WO2019114762A1 - Utilisation combinée de cellules immunes effectrices et de rayonnement pour le traitement de tumeurs - Google Patents

Utilisation combinée de cellules immunes effectrices et de rayonnement pour le traitement de tumeurs 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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Chinese (zh)
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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

L'invention concerne un procédé de traitement de tumeurs, en particulier, un procédé de traitement d'un individu présentant une tumeur au moyen de l'administration de cellules immunes effectrices en combinaison avec un rayonnement local. Selon le procédé, la clairance des lymphocytes n'est pas effectuée chez l'individu, et les cellules immunes effectrices contiennent des récepteurs reconnaissant les antigènes tumoraux de la tumeur. Le procédé de traitement de tumeurs présente des effets antitumoraux sur des tumeurs solides, et sans le prétraitement de la clairance des lymphocytes, le traitement par cellules CAR-T, en combinaison avec une radiothérapie locale et sans clairance des lymphocytes, permet d'obtenir un meilleur effet thérapeutique que le traitement par CAR-T avec la clairance des lymphocytes, et d'améliorer l'efficacité antitumorale.
PCT/CN2018/120679 2017-12-12 2018-12-12 Utilisation combinée de cellules immunes effectrices et de rayonnement pour le traitement de tumeurs WO2019114762A1 (fr)

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WO2023274303A1 (fr) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Polypeptide chimérique pour la régulation de l'activité physiologique cellulaire

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WO2022028623A1 (fr) 2020-08-07 2022-02-10 佧珐药业有限公司 Cellules modifiées et procédé de modification de cellules
WO2023274303A1 (fr) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Polypeptide chimérique pour la régulation de l'activité physiologique cellulaire

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