Combined tumor immunotherapy

Abstract

A method for treating tumors is provided, which includes administering immune effector cells and a PARP inhibitor to an individual suffering from a tumor, and the immune effector cells express receptors that recognize tumor antigens. A kit for treating tumors is also provided.

Description

Technical Field

[0001] This application belongs to the field of cellular immunotherapy, and particularly relates to the combined anti-tumor immunotherapy targeting tumor-specific antigens and PARP inhibitors. Background Art

[0002] In recent years, immunocyte therapy, especially CAR-T cell therapy, has shown amazing therapeutic effects in the treatment of B-cell tumors. Currently, there are more than 200 clinical trials of CAR-T cells for the treatment of hematological tumors (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).

[0003] However, due to the complexity of the biological body, especially the microenvironment of solid tumors, candidate drugs that show excellent effects in vitro often fail to show corresponding effects in vivo. In other words, the in vitro results of candidate drugs cannot reasonably predict the in vivo effects. In addition, the same antibody also has different effects on tumors in different parts with the same target. For example, trastuzumab has a good therapeutic effect when applied to HER2-positive breast cancer, but has no effect when applied to HER2-positive gastric cancer (Fu Qiang, HER2 signaling pathway in gastric cancer and clinical application progress of trastuzumab, Drug Evaluation, 2012, 9(27): 8 - 12).

[0004] Although immune effector cells have promising prospects in tumor immunotherapy, in the application of solid tumors, CAR-T cells face many problems. Among them, homing cells to the tumor local area and activating proliferation are the key steps for CAR-T cell therapy to take effect.

[0005] In recent years, PARP inhibitors have made breakthrough progress in the research and development of anti-cancer drugs. The anti-cancer mechanisms of PARP inhibitors mainly include: (1) blocking DNA damage repair, causing DNA damage accumulation, and ultimately killing tumor cells; (2) increasing the sensitivity of cells to other exogenous and endogenous DNA damage factors; (3) inhibiting angiogenesis; (4) enhancing the immunity of normal cells to resist the invasion of cancer cells. Currently, many PARP inhibitors have entered clinical trials. Among them, Olaparib (AZD2281) is the most widely studied 3rd-generation PARP inhibitor to date, and it is a potent PARP-1 inhibitor. However, some patients are still insensitive to this type of drug.

[0006] Therefore, the purpose of this study is to combine appropriate small molecule drugs to enhance the immunogenicity of tumor cells, reduce tumor immunosuppressive factors, promote the homing and continuous activation of CART cells, thereby enhancing the antitumor activity of CAR-T cells themselves. Summary of the Invention

[0007] The purpose of this application is to provide a tumor treatment method in combination with small molecule drugs to improve the anti-tumor application effect of immunocyte therapy, especially CAR-T cell therapy, in solid tumors.

[0008] In one aspect of this application, a method for treating tumors is provided, which includes: administering an immune effector cell and a PARP inhibitor to an individual suffering from a tumor, wherein the immune effector cell expresses a receptor that recognizes a tumor antigen.

[0009] In one aspect of this application, a method for reducing cancer cell growth, survival, or viability or all is provided, which includes: administering an immune effector cell and a PARP inhibitor to an individual suffering from a tumor, wherein the immune effector cell expresses a receptor that recognizes a tumor antigen.

[0010] In at least one specific aspect, the PARP inhibitor is selected from any of talazoparib, niraparib, olaparib, rucaparib, Niraparib, Pamiparib, Fluzoparib, Mefuparib, Ximingpiperid.

[0011] In at least one specific aspect, the PARP inhibitor is olaparib.

[0012] In at least one specific aspect, the therapeutic effect of the immune effector cell and the PARP inhibitor is greater than the effect of either the immune effector cell or the PARP inhibitor used alone.

[0013] In at least one specific aspect, the method of this application described above increases the CAR-T copy number in tumor tissue.

[0014] In at least one specific aspect, the method of this application described above increases the expression level of IFN-γ in tumor tissue.

[0015] In at least one specific aspect, the method of this application described above increases the infiltration of CD8+ T cell immune cells in tumor tissue.

[0016] In at least one specific aspect, the method of this application described above increases the infiltration of CD45+ immune cells.

[0017] In at least one specific aspect, the method of the present application reduces the infiltration of CD11b+Ly6G+ MDSCs cells.

[0018] In at least one specific aspect, the method of the present application reduces the infiltration of CD31+ cells.

[0019] In at least one specific aspect, the method of the present application does not cause a significant decrease in the expression of PD1, LAG3, and / or TIM3.

[0020] In at least one specific aspect, in the method of the present application, the way of administering immune effector cells and PARP inhibitors to an individual with a tumor is selected from any of the following: (1) administering PARP inhibitor first and then immune effector cells, (2) administering immune effector cells and PARP inhibitor simultaneously, and (3) administering immune effector cells first and then PARP inhibitor.

[0021] In at least one specific aspect, in the method of the present application, PARP inhibitor is administered first and then immune effector cells.

[0022] In at least one specific aspect, in the method of the present application, immune effector cells and PARP inhibitor are administered simultaneously.

[0023] In at least one specific aspect, in the method of the present application, immune effector cells are administered first and then PARP inhibitor.

[0024] In at least one specific aspect, the receptor is selected from: chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), or a combination thereof.

[0025] In at least one specific aspect, the tumor antigen is selected from EGFR or EGFRvIII.

[0026] In at least one specific aspect, the chimeric antigen receptor has: (i) an antibody or a fragment thereof that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, and CD3ζ; or (ii) an antibody or a fragment thereof that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD137, and CD3ζ; or (iii) an antibody or a fragment thereof that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, a co-stimulatory signal domain of CD137, and CD3ζ.

[0027] In at least one specific aspect, the antibody specifically recognizing a tumor antigen is an antibody targeting EGFR or EGFRvIII. Further preferably, the antibody has the following amino acid sequence, and the amino acid sequence is selected from any one of SEQ ID NO: 11 to SEQ ID NO: 29.

[0028] In at least one specific aspect, the chimeric antigen receptor has the following amino acid sequence, and the amino acid is selected from any one of SEQ ID NO: 30 to SEQ ID NO: 51, and SEQ ID NO: 55 to SEQ ID NO: 87.

[0029] In at least one specific aspect, the above-mentioned tumors include: breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvic cancer, spinal tumor, glioma, pituitary adenoma, Kaposi's sarcoma, combinations of the cancers and metastatic lesions of the cancers.

[0030] In at least one specific aspect, the above-mentioned immune effector cells are selected from: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytes or combinations 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.

[0031] In at least one specific aspect, the above-mentioned PARP inhibitor is administered orally, intraperitoneally and / or by injection.

[0032] In at least one specific aspect, in the above method, lymphocytapheresis is not performed on the individual.

[0033] In another aspect, the present application provides the use of immune effector cells expressing a receptor recognizing a tumor antigen and a PARP inhibitor in the preparation of a drug, wherein the drug is a drug for treating tumors or a drug for reducing the growth, survival or viability of cancer cells.

[0034] In at least one specific aspect, the therapeutic effect of the immune effector cells and the PARP inhibitor is greater than the effect of either the immune effector cells or the PARP inhibitor used alone.

[0035] In another aspect, the present application provides the use of immune effector cells expressing a receptor that recognizes a tumor antigen in the preparation of a medicament, wherein the medicament contains the cells and a PARP inhibitor and is used for treating tumors or reducing cancer cell growth, survival or viability in a human patient.

[0036] In at least one specific aspect, the therapeutic effect of the immune effector cells and the PARP inhibitor is greater than the effect of either the immune effector cells or the PARP inhibitor used alone.

[0037] In at least one specific aspect, the PARP inhibitor includes: talazoparib, niraparib, olaparib, rucaparib, Niraparib, Pamiparib, Fluzoparib, Mefuparib, and / or Ximiparib.

[0038] In at least one specific aspect, the immune effector cells and the PARP inhibitor can be administered in any order, the PARP inhibitor can be administered first and then the immune effector cells, they can be administered simultaneously, or the immune effector cells can be administered first and then the PARP inhibitor; preferably, the PARP inhibitor is olaparib.

[0039] In at least one specific aspect, the receptor is selected from: chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), or a combination thereof.

[0040] In at least one specific aspect, the chimeric antigen receptor has: (i) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, and CD3ζ; or (ii) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD137, and CD3ζ; or (iii) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, a co-stimulatory signal domain of CD137, and CD3ζ.

[0041] In at least one specific aspect, the above-mentioned tumors include: breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvic cancer, spinal tumor, glioma, pituitary adenoma, Kaposi's sarcoma, combinations of the cancers and metastatic lesions of the cancers.

[0042] In at least one specific aspect, the immune effector cells include: T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytes or combinations 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.

[0043] In at least one specific aspect, when using the drugs or kits of the present application, the PARP inhibitor is administered orally, intraperitoneally and / or by injection, and preferably, the PARP inhibitor is olaparib.

[0044] In at least one specific aspect, when using the drugs or kits of the present application, lymphocyte depletion is not performed on the individual.

[0045] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0046] Figure 1A It is a plasmid map of the recombinant vector MSCV-806-mCD28z; Figure 1B It is for the detection of the positive rate of CAR-T infection.

[0047] Figure 2A It is for the detection of EGFRvIII-positive cells; Figure 2B The expression of PARP1 in cells treated with olaparib was detected; Figure 2C It shows the cytotoxic effect of olaparib on breast cancer cells and CAR-T cells detected by the CCK8 assay.

[0048] Figure 3A It shows the killing effect of CAR-T treatment after olaparib pretreatment or the combined use of olaparib and CART on tumor cells detected by the cytotoxicity assay; Figure 3BIt shows the effects of olaparib on the secretion of cytokines TNFα, IL-2, and IFN-γ after the activation of CAR-T cells detected by ELISA experiment.

[0049] Figure 4A It shows the effects on cell proliferation after co-incubation of CAR-T in the olaparib pretreatment group in Example 4 with target cells; Figure 4B It shows the changes in the expression of proteins PD1, LAG3, and TIM3 related to exhaustion on the surface of CAR-T cells after co-incubation of CAR-T in the olaparib pretreatment group in Example 4 with target cells.

[0050] Figure 5 shows the experiment of combined treatment of olaparib and CAR-T on orthotopic breast cancer xenografts in mice: Figure 5A . Experimental flow chart; Figure 5B . Tumor volume detection results; Figure 5C . Detection of changes in mouse body weight; Figure 5D . Detection of mouse tumor weight; Figure 5E . Detection of CAR copy number in tumor tissue; Figure 5F . Immunohistochemical detection of CD4 / CD8 / CD31 in tumor tissue; Figure 5G . Detection of MDSC cells in tumor tissue.

[0051] Figure 6 shows the experiment of combined treatment of olaparib and CAR-T on orthotopic breast cancer xenografts in mice: Figure 6A . Experimental flow chart; Figure 6B . Tumor volume detection results; Figure 6C . Detection of changes in mouse body weight; Figure 6D . Detection of mouse tumor weight; Figure 6E . Detection of CAR copy number in tumor tissue; Figure 6F . Immunohistochemical detection of CD4 / CD8 / CD31 in tumor tissue; Figure 6G . Detection of MDSC cells in tumor tissue. Detailed implementation manners

[0052] This application relates to the combined use of immune effector cells and PARP inhibitors in the treatment of tumors. It should be understood that the present invention is not limited to the described methods and experimental conditions. Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics, molecular biology, and medicinal chemistry.

[0053] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, unless otherwise specified, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0054] Unless otherwise indicated, the practice of this application will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are all within the skill of the art. These techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: 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. Higgins eds. 1984); Transcription And Translation (B.D. Hames & S.J. Higgins eds. 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 and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, “Gene Expression Technology” (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0055] The present disclosure at least in part stems from the pioneering recognition that a combination treatment regimen comprising one or more cycles and / or doses of administering low-dose olaparib and immunotherapeutic cell treatment, either continuously, in any order, or substantially simultaneously, can be more effective in treating cancer in some subjects, and / or can initiate, enable, increase, enhance, or prolong the activity and / or number of immune cells, or a medically beneficial response of the tumor.

[0056] The term "PARP (poly(ADP)-ribose polymerase)" is a poly ADP-ribose polymerase that is involved in chromosomal remodeling, regulation of apoptosis, and cell division. PARP also plays a role in the immune response. To date, 17 family members have been discovered, including PARP1, PARP2, PARP3, Vault-PARP, and Tankyrase1. Among them, PARP1 is the most abundant, earliest discovered, and most extensively studied member of the PARP protein family, accounting for more than 80% of the total intracellular PARP activity. The biological function of PARP1 is that activated PARP1 forms a homodimer, cleaves the carbon-hydrogen bond of NAD+, generates nicotinamide and adenosine diphosphate ribose, further transfers and adds the latter to specific amino acid residues of specific nuclear receptor proteins (including itself), and continuously adds to form a straight-chain or branched-chain PARP long chain. PARP1 plays an important role in physiological processes such as DNA damage repair, gene transcription and expression, maintenance of genomic stability, cell cycle regulation, and apoptosis. Currently, three PARP inhibitor drugs have been approved by the US Food and Drug Administration (FDA) for marketing.

[0057] As an oral inhibitor of PARP1 and PARP2, Olaparib was granted accelerated approval by the FDA in December 2014 and marketed under the trade name Lynparza, becoming the world's first marketed PARP inhibitor. It is used for monotherapy in the treatment of advanced ovarian cancer with BRCA mutations that has been treated with 3 or more drugs. Currently, Olaparib is in Phase III clinical trials for advanced ovarian cancer that has relapsed after platinum-based chemotherapy and triple-negative breast cancer with BRAC deficiency, and its combination with paclitaxel in the treatment of gastric cancer is also in Phase III clinical trials. In addition, the treatment of prostate cancer has entered Phase II clinical trials. However, some patients are still insensitive to this type of drug.

[0058] In the specific embodiments, the PARP inhibitors used in this application include, but are not limited to, talazoparib, niraparib, olaparib, rucaparib, veliparib, Niraparib, Pamiparib, Fluzoparib, Mefuparib, and / or Ximingpiperidol, which are used in combination with CAR-T cells to treat breast cancer.

[0059] The applicant found in in vitro experiments that: Olaparib at a concentration higher than 1 μM can inhibit the growth of tumor cells, and Olaparib at a concentration higher than 2.5 μM can inhibit the growth of CAR-T cells; Olaparib can significantly enhance the killing effect of CAR-T cells on E0771-EGFRvIII cells and / or 4T1-EGFRvIII cells. Especially in the 5 μM Olaparib pretreatment group, the promotion of the killing effect is the most significant, which is better than the killing effect of the 5 μM Olaparib combination treatment group, suggesting that a higher concentration of Olaparib may cause a certain degree of inhibition of the growth of CAR-T; compared with the CAR-T single treatment group, the level of IFN-γ in the cell culture supernatant is significantly increased in the Olaparib pretreatment group and the Olaparib combination treatment group.

[0060] In this application, the use of Olaparib can significantly enhance the killing effect of CAR-T cells on E0771-EGFRvIII and 4T1-EGFRvIII cells. Especially in the 5 μM Olaparib pretreatment group (that is, first treating with Olaparib and then providing CAR-T cells for treatment), the promotion of the killing effect is the most significant, which is better than the killing effect of the 5 μM Olaparib combination treatment group (that is, first treating with Olaparib and then treating with both Olaparib and CAR-T cells simultaneously), indicating that a higher concentration of Olaparib may cause a certain degree of inhibition of the growth of CAR-T. However, compared with the CAR-T single treatment group, the 5 μM Olaparib combination treatment group still has a higher synergistic lethal effect.

[0061] Compared with the olaparib pretreatment group, the olaparib combination therapy group and the CAR-T monotherapy group, the level of IFN-γ in the co-culture supernatant of E0771-EGFRvIII cells and CAR-cells was significantly increased, indicating that olaparib can effectively improve the killing ability of CAR-T cells against tumor cells. After the CAR-T cells in the olaparib pretreatment group were co-incubated with cancer cells, the proliferation of CAR-T cells was not significantly affected; while in the olaparib combination therapy group, due to the presence of olaparib in the co-culture environment, the proliferation of CAR-T cells was slightly inhibited, but there was no statistical significance. After the CAR-T cells in the olaparib pretreatment group were co-incubated with cancer cells, the expression of exhaustion-related proteins PD1, LAG3, and TIM3 on the surface of CAR-T cells did not change significantly; in the olaparib combination therapy group, the expression of exhaustion-related proteins PD1, LAG3, and TIM3 on the surface of CAR-T cells also did not change significantly. This suggests that olaparib treatment does not lead to the exhaustion of CAR-T, thus not affecting the killing effect of effector T cells, indicating the combined use of olaparib and CAR-T cells.

[0062] The applicant found in the experiment that using the method, pharmaceutical preparation or kit of the present application can effectively increase the CAR-T copy number in tumor tissues, increase the expression level of IFN-γ in tumor tissues, increase the infiltration of CD8+ T cell immune cells in tumor tissues, increase the infiltration of CD45+ immune cells, and reduce the infiltration of MDSCs cells with CD11b + Gr1 + and reduce the infiltration of CD31+ cells.

[0063] In further in vivo experiments, it was found that the combination of CAR-T cells and 50 mg / kg olaparib could significantly increase the copy number of CAR in tumor tissues in mice, improve the tumor immunosuppressive environment, and did not affect the body weight of mice, but could significantly inhibit the tumor volume and tumor weight of murine breast cancer, indicating that olaparib may significantly enhance the tumor cell growth inhibitory effect of CAR-T cells by homing CAR-T cells to the tumor local area and activating their proliferation. In in vitro experiments, another PARP inhibitor, veliparib, could affect the proliferation level of E0771 ERVIII cells at a concentration of 10 μM and above, and could significantly inhibit the proliferation of E0771 ERVIII cells only at a concentration of 20 μM and above, but caused a significant inhibitory effect on the proliferation of murine CAR-T cells at this concentration; in the LDH cell killing experiment, it was found that 10 μM veliparib could not enhance the killing effect of CART cells. Another small molecule drug, JQ1, is a BET bromodomain inhibitor that acts on BRD4. By targeting and inhibiting BRD4, it can induce tumor cell apoptosis, i.e., slow down proliferation, thereby achieving an anti-tumor effect. In in vitro experiments, after treating E0771 ERVIII cells with different concentrations of JQ1 for 48 hours, it was found that JQ1 could not inhibit the proliferation of E0771 ERVIII cells, but caused a significant inhibitory effect on the proliferation of murine T cells and CAR-T cells, and showed a dose-dependent manner.

[0064] The applicant also found that this application can not only improve the anti-cancer effect of refractory cancers, but also does not require lymphocyte depletion when using CAR-T cells, thus greatly reducing the low anti-cancer treatment effect caused by lymphocyte depletion and the toxic side effects caused by reducing the damage to normal tissues, especially the severe inhibition of the bone marrow.

[0065] In a specific embodiment, the PARP inhibitor can be safely administered orally or non-orally by itself, or in the form of a compound formed with a pharmaceutically acceptable carrier excipient and other additives (such as tablets, sustained-release preparations, capsules, injections, solutions) orally or non-orally in a safe manner. When administered orally, the composition can be formulated into tablets, dragees or capsules. Lactose or starch can be used as a carrier for preparing the oral composition, and gelatin, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, etc. are suitable binders or granulating agents. Starch or microcrystalline cellulose can be selected as a disintegrant, and talc powder, colloidal silica, glyceryl stearate, calcium stearate or magnesium stearate, etc. are often used as suitable anti-adhesives and lubricants. For example, tablets can be prepared by compressing wet granules. The active ingredient, together with the carrier and optionally a disintegrating additive, forms a mixture, which is granulated with an aqueous solution, alcoholic or aqueous-alcoholic solution of the binder in a suitable apparatus, and the dried granules are then added with other disintegrants, lubricants and anti-adhesives to press the mixture into tablets. To increase solubility, the heterocyclic derivative can be freed and made into a pharmaceutically acceptable organic acid, preferably methanesulfonic acid, fumaric acid, etc., to facilitate administration in the form of an injection. Although the dosage varies depending on the treatment subject, administration method, symptoms and other factors.

[0066] In a specific embodiment, the PARP inhibitor is the oral chemotherapy drug Olaparib (Olaparib, AZD2281) for treating advanced breast cancer, ovarian cancer, etc.

[0067] In certain embodiments, the daily oral dose of Olaparib for an individual with a tumor is 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.

[0068] In this application, the administration times of immune effector cells and PARP inhibitors are not sequential; PARP inhibitors can be administered first followed by immune effector cells; they can also be administered simultaneously; or immune effector cells can be administered first followed by PARP inhibitors. In certain embodiments, the immune effector cell therapy is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 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 before the administration of PARP inhibitors. In certain embodiments, the immune effector cell therapy is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 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 after the administration of PARP inhibitors.

[0069] The term "immune effector cell" refers to cells that participate in the immune response and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes. Preferably, the immune effector cells are T cells, NK cells, and NKT cells.

[0070] In a specific embodiment, the T cells are autologous T cells, xenogeneic T cells, or allogeneic T cells.

[0071] In a specific embodiment, the natural killer cells are allogeneic NK cells.

[0072] The term "immune effector cell activity" refers to the ability of immune effector cells to respond to antigen stimulation, proliferate to form immune effector substances, and carry out specific immune responses. For example, promoting the killing of target cells or inhibiting their growth or proliferation.

[0073] The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and refer to the amount of a compound, preparation, substance or composition that effectively achieves a particular biological result, such as but not limited to an amount or dose sufficient to promote a T cell response. When indicating an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount" or "therapeutically effective amount", the precise dosage of the immune effector cells or therapeutic agent of the present application can be determined by a physician taking into account the individual's age, weight, tumor size, degree of metastasis and the condition of the patient (subject). An effective amount of immune effector cells refers to but is not limited to an amount of immune effector cells that can increase, enhance or prolong the anti-tumor activity of immune effector cells; an increase in the number of anti-tumor immune effector cells or activated immune effector cells; promotion of IFN-γ secretion; tumor regression, tumor shrinkage, tumor necrosis.

[0074] The terms "therapeutically effective amount" and "effective amount" are used interchangeably herein and refer to the amount of a compound, preparation, substance or composition that effectively achieves a particular biological result, such as but not limited to an amount or dose sufficient to promote a T cell response. When indicating an "immunologically effective amount", "anti-tumor effective amount", "tumor inhibitory effective amount" or "therapeutically effective amount", the precise dosage of the immune effector cells or therapeutic agent of the present application can be determined by a physician taking into account the individual's age, weight, tumor size, degree of metastasis and the condition of the patient (subject). An effective amount of immune effector cells refers to but is not limited to an amount of immune effector cells that can increase, enhance or prolong the anti-tumor activity of immune effector cells; an increase in the number of anti-tumor immune effector cells or activated immune effector cells; promotion of IFN-γ secretion; tumor regression, tumor shrinkage, tumor necrosis.

[0075] The term "non-lymphodepletion" or "not performing lymphocyte depletion" means not depleting lymphocytes in the subject. This includes but is not limited to not administering lymphocyte depletion agents, total body radiation therapy or combinations thereof or other means that cause depletion of lymphocyte numbers; however, after administering lymphocyte depletion agents, total body radiation therapy or combinations thereof or other means that cause depletion of lymphocyte numbers, when the lymphocyte depletion rate in the subject is less than 60%, we believe that although lymphocyte depletion treatment has been administered, the effect of lymphocyte depletion has not been achieved, and it will also fall within the scope of "non-lymphodepletion" of the present application equivalently.

[0076] As used herein, a "chimeric receptor", a fusion molecule formed by ligating DNA fragments or corresponding cDNAs of proteins from different sources using genetic recombination techniques, includes an extracellular domain, a transmembrane domain and an intracellular domain. Chimeric receptors include but are not limited to: chimeric antigen receptors (CARs), modified T cell (antigen) receptors (TCRs), T cell fusion proteins (TFPs), T cell antigen couplers (TACs).

[0077] As used herein, "chimeric antigen receptor" or "CAR" refers to a group of polypeptides that, when present in an immune effector cell, confer specificity for a target cell (usually a cancer cell) to the cell and have intracellular signal generation. A 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"), which includes functional signaling domains derived from the stimulatory molecule and / or costimulatory molecule as defined below. In some aspects, the group of polypeptides are adjacent to each other. The group of polypeptides includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, e.g., that can couple the antigen-binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is the ζ chain that binds to the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, the CAR includes a chimeric fusion protein that comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that includes a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein that comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain that includes a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein that comprises an extracellular antigen-binding domain, a transmembrane domain, and includes two functional signalings derived from one or more costimulatory molecules.

[0078] As used herein, a "transmembrane domain" refers to a region of a protein sequence that spans the cell membrane and may include additional amino acids adjacent to one or more transmembrane regions, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is a domain related to one of the other domains of the chimeric receptor. For example, in one embodiment, the transmembrane domain may be from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain is derived. In certain cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid the binding of such a domain to the transmembrane domains of the same or different surface membrane proteins, for example, to minimize the interaction with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another chimeric receptor on the cell surface expressing the chimeric receptor. The transmembrane domain may be derived from natural or recombinant sources. When the source is natural, the domain may be derived from any membrane-bound protein or transmembrane protein. In one aspect, the transmembrane domain is capable of transducing a signal to the intracellular domain whenever the chimeric receptor binds to the target. Transmembrane domains specifically used in the present application may include at least the following transmembrane domains: for example, the α, β, or ζ chain of the T-cell receptor, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.In certain embodiments, the transmembrane domain may include at least the following transmembrane regions: for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, 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.

[0079] In some cases, the transmembrane domain may be linked to the extracellular region of the CAR, such as the antigen-binding domain of the CAR, via a hinge (e.g., a hinge from a human protein). Optionally, a short oligopeptide or polypeptide linker having a length between 2 and 10 amino acids may form a bond between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine diad provides a particularly suitable linker.

[0080] As used herein, the "intracellular domain" has the same meaning as the "cytoplasmic domain" and includes an intracellular signaling domain. The intracellular signaling domain is generally responsible for activating at least one of the normal immune effector functions of an immune cell into which a chimeric receptor has been introduced. The term "effector function" refers to the specialized function of a cell. The immune effector functions of T immune cells can be, for example, cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces signals for immune effector functions and directs the cell to perform a specific function. Although it is generally possible to apply the entire intracellular signaling domain, it is not necessary to use the entire chain in many cases. In terms of using truncated portions of the intracellular signaling domain, such truncated portions can be used in place of the full chain as long as they transduce signals for immune effector functions. Thus, the term intracellular signaling domain is meant to include truncated portions of the intracellular signaling domain that are sufficient to transduce signals for immune effector functions.

[0081] Specifically, the chimeric antigen receptor used in this application has: (i) an antibody or fragment thereof that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody or fragment thereof that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signaling domain of CD137, and CD3ζ; or (iii) an antibody or fragment thereof that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a co-stimulatory signaling domain of CD28, a co-stimulatory signaling domain of CD137, and CD3ζ.

[0082] In a specific embodiment, a murine CD8α signal peptide, a murine CD8α hinge region and transmembrane region, a murine CD28 intracellular domain, and a murine CD3ζ intracellular domain are employed.

[0083] In a specific embodiment, a human CD8α signal peptide, a human CD8α hinge region and transmembrane region, a human CD28 transmembrane domain, a human CD28 intracellular domain, and a human CD3ζ intracellular domain can be employed.

[0084] It is well known that the signals generated by a single TCR alone are not sufficient to fully activate T cells, and secondary and / or co-stimulatory signals are also required. Thus, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, such as co-stimulatory domains).

[0085] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence, which regulates the activation of immune cells in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated, for example, by the binding of the TCR / CD3 complex to the MHC-antigen peptide complex, and it leads to mediating T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as the "primary signaling domain") that acts in a stimulatory manner can contain a signaling motif called the immunoreceptor tyrosine-based activation motif or ITAM. Examples of cytoplasmic signaling sequences containing ITAM that are particularly useful for the present application include, but are not limited to, those derived from: CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. The intracellular signaling domain in any of the CARs of the present application includes an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ. In the specific CARs of the present application, the primary signaling sequence of CD3-ζ is an equivalent residue from human or non-human species such as mouse, rodent, monkey, ape, etc.

[0086] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence, which regulates the activation of immune cells in a stimulatory manner for at least some aspects of the immune cell signaling pathway. In one aspect, the signal is a primary signal initiated, for example, by the binding of the TCR / CD3 complex to the MHC-antigen peptide complex, and it leads to mediating T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as the "primary signaling domain") that acts in a stimulatory manner can contain a signaling motif called the immunoreceptor tyrosine-based activation motif or ITAM. Examples of cytoplasmic signaling sequences containing ITAM that are particularly useful for the present application include, but are not limited to, those derived from: CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. The intracellular signaling domain in any of the CARs of the present application includes an intracellular signaling sequence, such as the primary signaling sequence of CD3-ζ. In the specific CARs of the present application, the primary signaling sequence of CD3-ζ is an equivalent residue from human or non-human species such as mouse, rodent, monkey, ape, etc.

[0087] The term "costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as but not limited to proliferation. A costimulatory molecule is a cell surface molecule other than the 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). Further examples of such costimulatory molecules include 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, and the ligand that specifically binds CD83.

[0088] The co-stimulatory intracellular signaling domain can be the intracellular portion of a co-stimulatory molecule. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecule (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, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD5, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds to CD83, etc.

[0089] The intracellular signaling domain can include the entire intracellular portion of the molecule or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0090] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided in GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.; and the "4-1BB co-stimulatory 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, apes, etc. In one aspect, the "4-1BB co-stimulatory domain" is equivalent residues from humans or from non-human species such as mice, rodents, monkeys, apes, etc.

[0091] The term "scFv" refers to a fusion protein comprising at least one antibody fragment including the variable region of a light chain and at least one antibody fragment including the variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, the scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), the scFv can include VL-linker-VH or can include VH-linker-VL.

[0092] The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. An antibody can be polyclonal or monoclonal, multichain or single-chain, or a full immunoglobulin, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0093] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies (such as sdAb), camelid VHH domains, multispecific antibodies formed from antibody fragments (e.g., a bivalent fragment including two Fab fragments linked by a disulfide bond in the hinge region), and isolated CDRs or other epitope-binding fragments of an antibody. The term "scFv" refers to a fusion protein comprising at least one antibody fragment including the variable region of a light chain and at least one antibody fragment including the variable region of a heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, the scFv can have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), the scFv can include VL-linker-VH or can include VH-linker-VL.

[0094] The term "antibody heavy chain" refers to the larger of the two polypeptide chains that exist in an antibody molecule in its naturally occurring conformation and that generally determines the class to which the antibody belongs.

[0095] The term "antibody light chain" refers to the smaller of the two polypeptide chains that exist in an antibody molecule in its naturally occurring conformation. κ (kappa) and λ (lambda) light chains refer to the two major isotypes of antibody light chains.

[0096] The term "recombinant antibody" refers to an antibody produced using recombinant DNA techniques, such as an antibody expressed by a phage or yeast expression system. The term should also be construed to refer to an antibody that has been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or by specifying the amino acid sequence of the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques available and well known in the art.

[0097] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response can involve antibody production or the activation of cells with specific immune capabilities or both. Those skilled in the art should understand that any macromolecule that includes virtually all proteins or peptides can act as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. When this term is used herein, those skilled in the art should understand that any DNA that includes a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response is capable of encoding an "antigen". In addition, those skilled in the art should understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is obvious that the present application includes, but is not limited to, using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode polypeptides that elicit a desired immune response. Those skilled in the art should understand that an antigen need not be encoded by a "gene" at all. It is obvious that an antigen can be produced synthetically, or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.

[0098] The term "recombinant antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a phage or yeast expression system. This term should also be interpreted to refer to an antibody that has been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or the amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence techniques available and well-known in the art.

[0099] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response can involve antibody production or the activation of cells with specific immune capabilities or both. Those skilled in the art should understand that any macromolecule that includes virtually all proteins or peptides can act as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. When this term is used herein, those skilled in the art should understand that any DNA that includes a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response is capable of encoding an "antigen". In addition, those skilled in the art should understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is obvious that the present application includes, but is not limited to, using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode polypeptides that elicit a desired immune response. Those skilled in the art should understand that an antigen need not be encoded by a "gene" at all. It is obvious that an antigen can be produced synthetically, or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.

[0100] "Tumor antigen" refers to an antigen that newly appears or is overexpressed during the occurrence and development of hyperplastic diseases. In some aspects, the hyperplastic disorder of the present application refers to a tumor. The tumor antigen of the present application is EGFR or EGFRvIII. EGFR is overexpressed or mutated in many tumors. So far, antibodies against the EGFR287-302 epitope are considered to be able to achieve the purpose of recognizing EGFR, EGFRvIII, and de4EGFR overexpressed on the tumor surface.

[0101] In at least one specific embodiment of the present application, the antibody that specifically recognizes the tumor antigen is an antibody targeting EGFR or EGFRvIII. In at least one specific embodiment of the present application, the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:11, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:12, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:13, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:14, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:15, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:16, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:16, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:17, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:18, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:19, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:20, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:21, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:22, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:23, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:24, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:25, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:26, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:27, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:28, or the amino acid sequence of the antibody that specifically recognizes the tumor antigen is as shown in SEQ ID NO:29.

[0102] In at least one specific embodiment of the present application, the amino acid sequence of the chimeric antigen receptor of the present application is as shown in SEQ ID NO: 30, or as shown in SEQ ID NO: 31, or as shown in SEQ ID NO: 32, or as shown in SEQ ID NO: 33, or as shown in SEQ ID NO: 34, or as shown in SEQ ID NO: 35, or as shown in SEQ ID NO: 36, or as shown in SEQ ID NO: 37, or as shown in SEQ ID NO: 38, or as shown in SEQ ID NO: 39, or as shown in SEQ ID NO: 40, or as shown in SEQ ID NO: 41, or as shown in SEQ ID NO: 42, or as shown in SEQ ID NO: 43, or as shown in SEQ ID NO: 44, or as shown in SEQ ID NO: 45, or as shown in SEQ ID NO: 46, or as shown in SEQ ID NO: 47, or as shown in SEQ ID NO: 48, or as shown in SEQ ID NO: 49, or as shown in SEQ ID NO: 50, or as shown in SEQ ID NO: 51, or as shown in SEQ ID NO: 55, or as shown in SEQ ID NO: 56, or as shown in SEQ ID NO: 57, or as shown in SEQ ID NO: 58, or as shown in SEQ ID NO: 59, or as shown in SEQ ID NO: 60, or as shown in SEQ ID NO: 61, or as shown in SEQ ID NO: 62, or as shown in SEQ ID NO: 63, or as shown in SEQ ID NO: 64, or as shown in SEQ ID NO: 65, or as shown in SEQ ID NO: 66, or as shown in SEQ ID NO: 67, or as shown in SEQ ID NO: 68, or as shown in SEQ ID NO: 69, or as shown in SEQ ID NO: 70, or as shown in SEQ ID NO: 71, or as shown in SEQ ID NO: 72, or as shown in SEQ ID NO: 73, or as shown in SEQ ID NO: 74, or as shown in SEQ ID NO: 75, or as shown in SEQ ID NO: 76, or as shown in SEQ ID NO: 77, or as shown in SEQ ID NO: 78, or as shown in SEQ ID NO: 79, or as shown in SEQ ID NO: 80, or as shown in SEQ ID NO: 81, or as shown in SEQ ID NO: 82, or as shown in SEQ ID NO: 83, or as shown in SEQ ID NO: 84, or as shown in SEQ ID NO: 85, or as shown in SEQ ID NO: 86, or as shown in SEQ ID NO: 87.

[0103] The term "cancerous tumor" refers to a broad category of disorders characterized by excessive proliferative cell growth in vitro (e.g., transformed cells) or in vivo. Conditions that can be treated or prevented by the methods of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like. The methods of the present application can achieve inhibition and / or reversal of the unwanted excessive proliferative cell growth involved in such conditions. Specific examples of cancers include, but are not limited to: breast cancer, prostate cancer, leukemia, lymphoma, nasopharyngeal cancer, blood cancers, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or intraocular, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, renal or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis angiosarcoma, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T cell lymphoma, environmentally induced cancers, combinations of the foregoing cancers, and metastatic lesions of the foregoing cancers.

[0104] The terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include primary subject cells and their progeny.

[0105] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., tumor antigen) protein present in a sample, but that antibody or ligand substantially does not recognize or bind to other molecules in the sample.

[0106] The term "refractory" refers to a disease, e.g., a tumor, that does not respond to treatment. In embodiments, a refractory tumor can be resistant to treatment prior to or at the start of treatment. In other embodiments, a refractory tumor can become resistant during treatment. A refractory tumor is also referred to as a resistant tumor. In the present application, refractory tumors include, but are not limited to, tumors that are radioresistant, recur after radiotherapy, are chemoresistant, recur after chemotherapy, are insensitive to CAR-T therapy or recur after treatment. Refractory or recurrent malignancies can be treated using the treatment regimens described herein.

[0107] As used herein, "recurrent" refers to the return of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement, e.g., after a previous treatment with a therapy, e.g., a cancer therapy.

[0108] The terms "individual" and "subject" are used interchangeably herein and can refer to humans and animals from other species.

[0109] The term "enhanced" refers to allowing a subject or tumor cell to improve its ability to respond to a treatment disclosed herein. For example, an enhanced response can include an increase of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more in responsiveness. As used herein, "enhanced" can also refer to increasing the number of subjects responding to a treatment such as adoptive cell therapy. For example, an enhanced response can refer to the total percentage of subjects responding to a treatment, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more.

[0110] In one aspect, treatment is determined by clinical outcomes; an increase, enhancement or prolongation of anti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells as compared to the number prior to treatment, promotion of IFN-γ, TNFα secretion, or a combination thereof. In another aspect, the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; an anti-tumor response by the immune system; tumor growth, recurrence or spread or a combination thereof. In a further aspect, the treatment effect is predicted by the presence of T cells, the presence of gene markers indicative of T cell inflammation, promotion of IFN-γ, TNFα secretion, or a combination thereof.

[0111] The adoptive immune cells disclosed herein can be administered to an individual by a variety of routes, including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, rectally, intracisternally, intratumorally, intravasally, intradermally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis.

[0112] The total amount of a reagent to be administered in practicing the methods of the present application can be administered to a subject as a single dose by bolus injection or by infusion over a relatively short period of time, or can be administered using a fractionated treatment regimen, where multiple doses are administered over an extended period of time. Those skilled in the art will know that the amount of a composition for treating a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, the skilled person will adjust the specific dose as needed. Generally, initially, the formulation of the composition as well as the route and frequency of administration are determined using Phase I and Phase II clinical trials.

[0113] Scope: Throughout the disclosure, various aspects of the present application may exist in a range form. It should be understood that the description in range form is for convenience and brevity only and should not be regarded as an immutable limitation on the scope of the present application. Thus, the description of a range should be considered to specifically disclose all possible sub-ranges and the individual values within that range. For example, a range description such as from 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual values within that range, such as 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95 - 99% identity includes ranges having 95%, 96%, 97%, 98%, or 99% identity, and includes sub-ranges such as 96 - 99%, 96 - 98%, 96 - 97%, 97 - 99%, 97 - 98%, and 98 - 99% identity. This applies regardless of the width of the range.

[0114] According to the present disclosure, those skilled in the art should understand that many changes or alterations can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. The invention is not limited in scope to the specific embodiments described herein (which are only intended to be illustrative of aspects of the invention), and methods and components that are functionally equivalent are within the scope of the invention.

[0115] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual (Third Edition)" edited by J. Sambrook et al. (Science Press, 2002), or according to the conditions recommended by the manufacturer.

[0116] Exemplary antigen receptors of the present application, including CARs, and methods for engineering and introducing the receptors into cells, are referred to those disclosed in, for example, Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication Nos. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018018958A1.

[0117] Example 1 Construction of CAR-T cells

[0118] In this example, a second-generation CAR targeting EGFRvIII was used. For the needs of later animal experiments, the transmembrane domain and intracellular domain of CAR were constructed using mouse gene sequences.

[0119] The coding sequence of mouse CD8α signal peptide (SEQ ID NO: 1), the coding sequence of anti-EGFRvIII monoclonal antibody (SEQ ID NO: 2), the coding sequence of mouse CD8α hinge region and transmembrane region (SEQ ID NO: 3), the coding sequence of mouse CD28 intracellular domain (SEQ ID NO: 4), and the coding sequence of mouse CD3ζ intracellular domain (SEQ ID NO: 5) were connected in sequence. The 806-mCD28z gene fragment was obtained by in vitro gene synthesis, and the IRES-GFP fragment in the retroviral vector MSCV-IRES-GFP (purchased from Addgene) was replaced with Mlu Ⅰ and Sal Ⅰ double digestion sites to obtain the recombinant vector MSCV-806-mCD28z( Figure 1A ). In Example 1, the amino acid sequence of the antibody encoded by SEQ ID NO: 2 is as shown in SEQ ID NO: 12, and the amino acid sequence of the chimeric antigen receptor of the CAR-T cells constructed in Example 1 is as shown in SEQ ID NO: 30.

[0120] The 293T cells (purchased from ATCC) were infected with the recombinant vector MSCV-806-mCD28z to obtain the packaged retrovirus. The infection method is a conventional infection method in the preparation process of T cells expressing chimeric antigen receptors in the art.

[0121] The spleen of C57BL / 6 mice was ground to obtain lymphocytes. After treatment with the CD3+ mouse T cell negative selection kit, the obtained mouse CD3+ T lymphocytes were added to Dynabeads Mouse T-activator CD3 / CD28 magnetic beads for activation and stimulation at a volume ratio of 1:1, and placed in a cell culture incubator. The culture medium was RPMI1640 complete medium (10% FBS + 50 μM β-mercaptoethanol + 100 U / mL of IL-2 + 1 ng / mL IL-7).

[0122] The activated mouse spleen CD3+ T lymphocytes were inoculated into a 24-well plate coated with Retronectin (5 μg / mL). After adding the retrovirus and infecting for 24 h, the fresh medium was replaced to obtain mouse 806-mCD28z-CAR-T cells. Flow cytometry was used to detect the positive rate of CAR-T cell infection, and the positive rate of 806-mCD28z-CAR-T cell infection was 70.7%( Figure 1B ).

[0123] Example 2: Detecting the toxicity of olaparib to breast cancer cells and CAR-T cells by CCK8 assay

[0124] Since the EGFR287-302 epitope is only exposed in tumors with EGFRvIII or overexpressed EGFR, and this epitope is hidden in normal tissues (Gan HK et al., Targeting of a conformationally exposed, tumor-specific epitope of EGFR as a strategy for cancer therapy. Cancer Res, 2012, 72(12):2924-2930.). Therefore, murine breast cancer cell models of E0771 and 4T1 overexpressing murine EGFR with the human EGFR amino acid epitope at positions 287-302 (E0771-EGFRvIII, 4T1-EGFRvIII) were established using conventional molecular biology methods. Flow cytometry was used to sort and screen cells positive for the EGFRvIII target for subsequent research ( Figure 2A ). E0771 and 4T1 cells were purchased from the American Type Culture Collection (ATCC).

[0125] As an effective inhibitor of PARP1, olaparib can inhibit the DNA damage repair process involving PARP1. Different levels of PARP1 expression were detected in murine breast cancer cell lines, murine T cells, and CART cells ( Figure 2B ), so CCK8 proliferation assays were needed to detect the effect of olaparib on the proliferation of tumor cells and CART cells.

[0126] Take the 806-m28Z CAR-T cells from Example 1 and seed them in 96-well plates at 1×10 4 cells per well with 100 μl of medium. Seed breast cancer cells E0771-EGFRvIII and 4T1-EGFRvIII in 96-well plates at 1×104 cells per well with 100 μl of medium. Add different concentrations of olaparib to the cells to make 10 concentration gradients (i.e., the olaparib treatment concentrations are 50 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM, 0 μg / ml). After 48 h of drug treatment for different groups of cells, add 10 μl of CCK8 substrate chromogenic reagent (Dojindo) to each well. After incubation at 37 °C for 1 h, measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell viability respectively.

[0127] The formula for calculating cell viability is as follows:

[0128] Cell viability (%) = [A (drug added) - A (blank)] / [A (0 drug added) - A (blank)]

[0129] The results are as Figure 2C shown. After treating 806-mCD28z-CAR-T cells, 4T1-EGFRvIII cells, and E0771-ERVIII cells with different concentrations of olaparib respectively, the growth of 806-mCD28z-CAR-T cells and E0771-EGFRVIII cells was inhibited to a certain extent. Among them, the proliferation of 806-mCD28z-CAR-T cells was inhibited to a certain extent under the treatment of 2.5 μM and higher concentrations of olaparib, and the inhibition rate was nearly 80%; while the proliferation of the E0771-EGFRVIII cell line was inhibited after treatment with 1 μM and higher concentrations of olaparib, and the inhibition rate was 65%-90%; the proliferation of the 4T1-EGFRvIII cell line was inhibited after treatment with 1 μM and higher concentrations of olaparib, and the inhibition rate was 30-40%( Figure 2C ). This indicates that the effective concentrations of olaparib on the growth of 806-mCD28z-CAR-T cells, E0771-EGFRVIII cells, and 4T1-EGFRvIII cells are different, and it also suggests that appropriate concentrations of olaparib should be selected for subsequent research. Since the proliferation of E0771-EGFRVIII cells and 4T1-EGFRvIII cells was inhibited under the treatment of 1 μM olaparib, but the proliferation of 806-mCD28z-CAR-T cells was not affected; while under the treatment of 5 μM olaparib, the proliferation of E0771-EGFRVIII cells, 4T1-EGFRvIII cells, and 806-mCD28z-CAR-T cells was all inhibited. Therefore, two concentrations of olaparib were selected to clarify the effect of the combination of olaparib and CAR-T in the treatment of breast cancer under different concentrations and different cell response states. Therefore, in subsequent experiments, 1 μM and 5 μM olaparib were selected for research.

[0130] Example 3: Cytotoxicity experiment to detect the killing effect of olaparib pretreatment followed by CAR-T treatment or the combined use of olaparib and CAR-T on tumors, and ELISA experiment to detect the effect of olaparib on the secretion of cytokines granzyme B, IL-2, and IFN-γ

[0131] Experimental grouping for co-incubation with antigen target-negative cells: divided into UTD group and 806-mCD28z-CAR-T group (or denoted as 806-28Z). Take the untreated mouse T cells and 806-mCD28z-CAR T cells in Example 1 and co-incubate them with E0771 or 4T1 cells at ratios of 1:3, 1:1, and 3:1 respectively. After 16 h, use Cytox 96 Non-Radioactive Cytotoxicity Assay to detect the secretion of LDH in the supernatant, and calculate the killing toxicity of 806-mCD28z CAR T cells against tumor cells. As Figure 3A shown, 806-mCD28z-CAR T cells have no killing effect on antigen-negative target cells

[0132] Experimental grouping for co-incubation with antigen target-positive cells: divided into UTD group, 806-mCD28z-CAR-T group (or denoted as 806-28Z), olaparib pretreatment group, and olaparib combination treatment group;

[0133] Among them, the UTD group is untreated mouse T cells;

[0134] The olaparib pretreatment group includes: UTD + 1 μM olaparib pretreatment group (or denoted as UTD + Olaparib 1 μM pretreatment), UTD + 5 μM olaparib pretreatment group (or denoted as UTD + Olaparib 5 μM pretreatment), 806-mCD28z-CAR-T + 1 μM olaparib pretreatment group (or denoted as 806-28Z + Olaparib 1 μM pretreatment), 806-mCD28z-CAR-T + 5 μM olaparib pretreatment group (or denoted as 806-28Z + Olaparib 5 μM pretreatment);

[0135] The olaparib combination treatment group: UTD + 1 μM olaparib combination treatment group (or denoted as UTD + Olaparib 1 μM), UTD + 5 μM olaparib combination treatment group (or denoted as UTD + Olaparib 5 μM), 806-mCD28z-CAR-T + 1 μM olaparib combination treatment group (or denoted as 806-28Z + Olaparib 1 μM), 806-mCD28z-CAR-T + 5 μM olaparib combination treatment group (or denoted as 806-28Z + Olaparib 5 μM).

[0136] Olaparib pretreatment group: Different concentrations of olaparib (1 μM, 5 μM) were added to the culture media of E0771-EGFRVIII or 4T1-EGFRvIII tumor cells respectively. After culturing at 37°C for 24 hours, the cells were digested with trypsin and resuspended in fresh culture media. The untreated mouse T cells and 806-mCD28z-CAR T cells from Example 1 were co-incubated with E0771-EGFRvIII or 4T1-EGFRvIII cells pretreated with different concentrations of olaparib at ratios of 1:3, 1:1, and 3:1 respectively. After 16 hours, the secretion of LDH in the supernatant was detected using Cytox 96 Non-Radioactive Cytotoxicity Assay, and the killing toxicity of 806-mCD28z CAR T cells against tumor cells pretreated with different concentrations of olaparib was calculated. The specific detection steps and calculation method can be found in the Promaga Cytox 96 Non-Radioactive Cytotoxicity Assay instruction manual (Promaga Corporation, REF: G1782); meanwhile, the ELISA kit (Linker Biotechnology CAT: 1822) was used to detect the secretion of cytokines Granzyme B, IFN-γ, and IL-2 in the supernatant, and the specific experimental steps refer to the Linker Biotechnology Mouse Granzyme B, IFN-γ, IL-2 ELISA kit instruction manual.

[0137] Olaparib combination treatment group: Different concentrations of olaparib (1 μM, 5 μM) were added to the culture media of E0771-EGFRVIII or 4T1-EGFRvIII tumor cells respectively. After culturing at 37°C for 24 h, the cells were digested with trypsin and resuspended in fresh culture media. The untreated mouse T cells and 806-mCD28z-CAR-T cells in Example 1 were co-incubated with E0771-EGFRvIII cells or 4T1-EGFRvIII cells treated with different concentrations of olaparib at ratios of 1:3, 1:1, and 3:1 respectively, and the corresponding concentrations of olaparib (1 μM, 5 μM) were added to the culture media for continued treatment for 16 h. Then, the secretion of LDH in the supernatant was detected using Cytox 96 Non-Radioactive Cytotoxicity Assay, and the killing toxicity of 806-mCD28z CAR-T cells against tumor cells pretreated with different concentrations of olaparib was calculated. The specific detection steps and calculation methods are described in the Promaga Cytox 96 Non-Radioactive Cytotoxicity Assay instruction manual (Promaga Corporation, REF: G1782); meanwhile, the ELISA kit (Linker Biotechnology CAT: 1822) was used to detect the secretion of cytokines Granzyme B, IFN-γ, and IL-2 in the supernatant, and the specific experimental steps refer to the Linker Biotechnology Mouse Granzyme B, IFN-γ, IL-2 ELISA kit instruction manual.

[0138] As Figure 3A shown, the use of olaparib can significantly enhance the killing effect of CAR-T cells against E0771-EGFRvIII and 4T1-EGFRvIII cells. Especially in the group pretreated with 5 μM olaparib, the promotion of the killing effect is the most significant, which is better than the killing effect of the 5 μM olaparib combination treatment group. This suggests that a higher concentration of olaparib may cause a certain degree of inhibition of the growth of CAR-T. Compared with the CAR-T single treatment group, the 5 μM olaparib combination treatment group still has a higher synergistic lethal effect.

[0139] As Figure 3BAs shown, compared with the olaparib pretreatment group, the olaparib combination treatment group and the CAR-T single treatment group, the level of IFN-γ in the co-culture supernatant of E0771-EGFRvIII cells and CART cells increased significantly (Unpaired ttest, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, ns indicates no statistical difference); the levels of Granzyme B and IL-2 did not increase significantly. This experiment further confirmed that olaparib can significantly improve the killing ability of CAR-T cells. No increase in cytokine levels was observed in the co-culture supernatant of 4T1-EGFRvIII cells and CAR-T cells, indicating that the enhanced killing effect comes from the combined action of olaparib and CAR-T cells.

[0140] Example 4 Effect of Olaparib on the Expression of Proteins Related to the Proliferation and Exhaustion of CAR-T Co-Cultured with Mouse Breast Cancer Cells

[0141] Experimental grouping: 806-mCD28z-CAR-T group (or denoted as 806-28Z), olaparib pretreatment group, and olaparib combination treatment group;

[0142] The olaparib pretreatment group includes: 806-mCD28z-CAR-T + 1 μM olaparib pretreatment group (or denoted as 806-28Z + Olaparib 1 μM pretreatment), 806-mCD28z-CAR-T + 5 μM olaparib pretreatment group (or denoted as 806-28Z + Olaparib 5 μM pretreatment);

[0143] The olaparib combination treatment group: 806-mCD28z-CAR-T + 1 μM olaparib combination treatment group (or denoted as 806-28Z + Olaparib 1 μM), 806-mCD28z-CAR-T + 5 μM olaparib combination treatment group (or denoted as 806-28Z + Olaparib 5 μM).

[0144] Before performing the co-incubation experiment, 806-mCD28z-CAR-T cells need to be stained with Cell Trace. The specific operation method is as follows: For every 10 6 cells, add 1 μL of CellTrace dye to 1 mL of 1640 medium, and incubate at 37 °C in the dark for 20 min; after 20 min, add complete medium and incubate for 5 min, then centrifuge and discard the supernatant.

[0145] Olaparib pretreatment group: Different concentrations of olaparib (1 μM, 5 μM) were added to the culture media of E0771-EGFRvIII or 4T1-EGFRvIII tumor cells respectively. After culturing at 37°C for 24 h, the cells were digested with trypsin and resuspended in fresh culture media. The 806-mCD28z-CAR T cells in Example 1 were co-incubated with E0771-EGFRvIII or 4T1-EGFRvIII cells pretreated with different concentrations of olaparib at a ratio of 1:1. After 16 h, flow cytometry was used to detect the proliferation of CAR-T cells and the expression levels of proteins PD1, LAG3, and TIM3 related to T cell exhaustion on the surface of CAR-T cells.

[0146] Olaparib combination treatment group: Different concentrations of olaparib (1 μM, 5 μM) were added to the culture media of E0771-EGFRVIII or 4T1-EGFRvIII tumor cells respectively. After culturing at 37°C for 24 h, the cells were digested with trypsin and resuspended in fresh culture media. The 806-mCD28z-CAR T cells in Example 1 were co-incubated with E0771-EGFRvIII or 4T1-EGFRvIII cells pretreated with different concentrations of olaparib at a ratio of 1:1, and the corresponding concentrations of olaparib (1 μM, 5 μM) were added to the culture media. After 16 h, flow cytometry was used to detect the proliferation of CAR-T cells and the expression levels of proteins PD1, LAG3, and TIM3 related to T cell exhaustion on the surface of CAR-T cells.

[0147] As Figure 4A shown, after co-incubating the CART in the olaparib pretreatment group with target cells, the proliferation of CART cells was not significantly affected; while in the olaparib combination treatment group, due to the presence of olaparib in the co-culture environment, the proliferation of CART cells was slightly inhibited, but there was no statistical significance.

[0148] As Figure 4B shown, after co-incubating the CART in the olaparib pretreatment group with target cells, the expression of proteins PD1, LAG3, and TIM3 related to exhaustion on the surface of CART cells did not change significantly; in the olaparib combination treatment group, the expression of proteins PD1, LAG3, and TIM3 related to exhaustion on the surface of CART cells did not change significantly, which indicates that olaparib treatment does not lead to the exhaustion of CART, thus not affecting the killing effect of effector T cells.

[0149] Example 5 Olaparib combined with CAR-T for treating E0771-EGFRvIII orthotopic breast cancer xenografts in mice

[0150] (1) Establishment of mouse orthotopic breast cancer model and grouped treatment:

[0151] Collect E0771-EGFRvIII cells in the logarithmic growth phase with good growth, and inoculate 5×10 5 target cells at the position of the third pair of auxiliary mammary glands on the right side of C57BL / 6 mice (mice with normal immune systems). The tumor cell inoculation date is recorded as the first day (i.e., Day1).

[0152] On the 10th day after tumor inoculation (i.e., Day10), take mouse T cells and construct the 806-mCD28z-CAR-T cell line according to the steps in Step 1 of this example.

[0153] On the 14th day after tumor inoculation (i.e., Day14), the tumor volume reaches approximately 150mm 3 Perform grouping:

[0154] Olaparib stock solution formulation: Add olaparib to 4% DMSO + 30% PEG300 + 66% ddH2O in sequence, and use 5mg / mL as the stock solution;

[0155] The administration method of olaparib is intraperitoneal injection. The injection volume for each mouse is 50mg / kg, and the mouse weight is about 20g. Therefore, each mouse is injected with 200μL; in the control group, 200μL of solvent treatment (4% DMSO + 30% PEG 300 + 66% ddH2O) is given.

[0156] Divide the mice into 6 groups, with 6 mice in each group. The specific in vivo experimental process is as Figure 5A shown:

[0157] UTD group: Intraperitoneally infuse 200μL of solvent per day on Day 14, once a day for 5 consecutive days, and then intraperitoneally infuse 200μL of solvent per day after a 2-day interval, once a day for 5 consecutive days; on Day 21, intravenously infuse mouse T cells that have not been infected with virus at 5×10 6 ;

[0158] Olaparib (50mg / kg) group: Intraperitoneally infuse 50mg / kg of olaparib per day on Day 14, once a day for 5 consecutive days, and then intraperitoneally infuse 50mg / kg of olaparib per day after a 2-day interval, once a day for 5 consecutive days; on Day 21, intravenously infuse mouse T cells that have not been infected with virus at 5×10 6 ;

[0159] CAR-T (2×10 6 ) group: Intraperitoneally infuse 200μL of solvent per day on Day 14, once a day for 5 consecutive days, and then intraperitoneally infuse 200μL of solvent per day after a 2-day interval, once a day for 5 consecutive days; on Day 21, intravenously infuse 2×10 6 806-mCD28z-CAR-T cells;

[0160] Olaparib (50 mg / kg) + CAR-T (2×10 6 ) group: On Day 14, intraperitoneal injection of 50 mg / kg olaparib once a day for 5 consecutive days, with a 2-day interval, then intraperitoneal injection of 50 mg / kg olaparib once a day for 5 consecutive days; on Day 21, intravenous injection of 2×10 6 806-mCD28z-CAR-T cells;

[0161] CAR-T (5×10 6 ) group: On Day 14, intraperitoneal injection of 200 μL solvent once a day for 5 consecutive days, with a 2-day interval, then intraperitoneal injection of 200 μL solvent once a day for 5 consecutive days; on Day 21, intravenous injection of 5×10 6 806-mCD28z-CAR-T cells;

[0162] Olaparib (50 mg / kg) + CAR-T (5×10 6 ) group: On Day 14, intraperitoneal injection of 50 mg / kg olaparib once a day for 5 consecutive days, with a 2-day interval, then intraperitoneal injection of 50 mg / kg olaparib once a day for 5 consecutive days; on Day 21, intravenous injection of 5×10 6 806-mCD28z-CAR-T cells.

[0163] (2) Detection of tumor volume. Continuously observe and measure the changes in the tumor volume of mice, and record three times a week. The formula for calculating the tumor volume is: Tumor volume = (tumor length × tumor width 2 ) / 2.

[0164] The detection results of the tumor volume of mice are as Figure 5B shown. The results show that: on the 35th day after tumor inoculation, compared with the CAR-T (2×10 6 ) group, the tumor volume of mice in the Olaparib (50 mg / kg) + CAR-T (2×10 6 ) group was significantly reduced; compared with the CAR-T (5×10 6 ) group, the tumor volume of mice in the Olaparib (50 mg / kg) + CAR-T (5×10 6 ) group was significantly reduced. The above results suggest that CAR-T cells combined with olaparib can effectively inhibit the growth of breast cancer in mice ((Two-way ANOVA with Bonferroni post-tests, * indicates p < 0.05, ** indicates p < 0.01)).

[0165] At the same time, it was detected that the changes in the body weight of mice in each group were not obvious (as Figure 5CAs shown, it indicates that the combination of olaparib does not cause obvious toxic effects on the combined treatment with CAR-T cells.

[0166] (3) Measurement of tumor inhibition rate and tumor weight. On the 35th day after tumor inoculation, the mice were euthanized, and the orthotopic mammary tumors of the mice were dissected and weighed. The specific statistical results are as Figure 5D shown. Compared with the CAR-T (2×10 6 ) group, the tumor inhibition efficiency and tumor weight of the Olaparib (50mg / kg)+CAR-T (2×10 6 ) group of mice were significantly reduced (*** indicates p〈0.001, Unpaired t test); compared with the CAR-T (5×10 6 ) group, the tumor inhibition efficiency and tumor weight of the Olaparib (50mg / kg)+CAR-T (5×10 6 ) group of mice were significantly reduced (*** indicates p〈0.001, Unpaired t test).

[0167] This indicates that the combination of olaparib can enhance the tumor growth inhibitory effect of CAR-T cells and has a better anti-tumor effect on mouse breast cancer.

[0168] (4) Detection of CAR-T copy number in tumor tissues. The tumor tissues of each group of mice were ground to extract DNA, and the Taqman probe method was used to detect the retroviral vector fragment sequence to determine the DNA copy number of 806-mCD28z-CAR;

[0169] The primer sequences of the Taq probe method are as follows:

[0170] Forward primer Forward: 5’-GACGTTGGGTTACCTTCTG C-3’ (SEQ ID No.52)

[0171] Reverse primer Reverse: 5’-TTCCCAGGTCACGATGTAGG-3’ (SEQ ID No.53)

[0172] Probe primer sequence: 5’-(FAM)-ATGGCCGCGA GACGGCACCT-(BHQ1)-3’ (SEQ ID No.54)

[0173] The experimental results are as Figure 5E shown. Compared with the CAR-T (2×10 6 ) group, the Olaparib (50mg / kg)+CAR-T (2×10 6) The copy number of CAR in the tumors of the [group of mice] increased significantly (Unpaired t test, * indicates p < 0.05); compared with the CAR-T (5×10 6 ) group, the copy number of CAR in the tumors of the Olaparib (50mg / kg) + CAR-T (5×10 6 ) group of mice increased significantly (Unpaired t test, ** indicates p < 0.01). The above results indicate that the combination of olaparib enhances the survival and expansion of CAR-T cells in tumor tissues, playing an anti-tumor role.

[0174] (5) Immunohistochemical detection of CD4 / CD8 / CD31 in tumor tissues. Immunohistochemical staining was performed on paraffin sections of the tumor tissues of each group of mice. CD4 and CD8 can reflect the infiltration of T cells into tissues, and CD31 can reflect the level of angiogenesis in tumor tissues. As Figure 5F shown, there was no significant difference in the infiltration of CD4 + T cells in the tumor tissues of each group of mice, and there was no statistical difference; however, compared with the CAR-T (5×10 6 ) group, the infiltration of CD8 6 T in the tumors of the Olaparib (50mg / kg) + CAR-T (5×10 + ) group of mice increased significantly (Unpaired t test, * indicates p < 0.05). In addition, compared with the CAR-T (5×10 6 ) group, the CD31 6 cells in the tumor tissues of the Olaparib (50mg / kg) + CAR-T (5×10 + ) group of mice were also fewer, indicating that new blood vessels were also inhibited. The above results indicate that the combination of olaparib can increase the infiltration of CD8 + T cells with killing ability in tumor tissues, thus playing an anti-tumor role.

[0175] (6) Detection of MDSC cells (Myeloid-dereved suppressor cells) in tumor tissues. The tumor tissues of each group of mice were ground, digested with enzymes, and then flow cytometry was used to analyze the changes in the proportion of MDSC cells. As Figure 5G shown, compared with the CAR-T (2×10 6 ) group, the infiltration of CD45 6 immune cells in the tumor tissues of the Olaparib (50mg / kg) + CAR-T (2×10 + ) group of mice increased significantly, but CD11b + Gr1 +The infiltration of MDSCs cells was significantly reduced; compared with the CAR-T (5×10 6 ) group, the infiltration of CD45+ immune cells in the tumor tissues of mice treated with Olaparib (50mg / kg)+CAR-T (5×10 6 ) was significantly increased, but the infiltration of CD11b + Gr1 + MDSCs cells was significantly reduced. MDSCs cells can produce a tumor immunosuppressive microenvironment, which is not conducive to the anti-tumor effect of CART. Therefore, the above results indicate that the combination of Olaparib can improve the tumor immunosuppressive environment, thereby promoting the anti-tumor effect of CAR-T.

[0176] Example 6: Combined treatment of Olaparib and CAR-T on 4T1-EGFRvIII orthotopic breast cancer xenografts in mice

[0177] (1) Establishment of a mouse orthotopic breast cancer model and grouped treatment:

[0178] Collect 4T1-EGFRvIII cells in the logarithmic growth phase with good growth, and inoculate 5×10 5 target cells at the position of the third pair of accessory mammary glands on the right side of BABL / C mice (mice with normal immune systems). The diary of tumor cell inoculation is the 1st day (i.e., Day1).

[0179] On the 10th day after tumor inoculation (i.e., Day10), take mouse T cells and construct the 806-mCD28z-CAR-T cell line according to the steps 1 described in this example.

[0180] On the 14th day after tumor inoculation (i.e., Day14), when the tumor volume reaches about 150mm 3 grouping is carried out:

[0181] Olaparib stock solution formulation: Olaparib is sequentially added with 4% DMSO + 30% PEG300 + 66% ddH2O, and used as a stock solution at 5mg / mL;

[0182] The administration method of Olaparib is intraperitoneal injection, with an injection volume of 50mg / kg for each mouse. The weight of the mouse is about 20g, so each mouse is injected with 200μL; in the control group, 200μL of solvent treatment (4% DMSO + 30% PEG 300 + 66% ddH2O) is given.

[0183] The mice are divided into 4 groups, with 6 mice in each group. The specific in vivo experimental procedure is as Figure 6A shown:

[0184] UTD group: Intraperitoneal infusion of 200 μL of solvent per day on Day 14, once a day for 5 consecutive days, followed by a 2-day interval, and then intraperitoneal infusion of 200 μL of solvent per day on Day 17, once a day for 5 consecutive days; on Day 21, intravenous injection of mouse T cells that were not virus-infected at 5×10 6 ;

[0185] Olaparib (50 mg / kg) group: Intraperitoneal infusion of 50 mg / kg of olaparib per day on Day 14, once a day for 5 consecutive days, followed by a 2-day interval, and then intraperitoneal infusion of 50 mg / kg of olaparib per day on Day 17, once a day for 5 consecutive days; on Day 21, intravenous injection of mouse T cells that were not virus-infected at 5×10 6 ;

[0186] CAR-T (5×10 6 ) group: Intraperitoneal infusion of 200 μL of solvent per day on Day 14, once a day for 5 consecutive days, followed by a 2-day interval, and then intraperitoneal infusion of 200 μL of solvent per day on Day 17, once a day for 5 consecutive days; on Day 21, intravenous injection of 5×10 6 806-mCD28z-CAR-T cells;

[0187] Olaparib (50 mg / kg) + CAR-T (5×10 6 ) group: Intraperitoneal infusion of 50 mg / kg of olaparib per day on Day 14, once a day for 5 consecutive days, followed by a 2-day interval, and then intraperitoneal infusion of 50 mg / kg of olaparib per day on Day 17, once a day for 5 consecutive days; on Day 21, intravenous injection of 5×10 6 806-mCD28z-CAR-T cells.

[0188] (2) Detection of tumor volume. Continuously observe and measure the changes in the tumor volume of mice, and record three times a week. The formula for calculating the tumor volume is: Tumor volume = (tumor length × tumor width 2 ) / 2.

[0189] The detection results of the tumor volume of mice are as Figure 6B shown. The results show that: on the 35th day after tumor inoculation, compared with the CAR-T (5×10 6 ) group, the tumor volume of mice in the Olaparib (50 mg / kg) + CAR-T (5×10 6 ) group was significantly reduced. The above results suggest that CAR-T cells combined with olaparib can effectively inhibit the growth of breast cancer in mice (Two-way ANOVA with Bonferroni post-tests, * indicates p < 0.05).

[0190] At the same time, it was detected that the changes in the body weights of mice in each group were not obvious (as Figure 6CAs shown, it is indicated that the combination of olaparib does not cause obvious toxic effects on the combined treatment with CAR-T cells.

[0191] (3) Measurement of tumor inhibition rate and tumor weight. On the 35th day after tumor inoculation, the mice were euthanized, and the orthotopic mammary tumors of the mice were dissected and weighed. The specific statistical results are as Figure 6D shown. Compared with the CAR-T (5×10 6 ) group, the tumor inhibition efficiency and tumor weight of Olaparib (50 mg / kg) + CAR-T (5×10 6 ) mice were significantly reduced (* indicates p < 0.05, Unpaired t test).

[0192] This indicates that the combination of olaparib can enhance the tumor growth inhibitory effect of CAR-T cells and has a better anti-tumor effect on mouse breast cancer.

[0193] (4) Detection of CAR-T copy number in tumor tissues. The tumor tissues of each group of mice were ground to extract DNA, and the Taqman probe method was used to detect the retroviral vector fragment sequence to determine the DNA copy number of 806-mCD28z-CAR;

[0194] The primer sequences of the Taq probe method are as follows:

[0195] Forward primer Forward: 5’-GACGTTGGGTTACCTTCTG C-3’ (SEQ ID No.52)

[0196] Reverse primer Reverse: 5’-TTCCCAGGTCACGATGTAGG-3’ (SEQ ID No.53)

[0197] Probe primer sequence: 5’-(FAM)-ATGGCCGCGA GACGGCACCT-(BHQ1)-3’ (SEQ ID No.54)

[0198] The experimental results are as Figure 6E shown. Compared with the CAR-T (5×10 6 ) group, the copy number of CAR in the tumors of Olaparib (50 mg / kg) + CAR-T (5×10 6 ) group mice was significantly increased (Unpaired t test, ** indicates p < 0.01). The above results show that the combination of olaparib enhances the survival and expansion of CAR-T cells in tumor tissues and plays an anti-tumor role.

[0199] (5) Immunohistochemical detection of CD4 / CD8 / CD31 in tumor tissues. Immunohistochemical staining was performed on paraffin sections of tumor tissues from mice in each group. CD4 and CD8 can reflect the infiltration of T cells into tissues, and CD31 can reflect the level of angiogenesis in tumor tissues. As Figure 6F shown, there was no significant difference in the infiltration of CD4 + T cells in the tumor tissues of mice in each group, and there was no statistical difference; however, compared with the CAR-T (5×10 6 ) group, the infiltration of CD8 6 T in the tumors of mice in the Olaparib (50 mg / kg) + CAR-T (5×10 + ) group was significantly increased (Unpaired t test, * indicates p < 0.05). In addition, compared with the CAR-T (5×10 6 ) group, the CD31 6 cells in the tumor tissues of mice in the Olaparib (50 mg / kg) + CAR-T (5×10 + ) group were also fewer, indicating that angiogenesis was also inhibited. The above results show that the combination of olaparib can increase the infiltration of CD8 + T cells with killing ability in tumor tissues, thereby playing an anti-tumor role.

[0200] (6) Detection of MDSC cells in tumor tissues. The tumor tissues of mice in each group were ground, digested with enzymes, and then flow cytometry was used to analyze the changes in the proportion of MDSC cells. As Figure 6G shown, compared with the CAR-T (5×10 6 ) group, the infiltration of CD45 6 immunocytes in the tumor tissues of mice in the Olaparib (50 mg / kg) + CAR-T (5×10 + ) group was significantly increased, but the infiltration of MDSCs cells with CD11b + Gr1 + was significantly decreased. MDSCs cells can produce a tumor immunosuppressive microenvironment, which is not conducive to the killing of tumors by CART. Therefore, the above results show that the combination of olaparib can improve the tumor immunosuppressive environment, thereby promoting the anti-tumor effect of CART.

[0201] In the above embodiments, merely by way of example, CAR-T was prepared using a murine anti-EGFRvIII (SEQ ID NO: 11), the transmembrane domain and intracellular domain of a mouse, etc. When applied to human therapy, the commonly used coding sequence of the human CD8α signal peptide (SEQ ID NO: 6), the coding sequence of the human CD8α hinge region and transmembrane region (SEQ ID NO: 7), the coding sequence of the transmembrane domain of human CD28 (SEQ ID NO: 10), the coding sequence of the intracellular domain of human CD28 (SEQ ID NO: 8), and the coding sequence of the intracellular domain of human CD3ζ (SEQ ID NO: 9) can be selected for preparation. By way of example, the amino acid sequence of the chimeric antigen receptor targeting EFGRvIII is as shown in any one of SEQ ID NOs: 30-51 or SEQ ID NOs: 55-87.

[0202] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

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Claims

1. Use of immune effector cells expressing a receptor for recognizing tumor antigens and PARP inhibitors in the preparation of a medicament, wherein, The drug is a drug for treating tumors or a drug for reducing the growth, survival or viability of cancer cells; wherein, the tumor antigen is EGFRvIII; wherein, the PARP inhibitor is olaparib; The receptor is a chimeric antigen receptor (CAR), the immune effector cell is a CAR-T cell, and the CAR-T cell specifically recognizes EGFRvIII; the CAR-T cell has anti-tumor activity; the tumor is breast cancer; The chimeric antigen receptor has an antibody that specifically recognizes EGFRvIII; the antibody that specifically recognizes EGFRvIII is the following amino acid sequence, and the amino acid sequence is selected from any one of SEQ ID NO:11 to SEQ ID NO:

29.

2. The application according to claim 1, characterized in that The CAR-T cells selected from autologous T cells, allogeneic T cells.

3. The application according to claim 1, wherein The CAR-T cell is an autologous T cell.

4. The application according to any one of claims 1 to 3, characterized in that, The chimeric antigen receptor has: (i) an antibody that specifically recognizes EGFRvIII, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, and CD3ζ; or (ii) an antibody that specifically recognizes EGFRvIII, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD137, and CD3ζ; or (iii) an antibody that specifically recognizes EGFRvIII, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, a co-stimulatory signal domain of CD137, and CD3ζ.

5. The application according to claim 4, characterized in that The chimeric antigen receptor is the following amino acid sequence, and the amino acid is selected from any one of SEQ ID NO:30 to SEQ ID NO:51, and SEQ ID NO:55 to SEQ ID NO:

87.

6. The application according to any one of claims 1-3, wherein the application increases the CAR-T copy number in tumor tissues, and / or the application increases the expression level of IFN-γ in tumor tissues, and / or the application increases the infiltration of CD8+ T cell immune cells in tumor tissues, and / or the application increases the infiltration of CD45+ immune cells, and / or the application reduces the infiltration of CD11b+Ly6G+ MDSCs cells, and / or the application reduces the infiltration of CD31+ cells.

7. The application according to any one of claims 1-3, characterized in that, The application does not cause a significant decrease in the expression of PD1, LAG3, and / or TIM3.

8. The application according to any one of claims 1 to 3, characterized in that, The manner of administering CAR-T cells and olaparib is selected from any of the following: (1) olaparib is administered first and then CAR-T cells are administered, (2) CAR-T cells and olaparib are administered simultaneously, and (3) CAR-T cells are administered first and then olaparib is administered.

9. The application according to any one of claims 1 to 3, characterized in that, The olaparib is administered orally, intraperitoneally, and / or by injection.

10. The application according to any one of claims 1 to 3, characterized in that Lymphocyte depletion is not performed on the individual.

11. A kit for treating tumors, characterized in that, The kit contains: 1) immune effector cells expressing a receptor that recognizes a tumor antigen; 2) olaparib; 3) A container for containing the substances described in 1) and 2) above; and 4) An administration instruction manual for treating tumors using the kit; wherein, the therapeutic effect of the immune effector cells and olaparib is greater than the effect of either the immune effector cells or olaparib used alone; wherein, the tumor antigen is EGFRvIII; The receptor is a chimeric antigen receptor (Chimeric Antigen Receptor, CAR), the immune effector cells are CAR-T cells, and the CAR-T cells specifically recognize EGFRvIII; the CAR-T cells have anti-tumor activity; the tumor is breast cancer; The chimeric antigen receptor has an antibody that specifically recognizes EGFRvIII; the antibody that specifically recognizes EGFRvIII is the following amino acid sequence, and the amino acid sequence is selected from any one of SEQ ID NO:11 to SEQ ID NO:

29.

12. The kit according to claim 11, wherein The CAR-T cells are selected from autologous T cells and allogeneic T cells.

13. The kit according to claim 11, wherein, The CAR-T cells are autologous T cells.

14. The kit according to any one of claims 11-13, characterized in that, The chimeric antigen receptor has: (i) an antibody that specifically recognizes EGFRvIII, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, and CD3ζ; or (ii) an antibody that specifically recognizes EGFRvIII, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD137, and CD3ζ; or (iii) an antibody that specifically recognizes EGFRvIII, a transmembrane region of CD28 or CD8, a co-stimulatory signal domain of CD28, a co-stimulatory signal domain of CD137, and CD3ζ.

15. The kit according to claim 14, characterized in that, The chimeric antigen receptor is the following amino acid sequence, and the amino acids are selected from any one of SEQ ID NO:30 to SEQ ID NO:51, and SEQ ID NO:55 to SEQ ID NO:87.

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