Pharmaceutical combinations and uses for treating tumors

Abstract

The present disclosure relates to treating tumors with a combination of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor. The present disclosure also relates to treating tumors with a combination of a FAK inhibitor and a MEK inhibitor.

Description

[Technical field]

[0001] This application claims priority to Chinese Patent Application No. 202210071792.6 filed on January 21, 2022 and Chinese Patent Application No. 202211160420.7 filed on September 22, 2022, and the contents disclosed in the above Chinese patent application are incorporated herein by reference throughout this specification.

[0002] The present invention is in the field of medicinal chemistry. Specifically, the present invention relates to the combination of Focal Adhesive Kinase (FAK) inhibitors with other drugs to treat tumors. [Background technology]

[0003] Tumors are the second most threatening disease to people's health. Immunogenic cell death (ICD) is an additive effect based on programmed cell death. Cancer cells may activate stress signals within the cells upon contact with chemotherapy drugs or targeted therapeutic drugs, including endoplasmic reticulum stress (ER stress) and oxidative stress. Under the action of stress signals, cells first attempt to repair the stress, and when the damage caused by the stress exceeds the repair capacity, the cells initiate the programmed death process. This process is often accompanied by the release of damage associated molecular patterns (DAMPs). Such DAMPs are specifically recognized by the pattern recognition receptors of antigen-presenting cells (APCs) in the organism, induce APC maturation, differentiation and activation, and present them step by step to immune cells such as effector T cells, so that the immune cells form antigen memory and, when they encounter tumor cells of the same origin again, they specifically recognize and kill the tumor cells. The new specific immune response against tumors initiated by ICDs can enhance the action of immune checkpoint inhibitors (ICIs) by increasing their sensitivity, resulting in antitumor responses with durable immune memory.

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

[0005] MEK1, also known as MAP2K1, plays an important role in the MAPK / ERK (RAS-RAF-MEK-ERK) signal pathway. The signal pathway can be activated by various stimuli and regulate the proliferation, survival and differentiation of various types of eukaryotic cells, and the phenomenon of hyperactivation of the signal pathway appears in tumor cells in particular. Cobimetinib can selectively inhibit MEK1 and MEK2 to reduce ERK1 / 2 phosphorylation and inhibit the MAPK / ERK signal pathway, thereby inhibiting cell proliferation and promoting apoptosis of tumor cells. In 2014, the FDA approved the combination of Cobimetinib and vemurafenib (a B-RAF kinase inhibitor) for the treatment of unresectable or metastatic melanoma in patients with B-RAF V600E or V600K mutations. In clinical trials, Cobimetinib subjects showed adverse reactions such as diarrhea, nausea, vomiting, skin rash and fever.

[0006] It has been observed that some chemotherapy drugs induce ICD in cancer cells, and ICD is understood to be beneficial to the antitumor action of the therapy.Importantly, ICD can stimulate the immune system to attack cancer, and can provide sustained antitumor effects, especially when combined with anti-PD-1 or PD-L1.Although anti-PD-1 has achieved some success in combination with chemotherapy and targeted therapy, many clinical trials have failed, especially ovarian cancer (JAVELIN Ovarian 200) and lung cancer combined with Pembrolizumab and lenvatinib.Therefore, it is necessary to further search for drugs that can enhance the induction of ICD by chemotherapy drugs or targeted drugs, so as to increase the response rate and the possibility of obtaining long-term benefits from tumor therapy. Summary of the Invention

[0007] In one embodiment of the present disclosure, in the use of a FAK inhibitor, a MEK inhibitor, and an immune checkpoint inhibitor in the manufacture of a medicament for treating a tumor in a subject, the FAK inhibitor, the MEK inhibitor, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0008] According to another aspect of the present disclosure, in a pharmaceutical combination product of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor for treating a tumor in a subject, the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0009] According to yet another aspect of the present disclosure, there is provided a method for treating a tumor comprising administering to a subject in need thereof therapeutically effective amounts of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor, either simultaneously or sequentially.

[0010] A kit or pharma- ceutically acceptable composition according to another embodiment of the present disclosure includes (a) a FAK inhibitor, (b) a MEK inhibitor, and (c) an immune checkpoint inhibitor.

[0011] According to another aspect of the present disclosure, in the use of a FAK inhibitor and a MEK inhibitor in the manufacture of a medicament for treating a tumor, the FAK inhibitor enhances immunogenic cell death induced by the MEK inhibitor.

[0012] According to another embodiment of the present disclosure, a FAK inhibitor enhances immunogenic cell death induced by a MEK inhibitor in the treatment of tumors.

[0013] Another aspect of the present disclosure is a method for treating a tumor comprising administering to a subject in need thereof therapeutically effective amounts of a FAK inhibitor and a MEK inhibitor, either simultaneously or sequentially, wherein the FAK inhibitor enhances immunogenic cell death induced by the MEK inhibitor.

[0014] Another aspect of the present disclosure provides a use of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor in the manufacture of a combination drug for treating a tumor.

[0015] Another aspect of the present disclosure provides a use of a FAK inhibitor in the manufacture of a combination drug with a MEK inhibitor and an immune checkpoint inhibitor for treating a tumor.

[0016] Another aspect of the present disclosure provides the use of a MEK inhibitor together with a FAK inhibitor and an immune checkpoint inhibitor in the manufacture of a combination drug for treating a tumor.

[0017] Another aspect of the present disclosure provides the use of an immune checkpoint inhibitor together with a FAK inhibitor and a MEK inhibitor in the manufacture of a combination drug for treating a tumor.

[0018] Another aspect of the present disclosure provides the use of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor in combination in the manufacture of a medicament for treating a tumor.Another aspect of the present disclosure provides the use of a FAK inhibitor in combination with a MEK inhibitor and an immune checkpoint inhibitor in the manufacture of a medicament for treating a tumor.

[0019] Another aspect of the present disclosure provides the use of a MEK inhibitor in the manufacture of a medicament for treating a tumor in combination with a FAK inhibitor and an immune checkpoint inhibitor.

[0020] Another aspect of the present disclosure provides the use of an immune checkpoint inhibitor in combination with a FAK inhibitor and a MEK inhibitor in the manufacture of a medicament for treating a tumor.

[0021] According to another embodiment of the present disclosure, a kit includes a FAK inhibitor and instructions indicating that the FAK inhibitor can be used to treat tumors in combination with a MEK inhibitor and an immune checkpoint inhibitor.

[0022] According to another embodiment of the present disclosure, a kit includes a MEK inhibitor and instructions indicating that the MEK inhibitor can be used to treat a tumor in combination with a FAK inhibitor and an immune checkpoint inhibitor.

[0023] According to another aspect of the present disclosure, a kit includes an immune checkpoint inhibitor and instructions indicating that the immune checkpoint inhibitor can be used to treat a tumor in combination with a FAK inhibitor and a MEK inhibitor.

[0024] A method of treating a tumor according to another aspect of the present disclosure includes administering to a subject in need thereof a therapeutically effective amount of a FAK inhibitor and a MEK inhibitor.

[0025] A pharmaceutical combination product of a FAK inhibitor and a MEK inhibitor according to yet another aspect of the present disclosure treats tumors in a subject in need thereof.

[0026] Yet another aspect of the present disclosure provides the use of a FAK inhibitor and a MEK inhibitor in the manufacture of a combination medicament for treating a tumor.

[0027] Yet another aspect of the present disclosure provides the use of a FAK inhibitor together with a MEK inhibitor in the manufacture of a combination drug for treating a tumor.

[0028] Yet another aspect of the present disclosure provides the use of a MEK inhibitor together with a FAK inhibitor in the manufacture of a combination drug for treating a tumor.

[0029] Yet another aspect of the present disclosure provides the use of a FAK inhibitor and a MEK inhibitor in combination in the manufacture of a medicament for treating a tumor.

[0030] Yet another aspect of the present disclosure provides the use of a FAK inhibitor in the manufacture of a medicament for treating a tumor in combination with a MEK inhibitor.

[0031] Yet another aspect of the present disclosure provides the use of a MEK inhibitor in combination with a FAK inhibitor in the manufacture of a medicament for treating a tumor.

[0032] According to another embodiment of the present disclosure, a kit includes a FAK inhibitor and instructions indicating that the FAK inhibitor can be used in combination with a MEK inhibitor to treat a tumor.

[0033] According to another embodiment of the present disclosure, a kit includes a MEK inhibitor and instructions indicating that the MEK inhibitor can be used in combination with a FAK inhibitor to treat a tumor.

[0034] Optionally, the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably, IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably, IN10018 or a pharma- ceutically acceptable salt thereof, in particular, IN10018 tartrate, and the structure of IN10018 is as follows: [ka]

[0035] The defactinib is also called defactinib, and has a CAS number of 1345713-71-4, the GSK2256098 has a CAS number of 1224887-10-8, the PF-00562271 has a CAS number of 717907-75-0, the VS-4718 has a CAS number of 1061353-68-1, the APG-2449 is developed by Yashen Pharmaceutical, and the AMP945 has a CAS number of 1393653-34-3.

[0036] Optionally, the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0037] The trametinib is also called Trametinib / Mekinist and has a CAS number of 871700-17-3, the cobimetinib is also called Cobimetinib / Cotellic and has a CAS number of 934660-93-2, the binimetinib is also called Mektovi / binimetinib and has a CAS number of 606143-89-9, and the selumetinib is also called Koselugo / Selum. etinib, which has a CAS number of 606143-52-6, and its sulfate salt has a CAS number of 943332-08-9; PD-325901, which is also known as Mirdametinib or PD-0325901, which has a CAS number of 391210-10-9; TAK-733, which is also known as REC-4881, which has a CAS number of 1035555-63-5; and HL-085 (HL085) which is also known as Kechow Pharma Inc (Shanghai Kezhou Drug Research and Development Co., Ltd.), E-6201 has a CAS number of 603987-35-5, Pimasertib is also known as AS703026 and has a CAS number of 1236699-92-5, Refametinib is also known as RDEA119 and has a CAS number of 923032-37-5, and FCN-159 was developed by Fuxing Pharmaceuticals and its subsidiaries.

[0038] 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.

[0039] Optionally, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, for example, 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, in particular atezolizumab.

[0040] Optionally, the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, for example, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMMH-010, CA-170, ABSK043, or RRx-001.

[0041] Optionally, the immune checkpoint inhibitor is a TIGIT inhibitor, for example, 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.

[0042] Optionally, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colon cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, cervical cancer, malignant melanoma, uveal melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer, or laryngeal cancer. For example, the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further, acute myeloid leukemia, malignant melanoma, uveal melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0043] In order to more clearly describe the technical means in the embodiments of the present disclosure, the drawings of the embodiments are briefly described below. Obviously, the drawings in the following description are only related to some embodiments of the present disclosure, and do not limit the present invention. [Brief description of the drawings]

[0044] [Figure 1] The 72-hour injury curve and IC50 value for melanoma cells A375 in combination with Cobimetinib after FAK silencing are shown. [Diagram 2] Expression levels of endoplasmic reticulum stress-related proteins detected after FAK silencing followed by treatment of A375 with 10 nM and 30 nM Cobimetinib for 6 h are shown. [Diagram 3] Immunofluorescence staining of DAMPs following FAK silencing followed by treatment of A375 with 10 nM Cobimetinib for 48 h is shown. [Figure 4] 7 shows the 72-hour injury curves and IC50 values ​​of IN10018 and Cobimetinib against malignant melanoma cells A375. [Diagram 5]The combination of different doses of Cobimetinib and 3 μM IN10018 shows synergistic cytotoxicity against melanoma cells A375 for 72 hours (*p<0.05, **p<0.01, ***p<0.001). [Figure 6] The expression levels of endoplasmic reticulum stress-related proteins detected after 1 nM, 3 nM, 10 nM and 30 nM of Cobimetinib were combined with 3 μM IN10018 for 6 hours. [Figure 7] Immunofluorescence staining of DAMPs following combination treatment of 10 nM Cobimetinib and 3 μM IN10018 for 48 hours. [Figure 8] 1 shows changes in tumor growth after administration of different test substances to tumor-bearing mice in a BALB / c mouse homograft tumor model of mouse colon cancer CT-26. [Figure 9] 1 shows the animal survival rate after administration of different test substances to tumor-bearing mice in a BALB / c mouse homograft tumor model of mouse colon cancer CT-26. [Figure 10] 1 shows the weight change status of mice after administration of different doses of drugs and the weight change of mice after administration of different doses of drugs. [Figure 11] White light micrographs of cells taken after 48 hours of incubation with cells and drugs are shown. [Figure 12] The percentage of CRT positive CT26 cells after 48 hours of incubation with drugs (FIG. 12a) and the percentage of Annexin V positive CT26 cells after 48 hours of incubation with drugs (FIG. 12b) are shown. [Figure 13] 1 shows changes in tumor growth after administration of different test substances to tumor-bearing mice in a BALB / c mouse homograft tumor model of mouse colon cancer CT-26. [Figure 14] 1 shows the weight change status of mice after administration of different doses of drugs and the weight change of mice after administration of different doses of drugs. [Figure 15]The percentage of CRT positive 4T1 cells after 48 hours of incubation with drugs (FIG. 15a) and the percentage of Annexin V positive 4T1 cells after 48 hours of incubation with drugs (FIG. 15b) are shown. [Figure 16] The percentage of CRT positive CT26 cells after 48 hours of incubation with drugs (FIG. 16a) and the percentage of Annexin V positive CT26 cells after 48 hours of incubation with drugs (FIG. 16b) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] In order to make the objectives, technical means and advantages of the embodiments of the present disclosure clearer, the technical means in the embodiments of the present disclosure will be described clearly and completely below with reference to the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only some of the embodiments of the present disclosure, and not all of the embodiments. Based on the described embodiments of the present disclosure, any other embodiments obtained by a person skilled in the art without any creative effort are all within the scope of protection of the present invention.

[0046] The present invention may be embodied in other specific forms without departing from the essential attributes of the present invention. It should be understood that, unless inconsistent, any and all embodiments of the present invention may be combined with the technical features of any one or more other embodiments to obtain another embodiment. The present invention includes the other embodiment obtained from such combination.

[0047] All publications and patents mentioned in this disclosure are incorporated herein by reference in their entirety. If the terminology or usage used in any publication or patent incorporated by reference conflicts with the terminology or usage used in this disclosure, the terminology and usage in this disclosure shall control.

[0048] The section headings used herein are used for organizational purposes only and are not to be construed as limiting the subject matter discussed above.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the common meaning within the field to which the subject matter sought to be protected. In the event that a term has a plurality of definitions, those in this specification are the prevailing definition.

[0050] All numerical values ​​of quantitative nature, such as dosages, set forth in the specification and claims are to be understood in all instances to be modified by the term "about," except as otherwise expressly stated in the examples, and any numerical range recited herein is intended to include all subranges within that range and any combination of each endpoint of that range or subrange.

[0051] As used in this disclosure, words such as "comprise", "contain", or "comprise" and similar words are intended to cover the elements listed after the word and their equivalents, and not to exclude elements not listed. As used herein, the term "comprise" or "comprise" may be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0052] definition The following terms and symbols used in this application have the following meanings unless otherwise indicated in the context. The term "FAK inhibitor" as used herein refers to an effective inhibitor of FAK, 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: [ka]

[0053] The above-mentioned Defactinib is also called Defactinib, and has a CAS number of 1345713-71-4, and the above-mentioned GSK2256098 has a CAS number of 1224887-10-8. The above-mentioned PF-00562271 has a CAS number of 717907-75-0, the above-mentioned VS-4718 has a CAS number of 1061353-68-1, the above-mentioned APG-2449 is developed by Yashen Pharmaceutical, and the above-mentioned AMP945 has a CAS number of 1393653-34-3. In some embodiments, the above-mentioned FAK inhibitor is preferably IN10018, Defactinib, AMP945 or a pharmaceutical acceptable salt thereof, and in some embodiments, the above-mentioned FAK inhibitor is IN10018 or a pharmaceutical acceptable salt thereof, particularly IN10018 tartrate.

[0054] As used herein, the term "MEK" refers to a mitogen-activated protein kinase, a serine-threonine kinase, that mediates intracellular signal transduction involved in regulating protein and cellular functions related to membranes, intracellular and intercellular processes, transformation, proliferation / growth, differentiation, survival and death.

[0055] The term "MEK inhibitor" as used herein refers to a compound or drug that reduces or decreases MEK-dependent cell signaling / function and reduces or decreases MEK-associated tumor cell proliferation / growth. Examples of MEK inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159, or pharmaceutically acceptable salts thereof. In some embodiments, the MEK inhibitor is cobimetinib, HL-085, or pharmaceutically acceptable salts thereof.

[0056] The trametinib is also known as Trametinib / Mekinist, and has a CAS number of 871700-17-3; the cobimetinib is also known as Cobimetinib / Cotellic, and has a CAS number of 934660-93-2; the binimetinib is also known as Mektovi / binimetinib, and has a CAS number of 606143-89-9; the selumetinib is also known as Koselugo / Selumetinib, and has a CAS number of 606143-52-6, and its sulfate has a CAS number of 943332-08-9; the PD-325901 has a CAS number of 391210-10-9; the TAK-733 has a CAS number of 1035555-63-5; and the HL-085 (HL085) is manufactured by Kechow Pharma. Inc (Shanghai Kezhou Drug Research and Development Co., Ltd.); E-6201 has a CAS number of 603987-35-5; pimasertib is also known as AS703026 and has a CAS number of 1236699-92-5; refametinib is also known as RDEA119 and has a CAS number of 923032-37-5; and FCN-159 was developed by Fuxing Pharmaceuticals and its subsidiaries.

[0057] As used herein, the term "immune checkpoint inhibitor" refers to a drug that can improve immune system activity by modulating immune checkpoint pathways (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. The PD-1 inhibitor is also referred to as a PD-1 / PD-L1 inhibitor in this disclosure. For example, the PD-1 / PD-L1 inhibitor in the treatment methods, drugs and uses of the present disclosure is an anti-PD-1 / PD-L1 antibody, including, but not limited to, pembrolizumab (Keytruda / K drug), tislelizumab (Tislelizumab / Beigene), nivolumab (Nivolumab), toripalimab (Toripalimab / Takuei), durvalumab (Imfinzi / Durvalumab / durvalumab), avelumab (Avelumab / Bavencio), atezolizumab (MPDL3280A / Atezolizumab / Tecentriq / T drug), 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 preferred embodiments, the PD-1 inhibitor is atezolizumab. In some embodiments, the PD-1 inhibitor treats a subject that is human. In some embodiments, the PD-1 is human PD-1. PD-1 / PD-L1 inhibitors also include PD-1 / PD-L1 small molecule inhibitors, such as INCB-086550, lazertinib, IMMH-010, CA-170, ABSK043, or RRx-001. TIGIT (also known as WUCAM, Vstm3, VSIG9) is a receptor of the Ig superfamily and a novel immune checkpoint that follows PD-1 / PD-L1.For example, in the therapeutic methods, drugs, and uses of the present disclosure, the immune checkpoint inhibitor is a TIGIT inhibitor (e.g., a TIGIT antibody), and 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. In some embodiments, the TIGIT inhibitor treats a subject that is a human. For the avoidance of doubt, all references to antibodies herein include diabodies.

[0058] As used herein, a "pharmaceutical combination" or "pharmaceutical combination product" may refer to a fixed combination in one dosage unit form (e.g., all active pharmaceutical ingredients are present in one dosage form) or a kit of combined administration products, or may refer to the combination of one pharmaceutical agent with instructions for use of that agent in combination with one or more other pharmaceutical agents.

[0059] As used herein, "combination therapy" or "combination drug" refers to the use of one drug in combination with one or more other drugs to treat a disease, and includes not only the combination of one drug with one or more other drugs, but also the combination of one drug with instructions indicating that the drug can be used in combination with one or more other drugs.

[0060] "Administered simultaneously or sequentially" as used herein refers to administration of two or more drugs simultaneously or at a fixed time interval in one administration cycle (e.g., within 4 weeks, 3 weeks, 2 weeks, 1 week, or 24 hours), the administration mode of the drugs (e.g., oral, intravenous, intramuscular, or subcutaneous administration, etc.) may be the same or different, and the administration frequency / cycle of the two or more drugs may be the same or different. When the treatment method, product, or use of the present disclosure involves two drugs, the two drugs may be administered simultaneously or separately at a fixed time interval. When the treatment method, product, or use of the present disclosure involves three drugs, the three drugs may be administered at the same time, two drugs may be administered at a specific time and the remaining drug may be administered at another time, or all three drugs may be administered at different times.

[0061] 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 intravenously.

[0062] The ability of immune checkpoint inhibitors to treat cancer depends on the presence of tumor antigen-specific T cells in tumor tissue. This requires that tumor tissue expresses antigens that distinguish it from its non-transformed counterpart, for example, by novel protein products called neoantigens. Tumor neoantigen load strongly correlates with immunogenicity and sensitivity (e.g., sensitivity to checkpoint inhibitor therapy), meaning that tumors with poor immunogenicity should be highly resistant to these drugs. Therapies that release tumor antigens that can be taken up by APCs, such as those that induce immunogenic cell death (ICD), may effectively promote antitumor immunity, especially when combined with checkpoint inhibitors.

[0063] As used herein, the term "treatment" refers to the administration of one or more drug substances to a subject having a disease or symptoms of said disease to cure, alleviate, relieve, alter, treat, improve, treat or affect said disease or symptoms of said disease. In some embodiments, said disease is a tumor or cancer.

[0064] The term "tumor" as used herein refers to an abnormal lesion formed by the clonal abnormal proliferation of certain cells in local tissues that have lost normal proliferation control at the genetic level under the action of various carcinogenic factors. Examples include Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colon cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, uterine cancer, and ovarian cancer. The tumors include, but are not limited to, endometrial cancer, cervical cancer, malignant melanoma, uveal malignant melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further, the tumors are acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further, acute myeloid leukemia, malignant melanoma, uveal malignant melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0065] The term "subject" or "subject" as used herein refers to mammals and non-mammals. Mammals refer to any member of the mammalian genus, including, but not limited to, humans; non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats and pigs; livestock animals 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, and the like. The term "subject" is not limited to a particular age or sex. In some embodiments, the subject is a human.

[0066] As used herein, the term "pharmaceutical acceptable" refers to non-toxic, biologically tolerable, and suitable for administration to a subject.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to acid addition salts that are non-toxic, biologically acceptable and suitable for administration to a subject, and includes acid addition salts formed with inorganic acids, such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, and the like, as well as formates, acetates, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethanesulfonates, benzoates, salicylates, stearates, and salts of the formula HOOC-(CH2) n -COOH (wherein n is 0 to 4) formed with an alkanedicarboxylic acid, such as an acid addition salt formed with an organic acid; Alternatively, pharma- ceutically acceptable acid addition salts may be produced by dissolving the free base in a suitable solvent and treating the solution with an acid, following conventional procedures for preparing acid addition salts from basic compounds.Those skilled in the art can determine, without undue experimentation, various synthetic methods that may be used to prepare non-toxic pharma- ceutically acceptable acid addition salts.

[0068] The term "pharmaceutical acceptable composition" as used herein refers to a composition that must be chemically and / or toxicologically compatible with other components of the formulation and / or with the subject to be treated therewith. The term "therapeutically effective amount" as used herein generally refers to an amount sufficient to provide a beneficial therapeutic effect to the subject. The therapeutically effective amount of the present invention can be determined in a conventional manner (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., mode of administration, pharmacokinetics of the compound, severity and course of the disease, medical history of the subject, health status of the subject, responsiveness of the subject to the drug, etc.).

[0069] As used herein, the term "inhibition" refers to a reduction in the baseline activity of a biological activity or process.

[0070] The term "kit" as used herein refers to a box for containing chemical reagents for detecting chemical components, residual drugs, types of viruses, etc. The kit according to the present invention may include (i) one, two or three of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor, and (ii) instructions indicating that the FAK inhibitor, the MEK inhibitor and the 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 the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a MEK inhibitor and (ii) instructions indicating that the FAK inhibitor, the MEK inhibitor and the 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 indicating that the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit comprises (i) a FAK inhibitor, a MEK inhibitor, and an immune checkpoint inhibitor, and (ii) instructions indicating that the FAK inhibitor, the MEK inhibitor, and the immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit comprises (i) a FAK inhibitor, and (ii) instructions indicating that the FAK inhibitor and the MEK inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit comprises (i) a MEK inhibitor, and (ii) instructions indicating that the MEK inhibitor and the FAK inhibitor can be used to treat a tumor in a subject.

[0071] The compounds of the kit may be contained in separate containers. Optionally, two or more compounds may be contained in the same container. For example, the kit may include a first container, a second container, a third container, and a package insert, where the first container includes at least one dose of a drug including a FAK inhibitor, the second container includes at least one dose of a MEK inhibitor, and the third container includes at least one dose of an immune checkpoint inhibitor drug, and the package insert includes instructions for using the drug to treat a tumor in a subject. The first container, the second container, and the third container may include the same or different shapes (e.g., vials, syringes, and bottles) and / or materials (e.g., plastic or glass). The kit may further include other materials useful for administering the drug, such as diluents, filters, IV bags and pipelines, needles, and syringes.

[0072] The exact amount of FAK inhibitor, MEK inhibitor or immune checkpoint inhibitor administered to a subject depends on a variety of factors, such as the given drug or compound, drug formulation, route of administration, type of disease, symptoms, and identity of the subject or host being treated, but can still be routinely determined by one of ordinary skill in the art. For example, determining an effective amount also depends on the extent, severity and type of cell proliferation. Those of ordinary skill in the art can determine the appropriate dose depending on these and other factors.

[0073] The FAK inhibitor, MEK inhibitor or immune checkpoint inhibitor may be administered by an appropriate route, such as orally, intravenously, intramuscularly or subcutaneously.

[0074] For example, when administered orally, the drug may be administered orally with a pharma- ceutically acceptable carrier, such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the patient's food. For example, the drug may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, or wafers. Tablets, troches, pills, capsules, and the like may further include adhesives, such as gum tragacanth, gum arabic, corn starch, or gelatin; excipients, such as dibasic calcium phosphate; disintegrating agents, such as corn starch, potato starch, or alginic acid; lubricants, such as magnesium stearate; sweeteners, such as sucrose, fructose, lactose, or aspartame; or flavoring agents.

[0075] For example, for intravenous or intraperitoneal administration as an infusion or injection, solutions of the drug can be prepared in water, optionally mixed with a nontoxic surfactant.

[0076] Exemplary pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient suitable for the extemporaneous preparation of sterile injectable or infusion solutions or dispersions. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage.

[0077] Sterile injectable solutions may be prepared by incorporating the required amount of the drug in a suitable solvent with various other ingredients as described above, followed by filtration sterilization. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying technology, which can produce powders of the active ingredient plus any desired ingredients present after prior sterilization filtration.

[0078] The amount of FAK inhibitor, MEK inhibitor or immune checkpoint inhibitor required for treatment may vary depending on the particular agent selected, the route of administration, the nature of the disease being treated and the age and condition of the patient, and may ultimately be determined by the attending physician or clinician himself / herself. Generally, however, the dosage is in the range of about 0.1 to about 50 mg / kg body weight daily.

[0079] The FAK inhibitor is administered in a dosage range of 5 mg / day to 300 mg / day in an adult. In a specific embodiment, IN10018 or a pharma- ceutically acceptable salt thereof is administered in a dosage range of 5 mg / day to 100 mg / day in an adult, the dosages being calculated on the free base.

[0080] The MEK inhibitor is administered in a dose range of 2-250 mg daily in an adult. In a specific embodiment, cobimetinib or a pharma- ceutically acceptable salt thereof is administered in a dose range of 2-250 mg daily in an adult, said dose being calculated based on cobimetinib, and HL-085 or a pharma-ceutically acceptable salt thereof is administered in a dose range of 2-250 mg daily in an adult, said dose being calculated based on HL-085.

[0081] The immune checkpoint inhibitor is administered at a dose of 2 to 10 mg / kg or 50 to 1200 mg in an adult each time, once every 2 to 3 weeks. In a specific embodiment, the immune checkpoint inhibitor is administered at a dose of 3 to 10 mg / kg or 100 to 1200 mg in an adult each time, once every 2 to 3 weeks.

[0082] Technical and scientific terms used herein that are not specifically defined have the meaning commonly understood by one of ordinary skill in the art of the invention.

[0083] In some embodiments, the present disclosure further discloses the following embodiments:

[0084] 1. A FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor for use in a method for treating a tumor in a subject, wherein the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor are administered simultaneously or sequentially to the subject.

[0085] 2. The compound for use according to embodiment 1, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, wherein the structure of IN10018 is as follows: [ka]

[0086] 3. The compound for said use according to embodiment 1 or 2, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085 or a pharma- ceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharma- ceutically acceptable salt thereof.

[0087] 4. The compound for use according to any one of embodiments 1 to 3, 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.

[0088] 5. The compound for said use according to any one of embodiments 1 to 4, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma-ceutically 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 atezolizumab.

[0089] 6. The tumor is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, cervical cancer, malignant black lung cancer, and the like. The compound for use according to any one of embodiments 1 to 5, wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further still further, acute myeloid leukemia, malignant melanoma, uveal melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0090] 7. (a) FAK inhibitor; (b) a MEK inhibitor; and (c) an immune checkpoint inhibitor,

[0091] 8. The kit or composition according to embodiment 7, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of IN10018 is as follows: [ka]

[0092] 9. The kit or composition according to embodiment 7 or 8, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0093] 10. The kit or composition according to any one of embodiments 7 to 9, 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.

[0094] 11. The kit or composition according to any one of embodiments 7 to 10, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma-ceutically 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 atezolizumab.

[0095] 12. The kit or composition according to any one of embodiments 7 to 11, for use as a medicament.

[0096] 13. The kit or composition according to any one of embodiments 7 to 12, which is used to treat a tumor.

[0097] 14. The tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, foetal cancer, The kit or composition of embodiment 13, wherein the tumor is cervical cancer, malignant melanoma, uveal melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0098] 15. A method for treating a tumor in a subject, comprising simultaneously or sequentially administering to the subject the compounds of the kit or composition described in any one of embodiments 7 to 12.

[0099] 16. The tumor is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, and endometrial cancer. The method of embodiment 15, wherein the tumor is cancer, cervical cancer, melanoma, uveal melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or cholangiocarcinoma, and further still further wherein the tumor is acute myeloid leukemia, melanoma, uveal melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0100] 17, a FAK inhibitor that enhances immunogenic cell death induced by MEK inhibitors.

[0101] 18. The FAK inhibitor of the use according to embodiment 17, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0102] 19. The FAK inhibitor of the use according to embodiment 17 or 18, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of the IN10018 is as follows: [ka]

[0103] 20. The FAK inhibitor of any one of embodiments 17 to 19, wherein the FAK inhibitor and the MEK inhibitor are administered simultaneously or sequentially.

[0104] 21. A method for treating a tumor in a subject, comprising administering to the subject therapeutically effective amounts of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor simultaneously or sequentially.

[0105] 22. The method according to embodiment 21, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of IN10018 is as follows: [ka]

[0106] 23. The method according to embodiment 21 or 22, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0107] 24. The method of any one of embodiments 21 to 23, 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.

[0108] 25. The method according to any one of embodiments 21 to 24, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma- ceutically 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 atezolizumab.

[0109] 26. The tumor is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, cervical cancer, The method according to any one of embodiments 21 to 25, wherein the tumor is cancer, malignant melanoma, uveal malignant melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal malignant melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0110] 27. A FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor for use in a method for treating a tumor by increasing immunogenic cell death in a subject, wherein the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0111] 28. The compound for use according to embodiment 27, wherein the FAK inhibitor is IN10018, defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of IN10018 is as follows: [ka]

[0112] 29. The method according to embodiment 27 or 28, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0113] 30. The compound for use according to any one of embodiments 27 to 29, 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.

[0114] 31. The compound for use according to any one of embodiments 27 to 30, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma-ceutically 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 atezolizumab.

[0115] 32. The tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, cervical cancer, malignant and wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0116] 33. A method for treating a tumor in a subject by increasing immunogenic cell death, comprising administering to the subject therapeutically effective amounts of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor simultaneously or sequentially.

[0117] 34. The method according to embodiment 33, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of IN10018 is as follows: [ka]

[0118] 35. The method according to embodiment 33 or 34, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0119] 36. The method of any one of embodiments 33 to 35, 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.

[0120] 37. The method according to any one of embodiments 33 to 36, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma- ceutically 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 atezolizumab.

[0121] 38, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, The method according to any one of embodiments 33 to 37, wherein the tumor is cervical cancer, malignant melanoma, uveal malignant melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal malignant melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0122] 39. Use of a FAK inhibitor in the manufacture of a medicament for treating a tumor in a subject, wherein the FAK inhibitor, MEK inhibitor and immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0123] 40. The use according to embodiment 39, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, wherein the structure of IN10018 is as follows: [ka]

[0124] 41. The use according to embodiment 39 or 40, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0125] 42. The use according to any one of embodiments 39 to 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.

[0126] 43. The use according to any one of embodiments 39 to 42, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma-ceutically 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 atezolizumab.

[0127] 44, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, The use according to any one of embodiments 39 to 43, wherein the tumor is cervical cancer, malignant melanoma, uveal malignant melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal malignant melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0128] 45. Use of a MEK inhibitor in the manufacture of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, the MEK inhibitor and an immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0129] 46. ​​The use according to embodiment 45, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0130] 47. The use according to embodiment 45 or 46, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, wherein the structure of IN10018 is as follows: [ka]

[0131] 48. The use according to any one of embodiments 45 to 47, 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.

[0132] 49. The use according to any one of embodiments 45 to 48, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma-ceutically 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 atezolizumab.

[0133] 50, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, The use according to any one of embodiments 45 to 49, wherein the tumor is cervical cancer, malignant melanoma, uveal malignant melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal malignant melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0134] 51. Use of an immune checkpoint inhibitor in the manufacture of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, a MEK inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0135] 52. Use of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor in the manufacture of a combination drug for treating tumors.

[0136] 53. Use of a FAK inhibitor in the manufacture of a combination drug for treating tumors with a MEK inhibitor and an immune checkpoint inhibitor.

[0137] 54. Use of a MEK inhibitor in the manufacture of a combination drug for treating tumors with a FAK inhibitor and an immune checkpoint inhibitor.

[0138] 55. Use of an immune checkpoint inhibitor in the manufacture of a combination drug for treating tumors with a FAK inhibitor and a MEK inhibitor.

[0139] 56. Use of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor in combination in the manufacture of a drug for treating a tumor.

[0140] 57. Use of a FAK inhibitor in the manufacture of a drug for treating tumors in combination with a MEK inhibitor and an immune checkpoint inhibitor.

[0141] 58. Use of a MEK inhibitor in the manufacture of a drug for treating tumors in combination with a FAK inhibitor and an immune checkpoint inhibitor.

[0142] 59. Use of an immune checkpoint inhibitor in combination with a FAK inhibitor and a MEK inhibitor in the manufacture of a drug for treating a tumor.

[0143] 60. The use according to any one of embodiments 51 to 59, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular, IN10018 tartrate, and the structure of IN10018 is as follows: [ka]

[0144] 61. The use according to any one of embodiments 51 to 60, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharmaceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharmaceutically acceptable salt thereof.

[0145] 62. The use according to any one of embodiments 51 to 61, 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.

[0146] 63. The use according to any one of embodiments 51 to 62, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma- ceutically 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 atezolizumab.

[0147] 64, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, esophagogastric junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, The use according to any one of embodiments 51 to 63, wherein the tumor is cervical cancer, malignant melanoma, uveal malignant melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal malignant melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

[0148] Working Example To further illustrate the present invention, the following examples are provided, which should be understood to be merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0149] In the following examples, experimental methods for which specific conditions are not specified may be carried out according to standard conditions or conditions recommended by the manufacturer.

[0150] All experimental materials and reagents used in the following examples are commercially available unless otherwise specified. The meanings of the abbreviations used in the examples are as follows: English abbreviation Japanese name siRNA Small interfering RNA DMSO Dimethyl sulfoxide COMBO Combined or combined use HMGB1 High Mobility Group Box Protein 1 AAALAC Association for Assessment and Accreditation of Laboratory Animal Care International IACUC Laboratory Animal Care and Use Committee SPF Special Pathogen Free BIW twice a week BID 2 times a day BW Body Weight EDTA Ethylenediaminetetraacetic acid Good Laboratory Practice (GLP) - Standards for conducting non-clinical trials on drug safety po Intragastric administration iv Intravenous administration ip intraperitoneal injection DPBS Dulbecco's Phosphate Buffer DDH2O Double distilled water RT room temperature SEM standard error TGI Tumor growth inhibition rate TV tumor volume TW tumor weight FBS Fetal Bovine Serum PBS Phosphate buffer solution MC Methylcellulose CO2 Carbon Dioxide CRT Calreticulin Annexin-V-FITC Fluorescein isothiocyanate-labeled Annexin V protein PI Propidium Iodide

[0151] Example 1: Study of Cobimetinib immunogenic cell death by targeting FAK in malignant melanoma cells Experimental scheme: 1. Improved sensitivity to cobimetinib after FAK silencing In vitro experiments, different concentrations of Cobimetinib were administered and treated under the condition of FAK gene silencing. A cytotoxicity curve was made in A375 cells to determine the IC50 value. The cytotoxicity of Cobimetinib was compared with the control group and the FAK siRNA group to explore the effect of Cobimetinib cytotoxicity on FAK target.

[0152] In the experiment, under the condition of FAK gene silencing, the cytotoxicity curves of different concentrations of Cobimetinib in melanoma cells A375 were measured and the IC50 values ​​were determined.

[0153] 2. After FAK silencing, it can effectively induce ICD in melanoma cells A375 in combination with cobimetinib.

[0154] In vitro experiments were conducted to detect the effects of FAK silencing on endoplasmic reticulum stress-related proteins GRP78 and Phospho-eIF2α (Ser51) after 6 hours of combination with Cobimetinib, and whether it could effectively enhance the release of Calreticulin, the main target of ICD, by cells after 48 hours of combination.

[0155] Test materials: FAK siRNA was provided by GenePharma and has the sequence shown in the table below: F: CCUGUAUGCCUAUCAGCUUTT, R:AAGCUGAUAGGCAUACAGGTT Cobimetinib was purchased from MCE and is GDC-0973.

[0156] Antibodies used in this study: Recombinant Alexa Fluor® 647 fluorescent Anti-Calreticulin antibody (Abcam, ab196159), FAK antibody (CST, 3285S), GRP78 antibody (HUABIO, ER1706-50), Phospho-eIF2α (Ser51) (CST, 3398), eif2α (CST, 5324), HRP-labeled Alpha Tubulin (α-tubulin) antibody (Proteintech, HRP-66031).

[0157] Experimental equipment: Fluorescence microscope (Olympus U-HGLGPS). Chemiluminescence image (BIORAD chemidoc touch).

[0158] Test Results: 1. IC50 curve for A375 in combination with Cobimetinib after FAK silencing 5000 A375 cells / well were placed in a 96-well cell plate, and two groups were transfected with control siRNA or FAK siRNA. After 24 hours of transfection, the highest concentration of 1μM Cobimetinib was added, and diluted with a 3-fold concentration gradient to obtain a total of six concentrations, with the lowest concentration of Cobimetinib being 4.115nM. After incubating the drug and cells together for 72 hours, 10μl of CCK8 solution was added to each well, and the cells were incubated at 37℃ in a 5% carbon dioxide incubator for 2 hours, and then the absorbance wavelength was set to 450nm and read using a microplate reader. The values ​​read were compared with the DMSO control group and plotted using Graphpad8.0. The results are shown in Figure 1.

[0159] 2. After FAK silencing, the combination of FAK with Cobimetinib significantly increased endoplasmic reticulum stress in A375 cells.

[0160] A375 cells were transfected with control siRNA or FAK siRNA at a final concentration of 50 nM, respectively. 24 h after transfection, different concentrations of Cobimetinib were administered and treated for 6 h, and the protein level expression of FAK and endoplasmic reticulum stress-related proteins GRP78 and Phospho-eIF2α (phosphorylated-Phospho-eIF2α) (Ser51) was detected. The results are shown in Figure 2.

[0161] 3. The combination of cobimetinib and IN10018 enhanced the release and exposure of the ICD target calreticulin.

[0162] A375 cells were transfected with control siRNA or FAK siRNA at a final concentration of 50 nM, respectively. After 24 hours, A375 cells were treated with 10 nM Cobimetinib for 48 hours, followed by immunofluorescence detection of Calreticulin. The results are shown in Figure 3.

[0163] Test conclusion: After FAK silencing, it can significantly enhance the cytotoxicity of Cobimetinib on melanoma cell A375. This effect is related to the enhancement of endoplasmic reticulum stress. In the experiment of detecting ICD markers, it can significantly enhance the exposure and release of DAMPs marker Calreticulin after FAK silencing, showing that silencing of FAK target can effectively enhance the immunogenic cell death of melanoma cells.

[0164] Example 2: Study on enhancement of Cobimetinib immunogenic cell death of malignant melanoma cells by IN10018 Experimental scheme: 1. IN10018 and Cobimetinib Combined to Show Synergistic Tumor Damage In vitro experiments were performed to measure the cytotoxicity curves of IN10018 and Cobimetinib in malignant melanoma cells A375, and IC50 values ​​were determined. The cytotoxicity of different doses of Cobimetinib in combination with 3 μM IN10018 was explored.

[0165] 2. The combination of IN10018 and cobimetinib can effectively induce ICD in melanoma cells A375.

[0166] In vitro experiments were conducted to detect the effects of IN10018 and Cobimetinib on endoplasmic reticulum stress-related proteins GRP78, Phospho-eIF2α (Ser51), and DDIT3 after 6 hours of combination therapy, and whether they could effectively enhance the release of Calreticulin and Annexin A1, the main targets of ICD, by cells after 48 hours of combination therapy.

[0167] Test materials: 1) Drugs used in this experiment IN10018 was synthesized by the method in patent WO2010058032. Cobimetinib was purchased from MCE and is GDC-0973.

[0168] Antibodies used in this study: Recombinant Alexa Fluor® 647 fluorescent Anti-Calreticulin antibody (Abcam, ab196159), Annexin A1 rabbit antibody (Cell signaling technology, 32934S), Hoechst33342 (Invitrogen, H21492), GRP78 antibody (HUABIO, ER1706-50), DDIT3 (HUABIO, ET1703-05), Phospho-eIF2α (Ser51) (CST, 3398), eif2α (CST, 5324), p44 / 42 MAPK (Erk1 / 2) (CST, 4695), Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (CST, 4370), HRP-labeled Alpha tubulin antibody (Proteintech, HRP-66031).

[0169] Experimental equipment: Fluorescence microscope (Olympus U-HGLGPS). Chemiluminescence image (BIORAD chemidoc touch).

[0170] Test Results: 1. IC50 curves of single agents, IN10018 and Cobimetinib, against A375 5000 A375 cells / well were plated in a 96-well cell plate, and after 24 hours, the drugs Cobimetinib at the highest concentration of 10μM and IN10018 at the highest concentration of 30μM were added and diluted with a 3-fold concentration gradient to obtain a total of 9 concentrations, with the lowest concentration of Cobimetinib being 1.52nM and the lowest concentration of IN10018 being 4.57nM. After incubating the drugs with the cells for 72 hours, 10μL of CCK8 solution was added to each well, and the cells were incubated at 37℃ in a 5% carbon dioxide incubator for 2 hours, and then read on a microplate reader with the absorbance wavelength set to 450nm. The readings were compared with the DMSO control group and plotted using Graphpad8.0. The results are shown in Figure 4.

[0171] 2. Synergistic toxicity of different doses of cobimetinib in combination with 3 μM IN10018 against A375 cells 5000 A375 cells / well were plated on a 96-well cell plate, and after 24 hours, four subdoses were set on the left and right sides of the IC value of Cobimetinib as the initial point, and Cobimetinib was combined with 3μM IN10018 at these doses. After 72 hours, cell viability was measured using the CCK8 method. The results were plotted using Graphpad8.0. The results are shown in Figure 5.

[0172] 3. The combination of cobimetinib and IN10018 significantly increased endoplasmic reticulum stress in A375 cells.

[0173] A375 cells were treated with 1nM, 3nM, 10nM and 30nM Cobimetinib in combination with 3μM IN10018, and 6 hours later, the protein level expression of endoplasmic reticulum stress-related proteins GRP78, Phospho-eIF2α(Ser51), and DDIT3 was detected. As can be seen from the results, the expression levels of GRP78, Phospho-eIF2α(Ser51), and DDIT3 were significantly improved in the combination group compared to the single drug group, explaining that the combination of the two drugs significantly increased endoplasmic reticulum stress (shown in Figure 6).

[0174] 4. The combination of cobimetinib and IN10018 enhanced the release and exposure of ICD targets calreticulin and Annexin A1.

[0175] A375 cells were treated with 10 nM Cobimetinib and 3 μM IN10018 in combination, and 48 hours later, calreticulin and Annexin A1 antibodies were used for fluorescent staining. As can be seen from the results, the release and exposure of calreticulin and Annexin A1 were significantly enhanced in the combination group compared to the single drug group (Figure 7).

[0176] Test conclusion: IN10018 can significantly enhance the cytotoxicity of Cobimetinib on melanoma cell A375. This effect is related to the enhancement of endoplasmic reticulum stress. In the experiment of detecting ICD markers, it was found that IN10018 can significantly enhance the exposure and release of DAMPs markers Calreticulin and Annexin A1, indicating that IN10018 can effectively enhance the immunogenic cell death of melanoma cells.

[0177] Example 3: In vivo antitumor efficacy study of cobimetinib in colon cancer CT-26 cell BALB / c mouse subcutaneous allograft tumor model Test materials: Mice: 6-8 week-old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experiments were started after the animals were adapted to the experimental environment after purchase. The animals were kept in IVC (individually ventilated system) cages in an SPF breeding room (6 per cage). All cages, bedding and drinking water should be sterilized before use. All experimenters should wear protective clothing and latex gloves when operating in the breeding room. The animal information card of each cage should indicate the number of animals in the cage, sex, breed, date and time of receipt, administration scheme, experiment number, group and start date of the experiment. Cages, food and drinking water were changed twice a week. The breeding environment and light exposure conditions are as follows:

[0178] Temperature: 20-26oC, Humidity: 40-70%, Light exposure cycle: 12 hours light exposure, 12 hours no light exposure Cage: Made of polycarbonate, with a volume of 300 mm x 180 mm x 150 mm. Corn cobs were used as bedding and were changed twice a week.

[0179] Food: Experimental animals will have access to ad libitum (radiation-sterilized, dry particulate food) throughout the entire experimental phase.

[0180] Drinking water: Experimental animals were provided with sterile water ad libitum.

[0181] Cage Labels: Animal information cards in each cage should indicate the number of animals in the cage, sex, breed, date and time of receipt, dosing scheme, study number, group, and start date of the study.

[0182] Animal labeling: Laboratory animals are labeled with ear tags.

[0183] The compound information is shown in Table 1. Compound Information [Table 1]

[0184] Colon cancer cells CT-26 (originating from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60043) are maintained for passage by Yingshi Biotechnology (Nanjing) Co., Ltd. The cells were cultured in monolayer in vitro, and the culture conditions were RPMI-1640 medium supplemented with 10% fetal bovine serum, cultured at 37℃ in a 5% CO2 incubator. The cells were passaged 2-3 times a week with regular digestion treatment using pancreatin-EDTA. When the cells were in the exponential growth phase and reached 80-90% saturation, the cells were harvested, counted, and then inoculated.

[0185] Cell inoculation and grouping 3×10 5 A 0.1 mL cell suspension containing 10 cells was inoculated subcutaneously into the right hindquarters of each mouse. The tumor volume was ∼58 mm. 3 On the 10th day after inoculation, the mice were randomly divided into groups based on tumor volume and administered the drugs. The group information is shown in Table 2. Administration scheme of test substance to CT-26 mouse xenograft tumor model [Table 2] Note: 1. Number of mice in each group 2. Dosage volume: 10mL / kg based on mouse body weight. When the body weight loss exceeds 15%, the administration to the animals will be stopped, and when the body weight is restored to 10%, the administration will be resumed.

[0186] Details of the preparation of test substances are shown in Table 3. Preparation of test substances [Table 3]

[0187] Daily observation of laboratory animals The establishment of this experimental scheme and any modifications were both evaluated and approved by the Yunqiao Biological IACUC. The use and care of experimental animals were carried out in accordance with AAALAC regulations. The health and mortality of the animals were monitored daily, and regular inspections included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake (visual observation only), weight change, external signs, or other abnormal conditions. The number of deaths and side effects of animals in the groups were recorded based on the number of animals in each group.

[0188] Testing stopped The animal's health condition continues to deteriorate or the tumor volume exceeds 2500mm 3 If the weight of the animal exceeds 100 kg / m2, or if the animal is in severe disease or pain, the animal should be euthanized. If the animal is found to be obviously thin, with a weight loss of more than 20%, unable to freely access food and water, or if the average tumor volume in the control group is greater than 2500 mm3, the animal should be euthanized. 3 The animals showed clinical signs of persistent deterioration, including ruffled fur, hunched back, pale ears, nose, eyes or paws, shortness of breath, convulsions, persistent diarrhea, dehydration, slowness and vocalization.

[0189] Tumor Measurements and Laboratory Indices Tumor diameters were measured with a caliper three times a week. The formula for calculating tumor volume was V = 0.5 × a × b 2 where a and b represent the long and short diameters of the tumor, respectively.

[0190] The tumor growth on the first day after grouping was used as a reference to calculate the tumor growth inhibition rate TGI (%) according to the following formula: TGI (%) = [1-(mean tumor volume of a treatment group-mean tumor volume at the start of treatment of the treatment group) / (mean tumor volume of the solvent control group-mean tumor volume at the start of treatment of the solvent control group)] x 100%.

[0191] After 2 weeks of administration, the animals in groups G1, G2, and G3 were euthanized due to the large tumor volume, and the remaining groups were administered and the survival rate of the animals was monitored. The animals died naturally or the tumor volume reached 2500 mm3. 3The animals were considered to have died when the dose exceeded this limit, and the survival rate and survival period of the animals after administration were calculated based on this.

[0192] statistical analysis Statistical analysis was performed using Prism Graphpad software based on tumor volume and tumor weight at the end of the study. Comparisons between multiple groups were analyzed using Two-way ANOVA test, and P<0.05 was considered to be significant.

[0193] The survival rates of the animals were analyzed using the Gehan-Breslow-Wilcoxon test, and P<0.05 was considered to indicate significant differences.

[0194] Test Results: In vivo efficacy of the test substances Cobimetinib and / or PDL1 antibody in combination with IN10018 in a CT-26 mouse colon cancer cell line subcutaneous homograft tumor model in BALB / c mice. After inoculation of the cells, tumor growth was observed daily, and on the 10th day after inoculation, the tumors were divided into groups based on tumor volume. The average tumor volume was approximately 58 mm. 3 According to the tumor burden, the mice in the control group and each single drug group (except for the Cobimetinib 5mpk group) were euthanized on the 25th day after inoculation, i.e., on the 15th day after group administration, and the survival time of the animals in each remaining group was continuously observed. When the survival rate of the last group fell below 50%, all the experiments were terminated.

[0195] On the 15th day after group administration, the tumor volume in the control group was 3007.0 ± 799.4 mm 3 The tumor volumes in each treatment group, IN10018 (12.5 mg / kg) single agent, PDL1 antibody (5 mg / kg), Cobimetinib (7.5 mg / kg) single agent, Cobimetinib + IN10018 (7.5 + 12.5 mg / kg), Cobimetinib + PDL1 antibody (7.5 + 5 mg / kg), and Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg), were 2418.8 ± 436.5 mm, respectively. 3 , 2614.2±568.4mm3 , 1261.3±565.4mm 3 , 1016.5±271.3mm 3 , 1196.9±411.5mm 3 and 686.3±292.9mm 3 ,Details are shown in Table 4. When comparing the pooled tumor volumes with the control group, the tumor inhibition rates (TGI) for the treatment groups IN10018 (12.5mpk) single agent, PDL1 antibody (5mg / kg), Cobimetinib (7.5mg / kg) single agent, Cobimetinib+IN10018 (7.5+12.5mg / kg), Cobimetinib+PDL1 antibody (7.5+5mg / kg) and Cobimetinib+IN10018+PDL1 antibody (7.5+12.5+5mg / kg) were 19.9% ​​(p=0.0002), 13.3% (p=0.0241), 59.2% (p<0.0001), 67.5% (p<0.0001), 61.4% (p<0.0001) and 78.7% (p<0.0001), respectively. The tumor volumes were combined and compared with the Cobimetinib+IN10018+PDL1 antibody (7.5+12.5+5mg / kg) group for statistical analysis, and the P values ​​of the control group, IN10018 (12.5mg / kg) single agent, PDL1 antibody (5mg / kg), Cobimetinib (7.5mpk) single agent, Cobimetinib+IN10018 (7.5+12.5mg / kg) and Cobimetinib+PDL1 antibody (7.5+5mg / kg) groups were p<0.0001, p<0.0001, p=0.0002, p=0.0805 and p=0.0015, respectively, as shown in Table 4. The tumor volumes of each dose group at different time ranges are shown in Figure 8.

[0196] Evaluation of the tumor-inhibiting effect of the test substance on a BALB / c mouse tumor model in which mouse colon cancer CT-26 cells were transplanted (based on data on the 15th day after group administration) [Table 4] Note: 1. Calculated based on the number of days after group administration. Data are mean ± SEM. 2. Compared with the control group, *:p<0.05, ****:p<0.0001, Two-way ANOVA. 3. Compared to the cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg) group, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, Two-way ANOVA.

[0197] After 15 days of administration, the tumor volumes were too large, so the control group, the IN10018 (12.5 mg / kg) single drug group, and the PDL1 antibody (5 mg / kg) group were euthanized, and administration was stopped for the remaining Cobimetinib (7.5 mg / kg) single drug group, Cobimetinib + IN10018 (7.5 + 12.5 mg / kg), Cobimetinib + PDL1 antibody (7.5 + 5 mg / kg), and Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg) groups. The tumor volumes were observed and recorded, and when the tumor volume reached 2500 mm 3Any situation exceeding 10 days or natural death could be recorded as animal death, and changes in survival rate were observed until the survival rate of the group with the highest survival rate fell to 50% or less. On the 31st day after administration was stopped, the group with the highest survival rate was the Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg) group, with median survival time and survival rate of 30 days and 16.7%, respectively, and the remaining Cobimetinib (7.5 mg / kg) single drug, Cobimetinib + IN10018 (7.5 + 12.5 mg / kg) and Cobimetinib + PDL1 antibody (7.5 + 5 mg / kg) groups had median survival time and survival rate of 22 days and 16.7%, 24 days and 0%, and 22 days and 0%, respectively. Statistical analysis was performed using Gehan-Breslow-Wilcoxon test, and the P values ​​of the Cobimetinib (7.5 mg / kg) single agent, Cobimetinib + IN10018 (7.5 + 12.5 mg / kg) and Cobimetinib + PDL1 antibody (7.5 + 5 mg / kg) groups compared to the Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg) group were p = 0.0434, p = 0.0137 and p = 0.0029, respectively. Details are shown in Table 5. The survival rate of each dose group at different time ranges is shown in Figure 9.

[0198] Evaluation of tumor survival rate in BALB / c mouse tumor model in which the test substance is administered to mice containing mouse colon cancer CT-26 cells (based on data on the 31st day after group administration) [Table 5] Note: 1. Based on the number of days after group administration, the survival rate was calculated as the number of surviving animals / 6*100. 2. Compared with the Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg) group, *: p < 0.05, **: p < 0.01, Gehan-Breslow-Wilcoxon test

[0199] According to the administration scheme, the experiment was carried out, and during the course of the experiment, the animals' activities such as food and water intake were observed daily, and the animal's body weight was recorded three times a week, and the animal's body weight curve is shown in Figure 9. Throughout the entire administration cycle, the animals in each group did not lose weight obviously and were in good condition.

[0200] Test conclusion: Compared with the blank control group, each treatment group shows obvious tumor growth inhibition effect, and each has statistical difference compared with the control group. The average tumor volume of the triple combination group of Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg) is the smallest, and each group except for the Cobimetinib + IN10018 (7.5 + 12.5 mg / kg) group has statistical difference compared with the triple combination group, and the Cobimetinib + IN10018 (7.5 + 12.5 mg / kg) group has no statistical difference compared with the triple combination group of Cobimetinib + IN10018 + PDL1 antibody (7.5 + 12.5 + 5 mg / kg), but the average tumor volume is larger than the triple combination group. The above data explains that the triple combination group has higher efficacy than the single drug group and the double drug combination group.

[0201] According to the survival data, the survival rate and median survival time of the Cobimetinib+IN10018+PDL1 antibody (7.5+12.5+5mg / kg) triple combination group are better than those of the Cobimetinib (7.5mg / kg) single drug, Cobimetinib+IN10018 (7.5+12.5mg / kg) and Cobimetinib+PDL1 antibody (7.5+5mg / kg) groups, and there is a statistical difference between the three groups. The above data demonstrate that the Cobimetinib+IN10018+PDL1 antibody (7.5+12.5+5mg / kg) triple combination group can significantly reduce the deaths of animals.

[0202] The animals in each group did not obviously lose weight and were in good condition, which explains that the animals tolerated the administration of Cobimetinib+IN10018+PDL1 antibody (7.5+12.5+5 mg / kg).

[0203] Example 4: In vitro efficacy study of IN10018 and HL085 in colon cancer CT-26 cell line Test materials: 1) Drugs used in this experiment HL-085 was provided by Shanghai Kezhou Drug Research and Development Co., Ltd.

[0204] 2) Antibodies used in this experiment Recombinant Alexa Fluor® 647 Fluorescent Anti-Calreticulin Antibody (Abcam, Cat No: ab196159, Lot No: CR33676773). Annexin-Modulation Assay Kit (Beyotime, Cat No: Beyotime, Lot No.: 021921210811).

[0205] Testing Method: CT26 cells are cultured using RPMI 1640 (Shanghai Yuanpei, Cat No: L210KJ, Lot No: F210916) + 10% FBS (Gibco, Cat No: 10099-141c, Lot No: 2158737cp), and when the cells are in good condition, the culture plate is placed in a 24-well plate. After 24 hours of spreading the cells, four groups are set up, the first group is the control group, the medium is added, the second group is IN10018, the concentration is 5 μM, the third group is HL-085, the concentration is 1 μM, and the fourth group is the combination of IN10018 (5 μM) and HL-085 (1 μM). The drugs are mixed and cultured at 37 °C in a 5% CO2 incubator for 48 hours.

[0206] Experimental Results After the drug was allowed to act for 48 hours, the cells were observed under a microscope and photographed to confirm the cytotoxic effect. Then, the cells were collected for flow analysis. The cells were washed twice with flow buffer (PBS + 2% FBS) and 0.5 μl of AF647, and anti-calreticulin antibody (abcam) was added to each well and mixed. Incubated at 4°C under light-resistant conditions, and after 20 min incubation, flow buffer was added, and 195 μl of Annexin-V-FITC conjugate was added using Annexin staining kit (Beyotime), sprayed and mixed with the cells, 5 μl of Annexin-V-FITC antibody was added, mixed gently, and finally 10 μl of PI dye was added and mixed, incubated at room temperature in the dark for 15 min, and the sample was transferred to a flow cytometer for signal detection.

[0207] Observation of cells under a microscope showed that the HL-085 single drug group and the dual drug combination group had poor cell condition, while the dual drug combination group had the worst cell condition and more cell death. The HL-085 single drug group and the dual drug combination group had cytotoxicity, with the dual drug combination group showing the most significant cytotoxicity, while the control group and IN10018 group had good cell condition, as shown in Figure 11. Analysis of the flow cytometer showed that the CRT positive rate and Annexin-V positive rate of the dual drug combination group were significantly better than those of the single drug group and the control group, as shown in Figures 12a and 12b.

[0208] Example 5: In vivo antitumor efficacy study of HL085 in colon cancer CT-26 cells BALB / c mouse subcutaneous allograft tumor model Test materials: Mice: 6-8 week-old female BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The experiments were started after the animals were adapted to the experimental environment after purchase. The animals were kept in IVC (individually ventilated system) cages in an SPF breeding room (5 per cage). All cages, bedding and drinking water should be sterilized before use. All experimenters should wear protective clothing and latex gloves when operating in the breeding room. The animal information card of each cage should indicate the number of animals in the cage, sex, breed, date and time of receipt, administration scheme, experiment number, group and start date of the experiment. Cages, food and drinking water were changed twice a week. The breeding environment and light exposure conditions are as follows:

[0209] Temperature: 20~26℃, humidity: 40~70%, light exposure cycle: 12 hours on, 12 hours off.

[0210] Cage: Made of polycarbonate, with a volume of 300 mm x 180 mm x 150 mm. Corn cobs were used as bedding and were changed twice a week.

[0211] Food: Experimental animals will have access to ad libitum (radiation-sterilized, dry particulate food) throughout the entire experimental phase.

[0212] Drinking water: Experimental animals were provided with sterile water ad libitum.

[0213] Cage Labels: Animal information cards in each cage should indicate the number of animals in the cage, sex, breed, date and time of receipt, dosing scheme, study number, group, and start date of the study.

[0214] Animal labeling: Laboratory animals are labeled with ear tags.

[0215] Compound information is shown in Table 6. Compound Information [Table 6]

[0216] Colon cancer cells CT-26 (originating from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60043) are maintained for passage by Yingshi Biotechnology (Nanjing) Co., Ltd. The cells were cultured in monolayer in vitro, and the culture conditions were RPMI-1640 medium supplemented with 10% fetal bovine serum, cultured at 37℃ in a 5% CO2 incubator. The cells were passaged 2-3 times a week with regular digestion treatment using pancreatin-EDTA. When the cells were in the exponential growth phase and reached 80-90% saturation, the cells were harvested, counted, and then inoculated.

[0217] Cell inoculation and grouping 0.3×10 5 A 0.1 mL cell suspension containing 10 cells was subcutaneously inoculated into the right hindquarters of each mouse. The tumor volume was ∼59 mm. 3 When the tumor volume reached 12 days after inoculation, the mice were randomly divided into groups based on the tumor volume and administered the drugs. The group information is shown in Table 7. Administration scheme of test substance to CT-26 mouse xenograft tumor model [Table 7] Note: 1. Number of mice in each group 2. Dosage volume: 10mL / kg based on mouse body weight. When the body weight loss exceeds 15%, the administration to the animals will be stopped, and when the body weight is restored to 10%, the administration will be resumed.

[0218] Details of the preparation of test articles are shown in Table 8. Preparation of test substances [Table 8]

[0219] Daily observation of laboratory animals The establishment of this experimental scheme and any modifications were both evaluated and approved by the Yunqiao Biological IACUC. The use and care of experimental animals were carried out in accordance with AAALAC regulations. The health and mortality of the animals were monitored daily, and regular inspections included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake (visual observation only), weight change, external signs, or other abnormal conditions. The number of deaths and side effects of animals in the groups were recorded based on the number of animals in each group.

[0220] Testing stopped The animal's health condition continues to deteriorate or the tumor volume exceeds 2500mm 3 If the weight of the animal exceeds 100 kg / m2, or if the animal is in severe disease or pain, the animal should be euthanized. If the animal is found to be obviously thin, with a weight loss of more than 20%, unable to freely access food and water, or if the average tumor volume in the control group is greater than 2500 mm3, the animal should be euthanized. 3 The animals showed clinical signs of persistent deterioration, including ruffled fur, hunched back, pale ears, nose, eyes or paws, shortness of breath, convulsions, persistent diarrhea, dehydration, slowness and vocalization.

[0221] Tumor Measurements and Laboratory Indices Tumor diameters were measured with a caliper three times a week. The formula for calculating tumor volume was V = 0.5 × a × b 2 where a and b represent the long and short diameters of the tumor, respectively.

[0222] The tumor growth on the first day after grouping was used as a reference to calculate the tumor growth inhibition rate TGI (%) according to the following formula: TGI (%) = [1-(mean tumor volume of a treatment group-mean tumor volume at the start of treatment of the treatment group) / (mean tumor volume of the solvent control group-mean tumor volume at the start of treatment of the solvent control group)] x 100%.

[0223] statistical analysis Statistical analysis was performed using Prism Graphpad software based on tumor volume and tumor weight at the end of the study. Comparisons between multiple groups were analyzed using Two-way ANOVA test. P<0.05 was considered to indicate significant differences.

[0224] Experimental Results In vivo efficacy of the test substances HL085 and / or IN10018 in combination with PDL1 antibody in a CT-26 mouse colon cancer cell line subcutaneous homograft tumor model in BALB / c mice. After inoculation of the cells, tumor growth was observed daily, and on the 12th day after inoculation, the tumors were divided into groups based on tumor volume. The average tumor volume was approximately 59 mm. 3 Due to tumor burden, the control group was euthanized on the 28th day after inoculation, i.e., the 16th day after group administration, and all the remaining groups of mice continued to be administered and were euthanized on the 33rd day after inoculation, i.e., the 21st day after group administration, and all the experiments were terminated.

[0225] On the 16th day after group administration, the tumor volume in the control group was 3330.1 ± 1587.5 mm 3 The tumor volumes in the PDL1 antibody (10 mg / kg), HL085 + IN10018 (1 + 25 mg / kg), and HL085 + IN10018 + PDL1 antibody (1 + 25 + 10 mg / kg) treatment groups were 1723.0 ± 1122.8 mm 3 , 522.3±327.3mm 3 and 228.4±102.2mm 3 The details are shown in Table 9. The tumor volumes were integrated and compared with the control group, and the tumor inhibition rates TGI of the PDL1 antibody (10 mg / kg), HL085+IN10018 (1+25 mg / kg) and HL085+IN10018+PDL1 antibody (1+25+10 mg / kg) groups were 49.2% (p<0.0001), 85.8% (p<0.0001) and 94.8% (p<0.0001), respectively. The details are shown in Table 9. The tumor volumes of each dose group at different time ranges are shown in Figure 13.

[0226] After 16 days of administration, the control group was euthanized due to the large tumor volume, and the remaining PDL1 antibody (10 mg / kg), HL085 + IN10018 (1 + 25 mg / kg), and HL085 + IN10018 + PDL1 antibody (1 + 25 + 10 mg / kg) groups were administered the same treatment. The tumor volumes were observed and recorded until the 21st day after administration, and the tumor volumes were 3038.0 ± 1798.7 mm, respectively. 3 , 737.0±658.5mm 3 and 360.9±161.3mm 3 The tumor volumes were combined and compared with the HL085+IN10018+PDL1 antibody (1+25+10 mg / kg) group for statistical analysis, and the P values ​​of PDL1 antibody (10 mg / kg) and HL085+IN10018 (1+25 mg / kg) were p<0.0001 and p=0.0461, respectively, with details shown in Table 9. The tumor volumes of each dose group at different time ranges are shown in Figure 13.

[0227] Evaluation of the tumor-inhibiting effect of the test substance on a BALB / c mouse tumor model in which mouse colon cancer CT-26 cells were transplanted (based on data on the 16th and 21st days after group administration) [Table 9] Note: 1. Calculated based on the number of days after group administration. Data are mean ± SEM. 2. Compared with the control group, ****: p<0.0001, Two-way ANOVA. 3. Compared to the HL085+IN10018+PDL1 antibody (1+25+10 mg / kg) group, *: p<0.05, ****: p<0.0001, Two-way ANOVA.

[0228] According to the administration scheme, the experiment was carried out, and during the course of the experiment, the animals' activities such as food and water intake were observed daily, and the animal's body weight was recorded three times a week, and the animal's body weight curve is shown in Figure 14. Throughout the entire administration cycle, the animals in each group did not lose weight obviously and were in good condition.

[0229] conclusion Compared with the blank control group, the PD-L1 antibody (10mg / kg), HL085+IN10018 (1+25mg / kg) and HL085+IN10018+PDL1 antibody (1+25+10mg / kg) groups all showed obvious tumor growth inhibition effects, and all had statistical differences compared with the control group. Taking into account the entire administration cycle, the HL085+IN10018+PDL1 antibody (1+25+10mg / kg) group had much smaller tumor volumes than the PD-L1 antibody (10mg / kg) group and the HL085+IN10018 (1+25mg / kg) group, and on the last day, i.e., the 21st day after group administration, there was a statistical difference with either of these two groups, and the tumor growth inhibition effect was higher than that of the PD-L1 antibody (10mg / kg) single drug group and the HL085+IN10018 (1+25mg / kg) double drug combination group.

[0230] Example 6: Study of target induction of in vitro immunogenic cell death of mouse breast cancer 4T1 cells by the detection target compounds Cobimetinib and AMP-945 Test materials: 1) Drugs used in this experiment Cobimetinib is provided by MCE, Lot No:07394.

[0231] AMP-945 is provided by MCE, Lot No: 143253.

[0232] 2) Antibodies used in this experiment Recombinant Alexa Fluor® 647 Fluorescent Anti-Calreticulin Antibody (Abcam, Cat No: ab196159, Lot No: CR33676773). Annexin V-Apoptosis Detection Kit (Beyotime, Cat No: C1062L, Lot No: 122221220706).

[0233] Testing Method: 4T1 cells (originating from Nanjing Kebai Biotechnology Co., Ltd., Product No.: CBP60352) are cultured using RPMI 1640 (Shanghai Yuanpei, Cat No: L210KJ, Lot No: F210916) + 10% FBS (Gibco, Cat No: 10099-141c, Lot No: 2158737cp), with the culture conditions being 37°C and 5% CO2. They are passaged and processed by regular digestion using trypsin 2-3 times a week. When the cells are in exponential growth phase, the cells are harvested and plated when they are 80-90% confluent. After digesting 4T1 cells with trypsin, the cells are harvested and counted, and based on the counting results, the cells are diluted with RPMI1640+10%FBS to a dilution concentration of 50,000 cells per milliliter, and then plated into a 12-well cell culture plate, with 2 ml of cell suspension in each well, i.e., 100,000 cells. After plating, the cells are placed in a 37℃, 5% CO2 incubator for culture. After 24 hours of spreading the cells, six groups were set up, the first group was the control group, the medium was added, the second group was AMP-945 at a concentration of 3 μM, the third group was AMP-945 at a concentration of 6 μM, the fourth group was Cobimetinib (MCE) at a concentration of 3 μM, the fifth group was AMP-945 (3 μM) combined with Cobimetinib (3 μM), and the sixth group was AMP-945 (6 μM) combined with Cobimetinib (3 μM). The drugs were mixed and incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0234] Experimental Results After 48 hours of drug exposure, cells were collected for flow analysis. The cells were washed twice with running buffer (PBS + 2% FBS) and 0.5 μl of AF647, anti-calreticulin antibody (abcam) was added to each well and mixed. Incubated at 4°C under light-resistant conditions, incubated for 20 min, then running buffer was added, and 195 μl of Annexin-V-FITC conjugate was added using Annexin staining kit (Beyotime), sprayed and mixed with the cells, 5 μl of Annexin-V-FITC antibody was added, mixed gently, and finally 10 μl of PI dye was added and mixed, incubated at room temperature in the dark for 15 min, and the samples were transferred to a flow cytometer for signal detection.

[0235] The flow cytometer analysis results showed that the CRT positive rate and Annexin-V positive rate in the dual drug combination group were significantly superior to those in the single drug group and control group, as shown in Figures 15a and 15b.

[0236] Example 7: Study of target induction of in vitro immunogenic cell death of mouse colon cancer CT26 cells by the detection target compounds Cobimetinib and AMP-945 Test materials: 1) Drugs used in this experiment Cobimetinib is provided by MCE, Lot No:07394.

[0237] AMP-945 is provided by MCE, Lot No: 143253.

[0238] 2) Antibodies used in this experiment Recombinant Alexa Fluor® 647 Fluorescent Anti-Calreticulin Antibody (Abcam, Cat No: ab196159, Lot No: CR33676773). Annexin V-Apoptosis Detection Kit (Beyotime, Cat No: C1062L, Lot No: 122221220706).

[0239] Testing Method: CT26 cells (originating from Nanjing Kebai Biotechnology Co., Ltd., Product No.: CBP60043) are cultured using RPMI 1640 (Shanghai Yuanpei, Cat No: L210KJ, Lot No: F210916) + 10% FBS (Gibco, Cat No: 10099-141c, Lot No: 2158737cp), with the culture conditions being 37°C and 5% CO2. They are passaged and processed by regular digestion using trypsin 2-3 times a week. When the cells are in exponential growth phase, the cells are harvested and plated when they are 80-90% confluent. After digesting CT26 cells with trypsin, the cells are harvested and counted, and based on the counting results, the cells are diluted with RPMI1640+10%FBS to a dilution concentration of 50,000 cells per milliliter, and then plated into a 12-well cell culture plate, with 2 ml of cell suspension in each well, i.e., 100,000 cells. After plating, the cells are placed in a 37℃, 5% CO2 incubator for culture. After 24 hours of spreading the cells, six groups were set up, the first group was the control group, the medium was added, the second group was AMP-945 at a concentration of 0.75 μM, the third group was AMP-945 at a concentration of 1.5 μM, the fourth group was Cobimetinib (MCE) at a concentration of 3 μM, the fifth group was AMP-945 (0.75 μM) combined with Cobimetinib (3 μM), and the sixth group was AMP-945 (1.5 μM) combined with Cobimetinib (3 μM). The drugs were mixed and incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0240] Experimental Results After 48 hours of drug exposure, cells were collected for flow analysis. The cells were washed twice with running buffer (PBS + 2% FBS) and 0.5 μl of AF647, anti-calreticulin antibody (abcam) was added to each well and mixed. Incubated at 4°C under light-resistant conditions, incubated for 20 min, then running buffer was added, and 195 μl of Annexin-V-FITC conjugate was added using Annexin staining kit (Beyotime), sprayed and mixed with the cells, 5 μl of Annexin-V-FITC antibody was added, mixed gently, and finally 10 μl of PI dye was added and mixed, incubated at room temperature in the dark for 15 min, and the samples were transferred to a flow cytometer for signal detection.

[0241] The flow cytometer analysis results showed that the CRT positive rate and Annexin-V positive rate in the dual drug combination group were significantly superior to those in the single drug group and control group, as shown in Figures 16a and 16b.

[0242] All references mentioned in this application are incorporated herein by reference in their entirety as if each reference was individually cited. After reading the disclosure of this application, a person skilled in the art will understand that various changes or modifications can be made to the present invention, and the equivalents thereof are also included in the scope defined by the appended claims of this application.

Claims

1. 2. Use of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor in the manufacture of a medicament for treating a tumor in a subject, wherein the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

2. A pharmaceutical combination product of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor for treating a tumor in a subject, wherein the FAK inhibitor, the MEK inhibitor and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

3. A method for treating a tumor, comprising administering to a subject in need thereof therapeutically effective amounts of a FAK inhibitor, a MEK inhibitor and an immune checkpoint inhibitor, either simultaneously or sequentially.

4. 4. The use, pharmaceutical combination product or method according to any one of claims 1 to 3, wherein said FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of said IN10018 is as follows: 【Chemistry 1】

5. The use, pharmaceutical combination product or method according to any one of claims 1 to 4, wherein said MEK inhibitor is Trametinib, Cobimetinib, Binimetinib, Selumetinib, PD-325901, TAK-733, HL-085, E-6201, Pimasertib, Renametinib, FCN-159 or a pharma-ceutically acceptable salt thereof, preferably Cobimetinib, HL-085 or a pharma-ceutically acceptable salt thereof.

6. The use, pharmaceutical combination product or method according to any one of claims 1 to 5, wherein said immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor or a TIGIT inhibitor.

7. The use, pharmaceutical combination product or method according to any one of claims 1 to 6, wherein said FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, said MEK inhibitor is Cobimetinib, HL-085, E-6201, Pimasertib, Renametinib, FCN-159 or a pharma- ceutically acceptable salt thereof, and said immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular Atezolizumab.

8. The tumors include Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colon cancer, rectal cancer, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction adenocarcinoma, thymoma, pancreatic cancer, endometrial cancer, cervical cancer, and the like.

8. The use, pharmaceutical combination product or method according to any one of claims 1 to 7, wherein the tumor is cancer, malignant melanoma, uveal melanoma, skin cancer, germ cell cancer, nasopharyngeal cancer, pharyngeal cancer or laryngeal cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, thyroid cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or bile duct cancer, and further wherein the tumor is acute myeloid leukemia, malignant melanoma, uveal melanoma, pancreatic cancer, ovarian cancer, colon cancer or colorectal cancer.

9. (a) a FAK inhibitor; and (b) a MEK inhibitor; and (c) an immune checkpoint inhibitor,

10. 10. The kit or composition of claim 9, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of IN10018 is as follows: 【Chemistry 2】

11. The kit or composition according to claim 9, wherein the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733, HL-085, E-6201, pimasertib, refametinib, FCN-159 or a pharma- ceutically acceptable salt thereof, preferably cobimetinib, HL-085 or a pharma- ceutically acceptable salt thereof.

12. The kit or composition according to any one of claims 9 to 11, 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.

13. The kit or composition according to any one of claims 9 to 12, wherein the FAK inhibitor is IN10018 or a pharma- ceutically acceptable salt thereof, the MEK inhibitor is cobimetinib, HL-085 or a pharma- ceutically 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 atezolizumab.

14. The kit or composition according to any one of claims 9 to 13, for use as a medicament.

15. 23. Use of a FAK inhibitor and a MEK inhibitor in the manufacture of a medicament for treating a tumor, wherein the FAK inhibitor enhances immunogenic cell death induced by the MEK inhibitor.

16. FAK inhibitors enhance immunogenic cell death induced by MEK inhibitors in the treatment of tumors.

17. A method for treating a tumor, comprising administering therapeutically effective amounts of a FAK inhibitor and a MEK inhibitor simultaneously or sequentially to a subject in need thereof, wherein the FAK inhibitor enhances immunogenic cell death induced by the MEK inhibitor.

18. The use, FAK inhibitor or method according to any one of claims 15 to 17, wherein said MEK inhibitor is Trametinib, Cobimetinib, Binimetinib, Selumetinib, PD-325901, TAK-733, HL-085, E-6201, Pimasertib, Renametinib, FCN-159 or a pharma-ceutically acceptable salt thereof, preferably Cobimetinib, HL-085 or a pharma-ceutically acceptable salt thereof.

19. 19. The use, FAK inhibitor or method according to any one of claims 15 to 18, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharma- ceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharma- ceutically acceptable salt thereof, more preferably IN10018 or a pharma- ceutically acceptable salt thereof, in particular IN10018 tartrate, and the structure of IN10018 is as follows: 【Chemistry 3】

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