg；
联合组：(1)EGFRvIII-m28Z CAR-T+PD-L1抗体组(EGFRvIII-m28Z+PD-L1)：Day7尾静脉输注2×10 6EGFRvIII-m28Z CAR-T细胞；Day7、Day9、Day11分别腹腔注射抗PD-L1的抗体(Tecentriq)，每日一次，每次200μg；(2)肿瘤局部辐射治疗+EGFRvIII-m28Z CAR-T组(X-ray+EGFRvIII-m28Z)：Day7肿瘤局部接受2Gy X-ray辐射；Day9尾静脉输注2×10 6EGFRvIII-m28Z CAR-T细胞；(3)肿瘤局部辐射治疗+PD-L1抗体+EGFRvIII-m28Z CAR-T组(X-ray+PD-L1+EGFRvIII-m28Z)：Day7肿瘤局部接受2Gy X-ray辐射，当天(Day7)腹腔注射抗PD-L1的抗体(Tecentriq)200μg；Day9尾静脉输注2×10 6EGFRvIII-m28Z CAR-T细胞，当天(Day9)腹腔注射抗PD-L1的抗体(Tecentriq)200μg；Day11腹腔注射抗PD-L1的抗体(Tecentriq)200μg。
(2)观察记录肿瘤大小，参照对照UT组计算肿瘤抑制率。持续观察并测量小鼠肿瘤体积变化。瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2。结果如图10B所示，在肿瘤接种后的第13天(Day13)，EGFRvIII-m28Z组肿瘤抑制率是5.79％±6.40％，X-ray+EGFRvIII-m28Z组肿瘤抑制率是30.84％±3.95％，X-ray+PD-L1+EGFRvIII-m28Z组肿瘤抑制率是43.26％±14.51％。X-ray+EGFRvIII-m28Z组和X-ray+PD-L1+EGFRvIII-m28Z组相对于EGFRvIII-m28Z组有更好的抑制肿瘤生长的效果(EGFRvIII-m28Z vs.X-ray+EGFRvIII-m28Z P<0.01,One way ANOVA；EGFRvIII-m28Z vs. X-ray+PD-L1+EGFRvIII-m28Z P<0.0001,One way ANOVA)。X-ray+PD-L1+EGFRvIII-m28Z组相对于X-ray+EGFRvIII-m28Z组有相对较高的肿瘤抑制率(P<0.05,t-test)。上述结果说明PD-1通路的阻断能进一步提高肿瘤局部辐射和CAR-T细胞联合治疗的抑制肿瘤生长的效果。
在上述实施例中，仅仅是作为示例性的，采用了靶向EGFRvIII的鼠抗(SEQ ID NO：2)、鼠的跨膜域和胞内域等制备的CAR-T，当应用于人体治疗时，常用的可以选择人CD8α信号肽的编码序列(SEQ ID NO：6)、人CD8α铰链区及跨膜区的编码序列(SEQ ID NO：7)、人的CD28跨膜域的编码序列(SEQ ID NO：10)、人CD28胞内域的编码序列(SEQ ID NO：8)、人CD3ζ胞内域的编码序列(SEQ ID NO：9)进行制备。示例性的，所采用的靶向EFGRvIII的嵌合抗原受体的氨基酸序列如SEQ ID NO：20、21、22任一所示。
可替换的，采用的CAR可以是靶向其他抗原的，如GPC3(示例性的，靶向GPC3的嵌合抗原受体的氨基酸序列如SEQ ID NO：11、12、13、14、15任一所示)；CLD18A2(示例性的，靶向CLD18A2的嵌合抗原受体的氨基酸序列如SEQ ID NO：16、17、18、19任一所示)；间皮素(示例性的，靶向间皮素的嵌合抗原受体的氨基酸序列如SEQ ID NO：23、24、25、26任一所示)。

Claims (38)

  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肿瘤局部辐射治疗联合进行治疗,并且对所述的个体不进行淋巴细胞清除，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原的受体。
  2. 如权利要求1所述的方法，其特征在于，免疫效应细胞施用和肿瘤局部辐射治疗给予时间不分先后；可以先给予肿瘤局部辐射治疗再给予免疫效应细胞施用；也可以同时给与；还可以先给予免疫效应细胞施用再给予肿瘤局部辐射治疗。
  3. 如权利要求1或2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。
  4. 如权利要求1-3任一所述的方法，其特征在于，所述局部辐射治疗为采用辐射治疗设备对所述肿瘤进行辐射，
    优选的，所述的辐射治疗设备通过产生下述任一射线对所述肿瘤进行辐射：X射线、α射线、β射线、γ射线、中子。
  5. 如权利要求1至4任一所述的方法，其特征在于，所述辐射治疗设备产生X射线。
  6. 如权利要求5所述的方法，其特征在于，所述X射线对所述的肿瘤进行至少1次辐射，或多次小剂量辐射。
  7. 如权利要求1-6任一所述的方法，其特征在于，所述辐射治疗的射线剂量为介于不高于100Gy之间，优选地不高于80Gy，更为优选地为不高于70Gy。
  8. 如权利要求1至7任一所述的方法，其特征在于，所述的辐射治疗的能量源位于所述个体的体内或体外。
  9. 如权利要求3所述的方法，其特征在于，所述的嵌合抗原受体包括：
    (i)特异性结合所述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)特异性结合所述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)特异性结合所述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。
  10. 如权利要求1-9任一所述的方法，其特征在于：
    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,血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；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)。
  11. 如权利要求1-10任一所述的方法，其特征在于，所述的免疫效应细胞选自特异识别EGFR、EGFRvIII、GPC3、Claudin18.2的CAR-T细胞。
  12. 如权利要求1-11任一所述的方法，其特征在于，所述的特异性识别肿瘤抗原的抗体具有SEQ ID NO:2所示的氨基酸序列。
  13. 权利要求3-11所述的方法，其特征在于，所述的嵌合抗原受体具有SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或26所示的氨基酸序列。
  14. 如权利要求1-13任一所述的方法，其特征在于，所述的免疫效应细胞施用在所述的肿瘤局部辐射治疗之后当天，或1天、2天、或3天后。
  15. 如权利要求1-14任一所述的方法，其特征在于，还包括给所述个体施用免疫检查点(immune checkpoint)抑制剂；优选地，所述免疫检查点抑制剂是生物学治疗剂或小分子；更优选地，所述免疫检查点抑制剂选自单克隆抗体、人源化抗体、全人抗体、融合蛋白或其组合。
  16. 如权利要求15所述的方法，其中所述免疫检查点抑制剂针对的免疫检查点 选自下述的免疫检查点蛋白质：CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK1、CHK2、A2aR和B-7家族配体或其组合；优选地，所述免疫检查点抑制剂是PD-1或PDL-1抑制剂。
  17. 如权利要求15或16所述的方法，其特征在于，所述免疫检查点抑制剂与选自下述的免疫检查点蛋白质的配体相互作用：CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK 1、CHK2、A2aR和B-7家族配体或其组合。
  18. 如权利要求15-16任一所述的方法，其特征在于，所述免疫效应细胞选自特异识别EGFR、EGFRvIII、GPC3、Claudin18.2的CAR-T细胞，所述免疫检查点抑制剂为特异识别PD-1或PD-L1的单克隆抗体。
  19. 如权利要求15-18任一所述的方法，其特征在于，包括在所述肿瘤局部辐射治疗之前、同时或之后，或在所述免疫效应细胞治疗之前、同时或之后，给予所述个体施用所述免疫检查点抑制剂；优选地，所述免疫效应细胞和所述免疫检查点抑制剂同时施用。
  20. 如权利要求1-19任一所述的方法，其特征在于，所述方法的治疗效果通过免疫效应细胞的存在、或指示T细胞炎症的基因标记的存在或其组合预测，优选地，通过检测IFN-γ水平变化来预测。
  21. 如权利要求1-20任一所述的方法，其特征在于，其中所述肿瘤包括：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，脑胶质瘤，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。
  22. 如权利要求1-21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。
  23. 免疫效应细胞在制备治疗肿瘤的药物中的应用，其特征在于，所述免疫效应细胞包含识别所述肿瘤的肿瘤抗原的受体，所述药物与肿瘤局部辐射治疗联合施用，包括同时或先后施用，患有所述肿瘤的个体接受所述免疫效应细胞和/或所述肿瘤局部辐射治疗时体内淋巴细胞数目相对于入组所述肿瘤治疗时不低于40％。
  24. 如权利要求23所述的应用，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。
  25. 如权利要求23或24所述的应用，其特征在于，所述局部辐射治疗为采用辐射治疗设备对所述肿瘤进行辐射，
    优选的，所述的辐射治疗设备通过产生下述任一射线对所述肿瘤进行辐射：X射线、α射线、β射线、γ射线、中子。
  26. 如权利要求23至25任一所述的应用，其特征在于，所述辐射治疗设备产生X射线。
  27. 如权利要求26所述的应用，其特征在于，所述X射线对所述的肿瘤进行至少1次辐射，或多次小剂量辐射。
  28. 如权利要求23-27任一所述的应用，其特征在于，所述辐射治疗的射线剂量为介于不高于100Gy之间，优选地不高于80Gy，更为优选地为不高于70Gy。
  29. 如权利要求23至28任一所述的应用，其特征在于，所述的辐射治疗的能量源位于所述个体的体内或体外。
  30. 如权利要求24所述的应用，其特征在于，所述的嵌合抗原受体包括：
    (i)特异性结合所述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)特异性结合所述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)特异性结合所述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。
  31. 如权利要求23-30任一所述的应用，其特征在于，所述的免疫效应细胞选自特异识别EGFR、EGFRvIII、GPC3、Claudin18.2的CAR-T细胞。
  32. 如权利要求23-31任一所述的应用，其特征在于，所述的特异性识别肿瘤抗原的抗体具有SEQ ID NO:2所示的氨基酸序列。
  33. 权利要求24-32任一所述的应用，其特征在于，所述的嵌合抗原受体具有SEQ ID NO:11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或26所示的氨基酸序列。
  34. 如权利要求23-33任一所述的应用，其特征在于，所述药物施用在所述的肿瘤局部辐射治疗之后当天，或1天、2天、或3天后。
  35. 如权利要求23-34任一所述的应用，其特征在于，还包括给所述个体施用免疫检查点(immune checkpoint)抑制剂；优选地，所述免疫检查点抑制剂是生物学治疗剂或小分子；更优选地，所述免疫检查点抑制剂选自单克隆抗体、人源化抗体、全人抗体、融合蛋白或其组合。
  36. 如权利要求35所述的应用，其中所述免疫检查点抑制剂针对的免疫检查点选自下述的免疫检查点蛋白质：CTLA-4、PDL1、PDL2、PD1、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK1、CHK2、A2aR和B-7家族配体或其组合；优选地，所述免疫检查点抑制剂是PD-1或PDL-1抑制剂。
  37. 如权利要求23-36任一所述的应用，其特征在于，其中所述肿瘤包括：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，脑胶质瘤，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。
  38. 如权利要求23-37任一所述的应用，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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