WO2019149279A1 - 细胞免疫治疗的组合 - Google Patents

细胞免疫治疗的组合 Download PDF

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WO2019149279A1
WO2019149279A1 PCT/CN2019/074535 CN2019074535W WO2019149279A1 WO 2019149279 A1 WO2019149279 A1 WO 2019149279A1 CN 2019074535 W CN2019074535 W CN 2019074535W WO 2019149279 A1 WO2019149279 A1 WO 2019149279A1
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cancer
phenyl
cells
tumor
antigen
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PCT/CN2019/074535
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English (en)
French (fr)
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李宗海
吴秀奇
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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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Priority to JP2020542127A priority Critical patent/JP2021512875A/ja
Priority to US16/966,789 priority patent/US20210113614A1/en
Priority to EP19748023.9A priority patent/EP3747433A4/en
Priority to KR1020207025358A priority patent/KR20200143672A/ko
Publication of WO2019149279A1 publication Critical patent/WO2019149279A1/zh

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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/303—Liver or Pancreas
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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Definitions

  • the invention belongs to the field of cellular immunotherapy, and particularly relates to an anti-tumor treatment combined with an immune effector cell and a kinase inhibitor chemotherapeutic agent.
  • CAR-T cell therapy has shown remarkable therapeutic effects in the treatment of hematoma.
  • CAR-T cells have been used in the clinical trial of hematoma treatment (Clinical development of CAR T cells-challenges And opportunities in translating innovative treatment concepts, Jessica Hartmann et al., EMBO Molecule Medicine, Published on line, August 1, 2017).
  • the treatment of solid tumors is difficult to achieve the effect of hematoma treatment.
  • CAR-T cells can more easily contact tumor cells through vein input to achieve killing, while CAR-T cells are more difficult to homing to solid tumor tissue.
  • solid tumors usually have a complex dynamic tumor microenvironment, which can interact with tumor cells, benign cells, stromal cells, vascular cells, etc.
  • CAR-T treatment is usually not effective in the treatment of solid tumors.
  • the object of the present invention is to provide a tumor treatment method for improving the application effect of immune cell therapy, particularly CAR-T cell therapy, in solid tumors.
  • a method of treating a tumor wherein an immune effector cell and a second therapeutic agent are administered to an individual having a tumor, the immune effector cell expressing a receptor recognizing a tumor antigen, said The second therapeutic agent is a kinase inhibitor.
  • the second therapeutic agent is a compound of Formula I, or a pharmaceutically acceptable salt thereof,
  • Ar' is an unsubstituted or substituted phenyl group, and the substituent is selected from the group consisting of halogen and C1-10 alkyl.
  • M is one or more bridging groups selected from -O- or -S-,
  • Py(X) is an X-substituted pyridyl group, and X is -C(O)R x , wherein R x is NR a R b , wherein R a and R b are each:
  • R f is a C1-10 alkyl group
  • a method of reducing the growth, survival or viability of cancer cells characterized in that an immune effector cell and a second therapeutic agent are administered to an individual having a tumor, said immune effector cell expressing There are receptors that recognize tumor antigens,
  • the second therapeutic agent is a compound of formula I, or a pharmaceutically acceptable salt thereof,
  • Ar' is an unsubstituted or substituted phenyl group, and the substituent is selected from the group consisting of halogen and C1-10 alkyl.
  • M is one or more bridging groups selected from -O- or -S-,
  • Py(X) is an X-substituted pyridyl group, and X is -C(O)R x , wherein R x is NR a R b , wherein R a and R b are each:
  • R f is a C1-10 alkyl group
  • the above method of treating a tumor, or a method of reducing the growth, survival or viability of cancer cells is performed without prior to administration of immune effector cells to an individual having a tumor.
  • the therapeutic effect of the immune effector cell and the second therapeutic agent is greater than the immune effector cell and the second therapeutic agent. The effect of either use alone.
  • the Ar" is a substituted phenyl group, and the substituent is selected from any one or a combination of chlorine, bromine, fluorine, trifluoromethyl, methoxy and t-butyl.
  • said M is -O-.
  • each of Ra and Rb is H or C1-10 alkyl, preferably, Ra and Rb are H and methyl, respectively.
  • the pharmaceutically acceptable salt is selected from the group consisting of: a) a basic salt of an inorganic acid and an organic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid.
  • the second therapeutic agent is selected from the compounds listed below or a pharmaceutically acceptable salt thereof:
  • the second therapeutic agent is selected from the group consisting of a compound of Formula II or Formula III below, or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt of the second therapeutic agent is selected from the group consisting of tosylate, besylate, hydrochloride, and methanesulfonate.
  • the second therapeutic agent is a compound of formula II or a hydrate of a compound of formula II, preferably a monohydrate.
  • the second therapeutic agent is administered in an amount of from 100 to 1000 mg per day, preferably from 200 to 800 mg per day, more preferably, the daily dose is 400-800mg.
  • the second therapeutic agent is administered 1-3 times a day, preferably 2 times a day, to the subject individual.
  • the immune effector cells are administered in an amount of about 1 x 10 5 to 1 x 10 8 cells/kg of the subject's body weight per subject individual, and more preferably, each administration amount is about 1 x 10 5 ⁇ 1x10 7 cells/kg of subject weight.
  • the immune effector cells and the second therapeutic agent are administered in no time; the second therapeutic agent can be administered to the immune effector cells first; or simultaneously, and the immune effector cells can be administered first.
  • the second therapeutic agent preferably administers the immune effector cells during administration of the second therapeutic agent.
  • the second therapeutic agent is administered orally.
  • the receptor is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a T cell receptor (TCR), and a T cell fusion protein (TFP). , T cell antigen coupler (TAC) or a combination thereof.
  • CAR Chimeric Antigen Receptor
  • TCR T cell receptor
  • TCP T cell fusion protein
  • TAC T cell antigen coupler
  • 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 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 alpha (IL- 13R ⁇ ); interleukin 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; 1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; plate
  • TSHR
  • the tumor antigen is a solid tumor antigen.
  • the solid tumor antigen is vascular endothelial growth factor receptor, EGFR, EGFRvIII, GPC3, or Claudin 18.2. More preferably, the solid tumor antigen is GPC3, EGFR, EGFRvIII, or a vascular endothelial growth factor receptor.
  • the vascular endothelial growth factor receptor is vascular endothelial growth factor receptor 2 (VEGFR2).
  • the solid tumor antigen is GPC3.
  • the antibody that specifically recognizes a tumor antigen is an antibody that targets Glypican 3 (GPC3).
  • the antibody that specifically recognizes the tumor antigen comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 17, 18, 19, and LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 20, 21, 22. .
  • amino acid sequence of an antibody that specifically recognizes a tumor antigen has at least 90% identity to the sequence set forth in SEQ ID NO: 14.
  • amino acid sequence of the chimeric antigen receptor is at least 90% identical to the sequence set forth in SEQ ID NO: 21, 22, 23 or 24.
  • the treatment is caused by clinical results: an increase, increase or prolongation of anti-tumor activity by the immune effector cells; an increase in the number of anti-tumor immune effector cells or activated immune effector cells compared to the number before treatment, or The combination is determined.
  • the clinical result is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor enlargement, recurrence or spread, or a combination thereof.
  • the therapeutic effect 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 changes in IFN-[gamma], TNF[alpha] levels.
  • the tumor comprises: blood cancer, breast cancer, glioma, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer , pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer , vaginal cancer, vulva 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 children, bladder cancer , renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem gliom
  • CNS central
  • the immune effector cells comprise: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow derived phagocytic cells, or a combination thereof; preferably
  • the immune effector cells are selected from autologous T cells, allogeneic T cells or allogeneic NK cells, and more preferably, the T cells are autologous T cells.
  • the invention provides a drug delivery system comprising an immune effector cell and a second therapeutic agent, the immune effector cell expressing a receptor that recognizes a tumor antigen.
  • the second therapeutic agent is a compound of formula I, or a pharmaceutically acceptable salt thereof,
  • Ar' is an unsubstituted or substituted phenyl group, and the substituent is selected from the group consisting of halogen and C1-10 alkyl.
  • M is one or more bridging groups selected from -O- or -S-,
  • Py(X) is an X-substituted pyridyl group, and X is -C(O)R x , wherein R x is NR a R b , wherein R a and R b are each:
  • R f is a C1-10 alkyl group
  • the Ar" is a substituted phenyl group, and the substituent is selected from any one or a combination of chlorine, bromine, fluorine, trifluoromethyl, methoxy and t-butyl.
  • said M is -O-.
  • each of Ra and Rb is H or C1-10 alkyl, preferably, Ra and Rb are H and methyl, respectively.
  • the pharmaceutically acceptable salt is selected from the group consisting of: a) a basic salt of an inorganic acid and an organic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid.
  • the second therapeutic agent is selected from the compounds listed below or a pharmaceutically acceptable salt thereof:
  • the second therapeutic agent is selected from the group consisting of a compound of Formula II or Formula III below, or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt is a tosylate salt, a besylate salt, a hydrochloride salt, a methanesulfonate salt.
  • the second therapeutic agent is administered in an amount of from 100 to 1000 mg per day, preferably from 200 to 800 mg per day, more preferably, from 400 to 800 mg per day. .
  • the second therapeutic agent is administered 1-3 times a day, preferably 2 times a day, to the subject individual.
  • the immune effector cells are administered in an amount of about 1 x 10 5 to 1 x 10 8 cells/kg of the subject's body weight per subject individual, and more preferably, each administration amount is about 1 x 10 5 ⁇ 1x10 7 cells/kg of subject weight.
  • the immune effector cells and the second therapeutic agent are administered in no time; the second therapeutic agent can be administered to the immune effector cells first; or simultaneously, and the immune effector cells can be administered first.
  • the second therapeutic agent preferably administers the immune effector cells during administration of the second therapeutic agent.
  • the second therapeutic agent is administered orally.
  • the receptor is selected from the group consisting of a Chimeric Antigen Receptor (CAR), a T cell receptor (TCR), and a T cell fusion protein (TFP). , T cell antigen coupler (TAC) or a combination thereof.
  • CAR Chimeric Antigen Receptor
  • TCR T cell receptor
  • TCP T cell fusion protein
  • TAC T cell antigen coupler
  • 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 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 alpha (IL- 13R ⁇ ); interleukin 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; 1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; plate
  • TSHR
  • the tumor antigen is a solid tumor antigen, preferably the solid tumor antigen is a vascular endothelial growth factor receptor, EGFR, EGFRvIII, GPC3, or Claudin 18.2, and more preferably, the solid tumor antigen It is GPC3, EGFR, EGFRvIII, or vascular endothelial growth factor receptor.
  • the vascular endothelial growth factor receptor is vascular endothelial growth factor receptor 2 (VEGFR2).
  • the tumor antigen is GPC3.
  • the antibody that specifically recognizes a tumor antigen is an antibody that targets Glypican 3 (GPC3).
  • the antibody that specifically recognizes the tumor antigen comprises HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 15, 16, 17, and LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 18, 19, and 20. .
  • amino acid sequence of an antibody that specifically recognizes a tumor antigen has at least 90% identity to the sequence set forth in SEQ ID NO: 14.
  • amino acid sequence of the chimeric antigen receptor is at least 90% identical to the sequence set forth in SEQ ID NO: 21, 22, 23 or 24.
  • the treatment is caused by clinical results: an increase, increase or prolongation of anti-tumor activity by the immune effector cells; an increase in the number of anti-tumor immune effector cells or activated immune effector cells compared to the number before treatment, or The combination is determined.
  • the clinical result is selected from the group consisting of tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor enlargement, recurrence or spread, or a combination thereof.
  • the therapeutic effect 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 changes in IFN-[gamma], TNF[alpha] levels.
  • the tumor comprises: blood cancer, breast cancer, glioma, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer , pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer , vaginal cancer, vulva 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 children, bladder cancer , renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem glio
  • CNS central
  • the immune effector cells comprise: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow derived phagocytic cells, or a combination thereof; preferably
  • the immune effector cells are selected from autologous T cells, allogeneic T cells or allogeneic NK cells, and more preferably, the T cells are autologous T cells.
  • an immune effector cell expressing a receptor for recognizing a tumor antigen for the preparation of a medicament, characterized in that the medicament is used in combination with sorafenib.
  • the above application is for treating a tumor in a human patient, wherein the cell and sorafenib are formulated to provide better or greater effects obtained by using the cell alone and sorafenib.
  • kits for treating a tumor characterized in that the kit comprises:
  • the immune effector cells and sorafenib are formulated to provide a therapeutic effect greater than that of the respective agents when used alone; preferably, the immune effector cells are CAR T cells, more preferably, CAR T cells specifically recognize EGFR, EGFRvIII, phosphatidylinositol 3, claudin 18.2, BCMA.
  • the CAR T cells specifically recognize phosphatidylinositol 3.
  • an article for treating a tumor characterized in that the preparation comprises: an immune effector cell and a second therapeutic agent, the immune effector cell expressing a receptor recognizing a tumor antigen .
  • the article comprises:
  • the immune effector cells express a chimeric antigen receptor that recognizes a tumor antigen
  • the immune effector cells and the second therapeutic agent are the same as those defined in the first aspect, the second aspect, and the third aspect of the invention described above.
  • an immune effector cell expressing a receptor for recognizing a tumor antigen and a second therapeutic agent for the preparation of a medicament or article for treating a tumor in a human patient, wherein
  • the medicament formulated with the cell and the second therapeutic agent can provide a superior or greater therapeutic effect than when the cell and the second therapeutic agent are each used alone.
  • the immune effector cells and the second therapeutic agent are the same as those defined in the first aspect, the second aspect, and the third aspect of the invention described above.
  • a method of treating a tumor or reducing the growth, survival or viability of a cancer cell characterized in that an immune effector cell and sorafenib are administered to an individual having a tumor, said immune effect
  • the cells express a chimeric antigen receptor that recognizes a tumor antigen.
  • the chimeric antigen receptor has an antibody or fragment thereof that specifically recognizes a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain.
  • the antibody or fragment thereof specifically recognizing the tumor antigen comprises the HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 15, 16, 17, and the LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 18, 19, and 20.
  • the tumor is liver cancer
  • the cancer cell is a liver cancer cell.
  • a method of treating a tumor or reducing the growth, survival or viability of a cancer cell characterized in that an immune effector cell and regorafenib are administered to an individual having a tumor, said immune effect
  • the cells express a chimeric antigen receptor that recognizes a tumor antigen.
  • the chimeric antigen receptor has an antibody or fragment thereof that specifically recognizes a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain.
  • the antibody or fragment thereof specifically recognizing the tumor antigen comprises the HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 15, 16, 17, and the LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 18, 19, and 20.
  • the tumor is liver cancer
  • the cancer cell is a liver cancer cell.
  • an article or kit for treating a tumor comprising:
  • the immune effector cells express a chimeric antigen receptor that recognizes a tumor antigen.
  • the chimeric antigen receptor has an antibody or fragment thereof that specifically recognizes a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain.
  • the antibody or fragment thereof specifically recognizing the tumor antigen comprises the HCDR1, HCDR2, HCDR3 shown in SEQ ID NOs: 15, 16, 17, and the LCDR1, LCDR2, LCDR3 shown in SEQ ID NOs: 18, 19, and 20.
  • the tumor is liver cancer
  • the cancer cell is a liver cancer cell.
  • the combination of the compound of the formula I provided by the present invention and an immune effector cell can significantly improve the ability to kill tumor cells.
  • the treatment regimen of the present invention is capable of combating immunosuppression in the cancer microenvironment, thereby significantly enhancing the effect on solid tumors, and also having a good effect on refractory and progressive cancer.
  • Figure 1A is a PRRLSIN-hu9F2-28Z plasmid map
  • Figure 1B is a CAR T cell positive rate assay.
  • Figure 2 shows the toxicity of sorafenib on liver cancer cells and CAR T cells.
  • Figure 3 shows the in vitro ability of CAR T cells to detect tumor cells after sorafenib treatment.
  • Figure 4 shows the flow cytometric expression of GPC3 on Hepa 1-6 cells.
  • Figure 5 shows the inhibitory effect of sorafenib in combination with CAR T cells on the tumor volume of hepa 1-6-GPC3 subcutaneous neoplasms.
  • Figure 6 shows the inhibitory effect of sorafenib in combination with CAR T cells on the tumor weight of hepa 1-6-GPC3 subcutaneous neoplasms.
  • Figure 7 shows the in vitro ability to detect the degranulation capacity (Figure 7A), proliferative capacity (Figure 7B) and killing ability of CAR T cells after sorafenib treatment ( Figure 7C).
  • Figure 8 shows the effect of detecting sorafenib on cytokine secretion of human CAR T cells by IL-2 (Figure 8A), IFN- ⁇ (Figure 8B) and TNF- ⁇ ( Figure 8C).
  • Figure 9 shows tumor volume (9A) and tumor weight (9B) of a combination of sorafenib and human CAR T cells in the treatment of liver cancer.
  • Figure 10 shows the intratumoral infiltration of CAR T cells (Figure 10A) and apoptosis ( Figures 10B, 10C).
  • Figure 11 shows in vitro detection of sorafenib in combination with human CAR T cells synergistically promoting apoptosis in PLC/RPF/5 cells.
  • Figure 12 shows the results of treatment of mouse liver cancer xenografts with CAR T cells in combination with regofenib.
  • Figure 13 shows an imaging image of a subject after treatment with a combination of CAR T cells and sorafenib.
  • the present invention relates to the combination of an immune effector cell and a second therapeutic agent (a compound of formula I) for the treatment of a tumor, it being understood that the invention is not limited to the methods and experimental conditions described.
  • a second therapeutic agent a compound of formula I
  • all technical and scientific terms used have the same meaning as commonly understood by those skilled in the art of gene therapy, biochemistry, genetics, molecular biology, and pharmaceutical chemistry.
  • the invention resides, at least in part, from the recognition that a combination treatment regimen of one or more cycles and/or doses of a second therapeutic agent and immune effector cells is administered continuously, in either order or substantially simultaneously, at the treatment.
  • Some subjects may be more effective in increasing, enhancing or prolonging the activity and/or number of immune cells in order to achieve an anti-tumor effect.
  • the "dose” as referred to herein may be a dose calculated on a weight basis or a dose calculated on a body surface area (BSA) basis.
  • the dose calculated on a weight basis is the dose administered to the patient based on the patient's body weight, such as mg/kg.
  • BSA in doses are calculated based on the surface area of the patient based on the calculated dose to be administered to a patient, e.g. mg / m 2.
  • the second therapeutic agent employed in the present invention is a compound of Formula I, or a pharmaceutically acceptable salt thereof.
  • Ar' is an unsubstituted or substituted phenyl group, and the substituent is selected from the group consisting of halogen and C1-10 alkyl.
  • M is one or more bridging groups selected from -O- or -S-,
  • Py(X) is an X-substituted pyridyl group, and X is -C(O)R x , wherein R x is NR a R b , wherein R a and R b are each:
  • R f is a C1-10 alkyl group
  • C1-10 alkyl means any straight or branched chain group having 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, or Butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, tert-amyl, n-hexyl, n-heptyl, n-octyl, isooctyl, 2-ethylhexyl, n-decyl, isoindole Base, positive base, etc.
  • C1-10 alkoxy refers to any of the above C1-C10 alkyl groups which are attached to the remainder of the molecule through an oxygen atom (-O-).
  • halogen means a fluorine element, a chlorine element, a bromine element, or an iodine element.
  • haloalkyl refers to fluoroalkyl, chloroalkyl, bromoalkyl, iodoalkyl.
  • C3-12 cycloalkyl refers to a non-aromatic, saturated or unsaturated, monocyclic or bicyclic hydrocarbon ring having from 3 to 12 carbon atoms.
  • exemplary "cycloalkyl” include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • heteroaryl refers to an aromatic heterocyclic ring, typically a 5- to 8-membered heterocyclic ring having from 1 to 3 heteroatoms selected from N, O or S; heteroaryl rings may optionally be Further fused or linked to aromatic and non-aromatic carbocyclic and heterocyclic rings.
  • Non-limiting examples of such heteroaryl groups are, for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, fluorenyl, imidazolyl, thiazolyl, isothiazolyl, thiazolyl, pyrrolyl, benzene -pyrrolyl, furyl, phenyl-furanyl, oxazolyl, isoxazolyl, pyrazolyl, thienyl, benzothienyl, isoindoline, benzimidazolyl, carbazolyl , quinolyl, isoquinolyl, 1,2,3-triazolyl, 1-phenyl-1,2,3-triazolyl, 2,3-dihydroindenyl, 2,3-di Hydrobenzofuranyl, 2,3-dihydrobenzothienyl, benzopyranyl, 2,3-dihydrobenzoxazinyl, 2,3
  • any group whose name is a compound name, such as "arylamino”, shall mean a moiety conventionally derived therefrom, for example, an amino group substituted with an aryl group.
  • aryl group is as defined above.
  • the compounds of Formula I include, but are not limited to, the compounds of Formula II and Formula III.
  • the second therapeutic agent can be a pharmaceutically acceptable salt of a compound of formula II, such as a tosylate, a besylate, a hydrochloride, a methanesulfonate, and the like.
  • the compound of the formula III may also employ a hydrate thereof such as a monohydrate or a dihydrate; a pharmaceutically acceptable salt thereof such as a hydrochloride or the like may also be employed.
  • a second therapeutic agent such as Compound II
  • a second therapeutic agent not only promotes the secretion of the cytokine IL2 by CAR T cells, but also promotes the infiltration of CAR T cells in tumor tissues and enhances the anti-tumor effect.
  • a second therapeutic agent such as Compound II
  • immune cell therapy that targets tumor-specific antigens can significantly enhance anti-tumor effects.
  • the Applicant has also found that the present invention can not only improve the anticancer effect of refractory cancer, but also achieve a good anti-tumor effect even when lymphocyte depletion is not performed when using CAR-T cells, thereby greatly reducing the cause of clearing.
  • the anti-cancer treatment is low in effect and reduces the toxic side effects caused by damage to normal tissues, especially the severe inhibition of bone marrow.
  • the second therapeutic agent such as Compound II
  • a pharmaceutically acceptable carrier excipient and other additives eg, tablets, Release preparations, capsules, injections, solutions
  • the compositions may be formulated as tablets, dragees or capsules.
  • lactose or starch can be used as a carrier, and gelatin, sodium carboxymethylcellulose, methylcellulose polyvinylpyrrolidone and the like are suitable binders or granules.
  • starch or microcrystalline cellulose may be used, and talc powder, colloidal silica gel, glyceryl stearate, calcium stearate or magnesium is often used as a suitable anti-adhesive agent and lubricant.
  • tablets can be prepared by compressing wet granules.
  • the active ingredient is mixed with a carrier and optionally with a disintegrating additive.
  • the mixture is granulated with an aqueous solution of an adhesive, an alcoholic or aqueous alcoholic solution in a suitable apparatus, and the dried granules are subsequently added to other disintegration.
  • Agent, Lubricant and Anti-Adhesive This mixture is compressed.
  • the heterocyclic derivative can be freed to form a pharmaceutically acceptable organic acid, preferably methanesulfonic acid, fumaric acid or the like to facilitate administration as an injection, although the dose depends on the subject to be treated, The mode of administration, symptoms and other factors vary.
  • a pharmaceutically acceptable organic acid preferably methanesulfonic acid, fumaric acid or the like
  • the second therapeutic agent is Sorafenib (having the structure shown by the compound of Formula II), preferably, sorafenib tosylate.
  • the second therapeutic agent is Ragorafenib (having the structure shown by the compound of Formula III), preferably, sorafenib tosylate.
  • sorafenib due to the synergistic effect of sorafenib and CAR-T cells, even a low dose of sorafenib can achieve better results, so the technical solution of the present invention can reduce the side effects of sorafenib. .
  • the low dose of the second therapeutic agent refers to a dose lower than that of the single drug, and the low dose of sorafenib means that the therapeutic effect is lower than that of the sorafenib alone.
  • the dose specifically the recommended dose of clinical sorafenib: recommended to take sorafenib for each 0.4g (2x0.2g) twice daily; or refers to clinical trials below the compound II alone
  • the concentration in the patient is about 6.5 uM.
  • the dose of sorafenib in the present invention may be an effective dose lower than the clinical one.
  • the average concentration of sorafenib in an individual having a tumor is about 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.095, 0.09, 0.085, 0.08, 0.075, 0.07, 0.065, 0.06, 0.055, 0.05, 0.045, 0.04, 0.035, 0.034, 0.033, 0.032, 0.031, 0.03, 0.029, 0.028, 0.027, 0.026, 0.025, 0.024, 0.023, 0.022, 0.021, 0.02, 0.019, 0.018, 0.017, 0.016, 0.015, 0.014, 0.013,
  • the individual having the tumor has a daily dose of sorafenib of about 700, 650, 600, 550, 500, 450, 400, 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mg, preferably 400-800 mg/day.
  • sorafenib of about 700, 650, 600, 550, 500, 450, 400, 350, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mg, preferably 400-800 mg/day.
  • the sorafenib can be administered once a day or several times a day, such as twice a day.
  • the daily dose may be from 100 to 1000 mg, preferably from 200 to 800 mg per day, and more preferably from 400 to 800 mg per day.
  • the immune effector cells and the second therapeutic agent are administered in no time; the second therapeutic agent may be administered first and then the immune effector cells may be administered; or the immune effector cells may be administered simultaneously; and the second therapeutic agent may be administered to the immune effector cells first.
  • the immune effector cells are administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, before the second therapeutic agent is administered, 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.
  • the immune effector cells are administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, after the second therapeutic agent is administered, 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.
  • the second therapeutic agent is administered continuously until the physician assesses the need to discontinue the drug or may discontinue the drug, such as a physician's assessment of withdrawal after complete remission, or discontinuation of the disease progression. In certain embodiments, the second therapeutic agent is administered continuously for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 Months, 11 months, 12 months or even longer. .
  • the immune effector cells are administered during the continuous administration of the second therapeutic agent.
  • 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 effect 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.
  • therapeutically effective amount refers to a compound, formulation, substance or composition effective to achieve a particular biological result as described herein.
  • the amount for example, but not limited to, an amount or dose sufficient to promote a T cell response.
  • therapeutic agents of the present invention to be administered may be determined by a physician It is determined in consideration of the individual's age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).
  • An effective amount of immune effector cells means, but is not limited to, an increase, increase or prolongation of anti-tumor activity of the immune effector cells; an increase in the number of anti-tumor immune effector cells or activated immune effector cells; promotion of IFN- ⁇ , TNF ⁇ secretion; tumor regression, The number of immune effector cells with tumor shrinkage and tumor necrosis.
  • lymphocyte clearance means that lymphocytes in the subject are not cleared. This includes, but is not limited to, not administering lymphocyte depleting agents, systemic radiation therapy, or a combination thereof, or other means of causing clearance of lymphocytes; however, administration of lymphocyte depleting agents, systemic radiation therapy, or combinations thereof, or other causes of lymphocyte clearance
  • the in vivo lymphocyte clearance rate is 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% will also fall into the category of “unclear” in this application.
  • the lymphocyte clearance rate can be calculated by detecting the number of lymphocytes before clearing lymphocytes and detecting the number of lymphocytes that clear lymphocytes, such as the number of lymphocytes before administration of clearing collateral - lymphocytes after administration of clearing lysate Quantity / number of lymphocytes before administration of clearing pills. Lymphocyte detection can be detected by the number of lymphocytes commonly used by medical personnel, such as blood routine.
  • peptide refers to a compound consisting of amino acid residues covalently linked by a peptide bond.
  • the protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide.
  • a polypeptide includes any peptide or protein comprising two or more amino acids that are bonded to each other by a peptide bond.
  • a "chimeric receptor” that is, a fusion molecule obtained by linking a DNA fragment of different origin or a corresponding cDNA of a protein by a genetic recombination technique, includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • Chimeric receptors include, but are not limited to, chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC).
  • 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.
  • the invention contemplates the modification of the amino acid sequence of a starting antibody or fragment (eg, scFv) that produces a functionally equivalent molecule.
  • a VH or VL of an antigen binding domain of a cancer associated antigen described herein, such as an scFv contained in a CAR can be modified to retain the initial VH or VL framework of the antigen binding domain of a cancer associated antigen described herein.
  • the invention contemplates modifications of the entire CAR construct, such as modification of one or more amino acid sequences of multiple domains of a CAR construct to produce a functionally equivalent molecule.
  • the CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 of the starting CAR construct. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • a transmembrane domain may include one or more additional amino acids adjacent to a transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived ( For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids in the extracellular region) and/or associated with the extracellular region of the protein from which the transmembrane protein is derived One or more additional amino acids (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids in the intracellular region).
  • the transmembrane domain is a domain associated with one of the other domains of the chimeric receptor, eg, in one embodiment, the transmembrane domain can be derived from a signaling domain, costimulatory The same protein from which the domain or hinge domain is derived. In certain instances, the transmembrane domain can be selected or modified by amino acid substitutions to avoid binding of such domains to the transmembrane domain of the same or different surface membrane proteins, for example, to allow interaction with other members of the receptor complex. The interaction is minimized. In one aspect, the transmembrane domain is capable of homodimerization with another chimeric receptor on the cell surface of a cell expressing the chimeric receptor.
  • the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of the native binding partner present in cells expressing the same chimeric receptor.
  • Transmembrane domains can be derived from natural or recombinant sources. When the source is native, the domain may be derived from any membrane-bound protein or transmembrane protein. In one aspect, a transmembrane domain is capable of transmitting a signal to an intracellular domain as long as the chimeric receptor binds to the target antigen.
  • Transmembrane domains specifically used in the present invention may include at least the following transmembrane domains: for example, alpha, beta or scorpion chains of T-cell receptors, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • transmembrane domains for example, alpha, beta or scorpion chains of T-cell receptors, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can comprise at least the following transmembrane regions: eg, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6.
  • transmembrane regions eg, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44,
  • CD49f ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO) -3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.
  • a transmembrane domain can be linked to an extracellular region of a CAR, such as an antigen binding domain of a CAR, via a hinge (eg, a hinge from a human protein).
  • a hinge eg, a hinge from a human protein.
  • the hinge can be a human Ig (immunoglobulin) hinge (eg, an IgG4 hinge, an IgD hinge), a GS linker (eg, a GS linker as described herein), a KIR2DS2 hinge, or a CD8a hinge.
  • the transmembrane domain can be recombinant, in which case it will primarily comprise hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.
  • a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length can form a bond between the transmembrane domain of the CAR and the cytoplasmic region.
  • the glycine-serine duplex provides a particularly suitable linker.
  • cytoplasmic domain includes an intracellular signaling domain.
  • the intracellular signaling domain is typically responsible for the activation of at least one of the normal effector functions of immune cells into which the chimeric receptor has been introduced.
  • effector function refers to the specialized function of a cell.
  • the effector function of a T cell can be, for example, a cytolytic activity or a helper activity, including secretion of a cytokine.
  • intracellular signaling domain refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a particular function.
  • intracellular signaling domain In the case of a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the entire strand as long as it transduces an effector function signal.
  • intracellular signaling domain is meant to include a truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.
  • T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation by TCR (primary intracellular signaling domain) and antigen-independent manner Those that function to provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains).
  • 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 is expressed by immune cells (eg, T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that modulates the signaling pathway for immune cells in a stimulatory 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.
  • the co-stimulator 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, donkey, and the like.
  • 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 cells produced by antibodies or having specific immunity.
  • Those skilled in the art will appreciate and virtually any macromolecule of any protein or peptide can act 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.
  • the antigen need not be encoded only by the full length nucleotide sequence of the gene.
  • the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode a polypeptide that elicits a desired immune response.
  • the antigen does not need to be encoded by a "gene” at all.
  • the antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide.
  • 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 new organism or overexpression product expressed during the process of cell carcinogenesis.
  • 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; CD70; CD123; CD138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13R ⁇ ); interleukin 11 receptor ⁇ (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosine En
  • 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.
  • Cancers include, but are not limited to, breast cancer, glioma, hematological cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, Head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumor of children, bladder cancer, kidney or ureteral cancer, renal pelvis Cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem gli
  • transfection or “transduction” refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell.
  • a “transfected” 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.
  • 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.
  • the refractory cancer can be resistant during treatment.
  • Refractory cancer is also known as resistant 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
  • 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.
  • the treatment is by clinical outcome; the anti-tumor activity of the T cell is increased, enhanced or prolonged; the number of anti-tumor T cells or activated T cells is increased, and the secretion of IFN- ⁇ , TNFa is promoted, compared to the number before treatment, Or a combination of decisions.
  • 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], TNFa secretion, or a combination thereof.
  • treating a tumor comprises reducing the growth, survival or viability of the cancer cell by treatment.
  • the immune effector cells as disclosed herein can be administered to an individual by various routes including, for example, oral or parenteral, such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral. Passive or accelerated absorption through the skin, intravasally, intradermally or separately using, for example, a skin patch or transdermal iontophoresis.
  • 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, WO2018/018958A1.
  • the humanized antibody hu9F2 was expressed by a conventional molecular biology method in the art (the nucleotide sequence is shown as SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 14.
  • the antibody hu9F2 has the HCDR1, HCDR2, HCDR3 shown in SEQ ID NOS: 15, 16, 17, and the LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOS: 18, 19, and 20.
  • the Hu9F2-28Z sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 6), hu9F2scFv (SEQ ID NO: 5), CD8hinge (SEQ ID NO: 7), CD28 transmembrane region (SEQ ID NO: 10), and intracellular signaling.
  • the domain (SEQ ID NO: 8) and the intracellular domain CD3 (CD ID NO: 9) of CD3 are composed.
  • the PRLRSIN-hu9F2-28Z was transfected into 293T and packaged with lentivirus to obtain a lentivirus.
  • T cell activation Human PBMC were cultured in AIM-V medium, 2% human AB type serum was added, 500 U/mL recombinant human IL-2 was added, and CD3/CD28 antibody was added to activate magnetic beads for 48 h.
  • the obtained lentivirus was infected with activated T cells to obtain hu9F2-28Z CART cells, and the flow detection result is shown in Fig. 1B, and the sequence of the CAR was as shown in SEQ ID NO: 22.
  • the hu9F2-28Z CAR T cells of Example 1 were taken and plated in 96-well plates at 4 x 10 4 cells per well, 100 ul.
  • Liver cancer cells PLC/RPF/5 (low expression GPC3), SK-HEP-1 (non-expressing GPC3), Huh7 (high expression GPC3) were plated in 96-well plates at 4000 cells per well, 100 ul. .
  • Sorafenib with different solubility was added to the cells to make six gradients (ie 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 0 ⁇ M six kinds of solubility gradients, 10 ⁇ M, 5 ⁇ M, 1 ⁇ M, 0.5 ⁇ M 0.1 ⁇ M was the dosing group, 0 ⁇ M l was the 0 dosing group), and a set of wells with only medium was set up, which was a blank group. After 48 h, 10 ul of CCK8 reagent (Dojindo) was added to each well for 1 h at 37 ° C, and the absorbance at 450 nm was measured by a microplate reader to calculate the cell viability.
  • CCK8 reagent Dojindo
  • cell viability (%) [A (dosing) - A (blank)] / [A (0 dosing) - A (blank)]
  • sorafenib had no significant inhibitory effect on CAR T cells.
  • Example 3 Killing activity of CAR T cells against tumor cells pretreated with sorafenib
  • Example 4 Sorafenib combined with CAR T cells inhibits hepa1-6-GPC3 subcutaneous tumor
  • This example constructs the transmembrane domain and the intracellular domain of CAR using the gene sequence of the mouse.
  • C57BL/6 mice normal immune system mice
  • Vehicle group no group given to sorafenib
  • Sorafenib group sorafenib alone administration group
  • CAR T+vehicle group hu9F2-m28Z CAR T cells and solvent group were administered;
  • UTD+sorafenib group T cells given uninfected CAR and sorafenib group;
  • CAR T+sorafenib group hu9F2-m28Z CAR T cells and sorafenib group were administered.
  • Hepa1 cell model (Hepa1-6-GPC3) of mouse hepatoma cells overexpressed by human and mouse chimeric GPC3 (SEQ ID NO: 11) was established by conventional methods in molecular biology. As shown in Figure 4, Hepa 1-6 cells overexpressing GPC3 were confirmed by flow cytometry.
  • Hepa1-6-GPC3 cells in the logarithmic growth phase and well-growth were harvested, and 1 ⁇ 10 7 target cells were inoculated subcutaneously into the right axilla of C57BL/6 mice (normal mice with normal immune system).
  • mice spleen T lymphocytes were taken to construct hu9F2-m28Z CAR T cells.
  • the construction method was as follows:
  • Coding sequence of mouse CD8 ⁇ signal peptide (SEQ ID NO: 4), hu9F2 scFv (SEQ ID NO: 5), murine CD8 ⁇ hinge region and transmembrane region coding sequence (SEQ ID NO: 1), murine CD28 intracellular domain
  • the coding sequence (SEQ ID NO: 2), the coding sequence of the murine CD3 sputum intracellular domain (SEQ ID NO: 3) were ligated in turn, and the hu9F2-m28Z gene fragment was obtained by in vitro gene synthesis, and the cleavage sites were digested with MluI and SalI.
  • the IRES-GFP fragment in the retroviral vector MSCV-IRES-GFP (purchased from Addgene) was used to obtain the recombinant vector MSCV-hu9F2-m28Z. 293T cells were infected with MSCV-hu9F2-m28Z to obtain a retrovirus after packaging.
  • Mouse spleen T lymphocytes were obtained and activated with Dynabeads Mouse T-activator CD3/CD28. After activation, retrovirus infection was added for 12 hours to obtain hu9F2-m28Z CAR T cells.
  • sorafenib or solvent dissolve sorafenib in solvent (5% DMSO, 45% PEG400, 50% H20), for Sorafenib group, UTD+sorafenib group, CAR Mice in the T+sorafenib group were intragastrically administered, and the dose per mouse was 7.5 mg/kg.
  • the vehicle group and the CAR T+vehicle group were administered with a solvent.
  • Sorafenib's first dosing diary was day 0, administered once a day for 5 consecutive days.
  • the tumor volume calculation formula is: (length * width 2 ) / 2.
  • the tumor volume test results are shown in Fig. 5.
  • the CAR T+sorafenib group has a significant effect of inhibiting tumor growth (Two-way ANOVA with Bonferroni post-tests, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01, *** indicates p ⁇ 0.001).
  • Example 5 Detection of the effects of sorafenib on degranulation, proliferation and killing of CAR T cells
  • the above-treated hu9F2-28Z CAR T cells and PLC/RPF/5 cells were plated in a 24-well plate at a ratio of 1:1, and different concentrations of sorafenib (0, 1, 5, 10 ⁇ M) were added. After 24 hours of culture, each group was provided with 3 duplicate wells.
  • Experimental group plating The number of target cells was 10000/well, and the effector cells were hu9F2-28Z CAR T cells treated with sorafenib.
  • the effective target ratios were 20:1, 10:1, 5:1, and 2.5:1, respectively, and different numbers of effector cells were added according to different target ratios.
  • Each group has 5 duplicate holes.
  • Control plate plating In this experiment, other control groups were also needed to exclude the interference of spontaneous release of LDH in effector cells, target cells, and interference of LDH contained in the medium. They were: the LDH control group with the maximum release of target cells; the LDH control group was spontaneously released from the target cells; the LDH control group was spontaneously released from the effector cells; the empty culture background group was added; and the lysate group was added to the empty culture background. Each group has 5 duplicate holes.
  • the cells were incubated in a 37 ° C, 5% CO 2 incubator, and transferred to an enzyme plate for color development of the substrate.
  • the absorbance at 490 nm was measured by a microplate reader to calculate the cell killing toxicity.
  • cytotoxicity (%) (experimental group - effector cell spontaneous group - target cell spontaneous group - background group) / (target cell maximal group - target cell spontaneous group - lysate background group).
  • sorafenib does not affect the degranulation ability, proliferation and killing toxicity to CAR cells.
  • Example 1 The 1 ⁇ 10 5 hu9F2-28Z CAR T cells in Example 1 were plated in a 24-well plate of GPC3 protein-coated plates, and different concentrations of sorafenib (0, 1, 5, 10 ⁇ M) were added. The cells were cultured at 37 ° C in a 5% CO 2 incubator, and the supernatant of the cells was collected by centrifugation, and the concentrations of human cytokines IL2, IFN- ⁇ , and TNF- ⁇ were measured.
  • ns represents p>0.05; *p ⁇ 0.05; **p ⁇ 0.01; *p ⁇ 0.001, 1way ANOVA.
  • Example 7 Sorafenib and human CAR T cells in combination with liver cancer
  • Day 11 The volume of the subcutaneous tumor of the mouse was about 150 mm 3 , and the mice were randomly divided into 6 groups according to the tumor volume, 5-6 per group. Sorafenib was dissolved in a solvent (5% DMSO, 45% PEG400, 50% H2O). No solvent is given to the group of sorafenib.
  • Control group Day 11 was administered with a solvent once a day, and the administration volume was the same as that of the experimental group for two weeks.
  • Sora 7.5 Day 11 was administered to mice with 7.5 mg/kg of sorafenib once daily for two weeks.
  • Sora 30 Day 11 was administered to mice with 30 mg/kg of sorafenib once daily for two weeks.
  • CAR+Vehicle Day11 was administered to the solvent once a day, and the administration volume was the same as that of the experimental group for two weeks; on the same day (Day11), after the solvent administration, 2 ⁇ 10 6 hu9F2-28Z CAR T cells in Example 1 were injected into the tail vein.
  • CAR+Sora7.5 Day11 was administered to mice with 7.5 mg/kg of sorafenib once daily for two weeks; on the same day (Day11), after sorafenib administration, the tail vein was injected with 2 in Example 1. ⁇ 10 6 hu9F2-28Z CAR T cells.
  • CAR+Sora30 Day11 was administered to mice with 30 mg/kg of sorafenib once a day for two weeks; on the same day (Day11), after sorafenib administration, the tail vein was injected with 2 ⁇ 10 6 in Example 1. hu9F2-28Z CAR T cells.
  • tumor volume (tumor length ⁇ tumor width 2 )/2, and the results are shown in Figure 9A, compared with the treatment group alone. Compared with the combination of sorafenib and hu9F2-28Z CAR T cells, tumor growth was significantly inhibited (P ⁇ 0.001, 2way ANOVA).
  • Tumor inhibition rate was calculated according to the control group. On the 43rd day after tumor inoculation (Day43), the tumor inhibition rate of each group was 18.8% for Sora 7.5, 35.8% for Sora30, 25.3% for CAR+Vehicle, CAR+Sora7. 5 is 68.7%, and CAR+Sora30 is 75.2%.
  • mice On day 43 (Day 43), mice were euthanized, the subcutaneous tumors were exfoliated, and the tumor weight was weighed. The results are shown in Figure 9B. Compared with the treatment group alone, sorafenib and hu9F2-28Z CAR T cells were combined (7.5 Tumor weight was significantly reduced in mg/kg or 30 mg/kg) (*p ⁇ 0.05; **p ⁇ 0.01 or ***p ⁇ 0.001, 1way ANOVA).
  • mice treated with hu9F2-28Z CAR T cells and sorafenib were compared to mice treated with hu9F2-28Z CAR T cells alone.
  • There were more CD3+ human CAR T cells in the tissues (*p ⁇ 0.05; **p ⁇ 0.01, 1way ANOVA), and no staining of human CAR T cells with CD3+ was observed in the group not receiving hu9F2-28Z CAR T cell therapy. .
  • the tissue sections were subjected to immunohistochemical staining, and the nuclei in the tumor tissues were counterstained using hematoxylin.
  • the hu9F2-28Z was combined with the control group and the hu9F2-28Z CAR T cells alone.
  • Mice treated with CAR T cells and sorafenib (7.5 mg/kg or 30 mg/kg) had more cleaved caspase-3 positive cells in tumor tissues.
  • Example 8 In vitro detection of synergy between sorafenib and human CAR T cells
  • the human liver cancer cell PLC/RPF/5 was labeled with the CellTrace dye according to the CellTrace Violet kit experimental procedure.
  • the labeled PLC/RPF/5 was mixed with hu9F2-28z CAR T cells in a 1:1 ratio, and different concentrations of sorafenib (0, 1, 5, 10 ⁇ M) were added to the cell culture incubator for 48 hours.
  • the cells were centrifuged and collected, and the cell pellet was resuspended using the FITC Annexin V Apoptosis Detection Kit (purchased from BD) in staining buffer, and stained with Annexin V-FITC dye.
  • On-machine detection The stained cells were collected by centrifugation, the staining buffer was resuspended, and the flow was detected on the machine.
  • the purple light signal is collected in the BV421 channel and represents all tumor cells.
  • Green light is collected in the FITC channel, representing tumor cells that have undergone apoptosis.
  • FlowJo software processes the data.
  • Fig. 11 The results are shown in Fig. 11.
  • the apoptosis rate of tumor cells in combination with hu9F2-28Z CAR T cells and sorafenib was significantly increased, and the number of apoptosis in tumor cells was significantly increased (*p ⁇ 0.05; **p ⁇ 0.01). Or ***p ⁇ 0.001, 1way ANOVA).
  • Example 9 Effect of CAR T cells combined with regefenib on the treatment of transplanted liver cancer in mice
  • Example 4 Under the procedure of Example 4, a transplanted tumor model of Hepa1-6-chGPC3 cells C57BL/6 and hu9F2-m28Z CAR T cells were constructed.
  • mice On the 8th day after tumor cell implantation, the mice were randomly divided into three groups according to the tumor volume. The mice in the regofenib treatment group and the regofenib + CAR T combination group were intragastrically administered with 10 mg/kg of rego. Nie, the control group was given a vehicle once a day for ten days.
  • Example 10 Clinical study of the combination therapy of sorafenib and CAR T cells
  • Subject A was 60 years old, weighing 77 kg, height: 170 cm. After diagnosis for hepatocellular carcinoma, after 7 months of surgery, it was still in progress. Immunohistochemistry showed GPC3 positive (70%, ++ ⁇ +++). ). In this case, the patient receives the treatment of the technical solution of the present invention.
  • Example 2 Following the operation of Example 1, after lentivirus infection of T cells of a patient, hu9F2-28Z CAR T cells were prepared.
  • lymphocyte clearance treatment including fludarabine about 39 mg / day ⁇ 4 days (about 20 mg / m 2 / day)
  • the cyclophosphamide is about 1000 mg/day x 2 days (about 500 mg/m 2 /day).
  • AFP was reduced by 35% compared to that given to CAR-T cells, and on day 13 after CAR-T cell administration, AFP was reduced by 58.2% compared to before administration of CAR-T cells.
  • the CAR of the CAR-T cell targeting GPC3 is exemplified to have the amino acid sequence shown in SEQ ID NO: 22, and those skilled in the art can adopt the SEQ ID according to the teachings of the above examples. NO: a sequence shown by 21, 23, or 24.
  • G-cell-targeting CAR-T cells are employed, and those skilled in the art can use CAR-T cells targeting other targets, such as CARs targeting EGFR, according to the teachings of the present application.
  • -T cells exemplary, the sequence of the scFv of the EGFR-targeting CAR-T cells is as shown in SEQ ID NO: 12
  • CAR-T cells targeting CLD18A2 exemplary, CAR targeting CLD18A2
  • the sequence of the scFv of the T cell is as shown in SEQ ID NO: 13.
  • sequence information involved in the present invention is as follows:

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Abstract

本发明提供了一种治疗肿瘤的方法，对患有肿瘤的个体施用免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体，所述的第二治疗剂为式I所示的化合物，或其医药学上可接受的盐。

Description

细胞免疫治疗的组合 技术领域
本发明属于细胞免疫治疗领域，具体涉及免疫效应细胞和激酶抑制剂类化疗剂联合用于抗肿瘤治疗。
背景技术
近年来，细胞免疫治疗如CAR-T细胞治疗在血液瘤治疗中显示出惊人的治疗效果，目前已经超过200项CAR-T细胞用于血液瘤治疗的临床实验(Clinical development of CAR T cells-challenges and opportunities in translating innovative treatment concepts,Jessica Hartmann et al.,EMBO Molecule Medicine,Published on line,August 1,2017)。然而对于实体瘤的治疗却难以达到血液瘤治疗的效果。
这是因为对于血液瘤而言，CAR-T细胞通过静脉输入能够更容易的接触到肿瘤细胞实现杀伤，而CAR-T细胞想要归巢到实体瘤的肿瘤组织则具有更大的难度。并且，实体瘤通常具有复杂的动态的肿瘤微环境，能够使肿瘤细胞、良性细胞、基质细胞、血管细胞等相互作用，另外，肿瘤微环境中还存在由细胞因子和生长因子相互作用的网络，因此，CAR-T治疗在用于实体瘤治疗时，通常疗效不佳。
发明内容
本发明的目的在于提供一种肿瘤治疗方法，以提高免疫细胞治疗特别是CAR-T细胞治疗在实体瘤中应用效果。
在本发明得第一方面，提供了一种治疗肿瘤的方法，对患有肿瘤的个体施用免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体，所述第二治疗剂为激酶抑制剂。
在具体实施方式中，所述的第二治疗剂为式I所示的化合物，或其医药学上可接受的盐，
Figure PCTCN2019074535-appb-000001
其中，Ar’是非取代或取代的苯基，取代基选自卤素和C1-10烷基，
M是一个或多个桥连基，选自-O-或-S-，
Py(X)是X取代的吡啶基，X为-C(O)R x，其中，R x是NR aR b，其中的R a和R b各自是：
a)氢，
b)C1-10烷基，
c)羟基取代的C1-10烷基，
d)含1-3个N，S或O的杂原子的C3-12环烷基，或
e)-OSi(R f)3，R f是C1-10烷基，
Ar”是非取代或取代苯基，取代基选自卤素或Wn，n＝0-3，W选自：
a)C1-10烷基，
b)C1-10烷氧基，
c)C1-10卤代烷基
d)含1-3个N，S或O杂原子的C3-12杂芳基，该杂芳基可被C1-10烷基取代。
在本发明的第二方面，还提供了一种降低癌细胞生长、存活或活力的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体，
所述的第二治疗剂为式I所示的化合物，或其医药学上可接受的盐，
Figure PCTCN2019074535-appb-000002
其中，Ar’是非取代或取代的苯基，取代基选自卤素和C1-10烷基，
M是一个或多个桥连基，选自-O-或-S-，
Py(X)是X取代的吡啶基，X为-C(O)R x，其中，R x是NR aR b，其中的R a和R b各自是：
a)氢，
b)C1-10烷基，
c)羟基取代的C1-10烷基，
d)含1-3个N，S或O的杂原子的C3-12环烷基，或
e)-OSi(R f)3，R f是C1-10烷基，
Ar”是非取代或取代苯基，取代基选自卤素或Wn，n＝0-3，W选自：
a)C1-10烷基，
b)C1-10烷氧基，
c)C1-10卤代烷基
d)含1-3个N，S或O杂原子的C3-12杂芳基，该杂芳基可被C1-10烷基取代。
在一优选方案中，采用上述治疗肿瘤的方法，或者降低癌细胞生长、存活或活力的方法，在对患有肿瘤的个体施用免疫效应细胞之前，所述的个体不进行淋巴细胞清除。
在一优选方案中，采用上述治疗肿瘤的方法，或者降低癌细胞生长、存活或活力的方法时，所述免疫效应细胞和第二治疗剂的治疗效果大于所述免疫效应细胞和第二治疗剂任一单独使用的效果。
在另一优选方案中，所述的Ar”是取代苯基，取代基选自氯、溴、氟、三氟甲基，甲氧基和叔丁基中的任意一种或其组合。
在另一优选方案中，所述的M是-O-。
在另一优选方案中，所述的Ra和Rb各自是H或C1-10烷基，优选的，Ra和Rb分别为H和甲基。
在另一优选方案中，所述医药学上可接受的盐选自：a)无机酸和有机酸的碱式盐，所述酸选自：盐酸、氢溴酸、硫酸、磷酸、甲磺酸、三氟甲磺酸、苯磺酸，对甲苯磺酸，1-萘磺酸，2-萘磺酸，乙酸、三氟乙酸、苹果酸、酒石酸、柠檬酸、乳酸、草酸、琥珀酸、富马酸，马来酸、苯甲酸、水杨酸、苯基乙酸和杏仁酸；b)有机和无机碱的酸式盐，所述阳离子选自：碱金属阳离子，碱土金属阳离子，铵阳离子，脂族基取代的铵阳离子，和芳香基取代的铵阳离子。
在具体实施方式中，所述的第二治疗剂选自下列任一所示的化合物或其医药学上可接受的盐：
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶基硫)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(4-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-2-氟-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶基硫)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧 基))苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲。
在具体实施方式中，所述的第二治疗剂选自下列式II或式III所示的化合物，或其医药学上可接受的盐。
Figure PCTCN2019074535-appb-000003
在具体实施方式中，所述的第二治疗剂的医药学上可接受的盐选自甲苯磺酸盐、苯磺酸盐、盐酸盐、甲磺酸盐。
在具体实施方式中，所述的第二治疗剂为式II化合物或者式II化合物的水合物，优选的为一水合物。
在另一优选方案中，针对受试者个体，第二治疗剂每天的给药量为100-1000mg，优选的，每天的给药量为200-800mg，更优选的，每天的给药量为400-800mg。
在另一优选方案中，针对受试者个体，所述第二治疗剂每天给予1-3次，优选每天给予第二治疗剂2次。
在另一优选方案中，针对受试者个体，所述免疫效应细胞每次的给予量为约1x10 5～1x10 8细胞/千克受试者体重，进一步优选，每次的给予量为约 1x10 5～1x10 7细胞/千克受试者体重。
在另一优选方案中，免疫效应细胞和第二治疗剂给予时间不分先后；可以先给予第二治疗剂再给予免疫效应细胞；也可以同时给药；还可以先给予免疫效应细胞再给予第二治疗剂，优选在给予所述第二治疗剂的期间给予所述免疫效应细胞。
在具体实施方式中，所述的第二治疗剂为口服给药。
在另一优选方案中，所述受体选自：嵌合抗原受体(Chimeric Antigen Receptor，CAR)、T细胞受体(T cell receptor，TCR)、T细胞融合蛋白(T cell fusionprotein，TFP)、T细胞抗原耦合器(T cell antigen coupler，TAC)或其组合。
在具体的实施方式中，所述的嵌合抗原受体包括：
(i)特异性结合所述抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ的胞内域；或
(ii)特异性结合所述抗原的抗体或其片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ的胞内域；或
(iii)特异性结合所述抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ的胞内域。
在具体的实施方式中，所述肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相 关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
在具体实施方式中，所述肿瘤抗原为实体瘤抗原。优选的所述实体瘤抗原为血管内皮生长因子受体、EGFR、EGFRvIII、GPC3、或Claudin18.2。更优选的，所述实体瘤抗原为GPC3、EGFR、EGFRvIII、或血管内皮生长因子受体。
在具体实施方式中，所述的血管内皮生长因子受体为血管内皮生长因子受体2(VEGFR2)。
在具体实施方式中，所述实体瘤抗原为GPC3。
在具体实施方式中，所述特异性识别肿瘤抗原的抗体是靶向磷脂酰肌醇蛋白聚糖3(GPC3)的抗体。
在一优选方案中，特异性识别肿瘤抗原的抗体含有SEQ ID NO:17、18、19所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:20、21、22所示的LCDR1、LCDR2、LCDR3。
在具体实施方案中，特异性识别肿瘤抗原的抗体的氨基酸序列与SEQ ID NO:14所示的序列具有至少90％的同一性。
在具体实施方案中，所述嵌合抗原受体的氨基酸序列与SEQ ID NO:21、22、23或24所示的序列具有至少90％的同一性。
在一优选方案中，所述治疗由临床结果：通过免疫效应细胞的抗肿瘤活性增加、增强或延长；与治疗前的数目相比较，抗肿瘤免疫效应细胞或活化免疫效应细胞数目的增加，或其组合决定。
在具体的实施方式中，所述临床结果选自肿瘤消退；肿瘤缩小；肿瘤坏死；通过免疫系统的抗肿瘤应答；肿瘤扩大、复发或扩散或其组合。
在具体的实施方式中，治疗效应通过免疫效应细胞的存在、或指示T细胞炎症的基因标记的存在或其组合预测，优选地，通过检测IFN-γ、TNFα水平变化来预测。
在具体的实施方式中，其中所述肿瘤包括：血液癌症，乳腺癌，脑胶质瘤，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤， 肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶。优选的，选自肝癌、肾细胞癌、肺鳞癌、甲状腺癌。
在具体的实施方式中，所述的免疫效应细胞包括：T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞或其组合；优选地，所述免疫效应细胞选自自体T细胞、同种异体T细胞或同种异体NK细胞，更优选地，所述T细胞为自体T细胞。
在本发明的第三方面，本发明提供了一种给药系统，所述给药系统中包含有免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体。
所述的第二治疗剂为式I所示的化合物，或其医药学上可接受的盐，
Figure PCTCN2019074535-appb-000004
其中，Ar’是非取代或取代的苯基，取代基选自卤素和C1-10烷基，
M是一个或多个桥连基，选自-O-或-S-，
Py(X)是X取代的吡啶基，X为-C(O)R x，其中，R x是NR aR b，其中的R a和R b各自是：
a)氢，
b)C1-10烷基，
c)羟基取代的C1-10烷基，
d)含1-3个N，S或O的杂原子的C3-12环烷基，或
e)-OSi(R f)3，R f是C1-10烷基，
Ar”是非取代或取代苯基，取代基选自卤素或Wn，n＝0-3，W选自：
a)C1-10烷基，
b)C1-10烷氧基，
c)C1-10卤代烷基
d)含1-3个N，S或O杂原子的C3-12杂芳基，该杂芳基可被C1-10烷基取代。
在一优选方案中，所述的Ar”是取代苯基，取代基选自氯、溴、氟、三 氟甲基，甲氧基和叔丁基中的任意一种或其组合。
在另一优选方案中，所述的M是-O-。
在另一优选方案中，所述的Ra和Rb各自是H或C1-10烷基，优选的，Ra和Rb分别为H和甲基。
在另一优选方案中，所述医药学上可接受的盐选自：a)无机酸和有机酸的碱式盐，所述酸选自：盐酸、氢溴酸、硫酸、磷酸、甲磺酸、三氟甲磺酸、苯磺酸，对甲苯磺酸，1-萘磺酸，2-萘磺酸，乙酸、三氟乙酸、苹果酸、酒石酸、柠檬酸、乳酸、草酸、琥珀酸、富马酸，马来酸、苯甲酸、水杨酸、苯基乙酸和杏仁酸；b)有机和无机碱的酸式盐，所述阳离子选自：碱金属阳离子，碱土金属阳离子，铵阳离子，脂族基取代的铵阳离子，和芳香基取代的铵阳离子。
在具体实施方式中，所述的第二治疗剂选自下列任一所示的化合物或其医药学上可接受的盐：
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶基硫)苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(4-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-氯-3-(三氟甲基)苯基)-N’-2-氟-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶基硫)苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(4-溴-3-(三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲。
在具体实施方式中，所述的第二治疗剂选自下列式II或式III所示的化合物，或其医药学上可接受的盐。
Figure PCTCN2019074535-appb-000005
Figure PCTCN2019074535-appb-000006
式II或式III所示的化合物的化学名称分别是：
N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲、
N-(4-氯-3-(三氟甲基)苯基)-N’-2-氟-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲。
在具体实施方式中，所述的医药学上可接受的盐为甲苯磺酸盐、苯磺酸盐、盐酸盐、甲磺酸盐。
在另一优选方案中，针对受试者个体，第二治疗剂每天给药量为100-1000mg，优选的，每天给药量为200-800mg，更优选的，每天给药量为400-800mg。
在另一优选方案中，针对受试者个体，所述第二治疗剂每天给予1-3次，优选每天给予第二治疗剂2次。
在另一优选方案中，针对受试者个体，所述免疫效应细胞每次的给予量为约1x10 5～1x10 8细胞/千克受试者体重，进一步优选，每次的给予量为约1x10 5～1x10 7细胞/千克受试者体重。
在另一优选方案中，免疫效应细胞和第二治疗剂给予时间不分先后；可以先给予第二治疗剂再给予免疫效应细胞；也可以同时给药；还可以先给予免疫效应细胞再给予第二治疗剂，优选在给予所述第二治疗剂的期间给予所述免疫效应细胞。
在具体实施方式中，所述的第二治疗剂为口服给药。
在另一优选方案中，所述受体选自：嵌合抗原受体(Chimeric Antigen Receptor，CAR)、T细胞受体(T cell receptor，TCR)、T细胞融合蛋白(T cell fusionprotein，TFP)、T细胞抗原耦合器(T cell antigen coupler，TAC)或其组合。
在具体的实施方式中，所述的嵌合抗原受体包括：
(i)特异性结合所述抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ的胞内域；或
(ii)特异性结合所述抗原的抗体或其片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ的胞内域；或
(iii)特异性结合所述抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ的胞内域。
在具体的实施方式中，所述肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋 白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
在具体实施方式中，所述肿瘤抗原为实体瘤抗原，优选的所述实体瘤抗原为血管内皮生长因子受体、EGFR、EGFRvIII、GPC3、或Claudin18.2，更优选的，所述实体瘤抗原为GPC3、EGFR、EGFRvIII、或血管内皮生长因子受体。
在一实施方式中，所述的血管内皮生长因子受体为血管内皮生长因子受体2(VEGFR2)。
在具体实施方式中，所述肿瘤抗原为GPC3。
在具体实施方式中，所述特异性识别肿瘤抗原的抗体是靶向磷脂酰肌醇蛋白聚糖3(GPC3)的抗体。
在一优选方案中，特异性识别肿瘤抗原的抗体含有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。
在具体实施方案中，特异性识别肿瘤抗原的抗体的氨基酸序列与SEQ ID  NO:14所示的序列具有至少90％的同一性。
在具体实施方案中，所述嵌合抗原受体的氨基酸序列与SEQ ID NO:21、22、23或24所示的序列具有至少90％的同一性。
在一优选方案中，所述治疗由临床结果：通过免疫效应细胞的抗肿瘤活性增加、增强或延长；与治疗前的数目相比较，抗肿瘤免疫效应细胞或活化免疫效应细胞数目的增加，或其组合决定。
在具体的实施方式中，所述临床结果选自肿瘤消退；肿瘤缩小；肿瘤坏死；通过免疫系统的抗肿瘤应答；肿瘤扩大、复发或扩散或其组合。
在具体的实施方式中，治疗效应通过免疫效应细胞的存在、或指示T细胞炎症的基因标记的存在或其组合预测，优选地，通过检测IFN-γ、TNFα水平变化来预测。
在具体的实施方式中，其中所述肿瘤包括：血液癌症，乳腺癌，脑胶质瘤，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶。优选的，选自肝癌、肾细胞癌、肺鳞癌、甲状腺癌。
在具体的实施方式中，所述的免疫效应细胞包括：T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞或其组合；优选地，所述免疫效应细胞选自自体T细胞、同种异体T细胞或同种异体NK细胞，更优选地，所述T细胞为自体T细胞。
在本发明的第四方面，提供了表达有识别肿瘤抗原的受体的免疫效应细胞在制备药物中的应用，其特征在于，所述药物与索拉非尼联用。
在具体实施方式中，上述应用时用于在人类患者中治疗肿瘤，其中将所述细胞和索拉非尼配制成能提供单独使用所述细胞和索拉非尼获得的效果更好或更大的治疗效果，即所述细胞和索拉非尼配制成能提供比所述试剂各自单独使用时的效果更大的治疗效果。
在本发明的第五方面，提供了一种用于治疗肿瘤的试剂盒，其特征在于，所述试剂盒包含：
1)表达有识别肿瘤抗原的受体的免疫效应细胞；
2)索拉非尼；
3)用于包含以上1)和2)所述物质的容器；和
4)利用所述试剂盒治疗肿瘤的给药说明书；
其中，所述免疫效应细胞和索拉非尼配制成能提供比所述试剂各自单独使用时的效果更大的治疗效果；优选地，所述免疫效应细胞为CAR T细胞，更优选地，所述CAR T细胞特异性识别EGFR、EGFRvIII，磷脂酰肌醇蛋白聚糖3、claudin 18.2、BCMA。
在具体实施方式中，所述CAR T细胞特异性识别磷脂酰肌醇蛋白聚糖3。
在本发明的第六方面，提供了一种用于治疗肿瘤的制品，其特征在于，所述制品包含：免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体。
所述制品包含：
1)表达有识别肿瘤抗原的受体的免疫效应细胞；
2)索拉非尼式II所示的化合物；
3)用于包含以上1)和2)所述物质的容器；和
4)利用所述制品治疗肿瘤的给药说明书；
其中免疫效应细胞表达有识别肿瘤抗原的嵌合抗原受体，所述免疫效应细胞和第二治疗剂与上述本发明第一方面、第二方面、以及第三方面中涉及的定义相同。
在本发明的第六方面，提供表达有识别肿瘤抗原的受体的免疫效应细胞和第二治疗剂在制备药物或制品中的用途，所述药物用于在人类患者中治疗肿瘤，其中将所述细胞和第二治疗剂配制成的药物能提供比所述细胞和第二治疗剂各自单独使用时更优或更大的治疗效果。
免疫效应细胞和第二治疗剂与上述本发明第一方面、第二方面、以及第三方面中涉及的定义相同。
在本发明的一个具体的实施方式中，提供治疗肿瘤或者降低癌细胞生长、存活或活力的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞 和索拉非尼，所述免疫效应细胞表达有识别肿瘤抗原的嵌合抗原受体。该嵌合抗原受体具有特异性识别肿瘤抗原的抗体或其片段、跨膜结构域和细胞质信号传导结构域。其中所述特异性识别肿瘤抗原的抗体或其片段含有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。其中，所述肿瘤为肝癌，所述癌细胞为肝癌细胞。
在本发明的一个具体的实施方式中，提供治疗肿瘤或者降低癌细胞生长、存活或活力的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和瑞戈非尼，所述免疫效应细胞表达有识别肿瘤抗原的嵌合抗原受体。该嵌合抗原受体具有特异性识别肿瘤抗原的抗体或其片段、跨膜结构域和细胞质信号传导结构域。其中所述特异性识别肿瘤抗原的抗体或其片段含有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。其中，所述肿瘤为肝癌，所述癌细胞为肝癌细胞。
在本发明的一个具体的实施方式中，提供了一种用于治疗肿瘤的制品或试剂盒，其特征在于，所述制品或试剂盒包含：
1)免疫效应细胞，
2)索拉非尼或瑞戈非尼，
3)用于包含以上1)和2)所述物质的容器；和
4)利用所述制品治疗肿瘤的给药说明书；
其中，所述免疫效应细胞表达有识别肿瘤抗原的嵌合抗原受体。该嵌合抗原受体具有特异性识别肿瘤抗原的抗体或其片段、跨膜结构域和细胞质信号传导结构域。其中所述特异性识别肿瘤抗原的抗体或其片段含有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。其中，所述肿瘤为肝癌，所述癌细胞为肝癌细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
本发明的有益效果：
1、本发明所提供的式I所示的化合物和免疫效应细胞联用，能够显著 提高杀伤肿瘤细胞的能力。
2、采用本发明的治疗方案能够对抗癌症微环境中的免疫抑制，从而对于实体瘤的作用显著增强，对于难治性和进行性的癌症也具有较好的效果。
附图说明
图1A为PRRLSIN-hu9F2-28Z质粒图谱；图1B为CAR T细胞阳性率检测。
图2显示了索拉非尼对肝癌细胞及CAR T细胞的毒性检测。
图3显示了体外检测CAR T细胞对经索拉非尼处理后的肿瘤细胞的杀伤能力。
图4显示了流式检测Hepa1-6细胞上GPC3的表达。
图5显示了索拉非尼与CAR T细胞联用对hepa1-6-GPC3皮下瘤的肿瘤体积的抑制效果。
图6显示了索拉非尼与CAR T细胞联用对hepa1-6-GPC3皮下瘤的肿瘤重量的抑制效果。
图7显示了体外检测索拉非尼处理后的CAR T细胞的脱颗粒能力(图7A)、增殖能力(图7B)及杀伤能力(图7C)。
图8显示了检测索拉非尼对人CAR T细胞的细胞因子分泌IL-2(图8A)、IFN-γ(图8B)和TNF-α(图8C)的影响。
图9显示了索拉非尼和人CAR T细胞联合治疗肝癌的肿瘤体积(9A)和肿瘤重量(9B)。
图10显示了CAR T细胞瘤内浸润情况(图10A)和细胞凋亡情况(图10B、10C)。
图11显示了体外检测索拉非尼联合人CAR T细胞协同促进PLC/RPF/5细胞的凋亡情况。
图12显示了CAR T细胞与瑞戈非尼联用对小鼠肝癌移植瘤的治疗结果。
图13显示了利用CAR T细胞与索拉非尼联合治疗后受试者的影像学图像。
具体实施方式
本发明涉及免疫效应细胞和第二治疗剂(式I所示的化合物)联合应用于治疗肿瘤，应理解本发明并不限于所述的方法和实验条件。除非本文中专门定义，所使用的所有技术和科学术语具有在基因治疗、生物化学、遗传学、分子生物学、以及药物化学领域内的技术人员通常理解的相同含义。
Figure PCTCN2019074535-appb-000007
类似或等效于本文中描述的方法和材料都可以在本发明的实践或测试中使用。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书包括定义为准。此外，除非另有说明，材料、方法和实施例仅是说明性的，而并非进行限制。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。参见，例如，Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
本发明至少部分源于下述认知：连续地、以任一次序地或基本上同时地给予第二治疗剂和免疫效应细胞的一个或多个周期和/或剂量的组合治疗方案，在治疗一些受试者的癌症时可以是更有效的增加、增强或延长免疫细胞的活性和/或数目，从而达到抗肿瘤效果。
本文使用的术语约是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”或“包含”可意指按照在该领域中的实际的标准偏差在1以内或多于1。或者“约”或“包含”可意指至多10％(即±10％)的范围。例如，约5mg可包括在4.5mg与5.5mg之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”或“包含”应假定为在该特定值或组成的可接受误差范围内。
本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值，除非另外指出。
本文中所述的“剂量”，可以是以重量为基础计算的剂量或以体表面积(BSA)为基础计算的剂量表示。以重量为基础计算的剂量是以患者体重为基础所计算出的对于患者给予的剂量，例如mg/kg。以BSA为基础计算的剂量是以患者的表面积为基础所计算出的对患者给予的剂量，例如mg/m 2。
在具体实施方式中，本发明采用的第二治疗剂为式I所示的化合物，或其医药学上可接受的盐。
Figure PCTCN2019074535-appb-000008
其中，Ar’是非取代或取代的苯基，取代基选自卤素和C1-10烷基，
M是一个或多个桥连基，选自-O-或-S-，
Py(X)是X取代的吡啶基，X为-C(O)R x，其中，R x是NR aR b，其中的R a和R b各自是：
a)氢，
b)C1-10烷基，
c)羟基取代的C1-10烷基，
d)含1-3个N，S或O的杂原子的C3-12环烷基，或
e)-OSi(R f)3，R f是C1-10烷基，
Ar”是非取代或取代苯基，取代基选自卤素或Wn，n＝0-3，W选自：
a)C1-10烷基，
b)C1-10烷氧基，
c)C1-10卤代烷基
d)含1-3个N，S或O杂原子的C3-12杂芳基，该杂芳基可被C1-10烷基取代。
在本发明中，术语“C1-10烷基”指的是任意的含有1－10个碳原子的直链或支链基团，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、叔戊基、正己基、正庚基、正辛基、异辛基、2-乙基己基、正壬基、异壬基、正癸基等。
术语“C1-10烷氧基”指的是任意上述C1-C10烷基其通过氧原子(-O-)连接到分子的其余部分。
术语“卤素”是指氟元素、氯元素、溴元素、碘元素。
术语“卤代烷基”是指氟代烷基、氯代烷基、溴代烷基、碘代烷基。
术语“C3-12环烷基”是指具有3至12个碳原子的非芳族、饱和或不饱和、单环或双环烃环。示例“环烷基”包括但不限于环丙基、环丁基、环戊基、环己基和环庚基等。
术语"杂芳基"是指芳族的杂环，通常为具有1至3个选自N、O或S的杂原子的5-至8-元的杂环；杂芳基环可以任选地进一步稠合或连接于芳族和非芳族的碳环和杂环。所述杂芳基的非限制性的实例为例如吡啶基、吡嗪基、嘧啶基、哒嗪基、吲哚基、咪唑基、噻唑基、异噻唑基、噻噁唑基、吡咯基、苯基-吡咯基、呋喃基、苯基-呋喃基、噁唑基、异噁唑基、吡唑基、噻吩基、苯并噻吩基、异二氢吲哚基、苯并咪唑基、吲唑基、喹啉基、异喹啉基、1,2,3-三唑基、1-苯基-1,2,3-三唑基、2,3-二氢吲哚基、2,3-二氢苯并呋喃基、2,3-二氢苯并噻吩基、苯并吡喃基、2,3-二氢苯并噁嗪基、2,3-二氢喹喔啉基等。
从所有上述描述中，对本领域技术人员显而易见的是，其名称是复合名称的任意基团，例如“芳基氨基”，应该指的是常规地从其衍生的部分例如从被芳基取代的氨基来构建，其中芳基如上文所定义。
在具体实施方式中，式I所示的化合物包括但不限于为式II和式III所示的化合物。
Figure PCTCN2019074535-appb-000009
在具体实施方案中，第二治疗剂可以为式II化合物的医药学上可接受的盐，如甲苯磺酸盐、苯磺酸盐、盐酸盐、甲磺酸盐等。
在具体实施方案中，式III化合物还可采用其水合物，如一水合物、二水合物；还可采用其医药学上可接受的盐，如盐酸盐等。
申请人发现，第二治疗剂如化合物II不仅能促进CAR T细胞分泌细胞因子IL2，还能够促进CAR T细胞在肿瘤组织的浸润、还能提高抗肿瘤作用。因此，第二治疗剂如化合物II与靶向肿瘤特异性抗原的免疫细胞治疗联用可以显著提高抗肿瘤作用。
申请人还发现，本发明不仅可以提高难治型癌症的抗癌效果，在使用CAR-T细胞时，即使没有进行淋巴细胞清除，也能达到很好的抗肿瘤效果，从而大大减轻清淋造成的抗癌治疗效果低以及减轻对正常组织的损伤所造成的毒副作用，尤其是对骨髓的严重抑制。
在具体实施方式中，第二治疗剂如化合物II能以其本身安全的口服或非口服给药，或者以和药学上可接受的载体赋形剂及其他添加剂形成的化合物(如片剂、缓释制剂、胶囊剂、注射剂、溶液剂)安全的口服或非口服给药。当口服给药时，组合物可配制成片剂、糖衣剂或胶囊。为制备口服组合物可采用乳糖或淀粉做载体，明胶，羧甲基纤维素钠，甲基纤维素聚乙烯吡咯烷酮等是合适的结合剂或成颗剂。作为崩解剂可选用淀粉或微晶纤维素，常以滑石粉，胶体硅胶，硬脂酸甘油酯，硬脂酸钙或镁等作为合适的抗黏合剂和润滑剂。例如，可通过压制湿颗粒来制备 片剂。活性成分与载体以及选择性的与一份崩解添加剂组成混合物，该混合物与黏合剂的含水溶液，醇性或含水醇性溶液在合适的设备中进行颗粒化，干燥颗粒随后加入其他的崩解剂，润滑剂和抗黏剂将此混合物压片。为增加溶解性可将杂环类衍生物游离后制成药学上可接受的有机酸，较好地为甲磺酸，富马酸等以有利于以注射剂形式给药，虽然剂量依治疗对象、给药方式、症状及其它因素而改变。
在具体实施方式中，第二治疗剂为索拉非尼(Sorafenib)(具有式II化合物所示的结构)，优选的，为甲苯磺酸索拉非尼。
在具体实施方式中，第二治疗剂为瑞戈非尼(Ragorafenib)(具有式III化合物所示的结构)，优选的，为甲苯磺酸索拉非尼。
申请人发现，由于索拉非尼与CAR-T细胞的协同作用，即使使用低剂量的索拉非尼也能实现较好的效果，故本发明的技术方案可以降低索拉非尼的毒副作用。
在具体实施方式中，低剂量的第二治疗剂是指低于单独用药能达到临床治疗效果的剂量，如低剂量的索拉非尼是指低于索拉非尼单独用药能达到临床治疗效果的剂量，具体是指临床上索拉非尼的推荐剂量：推荐服用索拉非尼为每次0.4g(2x0.2g)，每日两次；或者是指低于化合物II单独用药后临床实验中病人体内大约的浓度6.5uM。
本发明中索拉非尼用药剂量可以为低于临床的有效剂量。在某些实施方案中，患有肿瘤的个体的体内索拉非尼平均浓度约为6、5.5、5、4.5、4、3.5、3、2.5、2、1.5、1、0.95、0.9、0.85、0.8、0.75、0.7、0.65、0.6、0.55、0.5、0.45、0.4、0.35、0.3、0.25、0.2、0.15、0.1、0.095、0.09、0.085、0.08、0.075、0.07、0.065、0.06、0.055、0.05、0.045、0.04、0.035、0.034、0.033、0.032、0.031、0.03、0.029、0.028、0.027、0.026、0.025、0.024、0.023、0.022、0.021、0.02、0.019、0.018、0.017、0.016、0.015、0.014、0.013、0.012、0.011、0.01、0.009、0.008、0.007、0.006、0.005、0.004、0.003、0.002或0.001uM。在某些实施方案中，患有肿瘤的个体每天口服索拉非尼的剂量约为700、650、600、550、500、450、400、350、300、290、280、270、260、250、240、230、220、210、200、190、180、170、160、150、140、130、120、110、100、90、80、70、60、50、40、30、20、10、9、8、7、6、5、4、3、2或1mg，优选的，为400-800mg/ 天。
本发明中索拉非尼用药可以一天一次，也可以一天多次，如一天两次。
就索拉非尼而言，每天给药量可以在100-1000mg，优选的，每天给药200-800mg，更有选的，每天给药400-800mg。
本发明中免疫效应细胞和第二治疗剂给予时间不分先后；可以先给予第二治疗剂再给予免疫效应细胞；也可以同时给药；还可以先给予免疫效应细胞再给予第二治疗剂。
在某些实施方案中，免疫效应细胞在第二治疗剂给予之前1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、1个月或其任何组合施用。
在某些实施方案中，免疫效应细胞在第二治疗剂给予之后1小时、2小时、3小时、4小时、5小时、6小时、7小时、8小时、9小时、10小时、11小时、12小时、1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、1个月或其任何组合施用。
在某些实施方案中，第二治疗剂持续给予直至医生评估需要停药或者可以停药，如医生评估达到完全缓解后停药，或者疾病进展停药。在某些实施例中，第二治疗剂持续给予1个月、2个月、3个月、4个月、5个月、6个月、7个月、8个月、9个月、10个月、11个月、12个月甚至更久。。免疫效应细胞细胞在第二治疗剂持续给予期间给予。
术语“免疫效应细胞”，是指参与免疫应答，例如，促进免疫效应的细胞。免疫效应细胞的实例包括T细胞，例如，α/β的T细胞和γ/δT细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。优选地，所述T细胞包括自体T细胞、异种T细胞、同种异体T细胞，所述的自然杀伤细胞是同种异体NK细胞。正如本文中使用的那样，术语“免疫效应功能或免疫效应应答”是指免疫效应细胞，例如增强或促进靶细胞的 免疫攻击的功能或反应。例如，免疫效应功能或应答是指促进靶细胞的杀伤或者抑制生长或增殖的T细胞或NK细胞的属性。
术语“治疗有效量”、“治疗有效的”、“有效量”在本文中可互换地使用，并且是指如本文中所述有效地实现特定生物学结果的化合物、制剂、物质或组合物的量，例如但不限于足以促进T细胞应答的量或剂量。当指示“免疫学上有效量”、“抗肿瘤有效量”、“抑制肿瘤有效量”或“治疗有效量”时，将要被施用的本发明的免疫效应细胞、治疗剂的精确数量可以由医师在考虑个体在年龄、体重、肿瘤大小、感染或转移的程度以及患者(受试者)的状况的情况下确定。有效量的免疫效应细胞是指但不限于能使免疫效应细胞抗肿瘤活性增加、增强或延长；抗肿瘤免疫效应细胞或活化免疫效应细胞数目的增加；促进IFN-γ、TNFα分泌；肿瘤消退、肿瘤缩小、肿瘤坏死的免疫效应细胞的数量。
术语“不清淋”或“不进行淋巴细胞清除”，即不清除受试者体内的淋巴细胞。包括但不限于不给予淋巴细胞清除剂、全身辐射治疗或其组合或其他引起淋巴细胞数量清除的手段；但是，在给予了淋巴细胞清除剂、全身辐射治疗或其组合或其他引起淋巴细胞数量清除的手段后，当受试者体内淋巴细胞清除率低于60％，例如，体内淋巴细胞清除率为55％、50％、45％、40％、35％、30％、25％、20％、15％或10％，也将以等同落入本申请“不清淋”的范畴。
在本文中，淋巴细胞清除率可通过检测清除淋巴细胞之前的淋巴细胞数量和检测清除淋巴细胞的淋巴细胞数量计算得到，如给予清淋药前的淋巴细胞数量-给予清淋药后的淋巴细胞数量/给予清淋药前的淋巴细胞数量。淋巴细胞的检测可以通过医务人员常用的淋巴细胞数量检测方法检测，如血常规等。
术语“肽”、“多肽”和“蛋白质”可互换地使用，并且是指由肽键共价连接的氨基酸残基组成的化合物。蛋白质或肽必须含有至少两个氨基酸，并且对于可以包括蛋白质或肽的序列的氨基酸的最大数量没限制。多肽包括含有彼此通过肽键结合的两个或多个氨基酸的任何肽或蛋白质。本文所用的“嵌合受体”,即用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子，包括胞外域、跨膜域和胞内域。嵌合受体包括但不限于：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋 白(TFP)、T细胞抗原耦合器(TAC)。
本文所用的“嵌合抗原受体”或“CAR”是指一组多肽，当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域、跨膜结构域和细胞质信号传导结构域(本文中也称为“胞内信号传导结构域”)，其包括来源于如下定义的刺激性分子和/或共刺激性分子的功能信号传导结构域。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链。在一个方面，细胞质信号传导结构域进一步包括一种或多种来源于至少一个如下定义的共刺激性分子的功能性信号传导结构域。在一个方面，共刺激性分子选自本文所述共刺激性分子，例如4-1BB(即CD137)、CD27和/或CD28。在一个方面，CAR包括嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于共刺激性分子的功能性信号传导结构域和来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面中，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于一个或更多个共刺激性分子的两个功能性信号传导。
在一个方面，本发明设想产生功能等同分子的起始抗体或片段(例如scFv)氨基酸序列的修饰。例如，本文所述的癌症相关抗原的抗原结合结构域的VH或VL，例如CAR中包含的scFv，可以被修饰以保留本文所述的癌症相关抗原的抗原结合结构域的起始VH或VL构架区(例如scFv)的至少约70％、71％、72％.73％、74％、75％、76％、77％、78％、79％、80％,81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。本发明设想整个CAR构建体的修饰，例如CAR构建体的多个结构域的一个或更多个氨基酸序列的修饰，以产生功能等同分子。CAR构建体可以被修饰以保留起始CAR构建体的至少约70％、71％、72％.73％、74％、75％、76％、77％、78％、79％、80％,81％、82％、83％、84％、85％、86％、87％、88％、89％、 90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。
本文所用的“跨膜结构域”，可以包括一个或更多个邻接跨膜区域的另外氨基酸，例如一个或更多个与所述跨膜所源自的蛋白质的胞外区域相关联的氨基酸(例如，胞外区域的1、2、3、4、5、6、7、8、9、10直至15个氨基酸)和/或与所述跨膜蛋白所源自的蛋白质的胞外区域相关联的一个或更多个另外的氨基酸(例如，胞内区域的1、2、3、4、5、6、7、8、9、10直至15个氨基酸)。在一个方面，跨膜结构域是与嵌合受体的其它结构域中的一个有关的结构域，例如，在一种实施方式中，所述跨膜结构域可以来自信号传导结构域、共刺激结构域或铰链结构域所源自的相同蛋白质。在某些情况下，跨膜结构域可以被选择或通过氨基酸取代修饰以避免这样的结构域与相同或不同表面膜蛋白的跨膜结构域结合，例如以使与受体复合体的其它成员的相互作用最小化。在一个方面，跨膜结构域能够与表达嵌合受体的细胞的细胞表面上的另一个嵌合受体同型二聚化。在一个不同的方面，跨膜结构域的氨基酸序列可以被修饰或取代，以便使与存在于表达相同嵌合受体的细胞中的天然结合配偶体的结合结构域的相互作用最小化。跨膜结构域可以来源于天然或重组来源。当所述来源是天然的时，所述结构域可以来源于任何膜结合的蛋白质或跨膜蛋白质。在一个方面，只要所述嵌合受体与所述靶抗原结合时，跨膜结构域能够向胞内结构域传导信号。在本发明中特别使用的跨膜结构域可以包括至少以下的跨膜结构域:例如，T-细胞受体的α、β或ζ链、CD28、CD27、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154。在某些实施方式中，跨膜结构域可以包括至少下述跨膜区域:例如KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、 LTBR、PAG/Cbp、NKG2D、NKG2C。
在某些情况下，跨膜结构域可以经由铰链(例如，来自人蛋白质的铰链)连接至CAR的胞外区域，例如CAR的抗原结合结构域。例如，在一种实施方式中，铰链可以是人Ig(免疫球蛋白)铰链(例如，IgG4铰链、IgD铰链)、GS接头(例如，本文所述的GS接头)、KIR2DS2铰链或CD8a铰链。在一个方面，跨膜结构域可以是重组的，在这样情况下，其将会主要包含疏水性残基，比如亮氨酸和缬氨酸。在一个方面，在重组跨膜结构域的每个末端可以发现苯丙氨酸、色氨酸和缬氨酸的三联体。任选地，长度在2至10个氨基酸之间的短的寡肽或多肽接头可以在CAR的跨膜结构域与细胞质区之间形成键。甘氨酸-丝氨酸二联体提供一种特别合适的接头。
本文所用的“细胞质结构域”，包括胞内信号传导结构域。胞内信号传导结构域通常负责其中已经引入嵌合受体的免疫细胞的正常效应子功能中至少一个的活化。术语“效应子功能”是指细胞的特化功能。T细胞的效应子功能例如可以是细胞溶解活性或辅助活性，包括分泌细胞因子。因此，术语“胞内信号传导结构域”是指转导效应子功能信号且引导细胞执行特定功能的蛋白质的部分。虽然通常可以应用全部胞内信号传导结构域，但是在许多情况下不必使用整个链。就使用胞内信号传导结构域的截短部分来说，可以使用这样的截短部分代替完整的链，只要其转导效应子功能信号。因此，术语胞内信号传导结构域意味着包括足以转导效应子功能信号的胞内信号传导结构域的截短部分。
众所周知通过单独的TCR产生的信号不足以完全活化T细胞，并且也需要二级和/或共刺激信号。因此，T细胞活化可以被称为是由两个不同种类的细胞质信号传导序列介导的:通过TCR引发抗原依赖性一级活化的那些(一级胞内信号传导结构域)以及以抗原独立方式起作用以提供二级或共刺激信号的那些(二级细胞质结构域，例如共刺激结构域)。
术语“刺激”是指由刺激性分子(例如，TCR/CD3复合体或CAR)与其同源配体(或在CAR的情况下为肿瘤抗原)的结合，由此介导信号转导事件(比如但不限于经由TCR/CD3复合体的信号转导或经由适合的NK受体或CAR的信号传导结构域的信号转导)而诱导的初次应答。刺激可以介导某些分子的改变的表达。
术语“刺激性分子”是指由免疫细胞(例如，T细胞、NK细胞、B细胞) 表达的提供细胞质信号传导序列的分子，该信号传导序列以刺激性方式调节用于免疫细胞信号传导途径的至少一些方面的免疫细胞的活化。在一个方面，信号是通过例如TCR/CD3复合体与负载有肽的MHC分子的结合启动的初级信号，并且其导致介导T细胞应答，包括，但不限于增殖、活化、分化等。以刺激方式起作用的一级细胞质信号传导序列(也称为“一级信号传导结构域”)可以含有被称为基于免疫受体酪氨酸的活化基序或ITAM的信号传导基序。特别地用于本发明的含有ITAM的细胞质信号传导序列的实例包括，但不限于来源于下述的那些：CD3ζ、常见的FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。在本发明的特异性CAR中，在本发明的任一个或更多个CAR中的胞内信号传导结构域包括细胞内信号传导序列，例如CD3-ζ的初级信号传导序列。在本发明的特异性CAR中，CD3-ζ的初级信号传导序列是来自人或非人类种类例如小鼠、啮齿类动物、猴、猿等的等同残基。
术语“共刺激性分子”是指T细胞上的同源结合配偶体，其特异性地结合共刺激配体，从而介导T细胞的共刺激反应，比如但不限于增殖。共刺激性分子为除了抗原受体或其配体之外的细胞表面分子，其促进有效的免疫应答。共刺激性分子包括但不限于MHC I类分子，BTLA和Toll配体受体，以及OX40、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)。这样的共刺激性分子的进一步实例包括CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a，以及特异性地结合CD83的配体。
共刺激性胞内信号传导结构域可以为共刺激性分子的细胞内部分。共刺 激性分子可以以下述蛋白质家族代表：TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导淋巴细胞性活化分子(SLAM蛋白质)、和NK细胞受体。这样的分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、GITR、CD30、CD40、ICOS、BAFFR、HVEM、ICAM-1、与淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CDS、CD7、CD287、LIGHT、NKG2C、NKG2D、SLAMF7、NKp80、NKp30、NKp44、NKp46、CD160、B7-H3、以及特异性地结合CD83的配体等。
胞内信号传导结构域可以包括分子的全部细胞内部分或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“4-1BB”是指具有如GenBank Accession No.AAA62478.2提供的氨基酸序列的TNFR超家族的成员，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基；并且“4-1BB共刺激结构域”被定义为GenBank Accession No.AAA62478.2的氨基酸残基214～255，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。在一个方面，“4-1BB共刺激结构域”为来自人或者来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
术语“抗体重链”是指以其天然存在的构型存在于抗体分子中且通常决定抗体所属类型的两种多肽链中较大者。
术语“抗体轻链”是指以其天然存在构型存在于抗体分子中的两种多肽链的较小者。κ(k)和λ(l)轻链是指两种主要的抗体轻链的同种型。
术语“重组抗体”是指使用重组DNA技术产生的抗体，比如例如由噬菌体或酵母菌表达系统表达的抗体。该术语也应当解释为指已经通过合成编码抗体的DNA分子(且其中DNA分子表达抗体蛋白质)或指定抗体的氨基酸序列产生的抗体，其中所述DNA或氨基酸序列已经使用重组DNA或本领域可 获得且熟知的氨基酸序列技术获得。
术语“抗原”或“Ag”是指引起免疫应答的分子。该免疫应答可以涉及抗体产生或有特异性免疫能力的细胞的活化。本领域技术人员应当理解并且实际上所有蛋白质或肽的任何大分子都可以充当抗原。此外，抗原可以来源于重组或基因组DNA。当在本文中使用该术语时，本领域技术人员应当理解包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA。此外，本领域技术人员应当理解抗原无需仅通过基因的全长核苷酸序列编码。本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列以不同组合排列以编码引发期望免疫应答的多肽。而且，本领域技术人员应当理解抗原根本无需由“基因”编码。抗原可以合成产生，或者可以来源于生物样品，或者可以是除了多肽之外的大分子。这样的生物样品可以包括，但不限于组织样品、肿瘤样品、具有其它生物组分的细胞或液体。
“肿瘤抗原”指细胞癌变过程中所表达的新生物或过量表达产物。在某些方面，本发明的过度增生性病症抗原源自癌症。本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD70；CD123；CD138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体,血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型 AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
术语“癌症”指特征在于在体外(例如经转化的细胞)或体内的过度增殖性细胞生长的广泛病症类别。可以通过本发明的方法治疗或预防的病况包括例如各种赘生物，包括良性或恶性肿瘤，各种增生等等。本发明的方法可以实现这样的病况中所牵涉的不希望有的过度增殖性细胞生长的抑制和/或逆转。癌症包括但不限于：乳腺癌，脑胶质瘤，血液癌症，结肠癌，直肠癌，肾细 胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶。
术语“转染”或“转导”是指外源性核酸转移或引入到宿主细胞中的过程。“转染的”或“转导的”细胞是已经用外源性核酸转染、转化或转导的细胞。所述细胞包括原发性受试者细胞及其后代。
术语“特异性地结合”、“特异性地识别”在本文中具有相同的含义，是指识别并且结合存在于样品中的抗原(例如肿瘤抗原)的抗体或配体，但是该抗体或配体基本上不会识别或结合样品中的其它分子。
这里使用的“难治”指的是一种疾病，例如，癌症，其不应答治疗。在实施方案中，难治性癌症可以是对治疗开始前或开始时的治疗有抗性。在其他实施方案中，难治性癌症可以成为治疗期间抗性的。难治性癌症也称为抗性癌症。在本发明中，难治性癌症包括但不限于放疗不敏感、放疗后复发、化疗不敏感、化疗后复发、对CAR-T治疗不敏感或治疗后复发的癌症。难治性或复发性恶性肿瘤可以使用本文中描述的治疗方案。
如本文所用“复发的”是指在一段改进期，例如，在疗法，例如癌症疗法的先前治疗后，返回疾病(例如癌症)或疾病如癌症的体征和症状。
术语“个体”和“受试者”在本文中具有同等含义，可以是人和来自其他种属的动物。
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强的应答可以包含应答性中5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫效应细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、 85％、90％、95％或98％更多。
在一个方面，治疗由临床结果；通过T细胞的抗肿瘤活性增加、增强或延长；与治疗前的数目相比较，抗肿瘤T细胞或活化T细胞数目的增加，促进IFN-γ、TNFa分泌，或其组合决定。在另一个方面，临床结果是肿瘤消退；肿瘤缩小；肿瘤坏死；通过免疫系统的抗肿瘤应答；肿瘤扩大，复发或扩散或其组合。在一个另外方面，治疗效应通过T细胞的存在、指示T细胞炎症的基因标记的存在，促进IFN-γ、TNFa分泌，或其组合预测。在一个方面，治疗肿瘤包括通过治疗降低癌细胞生长、存活或活力。
如本文公开的免疫效应细胞可以通过各种途径施用于个体，包括例如经口或肠胃外，例如静脉内、肌内、皮下、眶内、囊内、腹膜内、直肠内、脑池内、瘤内、鼻内(intravasally)、皮内或者分别使用例如皮肤贴片或透皮离子电渗疗法通过皮肤的被动或促进吸收。
在实践本发明的方法中待施用的试剂总量可以作为单一剂量以推注或通过在相对短时间段的输注，施用于受试者，或可以使用分级治疗方案进行施用，其中在延长时间段施用多个剂量。本领域技术人员将知道治疗受试者中的病理状况的组合物的量取决于许多因素，包括受试者的年龄和一般健康、以及施用途径和待施用的治疗次数。考虑到这些因素，技术人员将根据需要调整具体剂量。一般而言，最初，使用I期和II期临床试验测定组合物的配制以及施用途径和频率。
范围：在整个公开中，本发明的各个方面都可以以范围形式存在。应当理解，范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本发明的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。例如，范围的描述比如从1至6就应当被认为具体地公开了子范围比如1至3、1至4、1至5、2至4、2至6、3至6等，以及该范围内的单独数值，例如1、2、2.7、3、4、5、5.3、和6。作为另一个实例，范围比如95-99％的同一性包括具有95％、96％、97％、98％或99％同一性的范围，并且包括子范围比如96～99％、96～98％、96～97％、97～99％、97～98％和98～99％的同一性。不考虑范围的宽度，这均适用。
根据本公开内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精 神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等价的方法和组分在本发明的范围内。事实上，本发明的各种修饰加上本文显示且描述的那些，根据前述描述对于本领域技术人员将变得明显。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，《分子克隆实验指南(第三版)》(科学出版社，2002)中所述的条件，或按照制造厂商所建议的条件。
本发明的示例性的抗原受体，包括CAR，以及用于工程改造和将受体导入细胞中的方法，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1中公开的那些。
实施例一.CAR-T细胞的构建
参照图1A所示的质粒图，采用本领域常规分子生物学方法，构建了表达人源化抗体hu9F2(核苷酸序列如SEQ ID NO：5所示，氨基酸序列如SEQ ID NO：14所示)的二代嵌合抗原受体的慢病毒质粒PRRLSIN-hu9F2-28Z。
抗体hu9F2具有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。
Hu9F2-28Z序列由CD8α信号肽(SEQ ID NO:6)、hu9F2scFv(SEQ ID NO:5)、CD8hinge(SEQ ID NO:7)、CD28跨膜区(SEQ ID NO:10)和胞内信号传导结构域(SEQ ID NO:8)以及CD3的胞内段CD3ξ(SEQ ID NO:9)组成。将PRRLSIN-hu9F2-28Z转染293T包装慢病毒，得到慢病毒。
T细胞活化：取人PBMC培养于AIM-V培养基，添加2％人AB型血清，500U/mL重组人IL-2，并加入CD3/CD28抗体结合磁珠活化48h。将所得的慢病毒感染活化的T细胞，得到hu9F2-28Z CART细胞，流式检测结果如图1B所示，其CAR的序列如SEQ ID NO：22所示。
实施例二.CCK8实验检测索拉非尼对肝癌细胞、CAR-T细胞的毒性
取实施例1中的hu9F2-28Z CAR T细胞，将其铺于96孔板中，每孔4×10 4个细胞，100ul培基。取肝癌细胞PLC/RPF/5(低表达GPC3)、SK-HEP-1(不表达GPC3)、Huh7(高表达GPC3)，将其铺于96孔板中，每孔4000个细胞，100ul培基。分别取不同溶度的索拉非尼分别加入到细胞中，做成六个梯度(即10μM、5μM、1μM、0.5μM、0.1μM、0μM六种溶度梯度，10μM、5μM、1μM、0.5μM、0.1μM为加药组，0μM l为0加药组)，此外设立一组只有培养基的孔，为空白组。48h后每孔加入10ul CCK8试剂(Dojindo公司)37℃孵育1h后酶标仪测定450nm处吸光度，分别计算细胞活力。
计算公式为：细胞活力(％)＝[A(加药)-A(空白)]/[A(0加药)-A(空白)]
结果如图2所示，索拉非尼对CAR T细胞没有明显的抑制作用。
实施例三、CAR T细胞对索拉非尼预处理后的肿瘤细胞的杀伤活性
将含有Huh7细胞的培养基中分别加入不同浓度的索拉非尼(1μM、0.1μM、0μM)，37℃培养24h后，胰酶消化，PBS洗净残留的药物。取实施例1中的hu9F2-28Z CAR T细胞分别与上述不同浓度索拉非尼预处理后的Huh7细胞1:1共孵育，18h后使用Cytox 96 Non-Radioactive Cytotoxicity Assay(Promaga公司，REF:G1782)检测hu9F2-28Z CAR T细胞对不同浓度索拉非尼预处理后的肿瘤细胞的杀伤。
结果如图3所示，相比较不加索拉非尼的组别，经0.1μM索拉非尼处理后的肿瘤细胞对CAR T细胞的杀伤的敏感性增强了11％，经1μM索拉非尼处理后的肿瘤细胞对CAR T细胞的杀伤的敏感性增强了16.8％。
实施例四：索拉非尼与CAR T细胞联用对hepa1-6-GPC3皮下瘤的抑
制效果
本实施例采用鼠的基因序列构建CAR的跨膜域和胞内域。将C57BL/6小鼠(免疫系统正常的小鼠)分为5组：
Vehicle组：不给予索拉非尼组；
Sorafenib组：索拉非尼单独给药组；
CAR T+vehicle组：给予hu9F2-m28Z CAR T细胞和溶剂组；
UTD+sorafenib组：给予未感染CAR的T细胞和索拉非尼组；
CAR T+sorafenib组：给予hu9F2-m28Z CAR T细胞和索拉非尼组。
1、利用分子生物学常规手段建立人鼠嵌合的GPC3(SEQ ID NO:11)过表达的鼠肝癌细胞Hepa1细胞模型(Hepa1-6-GPC3)。如图4所示，通过流式细胞术确认过表达GPC3的Hepa1-6细胞。
2、收集处于对数生长期且生长良好的Hepa1-6-GPC3细胞，接种1×10 7靶细胞于C57BL/6小鼠(免疫系统正常的小鼠)的右侧腋部皮下。
3、接种肿瘤后第4天，取小鼠脾脏T淋巴细胞，构建hu9F2-m28Z CAR T细胞，构建方法如下：
将小鼠CD8α信号肽的编码序列(SEQ ID NO：4)、hu9F2scFv(SEQ ID NO：5)、鼠CD8α铰链区及跨膜区的编码序列(SEQ ID NO：1)、鼠CD28胞内域的编码序列(SEQ ID NO：2)、鼠CD3ζ胞内域的编码序列(SEQ ID NO：3)依次连接，通过体外基因合成的方法获得hu9F2-m28Z基因片段，并用MluⅠ和SalⅠ双酶切位点置换逆转录病毒载体MSCV-IRES-GFP(购自Addgene)中的IRES-GFP片段，获得重组载体MSCV-hu9F2-m28Z。用MSCV-hu9F2-m28Z感染293T细胞，得到包装后的逆转录病毒。
取小鼠脾脏T淋巴细胞，采用Dynabeads Mouse T-activator CD3/CD28活化，活化后加入逆转录病毒感染12小时，得到hu9F2-m28Z CAR T细胞。
4、接种肿瘤后第7天，给予索拉非尼或溶剂：将索拉非尼溶解于溶剂(5％DMSO、45％PEG400、50％H20)中，对Sorafenib组、UTD+sorafenib组、CAR T+sorafenib组小鼠进行灌胃，每只小鼠给药量为7.5mg/kg。Vehicle组和CAR T+vehicle组给予溶剂灌胃。索拉非尼第一次给药日记为第0天，每天给药一次，连续给药5天。
5、接种肿瘤后第8天，CAR T+vehicle组和CAR T+sorafenib组，每只小鼠给3*10 6个hu9F2-m28Z CAR T细胞；UTD+sorafenib组给予3*10 6未感染CAR的T细胞。
持续观察瘤体积变化，每周测量三次，瘤体积计算公式为：(长*宽 2)/2。肿瘤体积检测结果如图5所示，与其他组相比，CAR T+sorafenib组具有显著的抑制肿瘤生长的作用(Two-way ANOVA with Bonferroni post-tests，*表示p〈0.05，**表示p〈0.01，***表示p〈0.001)。
到治疗后的22天，CAR T+sorafenib联合治疗组6只小鼠中有4只小鼠肿瘤消失，而其他各组均没有小鼠肿瘤消失。在治疗后22天(即Day 22)， 将小鼠引颈处死，剥离小鼠皮下肿瘤并称量瘤重，结果如图6所示，结果表明相比较Vehicle组，Sorafenib组、CAR T+vehicle组，UTD+sorafenib组而言，CAR T+sorafenib联合治疗组具有显著的肿瘤抑制作用(Unpaired t test，*表示p〈0.05，**表示p〈0.01，***表示p〈0.001)。
实施例五：检测索拉非尼对CAR T细胞的脱颗粒、增殖及杀伤的影响
1、索拉非尼对脱颗粒标志物CD107a的影响
将实施例1中的hu9F2-28Z CAR T细胞和不同浓度索拉非尼及与PLC/RPF/5细胞共孵育24小时后，收取处理后的CAR T细胞，孵育抗人CD107a-PerCP抗体(eBioscience)及带GFP荧光蛋白的GPC3标签蛋白，流式检测PerCP及GFP的荧光强度，分别代表人CD107a及CAR的表达情况，结果如图7A所示，未加索拉非尼组和索拉非尼处理组的CD107a表达能力没有显著变化。
2、索拉非尼对CAR T细胞增殖的影响
采用CellTrace Violet试剂盒(Thermofisher)监测不同代数的细胞增殖情况。取hu9F2-28Z CAR T细胞，加入CellTrace染料工作液，37℃孵育，中和染料。
将上述处理后的hu9F2-28Z CAR T细胞和PLC/RPF/5细胞按照1:1的比例铺在24孔板中，分别加入不同浓度的索拉非尼(0、1、5、10μM)共培养24小时，各组均设3个复孔。
流式检测各组细胞，索拉非尼结果如图7B所示，未加索拉非尼组和索拉非尼处理组与表达GPC3的肿瘤细胞共孵育后,CAR T细胞增殖能力没有显著变化。
3、体外细胞毒性实验
a)以PLC/RPF/5细胞为靶细胞，实施例1中的hu9F2-28Z CAR T细胞为效应细胞。取适量hu9F2-28Z CAR T细胞，经不同浓度索拉非尼(0、1、5、10μM)处理4小时后，分别离心收集。
实验组铺板：靶细胞数量为10000/孔，效应细胞为索拉非尼处理后的hu9F2-28Z CAR T细胞。效靶比分别为20：1，10：1，5：1，2.5：1，根据不同效靶比加入不同数量效应细胞。各组均设5个复孔。
对照组铺板：本实验中还需要设置其他对照组来排除效应细胞、靶 细胞中自发性释放的LDH的干扰，以及培养基中包含的LDH的干扰。分别为：靶细胞最大释放LDH对照组；靶细胞自发释放LDH对照组；效应细胞自发释放LDH对照组；空培基背景组；空培基背景加入裂解液组。各组均设5个复孔。
上述细胞铺板完成后，37℃、5％CO 2恒温培养箱中孵育，转入酶标板中，进行底物显色。酶标仪测定490nm处吸光度，分别计算细胞杀伤毒性。
b)计算公式为：细胞毒性(％)＝(实验组－效应细胞自发组－靶细胞自发组－背景组)/(靶细胞最大组－靶细胞自发组－裂解液背景组)。
结果如图7C所示，索拉非尼处理后的hu9F2-28Z CAR T细胞对GPC3阳性的肿瘤细胞的杀伤毒性没有显著变化。
上述结果说明索拉非尼不影响CAR T细胞脱颗粒能力、增殖和对肿瘤细胞的杀伤毒性。
实施例六：索拉非尼对CAR T细胞的细胞因子分泌的影响
具体步骤参考cytometric bead array system(BD,USA)说明书中的步骤，简述如下：
a)收集上清：将实施例1中的1×10 5hu9F2-28Z CAR T细胞铺在GPC3蛋白包板的24孔板中，加入不同浓度索拉非尼(0、1、5、10μM)处理，37℃、5％CO 2培养箱培养后离心收集细胞上清，检测其中人的细胞因子IL2、IFN-γ、TNF-α的浓度。
b)取人细胞因子IL2、IFN-γ、TNF-α的捕获微球(cytometric bead array system,BD,USA)悬液混合。
c)标准品或上清样品添加捕获微球混合液以及检测抗体，室温避光孵育。孵育完成后用洗涤缓冲液洗涤，离心，弃上清。
流式检测IL-2、IFN-γ及TNF-α的分泌水平，结果从图8A、8B、8C所示，其中，ns表示p>0.05；*p<0.05；**p<0.01；***p<0.001,1way ANOVA。
实施例七：索拉非尼和人CAR T细胞联合治疗肝癌
1.建立人肝癌细胞移植瘤模型：取对数生长期的PLC/RPF/5细胞5×10 6细胞接种到NSG小鼠腋下皮肤中，接种日当天为第0天(Day0)。
2.第11天(Day11)：小鼠皮下瘤的体积约在150mm 3左右，将小鼠按照肿瘤体积随机分6组,每组5-6只。索拉非尼溶解于溶剂(5％DMSO、45％PEG400、50％H2O)中。不给索拉非尼的组别中给予溶剂。
(1)对照组(Vehicle)：Day11给予溶剂，每天一次，给药体积与实验组相同，持续两周。
(2)单独治疗组(共3组)：
Sora 7.5：Day11给予小鼠灌胃7.5mg/kg的索拉非尼，每天一次，持续两周。
Sora 30：Day11给予小鼠灌胃30mg/kg的索拉非尼，每天一次，持续两周。
CAR+Vehicle：Day11给予溶剂每天一次，给药体积与实验组相同，持续两周；当天(Day11)，溶剂给予后，尾静脉注射实施例1中的2×10 6hu9F2-28Z CAR T细胞。
(3)联合治疗组(共2组)：
CAR+Sora7.5：Day11给予小鼠灌胃7.5mg/kg的索拉非尼，每天一次，持续两周；当天(Day11)，索拉非尼给予后，尾静脉注射实施例1中的2×10 6hu9F2-28Z CAR T细胞。
CAR+Sora30：Day11给予小鼠灌胃30mg/kg的索拉非尼,每天一次，持续两周；当天(Day11)，索拉非尼给予后，尾静脉注射实施例1中的2×10 6hu9F2-28Z CAR T细胞。
3.每周测量并记录两次小鼠皮下瘤的体积变化情况，瘤体积计算公式为：肿瘤体积＝(肿瘤长×肿瘤宽 2)/2，结果如图9A所示，与单独治疗组相比，索拉非尼和hu9F2-28Z CAR T细胞联合治疗能显著抑制肿瘤生长(P<0.001，2way ANOVA)。
参照对照组计算肿瘤抑制率，在肿瘤接种后的第43天(Day43),各组的肿瘤抑制率为：Sora 7.5为18.8％，Sora30为35.8％，CAR+Vehicle为25.3％，CAR+Sora7.5为68.7％，CAR+Sora30为75.2％。
第43天(Day43)时给予小鼠安乐死处理，剥离皮下瘤，称量肿瘤重量，结果如图9B所示，相比单独治疗组，索拉非尼和hu9F2-28Z CAR  T细胞联合治疗(7.5mg/kg或30mg/kg)中肿瘤重量显著降低(*p<0.05；**p<0.01或***p<0.001，1way ANOVA)。
4.CAR T细胞瘤内浸润及瘤内细胞的凋亡情况
1)采用常规组织切片制备方法，将安乐死后小鼠的肿瘤组织制成组织切片。
2)采用抗人CD3抗体(Thermo Scientific)来检测步骤1)中制备好的肿瘤组织切片中人的CAR T细胞。结果如图10A所示，与hu9F2-28Z CAR T细胞单独治疗的小鼠相比，联合hu9F2-28Z CAR T细胞与索拉非尼(7.5mg/kg或30mg/kg)治疗的小鼠在肿瘤组织中有更多CD3+的人CAR T细胞(*p<0.05；**p<0.01，1way ANOVA)，没有接受hu9F2-28Z CAR T细胞治疗的组中没有观察到CD3+的人CAR T细胞的染色。结果说明索拉非尼能促进CAR T细胞在肿瘤组织的浸润。
3)采用抗cleaved caspase-3抗体(Cell Signaling Technology)检测步骤1中肿瘤组织切片中发生凋亡的细胞
将组织切片进行免疫组化染色，使用苏木素对肿瘤组织中的细胞核进行复染，结果如图10B所示，与对照组和hu9F2-28Z CAR T细胞单独治疗的小鼠相比，联合hu9F2-28Z CAR T细胞与索拉非尼(7.5mg/kg或30mg/kg)治疗的小鼠在肿瘤组织中有更多cleaved caspase-3阳性的细胞。
采用两只小鼠肿瘤切片中总计5个不同视野进行观察统计，结果如图10C所示索拉非尼和CAR T细胞联合使用时，肿瘤细胞发生凋亡数量显著增加(**p<0.01，***p<0.001，1way ANOVA)。
实施例八：体外检测索拉非尼与人CAR T细胞的协同作用
参照CellTrace Violet试剂盒实验操作，将人肝癌细胞PLC/RPF/5使用CellTrace染料标记。将标记好的PLC/RPF/5按照1：1比例与hu9F2-28z CAR T细胞混合，同时分别添加不同浓度索拉非尼(0、1、5、10μM)于细胞培养箱共孵育48小时，离心并收集细胞，使用FITC Annexin V凋亡检测试剂盒(购自BD)中染色缓冲液对细胞沉淀进行重悬，加入AnnexinV-FITC染料进行染色。上机检测：离心收集完成染色的细胞，染色缓冲液重悬，流式上机检测。BV421通道中收集紫光信号，代表所有的肿瘤细胞。FITC通道中收集绿光，代表发生凋亡的肿瘤细胞。FlowJo 软件处理数据。
结果如图11所示，联合hu9F2-28Z CAR T细胞与索拉非尼治疗后肿瘤细胞的凋亡比例明显升高，肿瘤细胞发生凋亡数量显著增加(*p<0.05；**p<0.01或***p<0.001，1way ANOVA)。
上述体外实验结果说明索拉非尼和CAR T细胞联合治疗能协同地促进肿瘤细胞的凋亡。
实施例九：CAR T细胞与瑞戈非尼联用对小鼠肝癌移植瘤的治疗的影响
参照实施例4的操作，构建Hepa1-6-chGPC3细胞C57BL/6的移植瘤模型和hu9F2-m28Z CAR T细胞。
肿瘤细胞植入后第8天，将小鼠按照肿瘤体积随机分为三组，瑞戈非尼治疗组及瑞戈非尼+CAR T联合组的小鼠灌胃给予10mg/kg的瑞戈非尼，对照组给予溶媒，每天一次，持续十天。
肿瘤细胞植入后第9天，对CAR T治疗组小鼠及瑞戈非尼+CAR T联合组尾静脉注射2×10 6CAR T细胞，此后每周两次测量并记录小鼠皮下瘤的体积变化，结果如图12所示。
实施例十：索拉非尼和CAR T细胞联合治疗的临床研究
受试者A年龄60岁，体重77kg，身高：170cm，诊断为肝细胞癌后经手术治疗7个月后，仍处于进展状态，免疫组化显示GPC3阳性(70％，++～+++)。在该情况下，患者接受了本发明的技术方案的治疗。
单采后给予患者持续口服索拉非尼，服用量为一日两次，一次200mg-400mg(以患者可耐受剂量为准，在该受试者中，一次200mg)，并持续每天给予。
参照实施例一的操作，将慢病毒感染患者的T细胞后，制备得到hu9F2-28Z CAR T细胞。
在开始给予索拉非尼两周后，且在给予CAR-T细胞前，患者先接受淋巴细胞清除处理，包括氟达拉滨约39mg/日×4日(约约20mg/m 2/日)，环磷酰胺约1000mg/日×2日(约500mg/m 2/日)。
淋巴细胞清除处理后的第3天开始，连续两天分别给予大约5×10 8个CAR-T细胞(约为6.49×10 6个CAR-T细胞/kg患者体重，总剂量约为0.13×10 8个CAR-T细胞)。
CAR-T细胞给予后第3天，AFP相比给予CAR-T细胞之前降低了35％，CAR-T细胞给予后第13天，AFP相比给予CAR-T细胞之前降低了58.2％。
治疗4周后CT检查，影像学如图13所示，相对于基线，3个可能的靶病灶都明显缩小，达到时间点部分缓解(PR)。
在上述实施例中，示例性的，采用了靶向GPC3的CAR-T细胞的CAR具有SEQ ID NO：22所示的氨基酸序列，本领域技术人员可以依据上述实施例的教导，采用如SEQ ID NO：21、23、或24所示的序列。
在上述实施例中，示例性的，采用了靶向GPC3的CAR-T细胞，本领域技术人员可以依据本申请的教导，采用靶向其他靶点的CAR-T细胞，如靶向EGFR的CAR-T细胞(示例性的，靶向EGFR的CAR-T细胞的scFv的序列如SEQ ID NO：12所示)，如靶向CLD18A2的CAR-T细胞(示例性的，靶向CLD18A2的CAR-T细胞的scFv的序列如SEQ ID NO：13所示)。
本发明所涉及的序列信息如下：
Figure PCTCN2019074535-appb-000010
Figure PCTCN2019074535-appb-000011
Figure PCTCN2019074535-appb-000012
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (30)

  1. 治疗肿瘤的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体，
    所述的第二治疗剂包括式I所示的化合物，或其医药学上可接受的盐，
    Figure PCTCN2019074535-appb-100001
    其中，Ar’是非取代或取代的苯基，取代基选自卤素和C1-10烷基，
    M是一个或多个桥连基，选自-O-或-S-，
    Py(X)是X取代的吡啶基，X为-C(O)R x，其中，R x是NR aR b，其中的R a和R b各自是：
    a)氢，
    b)C1-10烷基，
    c)羟基取代的C1-10烷基，
    d)含1-3个N，S或O的杂原子的C3-12环烷基，或
    e)-OSi(R f)3，R f是C1-10烷基，
    Ar”是非取代或取代苯基，取代基选自卤素或Wn，n＝0-3，W选自：
    a)C1-10烷基，
    b)C1-10烷氧基，
    c)C1-10卤代烷基
    d)含1-3个N，S或O杂原子的C3-12杂芳基，该杂芳基可被C1-10烷基取代。
  2. 降低癌细胞生长、存活或活力的方法，其特征在于，对患有肿瘤的个体施用免疫效应细胞和第二治疗剂，所述免疫效应细胞表达有识别肿瘤抗原的受体，
    所述的第二治疗剂为式I所示的化合物，或其医药学上可接受的盐，
    Figure PCTCN2019074535-appb-100002
    其中，Ar’是非取代或取代的苯基，取代基选自卤素和C1-10烷基，
    M是一个或多个桥连基，选自-O-或-S-，
    Py(X)是X取代的吡啶基，X为-C(O)R x，其中，R x是NR aR b，其中的R a和R b各自选自：
    a)氢，
    b)C1-10烷基，
    c)羟基取代的C1-10烷基，
    d)含1-3个N，S或O的杂原子的C3-12环烷基，以及
    e)-OSi(R f)3，R f是C1-10烷基，
    Ar”是非取代或取代苯基，取代基选自卤素或Wn，n＝0-3，W选自：
    a)C1-10烷基，
    b)C1-10烷氧基，
    c)C1-10卤代烷基，以及
    d)含1-3个N，S或O杂原子的C3-12杂芳基，该杂芳基可被C1-10烷基取代。
  3. 如权利要求1或2所述的方法，其特征在于，在对患有肿瘤的个体施用免疫效应细胞之前，对所述的个体不进行淋巴细胞清除。
  4. 如权利要求1或2所述的方法，其特征在于，所述免疫效应细胞和第二治疗剂的治疗效果大于所述免疫效应细胞和第二治疗剂任一单独使用的效果。
  5. 如权利要求1或2所述的方法，其特征在于，所述的Ar”是取代苯基，取代基选自氯、溴、氟、三氟甲基，甲氧基和叔丁基中的任意一种或其组合。
  6. 如权利要求1或2所述的方法，其特征在于，所述的M是-O-。
  7. 如权利要求1或2所述的方法，其特征在于，所述的Ra和Rb各自是H或C1-10烷基，优选的，Ra和Rb分别为H和甲基。
  8. 如权利要求1或2所述的方法，其特征在于，所述医药学上可接受的盐选自：a)无机酸和有机酸的碱式盐，所述酸选自：盐酸、氢溴酸、硫酸、磷酸、甲磺酸、三氟甲磺酸、苯磺酸，对甲苯磺酸，1-萘磺酸，2-萘磺酸，乙酸、三氟乙酸、苹果酸、酒石酸、柠檬酸、乳酸、草酸、琥珀酸、富马酸，马来酸、苯甲酸、水杨酸、苯基乙酸和杏仁酸；b)有机和无机碱的酸式盐，所述阳离子选自：碱金属阳离子，碱土金属阳离子，铵阳离子，脂族基取代的铵阳离子，和芳香基取代的铵阳离子。
  9. 如权利要求1-8所述的方法，其特征在于，所述的第二治疗剂选自下列任一所示的化合物或其医药学上可接受的盐：
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶基硫)苯基)脲，
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
    N-(2-甲氧基-(5-三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
    N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
    N-(4-氯-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(4-氯-3-(三氟甲基)苯基)-N’-(4-(2-氨基甲酰基-4-吡啶氧基)苯基)脲，
    N-(4-氯-3-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(4-氯-3-(三氟甲基)苯基)-N’-2-氟-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(4-溴-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(4-溴-3-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(4-溴-3-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶基硫)苯基)脲，
    N-(4-溴-3-(三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
    N-(4-溴-3-(三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
    N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(3-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(4-(2-(N-甲基氨基甲酰基)-4-吡啶氧基)苯基)脲，
    N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(2-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲，
    N-(2-甲氧基-4-氯-5-(三氟甲基)苯基)-N’-(3-氯-4-(2-(N-甲基氨基甲酰基)(4-吡啶氧基))苯基)脲。
  10. 如权利要求9所述的方法，其特征在于，所述的第二治疗剂选自下列式II或式III所示的化合物，或其医药学上可接受的盐：
    Figure PCTCN2019074535-appb-100003
    Figure PCTCN2019074535-appb-100004
  11. 如权利要求10所述的方法，其特征在于，所述的医药学上可接受的盐选自甲苯磺酸盐、苯磺酸盐、盐酸盐、甲磺酸盐。
  12. 如权利要求10所述的方法，其特征在于，所述的第二治疗剂为式III化合物或者式III化合物的水合物，优选的为一水合物。
  13. 如权利要求1-12任一所述的方法，其特征在于，所述的第二治疗剂每天给药100-1000mg，优选的，每天给药200-800mg，更优选的，每天给药400-800mg，
    优选所述第二治疗剂每天给予1-3次，
    进一步优选，每天给予第二治疗剂2次，
    再优选所述第二治疗剂持续给予直至医生评估需要停药或者可以停药，如医生评估达到完全缓解后停药，或者疾病进展停药。
  14. 如权利要求13所述的方法，其特征在于，所述免疫效应细胞每次的给予量为约1x10 5～1x10 8细胞/千克受试者体重，
    优选地，每次的给予量为约1x10 5～1x10 7细胞/千克受试者体重。
  15. 如权利要求1-14任一所述的方法，其特征在于，免疫效应细胞和第二治疗剂给予时间不分先后，
    优选在给予所述第二治疗剂的期间给予所述免疫效应细胞。
  16. 如权利要求1-15任一所述的方法，其特征在于，所述的第二治疗剂为口服给药。
  17. 如权利要求1-16任一所述的方法，其特征在于，所述受体选自：嵌合抗原受体(Chimeric Antigen Receptor，CAR)、T细胞受体(T cell receptor，TCR)、T细胞融合蛋白(T cell fusionprotein，TFP)、T细胞抗原耦合器(T cell antigen coupler，TAC)或其组合。
  18. 如权利要求17所述的方法，其特征在于，所述的嵌合抗原受体具有：
    (i)特异性识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ的胞内域；或
    (ii)特异性识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ的胞内域；或
    (iii)特异性识别肿瘤抗原的抗体或其片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ的胞内域。
  19. 如权利要求1-18任一所述的方法，其特征在于：
    所述肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4； 8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
  20. 如权利要求1-18任一所述的方法，其特征在于：所述肿瘤抗原为实体瘤抗原；优选的所述实体瘤抗原为血管内皮生长因子受体、EGFR、 EGFRvIII、GPC3、或Claudin18.2；更优选的，所述实体瘤抗原为GPC3、EGFR、EGFRvIII、或血管内皮生长因子受体。
  21. 如权利要求18所述的方法，其特征在于，所述特异性识别肿瘤抗原的抗体含有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。
  22. 如权利要求21所述的方法，其特征在于，所述的特异性识别肿瘤抗原的抗体的氨基酸序列与SEQ ID NO:14所示的序列具有至少90％的同一性。
  23. 如权利要求22所述的方法，其特征在于，具有所述特异性识别肿瘤抗原的抗体的嵌合抗原受体的氨基酸序列与SEQ ID NO:21、22、23或24所示的序列具有至少90％的同一性。
  24. 如权利要求17所述的方法，其特征在于，所述的嵌合抗原受体具有：
    特异性识别肿瘤抗原的抗体或其片段、跨膜结构域和细胞质信号传导结构域，
    其中所述特异性识别肿瘤抗原的抗体或其片段含有SEQ ID NO:15、16、17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。
  25. 如权利要求24所述的方法，其特征在于，所述的嵌合抗原受体具有：
    (i)特异性识别肿瘤抗原的抗体或其片段，CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ的胞内域；或
    (ii)特异性识别肿瘤抗原的片段、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ的胞内域；或
    (iii)特异性识别肿瘤抗原的片段、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ的胞内域；
    其中所述特异性识别肿瘤抗原的抗体或其片段含有SEQ ID NO:15、16、 17所示的HCDR1、HCDR2、HCDR3，和SEQ ID NO:18、19、20所示的LCDR1、LCDR2、LCDR3。
  26. 如权利要求24所述的方法，其特征在于，所述第二治疗剂为式II所示的化合物，或其医药学上可接受的盐：
    Figure PCTCN2019074535-appb-100005
    所述第二治疗剂每次的给药剂量是每天给药100-1000mg，优选的，每天给药200-800mg，更优选的，每天给药400-800mg，
    优选，所述第二治疗剂每天给药1-3次，进一步优选的，每天给药2次，
    再优选所述第二治疗剂持续给予直至医生评估需要停药或者可以停药，如医生评估达到完全缓解后停药，或者疾病进展停药。
  27. 如权利要求1-26任一所述的方法，其特征在于，其中所述肿瘤包括：乳腺癌，脑胶质瘤，血液癌症，结肠癌，直肠癌，肾细胞癌，肝癌，肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶；
    优选的，所述肿瘤选自肝癌、肾细胞癌、肺鳞癌、甲状腺癌。
  28. 如权利要求1-27任一所述的方法，其特征在于，所述的免疫效应细胞选自：T细胞、B细胞、自然杀伤细胞(NK细胞)、自然杀伤T细胞(NKT细胞)、肥大细胞或骨髓源性吞噬细胞或其组合；
    优选地，所述免疫效应细胞选自T细胞或NK细胞；
    进一步优选，所述免疫效应细胞为自体免疫效应细胞。
  29. 表达有识别肿瘤抗原的受体的免疫效应细胞在制备药物中的应用，其特征在于，所述药物与索拉非尼联用，用于治疗肿瘤，其中将所述药物和索拉非尼配制成能提供优于所述药物和索拉非尼各自单独使用时的效果。
  30. 一种用于治疗肿瘤的试剂盒，其特征在于，所述试剂盒包含：
    1)表达有识别肿瘤抗原的受体的免疫效应细胞；
    2)索拉非尼；
    3)用于包含以上1)和2)所述物质的容器；和
    4)利用所述试剂盒治疗肿瘤的给药说明书；
    其中，所述免疫效应细胞和索拉非尼配制成能提供优于所述免疫效应细胞和索拉非尼各自单独使用时的效果；
    优选地，所述免疫效应细胞为CAR T细胞，
    更优选地，所述CAR T细胞特异性识别EGFR、EGFRvIII，磷脂酰肌醇蛋白聚糖3、claudin 18.2、BCMA中的任一种或多种。
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