Pharmaceutical composition of FAK inhibitor and EGFR-TKI and application

Abstract

The FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor and the immune checkpoint inhibitor are combined to treat tumors, and the FAK inhibitor and the epidermal growth factor receptor tyrosine kinase inhibitor are combined to treat tumors.

Description

Drug combination and use of FAK inhibitor and EGFR-TKI

[0001] This application claims priority to Chinese patent application No. 202211077426.8 filed on September 5, 2022, Chinese patent application No. 202211166052.7 filed on September 23, 2022, and Chinese patent application No. 202310998195.2 filed on August 8, 2023, and the contents of the above-mentioned Chinese patent application disclosures are hereby incorporated by reference in their entirety as part of this application. Technical Field

[0002] The present invention belongs to the field of medicinal chemistry and specifically relates to the treatment of tumors by combining a focal adhesion kinase (FAK) inhibitor with other drugs. Background Art

[0003] Tumors are the second largest killer threatening human health. Immunogenic cell death (ICD) is based on the superposition effect of programmed cell death. When cancer cells are exposed to chemotherapy or targeted therapy drugs, they may activate stress signals inside the cells. This type of stress signal includes endoplasmic reticulum stress (ER stress) and reactive oxygen stress (oxidative stress). Under the action of stress signals, cells will first try to repair the stress. If the damage caused by the stress exceeds the ability to repair, the cells will initiate a programmed death process. This process is often accompanied by the release of a class of molecules called damage-associated molecular patterns (DAMPs). These molecules include calreticulin, annexin A1, high mobility group box 1 (HMGB1), etc. These DAMPs are specifically recognized by pattern recognition receptors on antigen-presenting cells (APCs) in the body, inducing APC maturation, differentiation, and activation. These DAMPs are then presented to effector T cells and other immune cells, allowing these immune cells to develop antigen memory. Upon encountering tumor cells of the same origin, these immune cells will specifically recognize and kill them. The new tumor-specific immune response initiated by ICDs can increase sensitivity to immune checkpoint inhibitors (ICIs), thereby enhancing the effects of these inhibitors and producing an anti-tumor response with long-lasting immune memory.

[0004] FAK, also known as protein tyrosine kinase 2 (PTK2), is a non-receptor tyrosine kinase and a key component of the focal adhesion complex. FAK plays an important role in mediating integrin and growth factor signaling to regulate tumor cell invasion, proliferation, and survival.

[0005] EGFR stands for Epidermal Growth Factor Receptor. The EGFR gene encodes and produces a receptor protein called the epidermal growth factor receptor. The EGFR receptor protein is a transmembrane protein consisting of three parts: one end located outside the cell, another located within the cell membrane, and the other end located inside the cell. This allows the EGFR receptor to bind to other proteins outside the cell (called ligands), helping the cell receive signals and respond to stimulation. The binding of receptors and ligands is like a key to a lock, so each has a specific binding "partner." When EGFR binds to a ligand, it attaches to another nearby EGFR receptor to form a complex (dimer), entering an activated state and activating signaling pathways within the cell. EGFR mutations primarily occur in exons 18 to 21, with deletion mutations in exon 19 and the L858R point mutation in exon 21 being the most common mutations, accounting for 90% of all mutations. When a pathogenic gene mutation occurs in EGFR, the EGFR receptor protein will be in a continuously activated state, which causes cells to continuously receive proliferation and survival signals, resulting in excessive cell growth and survival (unable to undergo normal apoptosis), leading to tumor formation.

[0006] Currently available targeted drugs for EGFR mutations include: first-generation drugs (icotinib, gefitinib, and erlotinib) targeting exon 19 and 21 mutations; second-generation drugs (afatinib) targeting exon 8 and 20 mutations; and third-generation drugs (osimertinib (also referred to herein as AZD9291), almonertinib, and alflutinib) targeting T790M mutations. Targeted drugs for ALK mutations include: first-generation drug (crizotinib); second-generation drugs (ceritinib, alectinib, and brigatinib); and third-generation drug (lorlatinib). However, resistance to these targeted drugs often develops approximately one year after treatment. Overcoming resistance to targeted drugs, or delaying the time of resistance and improving the possibility of cure are the main goals of drug development.

[0007] Therefore, there is still a need to find a way to improve the efficacy of single targeted drugs, increase the response rate, create conditions for immunotherapy through combination therapy, and further overcome drug resistance to provide the possibility of tumor cure.

[0008] Summary of the Invention

[0009] In one aspect, the present disclosure provides uses of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor in the preparation of a medicament for treating a tumor in a subject.

[0010] In another aspect, the present disclosure provides a pharmaceutical combination product of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor for use in treating a tumor in a subject.

[0011] In another aspect, the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor to a subject in need thereof.

[0012] In another aspect, the present disclosure provides a kit or a pharmaceutically acceptable composition comprising: (a) a FAK inhibitor; (b) an epidermal growth factor receptor tyrosine kinase inhibitor; and (c) an immune checkpoint inhibitor.

[0013] In another aspect, the present disclosure provides use of a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating tumors, wherein the FAK inhibitor is used to enhance the immunogenic cell death induced by the epidermal growth factor receptor tyrosine kinase inhibitor.

[0014] Yet another aspect of the present disclosure provides a FAK inhibitor for use in the treatment of tumors to enhance immunogenic cell death induced by epidermal growth factor receptor tyrosine kinase inhibitors.

[0015] In another aspect, the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor to a subject in need thereof, wherein the FAK inhibitor is used to enhance immunogenic cell death induced by the epidermal growth factor receptor tyrosine kinase inhibitor.

[0016] In another aspect, the present disclosure provides the use of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor in the preparation of a medicament for the combined treatment of tumors.

[0017] In another aspect, the present disclosure provides use of a FAK inhibitor in the preparation of a combination drug for treating tumors with an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor.

[0018] In another aspect, the present disclosure provides use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a combination drug for treating tumors with a FAK inhibitor and an immune checkpoint inhibitor.

[0019] In another aspect, the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a combination drug for treating tumors with a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0020] In another aspect, the present disclosure provides use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor.

[0021] In another aspect, the present disclosure provides use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor and an immune checkpoint inhibitor.

[0022] In another aspect, the present disclosure provides use of an immune checkpoint inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0023] In another aspect, the present disclosure provides a kit comprising: a FAK inhibitor; and instructions, wherein the instructions indicate that the FAK inhibitor can be used in combination with an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor to treat tumors.

[0024] In another aspect, the present disclosure provides a kit comprising: an epidermal growth factor receptor tyrosine kinase inhibitor; and instructions, wherein the instructions indicate that the epidermal growth factor receptor tyrosine kinase inhibitor can be used in combination with a FAK inhibitor and an immune checkpoint inhibitor to treat tumors.

[0025] In another aspect, the present disclosure provides a kit comprising: an immune checkpoint inhibitor; and instructions, wherein the instructions indicate that the immune checkpoint inhibitor can be used in combination with a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor to treat tumors.

[0026] Another aspect of the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor to a subject in need thereof.

[0027] Yet another aspect of the present disclosure provides a pharmaceutical combination product of a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor for use in treating a tumor in a subject in need thereof.

[0028] In another aspect, the present disclosure provides use of a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor in preparing a combined drug for treating tumors.

[0029] In another aspect, the present disclosure provides use of a FAK inhibitor in the preparation of a medicament for use in combination with an epidermal growth factor receptor tyrosine kinase inhibitor in treating tumors.

[0030] In another aspect, the present disclosure provides use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a drug for use in combination with a FAK inhibitor in treating tumors.

[0031] In another aspect, the present disclosure provides use of a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for combined treatment of tumors.

[0032] In another aspect, the present disclosure provides use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with an epidermal growth factor receptor tyrosine kinase inhibitor.

[0033] In another aspect, the present disclosure provides use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor.

[0034] In another aspect, the present disclosure provides a kit comprising: a FAK inhibitor; and instructions, wherein the instructions indicate that the FAK inhibitor can be used in combination with an epidermal growth factor receptor tyrosine kinase inhibitor to treat tumors.

[0035] In another aspect, the present disclosure provides a kit comprising: an epidermal growth factor receptor tyrosine kinase inhibitor; and instructions, wherein the instructions indicate that the epidermal growth factor receptor tyrosine kinase inhibitor can be used in combination with a FAK inhibitor to treat tumors.

[0036] Optionally, the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate. The structure of IN10018 is as follows:

[0037] Defactinib, also known as defatinib, has a CAS number of 1345713-71-4; GSK2256098 has a CAS number of 1224887-10-8; PF-00562271 has a CAS number of 717907-75-0; VS-4718 has a CAS number of 1061353-68-1; APG-2449 was developed by Ascentage Pharma; and AMP945 has a CAS number of 1393653-34-3.

[0038] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), ametinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof.

[0039] The CAS number of the gefitinib is 184475-35-2; the CAS number of the erlotinib is 183321-74-6; the CAS number of the icotinib is 610798-31-7; the CAS number of the afatinib is 850140-72-6; the CAS number of the crizotinib is 877399-52-5; the CAS number of the osimertinib (AZD9291) is 1421373- 65-0; the CAS number of Almonertinib is 1899921-05-1; the CAS number of Alflutinib (also known as Furmonertinib) is 1869057-83-9; the CAS number of EAI045 is 1942114-09-1; the CAS number of JBJ-04-125-02 is 2060610-53-7; the CAS number of BLU-945 is 2660250-10-0; BLU-701 is a product of Blueprint Pharmaceuticals The drug was jointly developed by TQB3804 and Zai Lab; the CAS number of TQB3804 is 2267329-76-8; BBT-176 was developed by Biliji Biotech Co., Ltd.; ES-072 was developed by Bosheng Pharmaceutical; BPI-361175 was developed by Betta Pharmaceuticals; and CH7233163 was developed by Chugai Pharmaceutical Co., Ltd.

[0040] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

[0041] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is Osimertinib or a pharmaceutically acceptable salt thereof.

[0042] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof.

[0043] Optionally, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof.

[0044] Optionally, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor or a TIGIT inhibitor.

[0045] Optionally, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0046] Optionally, the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043 or RRx-001.

[0047] Optionally, the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (Ociperlimab / BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

[0048] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0049] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0050] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0051] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0052] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0053] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0054] Optionally, the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0055] Optionally, the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, Liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastases from spindle cell carcinoma, anaplastic large cell lymphoma, anaplastic thyroid cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or hematological malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML).

[0056] Optionally, the tumor is preferably breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

[0057] Optionally, the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer).

[0058] Optionally, the tumor is non-small cell lung cancer or colon cancer (including colorectal cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, and are not intended to limit the present invention.

[0060] FIG1 shows a white light microscope photograph of lung cancer KPL cells taken after incubation with drugs for 48 hours in Example 1.

[0061] FIG2 a shows the percentage of CRT-positive KPL cells after the lung cancer KPL cells of Example 1 were incubated with drugs for 48 hours; FIG2 b shows the percentage of Annexin V-positive KPL cells after the lung cancer KPL cells of Example 1 were incubated with drugs for 48 hours.

[0062] FIG3 shows a white light microscope photograph of the lung cancer KPL cells taken after incubation with the drug for 48 hours in Example 2.

[0063] FIG4 a shows the percentage of CRT-positive KPL cells after the lung cancer KPL cells of Example 2 were incubated with drugs for 48 hours; FIG4 b shows the percentage of Annexin V-positive KPL cells after the lung cancer KPL cells of Example 2 were incubated with drugs for 48 hours.

[0064] FIG5 shows white light microscope pictures of lung cancer KPL cells taken after incubation with drugs for 48 hours in Example 3.

[0065] FIG6 a shows the percentage of CRT-positive KPL cells after the lung cancer KPL cells of Example 3 were incubated with drugs for 48 hours; FIG6 b shows the percentage of Annexin V-positive KPL cells after the lung cancer KPL cells of Example 3 were incubated with drugs for 48 hours.

[0066] FIG7 shows the early and late apoptosis of cells after FAK silencing and combined treatment with 0.3 nM AZD9291 for 48 hours.

[0067] FIG8 shows that FAK silencing combined with 0.3 nM AZD9291 treatment for 48 hours enhanced calreticulin release and exposure.

[0068] FIG9 shows the synergistic killing effect of AZD9291 at different doses in combination with 3 μM IN10018 and 5 μM IN10018 for 48 hours on non-small cell lung cancer cells HCC827.

[0069] Figure 10 shows the detection of cell apoptosis after 48 hours of co-administration of 0.3 nM AZD9291 and 3 μM IN10018.

[0070] FIG11 shows that the combination of AZD9291 and IN10018 significantly upregulated the endoplasmic reticulum stress of HCC827.

[0071] Figure 12 shows that the combination of AZD9291 and IN10018 enhances calreticulin release and exposure.

[0072] FIG13 shows the changes in tumor volume after administration of different test substances in a subcutaneous BALB / c mouse colon cancer CT26 cell homograft tumor model.

[0073] FIG14 shows the changes in body weight of mice in a subcutaneous BALB / c mouse model of colon cancer CT26 cells after administration of different test substances.

[0074] FIG15 shows the changes in tumor volume after administration of different test substances in a subcutaneous homograft tumor model of colon cancer MC38 cells in C57BL / 6 mice.

[0075] FIG16 shows the changes in body weight of mice in a subcutaneous homograft tumor model of colon cancer MC38 cells in C57BL / 6 mice after administration of different test substances.

[0076] FIG17 a shows the percentage of CRT-positive 4T1 cells after the breast cancer 4T1 cells of Example 8 were incubated with drugs for 48 hours; FIG17 b shows the percentage of Annexin V-positive 4T1 cells after the breast cancer 4T1 cells of Example 8 were incubated with drugs for 48 hours.

[0077] FIG18 a shows the percentage of CRT-positive 4T1 cells after the breast cancer 4T1 cells of Example 9 were incubated with drugs for 48 hours; FIG18 b shows the percentage of Annexin V-positive 4T1 cells after the breast cancer 4T1 cells of Example 9 were incubated with drugs for 48 hours.

[0078] FIG19 a shows the percentage of CRT-positive 4T1 cells after the breast cancer 4T1 cells of Example 10 were incubated with drugs for 48 hours; FIG19 b shows the percentage of Annexin V-positive 4T1 cells after the breast cancer 4T1 cells of Example 10 were incubated with drugs for 48 hours. DETAILED DESCRIPTION

[0079] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] The present invention may be implemented in other specific forms without departing from the essential attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or multiple other embodiments to produce additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0081] All publications and patents mentioned in this disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0082] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0083] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0084] Except in the working examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited herein is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0085] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0086] definition

[0087] The following terms and symbols used in this application have the meanings described below, unless otherwise indicated in the context in which they are used. The term "FAK inhibitor" as used herein refers to an effective inhibitor of FAK, which can be suitable for mammals, particularly humans. In some embodiments, the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, and the structure of IN10018 is as follows:

[0088] Defactinib, also known as defatinib, has a CAS number of 1345713-71-4; GSK2256098 has a CAS number of 1224887-10-8; PF-00562271 has a CAS number of 717907-75-0; VS-4718 has a CAS number of 1061353-68-1; APG-2449 was developed by Ascentage Pharma; and AMP945 has a CAS number of 1393653-34-3.

[0089] In some embodiments, the FAK inhibitor is preferably IN10018, AMP945, Defactinib or a pharmaceutically acceptable salt thereof. In some preferred embodiments, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate.

[0090] As used herein, the term "epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI)" refers to an agent that selectively and effectively inhibits epidermal growth factor receptor tyrosine kinase. Examples of the epidermal growth factor receptor tyrosine kinase inhibitors include, but are not limited to, gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or pharmaceutically acceptable salts thereof. In some embodiments, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0091] The CAS number of the gefitinib is 184475-35-2; the CAS number of the erlotinib is 183321-74-6; the CAS number of the icotinib is 610798-31-7; the CAS number of the afatinib is 850140-72-6; the CAS number of the dacomitinib is 1110813-31-4; the CAS number of the crizotinib is 877399-52-5; the CAS number of the osimertinib is AZD9 291) has a CAS number of 1421373-65-0; Almonertinib has a CAS number of 1899921-05-1; Alflutinib (also known as Furmonertinib) has a CAS number of 1869057-83-9; EAI045 has a CAS number of 1942114-09-1; JBJ-04-125-02 has a CAS number of 2060610-53-7; BLU-945 has a CAS number of 2660250-10-0; BLU-701 is a product of Blueprint Pharmaceuticals The drug was jointly developed by TQB3804 and Zai Lab; the CAS number of TQB3804 is 2267329-76-8; BBT-176 was developed by Biliji Biotech Co., Ltd.; ES-072 was developed by Bosheng Pharmaceutical; BPI-361175 was developed by Betta Pharmaceuticals; and CH7233163 was developed by Chugai Pharmaceutical Co., Ltd.

[0092] The term "immune checkpoint inhibitor" as used herein refers to a drug that can increase the activity of the immune system by regulating the immune checkpoint pathway (e.g., PD-1, TIGIT, CTLA-4, LAG-3, TIM-3, etc.). In some embodiments, the immune checkpoint inhibitor is an antagonist of the PD-1 / PD-L1 (programmed cell death protein 1) pathway (also referred to as a "PD-1 inhibitor") or a TIGIT inhibitor. PD-1 inhibitors are also referred to as PD-1 / PD-L1 inhibitors in this disclosure. For example, in the methods of treatment, drugs, and uses disclosed herein, the PD-1 / PD-L1 inhibitor is a PD-1 / PD-L1 antibody, including but not limited to pembrolizumab (Keytruda), tislelizumab (Tislelizumab / Baizean), nivolumab, toripalimab (Toripalimab / Tuoyi), durvalumab (Improvin / Durvalumab / Durvalumab), avelumab (Bavencio), atezolizumab (MPDL3280A / Atezolizumab / Tecentriq), BMS-936559 (fully human anti-PD-L1 IgG4 monoclonal antibody), GS-4224, AN-4005, or MX-10181. In some preferred embodiments, the PD-1 inhibitor is toripalimab. In some embodiments, the PD-1 inhibitor is used to treat human subjects. In some embodiments, PD-1 is human PD-1. PD-1 / PD-L1 inhibitors also include small molecule PD-1 / PD-L1 inhibitors, such as INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001. TIGIT (also known as WUCAM, Vstm3, or VSIG9) is a receptor of the Ig superfamily and a new immune checkpoint following PD-1 / PD-L1.For example, in the methods of treatment, medicines and uses disclosed herein, the immune checkpoint inhibitor is a TIGIT inhibitor, including but not limited to Ociperlimab / BGB-A1217, Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, HB0036 or IBI-321. In some embodiments, TIGIT inhibitors are used to treat human subjects. To avoid ambiguity, antibodies herein all include dual antibodies.

[0093] As used herein, "drug combination" or "drug combination product" may refer to a fixed combination in the form of one dosage unit (for example, all active pharmaceutical ingredients are present in one dosage form) or a kit of parts for combined administration, or it may refer to a combination of one drug and instructions indicating that the drug can be used in combination with one or more other drugs.

[0094] As used herein, "combination therapy" or "combination drug" refers to the use of a drug in combination with one or more other drugs to treat a disease, including both the combination of a drug with one or more other drugs and the combination of a drug with instructions indicating that the drug can be used in combination with one or more other drugs.

[0095] "Administered simultaneously or sequentially" in this application refers to the administration of two or more drugs simultaneously or successively at certain time intervals within a dosing cycle (e.g., within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours), the mode of drug administration (e.g., oral, intravenous, intramuscular or subcutaneous administration, etc.) may be the same or different, and the administration frequency / cycle of two or more drugs may be the same or different. When the method of treatment, product, or use disclosed herein involves two drugs, the two drugs may be administered separately at the same time or at certain time intervals. When the method of treatment, product, or use disclosed herein involves three drugs, the three drugs may be administered at the same time point, or two drugs may be administered at one time point and the remaining drug may be administered at another time point, or all three drugs may be administered at different time points.

[0096] In some embodiments, the PD-1 / PD-L1 inhibitor is administered intravenously (e.g., as an intravenous infusion) or subcutaneously, or orally. Preferably, the PD-1 / PD-L1 inhibitor is administered as an intravenous infusion.

[0097] In some embodiments, the TIGIT inhibitor is administered intravenously (eg, as an intravenous infusion) or subcutaneously, or orally. Preferably, the TIGIT inhibitor is administered as an intravenous infusion.

[0098] The ability of immune checkpoint inhibitors to treat cancer depends on the presence of tumor antigen-specific T cells within tumor tissue. This requires that tumor tissue express antigens that distinguish itself from its non-transformed counterparts, for example, through novel protein products called neoantigens. Tumor neoantigen load is closely related to immunogenicity and sensitivity (for example, sensitivity to checkpoint inhibitor therapy), which means that tumors with poor immunogenicity should be largely resistant to these drugs. Therapies for releasing tumor antigens that can be taken up by APCs, such as those that induce immunogenic cell death (ICD), may promote effective anti-tumor immunity, especially when further combined with checkpoint inhibitors.

[0099] As used herein, the term "treat" refers to administering one or more pharmaceutical substances to a subject suffering from a disease or symptoms of a disease in order to cure, alleviate, relieve, alter, cure, ameliorate, improve, or affect the disease or symptoms of the disease. In some embodiments, the disease is a tumor or cancer.

[0100] The term "tumor" as used herein refers to an abnormal lesion formed when the cells of local tissues lose normal regulation of their growth at the genetic level under the action of various tumorigenic factors, thereby causing abnormal proliferation of their clonal types. The tumors include, but are not limited to: bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastatic tumors caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin lymphoma, Hodgkin lymphoma , gliomas and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); in some embodiments, the tumor is preferably breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma and pancreatic cancer; in some embodiments, the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer); in some embodiments, the tumor is non-small cell lung cancer or colon cancer (including colorectal cancer).

[0101] As used herein, the term "subject" or "subject" refers to both mammals and non-mammals. Mammals refer to any member of the class mammalia, including but not limited to humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds. The term "subject" does not limit the subject to a particular age or sex. In some embodiments, the subject is a human.

[0102] As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerable, and suitable for administration to a subject.

[0103] As used herein, the term "pharmaceutically acceptable salt" refers to non-toxic, biologically tolerable acid addition salts suitable for administration to a subject, including, but not limited to, acid addition salts formed with inorganic acids, such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, and the like; and acid addition salts formed with organic acids, such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts with the formula HOOC-(CH2) n -COOH (wherein n is 0-4) and the like.

[0104] In addition, pharmaceutically acceptable acid addition salts can be obtained by dissolving the free base in a suitable solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can determine various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable acid addition salts without undue experimentation.

[0105] The term "pharmaceutically acceptable composition" as used herein means that it must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation, and / or compatible with the subject being treated therewith. The term "therapeutically effective amount" as used herein means an amount that is generally sufficient to produce a beneficial therapeutic effect on the subject. The therapeutically effective amount of the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., route of administration, pharmacokinetics of the compound, severity and course of the disease, medical history of the subject, health status of the subject, degree of response of the subject to the drug, etc.).

[0106] As used herein, the term "inhibit" refers to a decrease in the baseline activity of a biological activity or process.

[0107] The term "kit" as used herein refers to a box for containing chemical reagents for detecting chemical components, drug residues, virus species, etc. The kit of the present invention may include one, two or three of (i) a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a FAK inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) an epidermal growth factor receptor tyrosine kinase inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) an immune checkpoint inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor; and (ii) instructions indicating that a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a FAK inhibitor; and (ii) instructions indicating that a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) an epidermal growth factor receptor tyrosine kinase inhibitor; and (ii) instructions indicating that an epidermal growth factor receptor tyrosine kinase inhibitor and a FAK inhibitor can be used to treat a tumor in a subject.

[0108] The compound of test kit can be contained in a separate container.Alternatively, two or more compounds are contained in the same container.For example, test kit can include a first container, a second container, a third container and a package insert, wherein the first container includes at least one dose of the medicine including a FAK inhibitor, the second container includes at least one dose of an epidermal growth factor receptor tyrosine kinase inhibitor, the third container includes at least one dose of an immune checkpoint inhibitor, and the package insert includes instructions for using a drug to treat the tumor of an object. The first container, the second container and the third container can include the same or different shapes (for example, vials, syringes and bottles) and / or materials (for example, plastic or glass). Test kit can also include other materials that can contribute to the administration of the drug, such as diluents, filters, IV bags and pipelines, needles and syringes.

[0109] The precise amount of FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, or immune checkpoint inhibitor administered to a subject will depend on various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease, the condition, the identity of the subject or host being treated, etc., but can still be routinely determined by one skilled in the art. For example, determining an effective amount also depends on the extent, severity, and type of cell proliferation. A skilled person will be able to determine an appropriate dosage based on these and other factors.

[0110] FAK inhibitors, epidermal growth factor receptor tyrosine kinase inhibitors, or immune checkpoint inhibitors can be administered by appropriate routes such as oral, intravenous, intramuscular, or subcutaneous administration.

[0111] For example, when administered orally, the drug can be administered orally with a pharmaceutically acceptable carrier, such as an inert diluent or an assimilable edible carrier. They can be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or mixed directly with the patient's food. For example, the drug can be combined with one or more excipients and used in the form of an ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, or wafer. Tablets, lozenges, pills, capsules, etc. may further include: a binder, such as gum tragacanth, gum arabic, corn starch, or gelatin; an excipient, such as dicalcium phosphate; a disintegrant, such as corn starch, potato starch, alginic acid, etc.; a lubricant, such as magnesium stearate; or a sweetener, such as sucrose, fructose, lactose, or aspartame; or a flavoring agent.

[0112] For example, when administered intravenously or intraperitoneally by infusion or injection, solutions of the drug can be prepared in water, optionally mixed with a nontoxic surfactant.

[0113] Exemplary pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage.

[0114] Sterile injectable solutions can be prepared by incorporating the required amount of the drug into an appropriate solvent with the various other ingredients listed above as required, followed by filtered sterilization. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods may be vacuum drying and freeze drying techniques, which can produce a powder of the active ingredient plus any other desired ingredients that have been previously sterile filtered.

[0115] The amount of FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, or immune checkpoint inhibitor required for treatment may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. However, in general, the dosage may be in the range of about 0.1 to about 50 mg / kg body weight per day.

[0116] The FAK inhibitor is administered in a dosage range of 5 mg / day to 300 mg / day in adults. In a specific embodiment, IN10018 or a pharmaceutically acceptable salt thereof is administered in a dosage range of 5 mg / day to 100 mg / day in adults. In a specific embodiment, IN10018 or a pharmaceutically acceptable salt thereof is administered in a dosage range of 25 mg / day to 100 mg / day in adults, calculated as the free base.

[0117] The epidermal growth factor receptor tyrosine kinase inhibitor is administered in a dosage range of 2-250 mg per day in adults. In a specific embodiment, osimertinib or a pharmaceutically acceptable salt thereof is administered in a dosage range of 2-250 mg per day in adults, for example, 80 mg, calculated as osimertinib; almonertinib or a pharmaceutically acceptable salt thereof is administered in a dosage range of 2-250 mg per day in adults, for example, 110 mg, calculated as almonertinib; and alflutinib or a pharmaceutically acceptable salt thereof is administered in a dosage range of 2-250 mg per day in adults, for example, 80 mg, calculated as alflutinib.

[0118] The immune checkpoint inhibitor is administered at a dose of 2-10 mg / kg or 50-1200 mg in adults per administration, administered once every 2 to 3 weeks. In a specific embodiment, the immune checkpoint inhibitor is administered at a dose of 3-10 mg / kg or 100-1200 mg in adults per administration, administered once every 2 to 3 weeks.

[0119] Technical and scientific terms used herein without specific definition have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0120] In some embodiments, the present disclosure further discloses the following:

[0121] 1. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor for use in a method of treating a tumor in a subject.

[0122] 2. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to embodiment 1, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0123] 3. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in embodiments 1-2, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof.

[0124] 4. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-3, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0125] 5. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-4, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

[0126] 6. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor according to any one of embodiments 1-4, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is almonertinib or a pharmaceutically acceptable salt thereof.

[0127] 7. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor according to any one of embodiments 1-4, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is alflutinib or a pharmaceutically acceptable salt thereof.

[0128] 8. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-7, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT inhibitor.

[0129] 9. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1 to 8, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, tislelizumab, nivolumab, toripalimab, atezolizumab, durvalumab, avelumab, camrelizumab, sintilimab, cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

[0130] 10. The use, pharmaceutical combination product, or method according to any one of embodiments 1 to 8, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0131] 11. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor according to any one of embodiments 1 to 8, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036, or IBI-321.

[0132] 12. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0133] 13. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0134] 14. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0135] 15. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to any one of embodiments 1-14, wherein the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0136] 16. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 1-15, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), ), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma and pancreatic cancer.

[0137] 17. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor as described in any one of embodiments 1-16, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), especially non-small cell lung cancer or colon cancer (including colorectal cancer).

[0138] 18. A kit or pharmaceutically acceptable composition comprising:

[0139] (a) FAK inhibitors;

[0140] (b) epidermal growth factor receptor tyrosine kinase inhibitors; and

[0141] (c) Immune checkpoint inhibitors.

[0142] 19. The kit or composition according to embodiment 18, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

[0143] 20. The kit or composition of embodiments 18-19, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof.

[0144] 21. The kit or composition of any one of embodiments 18-20, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0145] 22. The kit or composition of any one of embodiments 18-21, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

[0146] 23. The kit or composition of any one of embodiments 18-21, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is almonertinib or a pharmaceutically acceptable salt thereof.

[0147] 24. The composition of any one of embodiments 18-21, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is alflutinib or a pharmaceutically acceptable salt thereof.

[0148] 25. The kit or composition of any one of embodiments 18-24, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT inhibitor.

[0149] 26. The kit or composition of any one of embodiments 18-25, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

[0150] 27. The use, pharmaceutical combination product, or method according to any one of embodiments 18 to 25, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0151] 28. The kit or composition of any one of embodiments 18-25, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036, or IBI-321.

[0152] 29. The kit or composition according to any one of embodiments 18-21, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0153] 30. The kit or composition according to any one of embodiments 18-21, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0154] 31. The kit or composition according to any one of embodiments 18-21, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0155] 32. The kit or composition of any one of embodiments 18-31, wherein the composition is for use in medicine.

[0156] 33. The kit or composition of embodiment 32, wherein the drug is used to treat a tumor, and the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, The invention relates to a method for treating a thyroid cancer of the present invention and the like. The method comprises the following steps: treating a thyroid cancer of the present invention and the like; wherein the thyroid cancer is ...

[0157] 34. The kit or composition of embodiment 33, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

[0158] 35. A method of treating a tumor in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor.

[0159] 36. The method according to embodiment 35, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

[0160] 37. The method according to embodiments 35-36, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof.

[0161] 38. The method according to any one of embodiments 35-37, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib or a pharmaceutically acceptable salt thereof.

[0162] 39. The method of any one of embodiments 35-38, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

[0163] 40. The method of any one of embodiments 35-38, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof.

[0164] 41. The method of any one of embodiments 35-38, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is alflutinib or a pharmaceutically acceptable salt thereof.

[0165] 42. The method of any one of embodiments 35-41, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT inhibitor.

[0166] 43. The method according to any one of embodiments 35 to 42, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0167] 44. The use, pharmaceutical combination product, or method according to any one of embodiments 35 to 42, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0168] 45. The method according to any one of embodiments 35 to 42, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

[0169] 46. ​​A method as described in any one of embodiments 35-38, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0170] 47. A method as described in any one of embodiments 35-38, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0171] 48. A method as described in any one of embodiments 35-38, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0172] 49. The method of any one of embodiments 35-48, wherein the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0173] 50. A method as described in any one of embodiments 35-49, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer Preferably, the tumor is a breast cancer, an ovarian cancer, a colon cancer (including colorectal cancer), a lung cancer (including small cell lung cancer and non-small cell lung cancer), a melanoma, or a pancreatic cancer.

[0174] 51. A method as described in any one of embodiments 35-50, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

[0175] 52. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor for use in a method of treating a tumor by increasing immunogenic cell death in a subject.

[0176] 53. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor according to embodiment 52, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0177] 54. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor as described in embodiments 52-53, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof.

[0178] 55. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor of any one of embodiments 52-54, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0179] 56. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor of any one of embodiments 52-55, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

[0180] 57. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 52-55, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof.

[0181] 58. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 52-55, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof.

[0182] 59. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor according to any one of embodiments 52-58, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor or a TIGIT inhibitor.

[0183] 60. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor according to any one of embodiments 52-59, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, tislelizumab, nivolumab, toripalimab, albuterol, or a combination thereof. Atezolizumab, durvalumab, avelumab, camrelizumab, sintilimab, cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

[0184] 61. The use, pharmaceutical combination product, or method according to any one of embodiments 52 to 59, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0185] 62. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 52-59, wherein the immune checkpoint inhibitor is TIGIT Inhibitor, further, the TIGIT inhibitor is Ociperlimab (Ociperlimab / BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

[0186] 63. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 52-55, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0187] 64. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 52-55, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0188] 65. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor as described in any one of embodiments 52-55, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0189] 66. The FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor, and immune checkpoint inhibitor of any one of embodiments 52-65, wherein the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0190] 67. A FAK inhibitor, epidermal growth factor receptor tyrosine kinase inhibitor and immune checkpoint inhibitor as described in any one of embodiments 52-66, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, Uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma and pancreatic cancer.

[0191] 68. A FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor as described in any one of embodiments 52-67, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

[0192] 69. A method of treating a tumor by increasing immunogenic cell death in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor.

[0193] 70. The method according to embodiment 69, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0194] 71. The method of embodiments 69-70, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof.

[0195] 72. The method of any one of embodiments 69-71, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib, or a pharmaceutically acceptable salt thereof.

[0196] 73. The method of any one of embodiments 69-72, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

[0197] 74. The method of any one of embodiments 69-72, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is almonertinib or a pharmaceutically acceptable salt thereof.

[0198] 75. The method of any one of embodiments 69-72, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is alflutinib or a pharmaceutically acceptable salt thereof.

[0199] 76. A method as described in any one of embodiments 69-75, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT inhibitor.

[0200] 77. The method of any one of embodiments 69-76, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0201] 78. The use, pharmaceutical combination product, or method according to any one of embodiments 69 to 76, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0202] 79. The method according to any one of embodiments 69 to 76, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

[0203] 80. A method as described in any one of embodiments 69-72, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0204] 81. A method as described in any one of embodiments 69-72, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0205] 82. The method according to any one of embodiments 69 to 72, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0206] 83. The method of any one of embodiments 69-82, wherein the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0207] 84. A method as described in any one of embodiments 69-83, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer Preferably, the tumor is a breast cancer, an ovarian cancer, a colon cancer (including colorectal cancer), a lung cancer (including small cell lung cancer and non-small cell lung cancer), a melanoma, or a pancreatic cancer.

[0208] 85. A method as described in any one of embodiments 69-84, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

[0209] 86. Use of a FAK inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein the FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor are administered to the subject.

[0210] 87. Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor are administered to the subject.

[0211] 88. Use of an immune checkpoint inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and the immune checkpoint inhibitor are administered to the subject.

[0212] 89. Use of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor in the preparation of a combination drug for treating tumors.

[0213] 90. Use of a FAK inhibitor in the preparation of a combination drug for treating tumors with an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor.

[0214] 91. Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a combination drug for treating tumors with a FAK inhibitor and an immune checkpoint inhibitor.

[0215] 92. Use of immune checkpoint inhibitors in the preparation of a combination drug for treating tumors with a FAK inhibitor and an epidermal growth factor receptor tyrosine kinase inhibitor.

[0216] 93. Use of FAK inhibitors, epidermal growth factor receptor tyrosine kinase inhibitors and immune checkpoint inhibitors in the preparation of drugs for combined treatment of tumors.

[0217] 94. Use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with an epidermal growth factor receptor tyrosine kinase inhibitor and an immune checkpoint inhibitor.

[0218] 95. Use of an epidermal growth factor receptor tyrosine kinase inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor and an immune checkpoint inhibitor.

[0219] 96. Use of immune checkpoint inhibitors in the preparation of drugs for treating tumors in combination with FAK inhibitors and epidermal growth factor receptor tyrosine kinase inhibitors.

[0220] 97. The use according to any one of embodiments 86-96, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0221] 98. The use according to any one of embodiments 86-97, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof.

[0222] 99. The use according to any one of embodiments 86-98, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib or a pharmaceutically acceptable salt thereof.

[0223] 100. The use according to any one of embodiments 86-99, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

[0224] 101. The use according to any one of embodiments 86-99, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof.

[0225] 102. The use according to any one of embodiments 86-99, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is alflutinib or a pharmaceutically acceptable salt thereof.

[0226] 103. The use according to any one of embodiments 86-102, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT inhibitor.

[0227] 104. The use according to any one of embodiments 86 to 103, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0228] 105. The use, pharmaceutical combination product, or method according to any one of embodiments 86 to 103, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0229] 106. The use according to any one of embodiments 86 to 103, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

[0230] 107. The use according to any one of embodiments 86-99, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0231] 108. The use according to any one of embodiments 86-99, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0232] 109. The use according to any one of embodiments 86-99, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

[0233] 110. The use according to any one of embodiments 86-109, wherein the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0234] 111. The use according to any one of embodiments 86 to 110, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, Neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastases caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma and pancreatic cancer.

[0235] 112. The use according to any one of embodiments 86 to 111, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

[0236] Example

[0237] The following examples are provided to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0238] The experimental methods in the following examples where specific conditions are not specified can be carried out according to conventional conditions of such reactions or according to the conditions recommended by the manufacturers. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0239] The abbreviations used in the examples have the following meanings:

[0240] Example 1: Study of IN10018 and AZD9291 in lung cancer KPL cells

[0241] KPL cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI 1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When the cells were in good condition, the culture medium was plated in a 24-well plate. After 24 hours of cell spreading, four groups were set up: the first group was a negative control group, with culture medium added; the second group was treated with IN10018 at a concentration of 10 μM; the third group was treated with AZD9291 at a concentration of 10 μM; and the fourth group was a combination of IN10018 and AZD9291, with IN10018 (10 μM) and AZD9291 (10 μM), respectively. The drugs were mixed and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0242] After 48 hours of drug treatment, the cells were observed under a microscope and photographed, and the photographs were saved. The cells were then collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl Conjugated GRP94 polyclonal antibody (Proteintech, catalog number: CL488-14700, lot number: 21006579) was added to each well, mixed, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow buffer (PBS + 2% FBS) and analyzed on a flow cytometer.

[0243] Microscopic observation of cells revealed poor cell status in the AZD9291 monotherapy group, the worst in the dual-drug combination group, with more cell death. Cells in the negative control group and IN10018 group were in better condition. Flow cytometry revealed higher CRT and Annexin V positivity rates in the dual-drug combination group than in the monotherapy group. See Figures 1, 2a, and 2b for details.

[0244] Example 2: Study of IN10018 and Almonertinib in Lung Cancer KPL Cells

[0245] KPL cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI 1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When the cells were in good condition, the culture medium was plated in a 24-well plate. After 24 hours of cell spreading, four groups were set up: the first group was a negative control group, with culture medium added; the second group was treated with IN10018 at a concentration of 10 μM; the third group was treated with almonertinib at a concentration of 4.7 μM; and the fourth group was a combination of IN10018 and almonertinib, with IN10018 (10 μM) and almonertinib (4.7 μM), respectively. The drugs were mixed and incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0246] After 48 hours of drug treatment, the cells were observed under a microscope and photographed, and the photographs were saved. The cells were then collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl Conjugated GRP94 polyclonal antibody (Proteintech, catalog number: CL488-14700, lot number: 21006579) was added to each well, mixed, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow buffer (PBS + 2% FBS) and analyzed on a flow cytometer.

[0247] Microscopic observation of cells revealed poor cell status in the almonertinib monotherapy group, the worst in the dual-drug combination group, with more cell death. Cells in the negative control group and IN10018 group were in better condition. Flow cytometry revealed higher CRT and Annexin V positivity rates in the dual-drug combination group than in the monotherapy group. See Figures 3, 4a, and 4b for details.

[0248] Example 3: Study of IN10018 and Alflutinib in Lung Cancer KPL Cells

[0249] KPL cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI 1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, the culture medium was plated in 24-well plates. After 24 hours of cell growth, four groups were set up: the first group served as a negative control group, receiving culture medium; the second group received IN10018 at a concentration of 10 μM; the third group received alflutinib at a concentration of 4.7 μM; and the fourth group received a combination of IN10018 and alflutinib, with IN10018 (10 μM) and alflutinib (4.7 μM), respectively. The drugs were mixed and incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0250] After 48 hours of drug treatment, the cells were observed under a microscope and photographed, and the photographs were saved. The cells were then collected for flow analysis and washed twice with flow buffer (PBS + 2% FBS). 0.5 μl Conjugated GRP94 polyclonal antibody (Proteintech, catalog number: CL488-14700, lot number: 21006579) was added to each well, mixed, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow buffer (PBS + 2% FBS) and analyzed on a flow cytometer.

[0251] Microscopic observation of cells revealed poor cell status in the alflutinib monotherapy group, the worst in the dual-drug combination group, with more cell death. Cells in the negative control group and IN10018 group were in better condition. Flow cytometry revealed higher CRT and Annexin V positivity rates in the dual-drug combination group than in the monotherapy group. See Figures 5, 6a, and 6b for details.

[0252] Example 4: Study on the Enhancement of Cell Death in Non-Small Cell Lung Cancer Cells Against the Immunogenicity of AZD9291 by FAK Target Inhibition

[0253] Experimental plan:

[0254] 1. FAK silencing combined with AZD9291 promotes apoptosis of lung cancer cells HCC827

[0255] In vitro experiments used siRNA technology to reduce the expression level of FAK and combined it with AZD9291 for 48 hours; flow cytometry was used to detect apoptosis in control and FAK-silenced cells.

[0256] 2. FAK silencing combined with AZD9291 can effectively promote ICD in lung cancer cells HCC827

[0257] In vitro experiments used siRNA technology to reduce the expression level of FAK and combined it with AZD9291 for 48 hours; flow cytometry was used to detect the expression of calreticulin, the main target of ICD.

[0258] Experimental antibody: recombinant Alexa Fluorescent Anti-Calreticulin antibody (Abcam, ab196159), Annexin V apoptosis detection kit (Invitrogen, A35110).

[0259] FAK siRNA was provided by Genema, and the sequence is shown in the following table:

[0260] F: CCUGUAUGCCUAUCAGCUUTT;

[0261] R:AAGCUGAUAGGCAUACAGGTT.

[0262] Experimental instruments:

[0263] Fluorescence microscope (Olympus U-HGLGPS) and chemiluminescence imager (BIORAD chemidoc touch)

[0264] Experimental results:

[0265] 1. FAK silencing combined with AZD9291 significantly increased apoptosis in HCC827 cells

[0266] HCC827 cells were transfected with control siRNA or FAK siRNA at a final concentration of 50 nM. Twenty-four hours after transfection, cells were treated with 0.3 nM AZD9291 for 48 hours. Cells were stained using the Annexin V kit and analyzed by flow cytometry. Cells were analyzed for early and late apoptosis and compared with the DMSO control group using Graphpad 8.0.

[0267] The results showed that compared with the control group, the late apoptosis in the FAK silencing group was significantly enhanced after treatment with AZD9291, as shown in FIG7 .

[0268] 2. FAK silencing combined with AZD9291 significantly upregulated the release and exposure of the ICD target calreticulin

[0269] HCC827 cells were transfected with either control siRNA or FAK siRNA at a final concentration of 50 nM. Twenty-four hours after transfection, cells were treated with 0.3 nM AZD9291 for 48 hours. The cells were fluorescently stained with a calreticulin antibody and analyzed by flow cytometry. FlowJo software analysis revealed that FAK silencing combined with AZD9291 significantly enhanced calreticulin release and exposure, as shown in Figure 8.

[0270] Example 5: Study on the effect of IN10018 on the cell death of non-small cell lung cancer cells against the immunogenicity of AZD9291

[0271] Experimental plan:

[0272] 1. IN10018 combined with AZD9291 produces synergistic tumor killing effects and induces cell apoptosis

[0273] In vitro killing curves of AZD9291 in lung cancer HCC827 cells were determined, and IC50 values ​​were determined. Cytotoxic effects of different doses of AZD9291 were examined in combination with 3μM and 5μM IN10018. Flow cytometry and Annexin V staining were also used to assess apoptosis in cells treated with either drug alone or in combination.

[0274] 2. IN10018 combined with AZD9291 can effectively promote the ICD effect in lung cancer cells HCC827

[0275] In vitro experiments used IN10018 and AZD9291 together for 6 hours to detect the activation of the endoplasmic reticulum stress signaling pathway; the two drugs were used together for 48 hours to detect the expression of calreticulin, the main target of ICD.

[0276] Experimental Antibodies:

[0277] Restructuring Alexa 647 fluorescent anti-calreticulin antibody (Abcam, ab196159), FAK antibody (CST, 3285S), Phospho-eIF2α (Ser51) (CST, 3398), eIF2α (CST, 5324), DDIT3 (HUABIO, ET1703-05), HRP-labeled Alpha tubulin antibody (Proteintech, HRP-66031), Annexin V apoptosis detection kit (Invitrogen, A35110)

[0278] Experimental instruments:

[0279] Fluorescence microscope (Olympus U-HGLGPS) and chemiluminescence imager (BIORAD chemidoc touch).

[0280] Experimental results:

[0281] 1. IN10018 combined with AZD9291 produces synergistic tumor killing effect

[0282] 5,000 HCC827 cells were plated per well in a 96-well plate. After 24 hours, a fixed concentration of IN10018 was added in combination with varying concentrations of AZD9291. IN10018 concentrations ranged from 3 μM to 5 μM. AZD9291 was diluted to a maximum concentration of 1 μM and then diluted three-fold to a total of nine concentrations. After 72 hours of incubation, 10 μM CCK8 solution was added to each well. After incubation at 37°C and 5% CO2 for 2 hours, the absorbance was read using a microplate reader at 450 nm. The values ​​were compared with the DMSO control and plotted using Graphpad 8.0. The results show a synergistic effect of AZD9291 at different doses, combined with 3 μM and 5 μM IN10018 for 48 hours against HCC827 non-small cell lung cancer cells, as shown in Figure 9.

[0283] 2. IN10018 combined with AZD9291 promotes cell apoptosis

[0284] HCC827 cells were treated with 0.3 nM AZD9291 and 3 μM IN10018. After 48 hours, cells were harvested, stained with an Annexin V kit, and analyzed by flow cytometry. Early and late apoptosis were compared with the DMSO control group and plotted using Graphpad 8.0. The results showed that both early and late apoptosis were significantly enhanced in the combination group compared to the single-drug group, as shown in Figure 10.

[0285] 3. IN10018 combined with AZD9291 can significantly upregulate the endoplasmic reticulum stress of HCC827

[0286] HCC827 cells were treated with 0.1nM and 0.3nM AZD9291, respectively, in combination with 3μM IN10018. Six hours later, the protein expression of the ER stress-related proteins Phospho-eIF2α (Ser51) and DDIT3 was measured. The results showed that compared with the single-drug group, the expression levels of Phospho-eIF2α (Ser51) and DDIT3 were significantly upregulated in the combination group, indicating that the combination of the two drugs significantly increased ER stress, as shown in Figure 11.

[0287] 4. AZD9291 combined with IN10018 enhances the release and exposure of the ICD target calreticulin

[0288] HCC827 cells were treated with 0.3 nM AZD9291 and 3 μM IN10018. After 48 hours, fluorescent staining with a calreticulin antibody was performed and analyzed by flow cytometry. FlowJo software analysis showed that the release and exposure of calreticulin were significantly enhanced in the combination group compared to the single-drug group, as shown in Figure 12.

[0289] Example 6: In vivo antitumor efficacy study of AZD9291 in a subcutaneous BALB / c mouse colon cancer CT26 cell homograft model

[0290] Test materials:

[0291] Mice: Female BALB / c mice aged 6-8 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. The experiment began after the animals were acclimated to the experimental environment upon arrival. The animals were housed in IVC (independent ventilation system) cages (5 per cage) in an SPF-grade animal room. All cages, bedding, and drinking water must be sterilized before use. All experimental personnel should wear protective clothing and latex gloves when operating in the animal room. The animal information card for each cage should indicate the number of animals in the cage, sex, strain, date of receipt, dosing regimen, experimental number, group, and start date of the experiment. Cages, feed, and drinking water were changed twice a week. The breeding environment and lighting conditions are as follows:

[0292] Temperature: 20-26℃

[0293] Humidity: 40-70%

[0294] Photoperiod: 12 hours of light, 12 hours of no light

[0295] Cages: Made of polycarbonate, 300 mm × 180 mm × 150 mm. Bedding is corn cobs, changed twice a week.

[0296] Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.

[0297] Drinking water: Experimental animals can drink sterile water freely.

[0298] Animal identification: Experimental animals were identified with ear tags.

[0299] Compound information is shown in Table 1

[0300] Table 1. Compound information

[0301] Colorectal cancer cells CT26 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60043) were maintained and passaged by Ins Biotechnology (Nanjing) Co., Ltd. Cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged using trypsin-EDTA two to three times weekly. When cells were in the exponential growth phase and at 80%-90% saturation, they were harvested, counted, and plated.

[0302] Cell seeding and grouping

[0303] 0.1 mL contains 3×10 5 A cell suspension of 100 cells was subcutaneously inoculated on the right back of each mouse. 3 At 12 days after cell inoculation, the patients were randomly divided into groups according to the tumor volume and the group information is shown in Table 2.

[0304] Table 2. Dosage regimen of the test substance in the CT26 mouse transplant tumor model

[0305] Note: 1. Number of mice in each group;

[0306] 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight decreases by more than 15%, stop dosing the animal; resume dosing when the body weight recovers to 10% of the decrease.

[0307] Preparation of test substances

[0308] See Table 3 for details

[0309] Table 3. Test substance configuration method

[0310] Daily observation of experimental animals

[0311] The development of this experimental protocol and any modifications were reviewed and approved by the Yunqiao Biotech IACUC. The use and welfare of experimental animals were carried out in accordance with AAALAC guidelines. Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight changes, physical signs, or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0312] Experiment terminated

[0313] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0314] Tumor measurements and experimental parameters

[0315] Tumor diameter was measured with a vernier caliper three times a week. Tumor volume was calculated using the formula: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0316] Referring to the tumor volume on the first day after grouping, the tumor growth inhibition rate TGI (%) was calculated according to the following formula.

[0317] TGI (%) = [1-(average tumor volume of a certain medication group-average tumor volume of the medication group at the start of treatment) / (average tumor volume of the solvent control group-average tumor volume of the solvent control group at the start of treatment)] x 100%.

[0318] Statistical analysis

[0319] Statistical analysis was performed using Prism GraphPad software based on tumor volume and weight at the end of the study. Multiple group comparisons were performed using two-way ANOVA with Fisher's LSD test. P < 0.05 was considered significant.

[0320] Experimental results

[0321] The in vivo efficacy of the test substances AZD9291 and / or PD-L1 antibody in combination with IN10018 in a BALB / c mouse subcutaneous transplant tumor model of CT26 colorectal cancer cells. After cell inoculation, tumor growth was observed daily. Groups were divided according to tumor volume on day 12 after inoculation. The average tumor volume in the group was approximately 63 mm. 3 Due to tumor burden, the control group was euthanized on day 27 after inoculation, which was the 15th day after group administration, and the entire experiment was terminated.

[0322] On the 15th day after group administration, the tumor volume of the control group was 2777.3±705.2mm 3The tumor volumes of the AZD9291+IN10018 (20+25 mg / kg), AZD9291+PDL1 antibody (20+10 mg / kg), and AZD9291+IN10018+PDL1 antibody (20+25+10 mg / kg) treatment groups were 1693.4±1207.5 mm 3 、1387.6±859.6mm 3 and 927.2±627.9mm 3 , see Table 4 for details. Comparing the comprehensive tumor volume with the control group, the tumor inhibition rates (TGI) for the AZD9291+IN10018 (20+25 mg / kg), AZD9291+PDL1 antibody (20+10 mg / kg), and AZD9291+IN10018+PDL1 antibody (20+25+10 mg / kg) groups were 40.0% (p<0.0001), 51.2% (p<0.0001), and 62.7% (p<0.0001), respectively. See Table 4 for details. Statistical analysis was performed comparing the overall tumor volume with that of the AZD9291 + IN10018 + PDL1 antibody (20 + 25 + 10 mg / kg) triple combination group. The P values ​​for the control group, AZD9291 + IN10018 (20 + 25 mg / kg), and AZD9291 + PDL1 antibody (20 + 10 mg / kg) treatment groups were p < 0.0001, p = 0.0032, and p = 0.0745, respectively. The tumor volumes of each dose group at different time periods are shown in Figure 13.

[0323] Table 4: Evaluation of the tumor inhibition effect of the test substance on the BALB / c mouse transplant tumor model of mouse colon cancer CT26 cells (based on the data on the 15th day after group administration)

[0324] Note: 1. Calculated based on the number of days after group administration. Data are mean ± standard error.

[0325] 2.****: p<0.0001, vs. control group, Two-way ANOVA.

[0326] 3. **: p < 0.01, ****: p < 0.0001, vs. AZD9291 + IN10018 + PDL1 antibody (20 + 25 + 10 mg / kg) group, Two-way ANOVA.

[0327] The experiment was conducted according to the dosing regimen. During the experiment, the animals' activities such as food intake and drinking were observed daily, and their body weights were recorded three times a week. The animal weight curves are shown in Figure 14. Throughout the dosing period, the animals in each group showed no significant weight loss and were in good condition.

[0328] in conclusion

[0329] Compared with the blank control group, the AZD9291+IN10018 (20+25 mg / kg), AZD9291+PDL1 antibody (20+10 mg / kg) and AZD9291+IN10018+PDL1 antibody (20+25+10 mg / kg) treatment groups all had significant tumor growth inhibition effects, and there were statistical differences compared with the control group. Comprehensively considering the entire dosing cycle, the tumor volume of the AZD9291+IN10018+PDL1 antibody (20+25+10 mg / kg) triple combination group was always smaller than that of the AZD9291+IN10018 (20+25 mg / kg) and AZD9291+PDL1 antibody (20+10 mg / kg) two-drug combination groups, and there were statistical differences compared with the AZD9291+IN10018 (20+25 mg / kg) group. Compared with the AZD9291+IN10018 (20+25 mg / kg) and AZD9291+PDL1 antibody (20+10 mg / kg) two-drug combination groups, it had a better effect in inhibiting tumor growth. At the same time, the animals' weight changes were good, and no abnormalities were found in their activities, drinking water, eating, and mental state throughout the entire dosing period, indicating that the animals tolerated the combination of AZD9291+IN10018+PDL1 antibody (20+25+10 mg / kg).

[0330] Example 7: In vivo antitumor efficacy study of AZD9291 in a subcutaneous homograft model of colon cancer MC38 cells in C57BL / 6 mice

[0331] Test materials:

[0332] Mice: Female C57BL / 6 mice aged 6-8 weeks were purchased from Shanghai Slake Laboratory Animal Co., Ltd. The experiment began after the animals were adapted to the experimental environment upon arrival. The animals were housed in IVC (independent ventilation system) cages (5 per cage) in an SPF-grade animal room. All cages, bedding, and drinking water must be sterilized before use. All experimenters should wear protective clothing and latex gloves when operating in the animal room. The animal information card for each cage should indicate the number of animals in the cage, sex, strain, date of receipt, dosing regimen, experimental number, group, and start date of the experiment. Cages, feed, and drinking water were changed twice a week. The breeding environment and lighting conditions are as follows:

[0333] Temperature: 20-26℃

[0334] Humidity: 40-70%

[0335] Photoperiod: 12 hours of light, 12 hours of no light

[0336] Cages: Made of polycarbonate, 300 mm × 180 mm × 150 mm. Bedding is corn cobs, changed twice a week.

[0337] Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.

[0338] Drinking water: Experimental animals can drink sterile water freely.

[0339] Animal identification: Experimental animals were identified with ear tags.

[0340] The compound information is shown in Table 5.

[0341] Table 5: Compound information

[0342] Colorectal cancer cell lines MC38 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60825) were maintained and passaged by Ins Biotechnology (Nanjing) Co., Ltd. Cells were cultured as monolayers in vitro in DMEM medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged using trypsin-EDTA two to three times weekly. When cells were in the exponential growth phase and at 80%-90% saturation, they were harvested, counted, and plated.

[0343] Cell seeding and grouping

[0344] 0.1 mL contains 2×10 5 A cell suspension of 100 cells was subcutaneously inoculated on the right back of each mouse. 3 At 15 days after cell inoculation, the patients were randomly divided into groups and given medication according to the tumor volume. The grouping information is shown in Table 6.

[0345] Table 6: Dosage regimen of the test substance in the MC38 mouse transplant tumor model

[0346] Note: 1. Number of mice in each group;

[0347] 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight decreases by more than 15%, stop dosing the animal; resume dosing when the body weight recovers to 10% of the decrease.

[0348] Preparation of test substances

[0349] See Table 7 for details

[0350] Table 7: Test substance preparation method

[0351] Daily observation of experimental animals

[0352] The development of this experimental protocol and any modifications were reviewed and approved by the Yunqiao Biotech IACUC. The use and welfare of experimental animals were carried out in accordance with AAALAC guidelines. Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight changes, physical signs, or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0353] Experiment terminated

[0354] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3 If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0355] Tumor measurements and experimental parameters

[0356] Tumor diameter was measured with a vernier caliper three times a week. Tumor volume was calculated using the formula: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0357] The tumor growth inhibition rate (TGI) (%) was calculated based on the tumor volume on the first day after grouping according to the following formula: TGI (%) = [1 - (average tumor volume of a given dosing group - average tumor volume of that dosing group at the start of treatment) / (average tumor volume of the vehicle control group - average tumor volume of the vehicle control group at the start of treatment)] × 100%.

[0358] Statistical analysis

[0359] Statistical analysis was performed using Prism GraphPad software based on tumor volume and weight at the end of the study. Multiple group comparisons were performed using two-way ANOVA with Fisher's LSD test. P < 0.05 was considered significant.

[0360] Experimental results

[0361] The in vivo efficacy of the test substances AZD9291 and IN10018 in combination with PD-L1 antibodies in a subcutaneous homograft tumor model of MC38 colorectal cancer cells in C57BL / 6 mice. After cell inoculation, tumor growth was observed daily. Groups were divided according to tumor volume on the 15th day after inoculation. The average tumor volume in the group was approximately 70 mm 3 Due to tumor burden, the control group was euthanized on day 29 after inoculation, which was 14 days after group administration, and the entire experiment was terminated.

[0362] On the 14th day after group administration, the tumor volume of the control group was 1984.4±537.8mm 3 The tumor volumes of the PD-L1 antibody (5 mg / kg), AZD9291 (20 mg / kg), IN10018+PD-L1 antibody (25+5 mg / kg), and AZD9291+IN10018+PD-L1 antibody (20+25+5 mg / kg) treatment groups were 1974.8±1629.9 mm 3 , 1518.1±728.8mm 3 , 1425.2±889.6mm 3 and 1292.9±934.6mm 3 , see Table 8 for details. The comprehensive tumor volume was compared with the control group. The tumor inhibition rates (TGI) of the PD-L1 antibody (5 mg / kg), AZD9291 (20 mg / kg), IN10018 + PD-L1 antibody (25 + 5 mg / kg), and AZD9291 + IN10018 + PD-L1 antibody (20 + 25 + 5 mg / kg) groups were 0.5% (p = 0.9728), 24.4% (p = 0.1012), 29.2% (p = 0.0498), and 36.1% (p = 0.0205), respectively. See Table 8 for details. The overall tumor volume was compared with the AZD9291 + IN10018 + PD-L1 antibody (20 + 25 + 5 mg / kg) combination group for statistical analysis. The P values ​​for the control group, PD-L1 antibody (5 mg / kg), AZD9291 (20 mg / kg), and IN10018 + PD-L1 antibody (25 + 5 mg / kg) were p = 0.0205, p = 0.0170, p = 0.4274, and p = 0.6408, respectively. The tumor volume of each dose group at different time periods is shown in Figure 15.

[0363] Table 8: Evaluation of the tumor inhibition effect of the test substance on the C57BL / 6 mouse transplant tumor model of mouse colon cancer MC38 cells (based on the data on the 14th day after group administration)

[0364] Note: 1. Calculated based on the number of days after group administration. Data are mean ± standard error.

[0365] 2.*: p<0.05, vs. control group, Two-way ANOVA.

[0366] 3.*: p<0.05, vs. AZD9291+IN10018+PD-L1 antibody (20+25+5 mg / kg) group, Two-way ANOVA.

[0367] The experiment was conducted according to the dosing regimen. During the experiment, the animals' activities such as food and water intake were observed daily, and their weights were recorded three times a week. The animal weight curves are shown in Figure 16. One mouse in the control group died on the 11th day after group dosing due to tumor rupture. One mouse in the AZD9291+IN10018+PD-L1 antibody (20+25+5 mg / kg) group died on the 8th day after group dosing due to an error in oral administration. The animals in the remaining groups showed no significant weight loss and remained in good condition throughout the dosing period, demonstrating tolerance to the different dosing methods.

[0368] in conclusion

[0369] Compared with the blank control group, the IN10018+PD-L1 antibody (25+5 mg / kg) and AZD9291+IN10018+PD-L1 antibody (20+25+5 mg / kg) groups both had significant tumor growth inhibition effects, and there were statistical differences compared with the control group. Comprehensively considering the entire dosing cycle, the tumor volume of the AZD9291+IN10018+PD-L1 antibody (20+25+5 mg / kg) group was always smaller than that of the other treatment groups, and there was a statistical difference compared with the PD-L1 antibody (5 mg / kg) single-drug group. Although there was no statistical difference between the three-drug combination group and the AZD9291 (20 mg / kg) and IN10018+PD-L1 antibody (25+5 mg / kg) groups, its average tumor volume was always smaller than that of the other treatment groups, which also showed that the AZD9291+IN10018+PD-L1 antibody (20+25+5 mg / kg) three-drug combination group had a better effect in inhibiting tumor growth. At the same time, except for the animals that died naturally due to tumor rupture or human operational errors, the weight of the animals in the other groups changed well, and no abnormalities were found in their activities, drinking water, eating, and mental state throughout the dosing cycle, indicating that the animals tolerated the combination of AZD9291+IN10018+PD-L1 antibody (20+25+5mg / kg).

[0370] Example 8: Study on the induction of immunogenic cell death targets by AZD9291 and AMP945 in mouse breast cancer 4T1 cells in vitro

[0371] Experimental Materials:

[0372] 1) Drugs used in this experiment

[0373] AZD9291 was provided by Shanghai Chaolan Chemical Technology Center, Lot No.: 202012-02

[0374] AMP945 provided by MCE, Lot No.:143253

[0375] 2) Antibodies used in this experiment

[0376] Restructuring Alexa 647 fluorescent anti-calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773). Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).

[0377] Experimental methods:

[0378] 4T1 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) were cultured in RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp) at 37°C and 5% CO2. Two to three times a week, cells were routinely digested and passaged using trypsin. When the cells were in the exponential growth phase and 80%-90% confluent, they were harvested and plated. After trypsinization, the 4T1 cells were harvested and counted. Based on the count results, the cells were diluted in RPMI 1640 + 10% FBS to a concentration of 50,000 cells / mL. The cells were then plated in 12-well cell culture plates, with 2 mL of cell suspension per well, i.e., 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell spreading, six groups were set up: the first group was a control group, supplemented with culture medium; the second group was treated with AMP945 at a concentration of 3 μM; the third group was treated with AMP945 at a concentration of 6 μM; the fourth group was treated with AZD9291 at a concentration of 10 μM; the fifth group was treated with a combination of AMP945 (3 μM) and AZD9291 (10 μM); and the sixth group was treated with a combination of AMP945 (6 μM) and AZD9291 (10 μM). The drugs were mixed and cultured at 37°C in a 5% CO2 incubator for 48 hours.

[0379] Experimental results

[0380] After 48 hours of drug action, cells were collected for flow analysis, and the cells were washed twice with flow buffer (PBS + 2% FBS) and 0.5 μl AF647. Anti-calreticulin antibody (abcam) was added to each well and mixed. Incubate at 4 ° C in the dark. After incubation for 20 minutes, flow buffer was added. An annexin staining kit (Beyotime) was used, 195 μl Annexin-V-FITC conjugate was added, and after mixing with the cells, 5 μl Annexin-V-FITC antibody was added and gently mixed. Finally, 10 μl PI dye was added and mixed. The mixture was incubated at room temperature in the dark for 15 minutes, and the sample was sent to a flow cytometer for signal measurement. The results of flow cytometric analysis showed that the CRT positivity rate and Annexin-V positivity rate of the two-drug combination group were significantly better than those of the single-drug group and the control group, as shown in Figures 17a and 17b.

[0381] Example 9: Study on the induction of immunogenic cell death targets by Alflutinib and AMP945 in mouse breast cancer 4T1 cells in vitro

[0382] Experimental Materials:

[0383] 1) Drugs used in this experiment

[0384] Alflutinib was provided by Shanghai MCE, Lot No.: 33805

[0385] AMP945 provided by MCE, Lot No.:143253

[0386] 2) Antibodies used in this experiment

[0387] Restructuring Alexa 647 fluorescent anti-calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773). Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).

[0388] Experimental methods:

[0389] 4T1 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) were cultured in RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp) at 37°C and 5% CO2. Two to three times a week, cells were routinely digested and passaged using trypsin. When the cells were in the exponential growth phase and 80%-90% confluent, they were harvested and plated. After trypsinization, the 4T1 cells were harvested and counted. Based on the count results, the cells were diluted in RPMI 1640 + 10% FBS to a concentration of 50,000 cells / mL. The cells were then plated in 12-well cell culture plates, with 2 mL of cell suspension per well, i.e., 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell spreading, six groups were set up: the first group served as the control group and received culture medium; the second group received AMP945 at a concentration of 3 μM; the third group received AMP945 at a concentration of 6 μM; the fourth group received alflutinib at a concentration of 10 μM; the fifth group received a combination of AMP945 (3 μM) and alflutinib (10 μM); and the sixth group received a combination of AMP945 (6 μM) and alflutinib (10 μM). The drugs were mixed and cultured at 37°C in a 5% CO2 incubator for 48 hours.

[0390] Experimental results

[0391] After 48 hours of drug action, cells were collected for flow analysis, and the cells were washed twice with flow buffer (PBS + 2% FBS) and 0.5 μl AF647. Anti-calreticulin antibody (abcam) was added to each well and mixed. Incubate at 4 ° C in the dark. After incubation for 20 minutes, flow buffer was added. Annexin staining kit (Beyotime) was used, 195 μl Annexin-V-FITC conjugate was added, and after mixing with the cells, 5 μl Annexin-V-FITC antibody was added and gently mixed. Finally, 10 μl PI dye was added and mixed. Incubate at room temperature in the dark for 15 minutes, and the sample was sent to the flow cytometer for signal measurement. The results of flow cytometric analysis showed that the CRT positivity rate and Annexin-V positivity rate of the two-drug combination group were significantly better than those of the single-drug group and the control group, as shown in Figures 18a and 18b.

[0392] Example 10: Study on the induction of immunogenic cell death targets by Almonertinib and AMP945 in mouse breast cancer 4T1 cells in vitro

[0393] Experimental Materials:

[0394] 1) Drugs used in this experiment

[0395] Almonertinib was provided by Shanghai MCE, Lot No.: 65966

[0396] AMP945 provided by MCE, Lot No.:143253

[0397] 2) Antibodies used in this experiment

[0398] Restructuring Alexa 647 fluorescent anti-calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773). Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).

[0399] Experimental methods:

[0400] 4T1 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) were cultured in RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp) at 37°C and 5% CO2. Two to three times a week, cells were routinely digested and passaged using trypsin. When the cells were in the exponential growth phase and 80%-90% confluent, they were harvested and plated. After trypsinization, the 4T1 cells were harvested and counted. Based on the count results, the cells were diluted in RPMI 1640 + 10% FBS to a concentration of 50,000 cells / mL. The cells were then plated in 12-well cell culture plates, with 2 mL of cell suspension per well, i.e., 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell spreading, six groups were set up: the first group served as the control group and received culture medium; the second group received AMP945 at a concentration of 3 μM; the third group received AMP945 at a concentration of 6 μM; the fourth group received almonertinib at a concentration of 2 μM; the fifth group received a combination of AMP945 (3 μM) and almonertinib (2 μM); and the sixth group received a combination of AMP945 (6 μM) and almonertinib (2 μM). The drugs were mixed and cultured at 37°C in a 5% CO2 incubator for 48 hours.

[0401] Experimental results

[0402] After 48 hours of drug action, cells were collected for flow analysis, and the cells were washed twice with flow buffer (PBS + 2% FBS) and 0.5 μl AF647. Anti-calreticulin antibody (abcam) was added to each well and mixed. Incubate at 4 ° C in the dark. After incubation for 20 minutes, flow buffer was added. Annexin staining kit (Beyotime) was used, 195 μl Annexin-V-FITC conjugate was added, and after mixing with the cells, 5 μl Annexin-V-FITC antibody was added and gently mixed. Finally, 10 μl PI dye was added and mixed. Incubate at room temperature in the dark for 15 minutes, and the sample was sent to the flow cytometer for signal measurement. The results of flow cytometric analysis showed that the CRT positivity rate and Annexin-V positivity rate of the two-drug combination group were significantly better than those of the single-drug group and the control group, as shown in Figures 19a and 19b.

[0403] All references mentioned in this application are incorporated herein by reference in their entirety, just as if each reference were listed separately. It should be understood that after reading the disclosure of this application, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the claims appended hereto.

Claims

1. Use of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor in the preparation of a medicament for treating a tumor in a subject.

2. A pharmaceutical combination product of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor for use in treating a tumor in a subject.

3. A method for treating a tumor, comprising administering to a subject a therapeutically effective amount of a FAK inhibitor, an epidermal growth factor receptor tyrosine kinase inhibitor, and an immune checkpoint inhibitor.

4. The use, pharmaceutical combination or method of any one of claims 1 to 3, wherein the FAK inhibitor and the epidermal growth factor receptor tyrosine kinase inhibitor induce immunogenic cell death (ICD).

5. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

6. The use, pharmaceutical combination or method according to any one of claims 1 to 5, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof.

7. The use, pharmaceutical combination or method according to any one of claims 1 to 6, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib or a pharmaceutically acceptable salt thereof.

8. The use, pharmaceutical combination or method according to any one of claims 1 to 7, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

9. The use, pharmaceutical combination or method according to any one of claims 1 to 8, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is almonertinib or a pharmaceutically acceptable salt thereof.

10. The use, pharmaceutical combination or method according to any one of claims 1 to 8, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is alflutinib or a pharmaceutically acceptable salt thereof.

11. The use, pharmaceutical combination product or method according to any one of claims 1 to 10, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor or a TIGIT inhibitor.

12. The use, pharmaceutical combination or method according to any one of claims 1 to 11, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, tislelizumab, nivolumab, toripalimab, atezolizumab, durvalumab, avelumab, camrelizumab, sintilimab, cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

13. The use, pharmaceutical combination, or method according to any one of claims 1 to 11, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

14. The use, pharmaceutical combination product or method according to any one of claims 1 to 11, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

15. The use, pharmaceutical combination product, or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular an anti-PD-1 / PD-L1 antibody.

16. The use, pharmaceutical combination product, or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

17. The use, pharmaceutical combination product or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular an anti-PD-1 / PD-L1 antibody.

18. The use, pharmaceutical combination or method of any one of claims 1 to 17, wherein the FAK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

19. The use, pharmaceutical combination or method of any one of claims 1 to 18, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, The invention relates to a method for treating a thyroid cancer of the present invention and the like. The method comprises the following steps: treating a thyroid cancer of the present invention and the like; wherein the thyroid cancer is a thyroid cancer, a thyroid cancer, a salivary gland cancer, a metastatic tumor caused by spindle cell carcinoma, an anaplastic large cell lymphoma, an undifferentiated thyroid cancer, a non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a glioma or a hematological malignancy, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

20. The use, pharmaceutical combination or method according to any one of claims 1 to 19, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

21. A kit or pharmaceutically acceptable composition comprising: (a) FAK inhibitors; (b) epidermal growth factor receptor tyrosine kinase inhibitors; and (c) Immune checkpoint inhibitors.

22. The kit or composition of claim 21, wherein the FAK inhibitor and the epidermal growth factor receptor tyrosine kinase inhibitor induce immunogenic cell death (ICD).

23. The kit or composition according to any one of claims 21 to 22, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

24. The kit or composition of any one of claims 21 to 23, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, alflutinib (also known as furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163, or a pharmaceutically acceptable salt thereof.

25. The kit or composition according to any one of claims 21 to 24, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib, almonertinib, alflutinib or a pharmaceutically acceptable salt thereof.

26. The kit or composition according to any one of claims 21 to 25, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof.

27. The kit or composition of any one of claims 21 to 25, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof.

28. The kit or composition according to any one of claims 21 to 25, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof.

29. The kit or composition of any one of claims 21-28, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT inhibitor.

30. The kit or composition of any one of claims 21 to 29, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

31. The kit or composition of any one of claims 21-29, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

32. The kit or composition of any one of claims 21-29, wherein the immune checkpoint inhibitor is a TIGIT inhibitor, and further, the TIGIT inhibitor is Ociperlimab (BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

33. The kit or composition of any one of claims 21-22, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is osimertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

34. The kit or composition of any one of claims 21-22, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Almonertinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

35. The kit or composition of any one of claims 21-22, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the epidermal growth factor receptor tyrosine kinase inhibitor is Alflutinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially an anti-PD-1 / PD-L1 antibody.

36. A kit or composition according to any one of claims 21 to 35 for use as a medicament.

37. The kit or composition of any one of claims 21 to 36, wherein the drug is used for a tumor, and the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroblastoma, Endocrine cancer, ovarian cancer, salivary gland cancer, metastases caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

38. The kit or composition of claim 37, wherein the tumor is lung cancer (including small cell lung cancer and non-small cell lung cancer) or colon cancer (including colorectal cancer), in particular non-small cell lung cancer or colon cancer (including colorectal cancer).

39. FAK inhibitors for use in the treatment of tumors to enhance immunogenic cell death induced by epidermal growth factor receptor tyrosine kinase inhibitors.

40. The FAK inhibitor according to claim 39, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, AMP945, Defactinib, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

41. The FAK inhibitor according to claim 39 or 40, wherein the epidermal growth factor receptor tyrosine kinase inhibitor is gefitinib, erlotinib, icotinib, afatinib, dacomitinib, crizotinib, osimertinib (AZD9291), almonertinib, afatinib, dapoxetine ... lflutinib, also known as Furmonertinib), EAI045, JBJ-04-125-02, BLU-945, BLU-701, TQB3804, BBT-176, ES-072, BPI-361175, CH7233163 or a pharmaceutically acceptable salt thereof; preferably, the epidermal growth factor receptor tyrosine kinase inhibitor is Osimertinib, Almonertinib, Alflutinib or a pharmaceutically acceptable salt thereof.

42. The FAK inhibitor of any one of claims 39-41, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, Ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

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