Pharmaceutical composition of FAK inhibitor and substance for inducing immunogenic cell death, and use thereof

Abstract

The invention also discloses application of the composition of the FAK inhibitor, the substance for inducing immunogen cell death and the immune checkpoint inhibitor in preparation of medicines for treating tumors. The substance for inducing death of the immunogen cells is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor. And the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small-molecule inhibitor or a TIGIT antibody. A pharmaceutical combination of an FAK inhibitor, an immunogenic cell death-inducing substance and an immune checkpoint inhibitor for use in the treatment of tumors. A FAK inhibitor for use in the treatment of tumors to enhance immunogenic cell death induced by a substance that induces immunogenic cell death.

Description

Pharmaceutical combination and use of FAK inhibitor and substance inducing immunogenic cell death

[0001] This application claims priority to Chinese patent application No. 202211211068.5 filed on September 30, 2022 and Chinese patent application No. 202311207394.3 filed on September 19, 2023, and the contents of the above-mentioned Chinese patent application disclosures are hereby incorporated by reference in their entirety as part of this application. Technical Field

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

[0003] Cancer is the second leading threat to human health. Immunogenic cell death (ICD) is based on the synergistic effects of programmed cell death. When cancer cells are exposed to chemotherapy or targeted therapy drugs, they may activate internal stress signals, including endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) stress. Under the influence of stress signals, cells first attempt to repair the stress. If the damage caused by stress exceeds the ability to repair, the cells initiate programmed cell death. This process is often accompanied by the release of a class of molecules called damage-associated molecular patterns (DAMPs). These DAMPs are specifically recognized by pattern recognition receptors on antigen-presenting cells (APCs) in the body, inducing APC maturation, differentiation, and activation. These DAMPs are then progressively presented to immune cells, such as effector T cells, thereby enabling immune cells to develop antigen memory. Upon encountering tumor cells of the same origin, these immune cells will specifically recognize and kill the tumor cells. The new tumor-specific immune response initiated by ICD can increase sensitivity to immune checkpoint inhibitors (ICIs), thereby enhancing the effects of immune checkpoint inhibitors and producing anti-tumor responses with long-lasting immune memory.

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

[0005] Currently, the effectiveness of substances that induce immunogenic cell death in clinical practice needs to be improved. Therefore, there is still a need to find a way to improve the efficacy of single-agent substances that induce immunogenic cell death and further overcome the problem of drug resistance.

[0006] Summary of the Invention

[0007] In one aspect, the present disclosure provides the use of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor in the preparation of a medicament for treating tumors in a subject, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0008] In another aspect, the present disclosure provides a pharmaceutical combination product of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor for treating tumors in a subject, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0009] In another aspect, the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor to a subject in need thereof, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0010] In another aspect, the present disclosure provides a kit or a pharmaceutically acceptable composition comprising: (a) a FAK inhibitor; (b) a substance that induces immunogenic cell death; and (c) an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0011] Another aspect of the present disclosure provides the use of a FAK inhibitor and a substance that induces immunogenic cell death in the preparation of a drug for treating tumors, wherein the FAK inhibitor is used to enhance the immunogenic cell death induced by the substance that induces immunogenic cell death, and the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0012] In another aspect, the present disclosure provides a FAK inhibitor for use in the treatment of tumors to enhance immunogenic cell death induced by a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0013] Yet another aspect of the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of a FAK inhibitor and a substance that induces immunogenic cell death to a subject in need thereof, wherein the FAK inhibitor is used to enhance the immunogenic cell death induced by the substance that induces immunogenic cell death, and the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0014] In another aspect, the present disclosure provides the use of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor in the preparation of a medicament for the combined treatment of tumors, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0015] In another aspect, the present disclosure provides the use of a FAK inhibitor in the preparation of a combination drug for treating tumors with a substance that induces immunogenic cell death and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0016] In another aspect, the present disclosure provides the use of a substance that induces immunogenic cell death in the preparation of a combination drug for treating tumors with a FAK inhibitor and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0017] In another aspect, the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a combination drug for treating tumors with a FAK inhibitor and a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0018] In another aspect, the present disclosure provides the use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with a substance that induces immunogenic cell death and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0019] In another aspect, the present disclosure provides the use of a substance that induces immunogenic cell death in the preparation of a drug for treating tumors in combination with a FAK inhibitor and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0020] In another aspect, the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor and a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0021] In another aspect, the present disclosure provides a kit comprising: a FAK inhibitor; and instructions, which indicate that the FAK inhibitor can be used to treat tumors in combination with a substance that induces immunogenic cell death and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0022] In another aspect, the present disclosure provides a kit comprising: a substance that induces immunogenic cell death; and an instruction manual, which indicates that the substance that induces immunogenic cell death can be used in combination with a FAK inhibitor and an immune checkpoint inhibitor to treat tumors, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0023] In another aspect, the present disclosure provides a kit comprising: an immune checkpoint inhibitor; and instructions, which indicate that the immune checkpoint inhibitor can be used to treat tumors in combination with a FAK inhibitor and a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0024] Another aspect of the present disclosure provides a method for treating tumors, comprising administering to a subject in need thereof a therapeutically effective amount of a FAK inhibitor and a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0025] In another aspect, the present disclosure provides a pharmaceutical combination product of a FAK inhibitor and a substance that induces immunogenic cell death, which is used to treat tumors in a subject in need thereof, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0026] In another aspect, the present disclosure provides the use of a FAK inhibitor and a substance that induces immunogenic cell death in the preparation of a combination drug for treating tumors, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0027] In another aspect, the present disclosure provides the use of a FAK inhibitor in the preparation of a combination drug for treating tumors with a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0028] In another aspect, the present disclosure provides the use of a substance that induces immunogenic cell death in the preparation of a combination drug for treating tumors with a FAK inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0029] In another aspect, the present disclosure provides the use of a FAK inhibitor and a substance that induces immunogenic cell death in the preparation of a drug for combined treatment of tumors, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0030] In another aspect, the present disclosure provides the use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0031] In another aspect, the present disclosure provides the use of a substance that induces immunogenic cell death in the preparation of a drug for treating tumors in combination with a FAK inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0032] In another aspect, the present disclosure provides a kit comprising: a FAK inhibitor; and instructions, which indicate that the FAK inhibitor can be used to treat tumors in combination with a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0033] In another aspect, the present disclosure provides a kit comprising: a substance that induces immunogenic cell death; and instructions, which indicate that the substance that induces immunogenic cell death can be used in combination with a FAK inhibitor to treat tumors, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0034] Optionally, the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate. The structure of IN10018 is as follows:

[0035] Defactinib, also known as defatinib, has a CAS number of 1345713-71-4; GSK2256098 has a CAS number of 1224887-10-8; PF-00562271 has a CAS number of 717907-75-0; VS-4718 has a CAS number of 1061353-68-1; APG-2449 was developed by Ascentage Pharma; and AMP945 has a CAS number of 1393653-34-3.

[0036] Optionally, the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0037] Optionally, the RNA polymerase II inhibitor is lurbinectedin, SEL-120 or a pharmaceutically acceptable salt thereof.

[0038] The CAS number of lurbinectedin is 497871-47-3, and the CAS number of SEL-120 is 1609522-33-9.

[0039] Optionally, the RNA polymerase II inhibitor is lurbinectedin.

[0040] Optionally, the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0041] Optionally, the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

[0042] The CAS number of crizotinib is 877399-52-5, SIM-0201 was developed by Simcere Pharmaceuticals, XZP-3621 was developed by Xuanzhu Biotechnology, TQ-B3139 was developed by Zhengda Tianqing, SAF-189s was jointly developed by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chongqing Fuchuang Pharmaceutical Research Co., Ltd., the CAS number of Ceritinib is 1032900-25-6, and the CAS number of Lorlatinib (PF-06463922) is 1454846-35-5 The CAS number of alectinib is 1256580-46-7, the CAS number of ensartinib is 1365267-27-1, APG-2449 was developed by Ascent Pharmaceuticals, the CAS number of brigatinib is 1197953-54-0, TQ-B3101 was developed by Zhengda Tianqing, the CAS number of entrectinib is 1108743-60-7, and the CAS number of repotrectinib is 1802220-02-5.

[0043] Optionally, the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0044] Optionally, the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0045] Optionally, the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, especially D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0046] The D-1553 was developed by Yifang Bio, ARS-3248 was developed by Araxes, GF-105 was developed by GenFleet Therapeutics, JAB-21822 was developed by Jacobs, and JDQ-443 was developed by Novartis; the CAS number of LY-3537982 is 2414198-64-2, the CAS number of Sotorasib (AMG510) is 2296729-00-3, the CAS number of Adagrasib (MRTX849) is 2326521-71-3, and the CAS number of GDC-6036 is 2417987-45-0.

[0047] Optionally, the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0048] Optionally, the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0049] Optionally, the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000 or a pharmaceutically acceptable salt thereof.

[0050] The CAS number of the MRTX1133 is 2621928-55-8, JAB-22000 was developed by Jacobs, and HRS-4642 was developed by Hengrui Medicine.

[0051] Optionally, the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0052] Optionally, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor or a TIGIT antibody.

[0053] Optionally, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Atezolizumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0054] Optionally, the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043 or RRx-001.

[0055] Optionally, the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody is Ociperlimab (Ociperlimab / BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321.

[0056] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0057] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0058] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0059] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0060] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0061] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0062] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor.

[0063] Optionally, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

[0064] Optionally, the FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0065] Optionally, the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, Liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastases from spindle cell carcinoma, anaplastic large cell lymphoma, anaplastic thyroid cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, or hematological malignancies such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML).

[0066] Optionally, the tumor is preferably breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), gastric cancer, melanoma or pancreatic cancer.

[0067] Optionally, the tumor is lung cancer, colon cancer (including colorectal cancer) or breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, and are not intended to limit the present invention.

[0069] FIG1 shows the curve of the inhibitory effect of Lurbinectedin and its combination with IN10018 on the proliferation of mouse colon cancer CT26 cells relative to drug concentration.

[0070] Figure 2 shows white light microscope pictures of mouse colon cancer CT26 cells taken after incubation with drugs for 48 hours.

[0071] FIG3 shows the percentage of CRT-positive cells after mouse colon cancer CT26 cells were incubated with drugs for 48 hours (3a) and the percentage of Annexin V-positive cells after mouse colon cancer CT26 cells were incubated with drugs for 48 hours (3b).

[0072] FIG4 shows a curve showing the inhibitory effect of MRTX1133 and its combination with IN10018 on the proliferation of mouse colon cancer CT26 cells relative to drug concentration.

[0073] FIG5 shows white light microscope pictures of mouse colon cancer CT26 cells taken after incubation with drugs for 48 hours.

[0074] FIG6 shows the percentage of CRT-positive cells after mouse colon cancer CT26 cells were incubated with drugs for 48 hours (6a) and the percentage of Annexin V-positive cells after mouse colon cancer CT26 cells were incubated with drugs for 48 hours (6b).

[0075] FIG7 shows a curve showing the inhibitory effect of MRTX1133 and its combination with IN10018 on the proliferation of mouse lung cancer KPL cells relative to drug concentration.

[0076] FIG8 shows white light microscope pictures of mouse lung cancer KPL cells taken after incubation with drugs for 48 hours.

[0077] FIG9 shows the percentage of CRT-positive cells after incubation of mouse lung cancer KPL cells with drugs for 48 hours ( 9a ) and the percentage of Annexin V-positive cells after incubation of mouse lung cancer KPL cells with drugs for 48 hours ( 9b ).

[0078] FIG10 shows the percentage of calreticulin (CRT)-positive cells (10a) and the percentage of Annexin-V-positive cells (10b) after incubation of mouse colon cancer CT26 cells with drugs for 48 hours. FIG10 shows the percentage of calreticulin (CRT)-positive cells (10a) and the percentage of Annexin-V-positive cells (10b) after incubation of mouse colon cancer CT26 cells with drugs for 48 hours.

[0079] Figure 11 shows the changes in body weight of tumor-bearing mice after administration of the test compound in a mouse colon cancer CT26-KRAS G12C homograft tumor model. Data points represent the mean body weight within the group, and error bars represent the standard error (SEM).

[0080] Figure 12 shows the relative weight change (%) of tumor-bearing mice in a mouse colon cancer CT26-KRAS G12C homograft model after administration of the test compound. Relative weight change is calculated based on the animal's weight at the start of dosing. Data points represent the mean percentage change in body weight within each group, and error bars represent the standard error (SEM).

[0081] Figure 13 shows the tumor growth curves of mice bearing the CT26-KRAS G12C colon cancer homograft model after administration of the test drug. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).

[0082] Figure 14 shows the tumor growth curve of the subcutaneous BALB / c mouse colorectal cancer CT26 cell transplant model after administration. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).

[0083] Figure 15 shows the tumor growth curve of the mouse colorectal cancer CT26 cell BABL / c mouse subcutaneous transplant tumor model after administration. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).

[0084] Figure 16 shows the body weight change curve of the BALB / c mouse subcutaneous transplant tumor model of mouse colorectal cancer CT26 cells after administration. The data points represent the mean body weight within the group, and the error bars represent the standard error (SEM).

[0085] FIG17 shows the percentage of Calreticulin (CRT)-positive cells (17a) and the percentage of Annexin-V-positive cells (17b) after incubation of mouse breast cancer 4T1 cells with drugs for 48 hours. ...

[0086] FIG18 shows the percentage of Calreticulin (CRT)-positive cells after mouse breast cancer 4T1 cells were incubated with drugs for 48 hours ( 18a ) and the percentage of Annexin-V-positive cells after mouse breast cancer 4T1 cells were incubated with drugs for 48 hours ( 18b ). DETAILED DESCRIPTION

[0087] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0088] The present invention may be implemented in other specific forms without departing from the essential attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features of any other embodiment or multiple other embodiments to produce additional embodiments. The present invention includes additional embodiments resulting from such combinations.

[0089] All publications and patents mentioned in this disclosure are hereby incorporated into the present disclosure in their entirety by reference. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0090] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0091] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0092] Except in the working examples or otherwise indicated, all numbers stating quantitative properties such as dosage in the specification and claims should be understood to be modified by the term "about" in all cases. It should also be understood that any numerical range recited herein is intended to include all subranges within the range and any combination of the various endpoints of the range or subrange.

[0093] As used in this disclosure, words such as "include," "comprising," or "including" mean that the elements preceding the word include the elements listed after the word and their equivalents, without excluding unlisted elements. The terms "comprising" or "including" as used herein may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0094] definition

[0095] The following terms and symbols used in this application have the meanings described below, unless otherwise indicated in the context in which they are used. The term "FAK inhibitor" as used herein refers to an effective inhibitor of FAK, which can be suitable for mammals, particularly humans. In some embodiments, the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, and the structure of IN10018 is as follows:

[0096] Defactinib, also known as defatinib, has a CAS number of 1345713-71-4; GSK2256098 has a CAS number of 1224887-10-8. PF-00562271 has a CAS number of 717907-75-0; VS-4718 has a CAS number of 1061353-68-1; APG-2449 was developed by Ascentage Pharmaceuticals; and AMP945 has a CAS number of 1393653-34-3. In some embodiments, the FAK inhibitor is preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof. In some preferred embodiments, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, particularly IN10018 tartrate.

[0097] The term "substance that induces immunogenic cell death" as used herein refers to a substance that induces immunogenic cell death (ICD) in an anti-tumor immune response. Immunogenic cell death (ICD) is a specific variant of regulated cell death (RCD) that is driven by stress and can induce adaptive immunity against dead cell antigens. The substance that induces immunogenic cell death described herein is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0098] In some embodiments, the substance that induces immunogenic cell death can be an inhibitor of RNA polymerase II. In some embodiments, the inhibitor of RNA polymerase II is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof. The CAS number of lurbinectedin is 497871-47-3, and the CAS number of SEL-120 is 1609522-33-9. In some embodiments, the inhibitor of RNA polymerase II is lurbinectedin.

[0099] In some embodiments, the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0100] In some embodiments, the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

[0101] The CAS number of crizotinib is 877399-52-5, SIM-0201 was developed by Simcere Pharmaceuticals, XZP-3621 was developed by Xuanzhu Biotechnology, TQ-B3139 was developed by Zhengda Tianqing, SAF-189s was jointly developed by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chongqing Fuchuang Pharmaceutical Research Co., Ltd., the CAS number of Ceritinib is 1032900-25-6, and the CAS number of Lorlatinib (PF-06463922) is 1454846-35-5 The CAS number of alectinib is 1256580-46-7, the CAS number of ensartinib is 1365267-27-1, APG-2449 was developed by Ascent Pharmaceuticals, the CAS number of brigatinib is 1197953-54-0, TQ-B3101 was developed by Zhengda Tianqing, the CAS number of entrectinib is 1108743-60-7, and the CAS number of repotrectinib is 1802220-02-5.

[0102] In some embodiments, the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0103] In some embodiments, the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0104] In some embodiments, the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0105] The D-1553 was developed by Yifang Bio, ARS-3248 was developed by Araxes, GF-105 was developed by GenFleet Therapeutics, JAB-21822 was developed by Jacobs, and JDQ-443 was developed by Novartis; the CAS number of LY-3537982 is 2414198-64-2, the CAS number of Sotorasib (AMG510) is 2296729-00-3, the CAS number of Adagrasib (MRTX849) is 2326521-71-3, and the CAS number of GDC-6036 is 2417987-45-0.

[0106] In some embodiments, the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments, the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0108] In some embodiments, the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

[0109] The CAS number of the MRTX1133 is 2621928-55-8, JAB-22000 was developed by Jacobs, and HRS-4642 was developed by Hengrui Medicine.

[0110] In some embodiments, the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0111] The term "immune checkpoint inhibitor" as used herein refers to a drug that can increase the activity of the immune system by regulating the immune checkpoint pathway (e.g., PD-1, PDL-1, TIGIT, CTLA-4, LAG-3, TIM-3, etc.). In some embodiments, the immune checkpoint inhibitor is an antagonist of the PD-1 / PD-L1 (programmed cell death protein 1) pathway (also referred to as a "PD-1 inhibitor") or a TIGIT inhibitor. PD-1 inhibitors are also referred to as PD-1 / PD-L1 inhibitors in this disclosure. For example, in the methods of treatment, drugs, and uses disclosed herein, the PD-1 / PD-L1 inhibitor is a PD-1 / PD-L1 antibody, including but not limited to pembrolizumab (Keytruda / K drug), tislelizumab (Tislelizumab / Baizean), nivolumab, toripalimab (Toripalimab / Tuoyi), atezolizumab (Atezolizumab / Tai Shengqi), durvalumab (Improvin / Durvalumab / Durvalumab), 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 embodiments, the PD-1 inhibitor is used to treat a human subject. In some embodiments, the PD-1 is human PD-1. PD-1 / PD-L1 inhibitors also include small molecule PD-1 / PD-L1 inhibitors, such as INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0112] TIGIT (also known as WUCAM, Vstm3, and VSIG9) is a receptor of the Ig superfamily and a new immune checkpoint after PD-1 / PD-L1. For example, in the methods of treatment, drugs and uses disclosed herein, the immune checkpoint inhibitor is a TIGIT inhibitor, including but not limited to Ociperlimab / BGB-A1217, Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI-321. In some embodiments, the TIGIT inhibitor is used to treat human subjects. To avoid ambiguity, antibodies herein include bispecific antibodies.

[0113] As used herein, "drug combination" or "drug combination product" may refer to a fixed combination in the form of one dosage unit (for example, all active pharmaceutical ingredients are present in one dosage form) or a kit of parts for combined administration, or it may refer to a combination of one drug and instructions indicating that the drug can be used in combination with one or more other drugs.

[0114] As used herein, "combination therapy" or "combination drug" refers to the use of a drug in combination with one or more other drugs to treat a disease, including both the combination of a drug with one or more other drugs and the combination of a drug with instructions indicating that the drug can be used in combination with one or more other drugs.

[0115] "Administered simultaneously or sequentially" in this application refers to the administration of two or more drugs simultaneously or successively at certain time intervals within a dosing cycle (e.g., within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours), the mode of drug administration (e.g., oral, intravenous, intramuscular or subcutaneous administration, etc.) may be the same or different, and the administration frequency / cycle of two or more drugs may be the same or different. When the method of treatment, product, or use disclosed herein involves two drugs, the two drugs may be administered separately at the same time or at certain time intervals. When the method of treatment, product, or use disclosed herein involves three drugs, the three drugs may be administered at the same time point, or two drugs may be administered at one time point and the remaining drug may be administered at another time point, or all three drugs may be administered at different time points.

[0116] In some embodiments, the PD-1 / PD-L1 inhibitor is administered intravenously (e.g., as an intravenous infusion) or subcutaneously, or orally. Preferably, the PD-1 / PD-L1 inhibitor is administered as an intravenous infusion.

[0117] In some embodiments, the TIGIT inhibitor is administered intravenously (eg, as an intravenous infusion) or subcutaneously, or orally. Preferably, the TIGIT inhibitor is administered as an intravenous infusion.

[0118] The ability of immune checkpoint inhibitors to treat cancer depends on the presence of tumor antigen-specific T cells within tumor tissue. This requires that tumor tissue express antigens that distinguish itself from its non-transformed counterparts, for example, through novel protein products called neoantigens. Tumor neoantigen load is closely related to immunogenicity and sensitivity (for example, sensitivity to checkpoint inhibitor therapy), which means that tumors with poor immunogenicity should be largely resistant to these drugs. Therapies for releasing tumor antigens that can be taken up by APCs, such as those that induce immunogenic cell death (ICD), may promote effective anti-tumor immunity, especially when further combined with checkpoint inhibitors.

[0119] As used herein, the term "treat" refers to administering one or more pharmaceutical substances to a subject suffering from a disease or symptoms of a disease in order to cure, alleviate, relieve, alter, cure, ameliorate, improve, or affect the disease or symptoms of the disease. In some embodiments, the disease is a tumor or cancer.

[0120] The term "tumor" as used herein refers to an abnormal lesion formed when the cells of local tissues lose normal regulation of their growth at the genetic level under the influence of various tumorigenic factors, thereby causing abnormal proliferation of their clonal types. The tumors include, but are not limited to: bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, smooth muscle In some embodiments, the tumor is preferably breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), gastric cancer, melanoma or pancreatic cancer; in some embodiments, the tumor is lung cancer or colon cancer (including colorectal cancer) or breast cancer.

[0121] As used herein, the term "subject" or "subject" refers to both mammals and non-mammals. Mammals refer to any member of the class mammalia, including but not limited to humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and pigs; livestock such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds. The term "subject" does not limit the subject to a particular age or sex. In some embodiments, the subject is a human.

[0122] As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerable, and suitable for administration to a subject.

[0123] As used herein, the term "pharmaceutically acceptable salt" refers to non-toxic, biologically tolerable acid addition salts suitable for administration to a subject, including, but not limited to, acid addition salts formed with inorganic acids, such as hydrochlorides, hydrobromides, carbonates, bicarbonates, phosphates, sulfates, sulfites, nitrates, and the like; and acid addition salts formed with organic acids, such as formate, acetate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, and salts with the formula HOOC-(CH2) n -COOH (wherein n is 0-4) and the like.

[0124] In addition, pharmaceutically acceptable acid addition salts can be obtained by dissolving the free base in a suitable solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds. Those skilled in the art can determine various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable acid addition salts without undue experimentation.

[0125] The term "pharmaceutically acceptable composition" as used herein means that it must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation, and / or compatible with the subject being treated therewith. The term "therapeutically effective amount" as used herein means an amount that is generally sufficient to produce a beneficial therapeutic effect on the subject. The therapeutically effective amount of the present invention can be determined by conventional methods (e.g., modeling, dose escalation studies, or clinical trials) in combination with conventional influencing factors (e.g., route of administration, pharmacokinetics of the compound, severity and course of the disease, medical history of the subject, health status of the subject, degree of response of the subject to the drug, etc.).

[0126] As used herein, the term "inhibit" refers to a decrease in the baseline activity of a biological activity or process.

[0127] The term "kit" as used herein refers to a box for containing chemical reagents such as chemical components, drug residues, and virus species for testing. The kit of the present invention may include one, two, or three of (i) a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor can be used to treat tumors in a subject; the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor. In one embodiment, the kit includes (i) a FAK inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor can be used to treat tumors in a subject. In one embodiment, the kit includes (i) a substance that induces immunogenic cell death; and (ii) instructions, the instructions indicating that a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor can be used to treat tumors in a subject. In one embodiment, the kit includes (i) an immune checkpoint inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a FAK inhibitor; and (ii) instructions, the instructions indicating that a FAK inhibitor and a substance that induces immunogenic cell death can be used to treat a tumor in a subject. In one embodiment, the kit includes (i) a substance that induces immunogenic cell death; and (ii) instructions, the instructions indicating that a substance that induces immunogenic cell death and a FAK inhibitor can be used to treat a tumor in a subject.

[0128] The compound of test kit can be contained in a separate container.Alternatively, two or more compounds are contained in the same container.For example, test kit can include a first container, a second container, a third container and a package insert, wherein the first container includes at least one dose of the medicine including a FAK inhibitor, the second container includes at least one dose of the substance inducing immunogenic cell death, the third container includes at least one dose of the medicine of the immune checkpoint inhibitor, and the package insert includes instructions for using the tumor of the drug treatment object. The first container, the second container and the third container can include the same or different shapes (for example, vials, syringes and bottles) and / or materials (for example, plastic or glass). Test kit can also include other materials that can contribute to the administration of the drug, such as diluents, filters, IV bags and pipelines, needles and syringes.

[0129] The precise amount of FAK inhibitor, substance that induces immunogenic cell death, or immune checkpoint inhibitor administered to a subject will depend on various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease, the condition, the identity of the subject or host being treated, etc., but can still be routinely determined by one skilled in the art. For example, determining an effective amount also depends on the extent, severity, and type of cell proliferation. A skilled person will be able to determine an appropriate dosage based on these and other factors.

[0130] The FAK inhibitor, the substance that induces immunogenic cell death, or the immune checkpoint inhibitor can be administered by a suitable route, such as oral, intravenous, intramuscular, or subcutaneous administration.

[0131] For example, when administered orally, the drug can be administered orally with a pharmaceutically acceptable carrier, such as an inert diluent or an assimilable edible carrier. They can be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or mixed directly with the patient's food. For example, the drug can be combined with one or more excipients and used in the form of an ingestible tablet, buccal tablet, lozenge, capsule, elixir, suspension, syrup, or wafer. Tablets, lozenges, pills, capsules, etc. may further include: a binder, such as gum tragacanth, gum arabic, corn starch, or gelatin; an excipient, such as dicalcium phosphate; a disintegrant, such as corn starch, potato starch, alginic acid, etc.; a lubricant, such as magnesium stearate; or a sweetener, such as sucrose, fructose, lactose, or aspartame; or a flavoring agent.

[0132] For example, when administered intravenously or intraperitoneally by infusion or injection, solutions of the drug can be prepared in water, optionally mixed with a nontoxic surfactant.

[0133] Exemplary pharmaceutical dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient, which are suitable for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage.

[0134] Sterile injectable solutions can be prepared by incorporating the required amount of the drug into an appropriate solvent with the various other ingredients listed above as required, followed by filtered sterilization. For sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods may be vacuum drying and freeze drying techniques, which can produce a powder of the active ingredient plus any other desired ingredients that have been previously sterile filtered.

[0135] The amount of FAK inhibitor, substance that induces immunogenic cell death, or immune checkpoint inhibitor required for treatment may vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient, and may ultimately be determined at the discretion of the attending physician or clinician. However, in general, the dosage may be in the range of about 0.1 to about 50 mg / kg body weight per day.

[0136] The FAK inhibitor is administered in a dosage range of 5 mg / day to 500 mg / day in adults. In a specific embodiment, IN10018 or a pharmaceutically acceptable salt thereof is administered in a dosage range of 5 mg / day to 100 mg / day in adults, for example, IN10018 or a pharmaceutically acceptable salt thereof is administered in a dosage range of 25 mg / day to 100 mg / day in adults, calculated as the free base.

[0137] The substance that induces immunogenic cell death is administered in a dosage range in adults according to each drug. In some embodiments, the inhibitor of RNA polymerase II is administered in an adult at a dose of 1-25 mg / m2 per 21 days (treatment cycle). 2 In some embodiments, Lurbinectedin is administered at a dose range of 1-5 mg / m2 per 21 days in adults. 2In some embodiments, the ALK / ROS1 inhibitor is administered in a dosage range of 2-1000 mg per day in adults. In some specific embodiments, crizotinib is administered in a dosage range of 50-500 mg per day in adults. In some specific embodiments, the KRAS G12C inhibitor is administered in a dosage range of 10-500 mg per day in adults. In some specific embodiments, D-1553 is administered in a dosage range of 10-250 mg per day in adults. In some embodiments, the KRAS G12D inhibitor is administered in a dosage range of 25-800 mg per day in adults. In some specific embodiments, MRTX1133 is administered in a dosage range of 25-600 mg per day in adults. The above dosages are all calculated as free base.

[0138] The immune checkpoint inhibitor is administered at a dose of 2-10 mg / kg or 50-1200 mg in adults per administration, administered once every 2 to 3 weeks. In a specific embodiment, the immune checkpoint inhibitor is administered at a dose of 3-10 mg / kg or 100-1200 mg in adults per administration, administered once every 2 to 3 weeks.

[0139] Technical and scientific terms used herein without specific definition have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0140] In some embodiments, the present disclosure further discloses the following:

[0141] 1. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor for use in a method for treating tumors in a subject, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0142] 2. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 1, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0143] 3. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 1 or 2, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0144] 4. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1 to 3, wherein the RNA polymerase II inhibitor is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof.

[0145] 5. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 3-4, wherein the inhibitor of RNA polymerase II is lurbinectedin.

[0146] 6. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1-2, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0147] 7. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor as described in embodiment 6, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib, or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib, or a pharmaceutically acceptable salt thereof.

[0148] 8. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in any one of embodiments 6-7, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0149] 9. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1-2, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0150] 10. The FAK inhibitor, the substance that induces immunogenic cell death and the immune checkpoint inhibitor as described in embodiment 9, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0151] 11. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 9-10, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0152] 12. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1-2, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0153] 13. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 12, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

[0154] 14. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 12-13, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0155] 15. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1-14, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

[0156] 16. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1 to 15, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, nivolumab, toripalimab, atezolizumab, durvalumab, avelumab, atezolizumab, camrelizumab, sintilimab, cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

[0157] 17. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1-15, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0158] 18. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1 to 15, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody is Ociperlimab (Ociperlimab / BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Be lrestotug, 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.

[0159] 19. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 1, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is lurbinectedin; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0160] 20. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 1, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0161] 21. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 1, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0162] 22. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in Embodiment 1, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

[0163] 23. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 1-22, wherein the FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0164] 24. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor as described in any one of embodiments 1-23, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, Nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

[0165] 25. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 24, wherein the tumor is lung cancer or colon cancer (including colorectal cancer) or breast cancer.

[0166] 26. A kit or pharmaceutically acceptable composition comprising:

[0167] (a) FAK inhibitors;

[0168] (b) substances that induce immunogenic cell death; and

[0169] (c) immune checkpoint inhibitors;

[0170] The substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0171] 27. The kit or composition according to embodiment 26, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

[0172] 28. The kit or composition of any one of embodiments 26-27, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0173] 29. The kit or composition of embodiment 28, wherein the inhibitor of RNA polymerase II is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof.

[0174] 30. The kit or composition of any one of embodiments 28-29, wherein the inhibitor of RNA polymerase II is lurbinectedin.

[0175] 31. The kit or composition of any one of embodiments 26-27, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0176] 32. The kit or composition according to embodiment 31, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

[0177] 33. The kit or composition of any one of embodiments 31-32, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0178] 34. The kit or composition of any one of embodiments 26-27, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0179] 35. The kit or composition of embodiment 34, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036, or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510), or a pharmaceutically acceptable salt thereof.

[0180] 36. The kit or composition of any one of embodiments 34-35, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0181] 37. The kit or composition of any one of embodiments 26-27, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0182] 38. The kit or composition of embodiment 37, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

[0183] 39. The kit or composition of any one of embodiments 37-38, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0184] 40. The kit or composition of any one of embodiments 26-39, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

[0185] 41. The kit or composition of any one of embodiments 26-40, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Atezolizumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

[0186] 42. The kit or composition according to any one of embodiments 26-40, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0187] 43. The kit or composition of any one of embodiments 26-40, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody 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.

[0188] 44. The kit or composition of embodiment 26, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0189] 45. The kit or composition of embodiment 26, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0190] 46. ​​The kit or composition of embodiment 26, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0191] 47. The kit or composition of embodiment 26, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

[0192] 48. The kit or composition of any one of embodiments 26-47, wherein the composition is for use in medicine.

[0193] 49. The kit or composition of embodiment 48, wherein the drug is used to treat a tumor, and the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, The invention relates to a method for treating a thyroid cancer of the present invention and the like. The method comprises the following steps: treating a thyroid cancer of the present invention and the like; wherein the thyroid cancer is a thyroid cancer, a thyroid cancer, a salivary gland cancer, a metastatic tumor caused by spindle cell carcinoma, an anaplastic large cell lymphoma, an undifferentiated thyroid cancer, a non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a glioma or a hematological malignancy, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

[0194] 50. The kit or composition of embodiment 49, wherein the tumor is lung cancer, colon cancer (including colorectal cancer), or breast cancer.

[0195] 51. A method for treating a tumor in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0196] 52. The method according to embodiment 51, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

[0197] 53. A method as described in any one of embodiments 51-52, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0198] 54. The method of embodiment 53, wherein the inhibitor of RNA polymerase II is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof.

[0199] 55. The method of any one of embodiments 53-54, wherein the inhibitor of RNA polymerase II is lurbinectedin.

[0200] 56. The method of any one of embodiments 51-52, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0201] 57. The method according to embodiment 56, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

[0202] 58. The method according to any one of embodiments 56-57, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0203] 59. A method as described in any one of embodiments 51-52, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0204] 60. A method as described in embodiment 59, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0205] 61. The method of any one of embodiments 59-60, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0206] 62. A method as described in any one of embodiments 51-52, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0207] 63. The method of embodiment 62, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

[0208] 64. The method of any one of embodiments 62-63, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0209] 65. A method as described in any one of embodiments 51-64, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

[0210] 66. The method of embodiment 65, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Atezolizumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0211] 67. The method according to embodiment 65, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043 or RRx-001.

[0212] 68. The method of embodiment 65, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody 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.

[0213] 69. The method according to embodiment 51, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0214] 70. The method according to embodiment 51, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0215] 71. The method according to embodiment 51, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0216] 72. The method according to embodiment 51, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is a TIGIT antibody.

[0217] 73. The method of any one of embodiments 51-72, wherein the FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0218] 74. A method as described in any one of embodiments 51-73, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer Preferably, the tumor is a breast cancer, an ovarian cancer, a colon cancer (including colorectal cancer), a lung cancer (including small cell lung cancer and non-small cell lung cancer), a melanoma or a pancreatic cancer.

[0219] 75. The method of embodiment 74, wherein the tumor is breast cancer, ovarian cancer, or colon cancer (including colorectal cancer).

[0220] 76. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor for use in a method for treating tumors by increasing immunogenic cell death in a subject, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0221] 77. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to embodiment 76, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0222] 78. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in any one of embodiments 76-77, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0223] 79. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 78, wherein the inhibitor of RNA polymerase II is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof.

[0224] 80. The FAK inhibitor, immunogenic cell death-inducing substance, and immune checkpoint inhibitor of any one of embodiments 78-79, wherein the RNA polymerase II inhibitor is lurbinectedin.

[0225] 81. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in any one of embodiments 76-77, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0226] 82. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor as described in embodiment 81, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib, or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib, or a pharmaceutically acceptable salt thereof.

[0227] 83. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in any one of embodiments 81-82, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0228] 84. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in any one of embodiments 76-77, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0229] 85. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 84, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0230] 86. The FAK inhibitor, immunogenic cell death-inducing substance, and immune checkpoint inhibitor of any one of embodiments 84-85, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0231] 87. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor as described in any one of embodiments 76-77, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0232] 88. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor of embodiment 87, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

[0233] 89. The FAK inhibitor, immunogenic cell death-inducing substance, and immune checkpoint inhibitor of any one of embodiments 87-88, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0234] 90. The FAK inhibitor, immunogenic cell death-inducing substance, and immune checkpoint inhibitor of any one of embodiments 76-89, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

[0235] 91. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 76-90, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, or the like. izumab), durvalumab, avelumab, atezolizumab, camrelizumab, sintilimab, cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

[0236] 92. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 76-90, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

[0237] 93. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor according to any one of embodiments 76-90, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody is Ociperlimab (Ociperlimab / BGB-A1217), Vibostolimab, domvanalimab (AB154), Tiragolumab, Be lrestotug, 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.

[0238] 94. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 76, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is lurbinectedin; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0239] 95. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 76, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, especially, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0240] 96. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 76, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0241] 97. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in Embodiment 76, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

[0242] 98. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in any one of embodiments 76-97, wherein the FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0243] 99. A FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor as described in any one of embodiments 76-98, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, Nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

[0244] 100. The FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor as described in embodiment 99, wherein the tumor is lung cancer or colon cancer (including colorectal cancer) or breast cancer.

[0245] 101. A method for treating a tumor by increasing immunogenic cell death in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0246] 102. The method according to embodiment 101, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

[0247] 103. A method as described in any one of embodiments 101-102, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0248] 104. The method of embodiment 103, wherein the inhibitor of RNA polymerase II is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof.

[0249] 105. The method of any one of embodiments 103-104, wherein the inhibitor of RNA polymerase II is lurbinectedin.

[0250] 106. The method of any one of embodiments 101-102, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0251] 107. The method according to embodiment 106, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

[0252] 108. The method according to any one of embodiments 106-107, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0253] 109. A method as described in any one of embodiments 101-102, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0254] 110. The method according to embodiment 109, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0255] 111. The method of any one of embodiments 109-110, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0256] 112. A method as described in any one of embodiments 101-102, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0257] 113. The method of embodiment 112, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

[0258] 114. The method of any one of embodiments 112-113, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0259] 115. A method as described in any one of embodiments 101-114, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

[0260] 116. The method of embodiment 115, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Atezolizumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0261] 117. The method according to embodiment 115, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043 or RRx-001.

[0262] 118. The method of embodiment 115, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody 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.

[0263] 119. The method according to embodiment 101, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0264] 120. The method according to embodiment 101, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0265] 121. The method according to embodiment 101, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor; in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0266] 122. The method according to embodiment 101, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

[0267] 123. The method of any one of embodiments 101-122, wherein the FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0268] 124. A method as described in any one of embodiments 101-123, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovariectomy, or ovarian cancer. Preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

[0269] 125. The method of embodiment 124, wherein the tumor is lung cancer, colon cancer (including colorectal cancer), or breast cancer.

[0270] 126. Use of a FAK inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein the FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor are administered to the subject, and the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0271] 127. Use of a substance that induces immunogenic cell death in the preparation of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, the substance that induces immunogenic cell death and an immune checkpoint inhibitor are administered to the subject, and the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0272] 128. Use of an immune checkpoint inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, a substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject, and the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0273] 129. Use of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor in the preparation of a combination drug for treating tumors, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0274] 130. Use of a FAK inhibitor in the preparation of a combination drug for treating tumors with a substance that induces immunogenic cell death and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0275] 131. Use of a substance that induces immunogenic cell death in the preparation of a combination drug for treating tumors with a FAK inhibitor and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0276] 132. Use of an immune checkpoint inhibitor in the preparation of a combination drug for treating tumors with a FAK inhibitor and a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0277] 133. Use of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor in the preparation of a drug for combined treatment of tumors, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

[0278] 134. Use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with a substance that induces immunogenic cell death and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0279] 135. Use of a substance that induces immunogenic cell death in the preparation of a drug for treating tumors in combination with a FAK inhibitor and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0280] 136. Use of an immune checkpoint inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor and a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

[0281] 137. The use according to any one of embodiments 126 to 136, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

[0282] 138. The use according to any one of embodiments 126-137, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

[0283] 139. The use according to embodiment 138, wherein the inhibitor of RNA polymerase II is lurbinectedin, SEL-120, or a pharmaceutically acceptable salt thereof.

[0284] 140. The use according to any one of embodiments 138-139, wherein the inhibitor of RNA polymerase II is lurbinectedin.

[0285] 141. The use according to any one of embodiments 126-137, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

[0286] 142. The use according to embodiment 141, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

[0287] 143. The use according to any one of embodiments 141-142, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

[0288] 144. The use according to any one of embodiments 126-137, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

[0289] 145. The use according to embodiment 144, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

[0290] 146. The use according to any one of embodiments 144-145, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

[0291] 147. The use according to any one of embodiments 126 to 137, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

[0292] 148. The use according to embodiment 147, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000 or a pharmaceutically acceptable salt thereof.

[0293] 149. The use according to any one of embodiments 147-148, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

[0294] 150. The use according to any one of embodiments 126-149, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

[0295] 151. The use according to embodiment 150, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Atezolizumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

[0296] 152. The use according to embodiment 150, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, Lazertinib, IMH-010, CA-170, ABSK043 or RRx-001.

[0297] 153. The use according to embodiment 150, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody 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.

[0298] 154. The use according to any one of embodiments 126-136, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0299] 155. The use according to any one of embodiments 126 to 136, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0300] 156. The use according to any one of embodiments 126 to 136, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof, and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

[0301] 157. The use according to any one of embodiments 126-136, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

[0302] 158. The use according to any one of embodiments 126-157, wherein the FAK inhibitor, the substance that induces immunogenic cell death, and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

[0303] 159. The use according to any one of embodiments 126 to 158, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovariectomy, or the like. Preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma or pancreatic cancer.

[0304] 160. The use according to embodiment 159, wherein the tumor is lung cancer, colon cancer (including colorectal cancer), or breast cancer.

[0305] Example

[0306] The following examples are provided to further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0307] The experimental methods in the following examples where specific conditions are not specified can be carried out according to conventional conditions of such reactions or according to the conditions recommended by the manufacturers. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0308] The abbreviations used in the examples have the following meanings:

[0309] Example 1: Synergistic effect of IN10018 and rubicatin in colon cancer CT26 cells

[0310] Mouse colon cancer CT26 cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI-1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, they were plated in 96-well plates at 3,000 cells / well. After 24 hours of cell growth, medium containing lupicatin (DC Chemicals, catalog number: DC12502, batch number: DC1250203) was added. Ten drug concentrations were set up, starting with a 0.1 μM concentration in a four-fold dilution and ending with a control with zero drug concentration. Three wells were plated for each drug concentration. Simultaneously, another set of cells was established using the same drug concentrations as above. The difference was that 6 μM IN10018 was added to each well, the drugs were mixed, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 72 hours.

[0311] After 72 hours of drug treatment, the cells were observed under a microscope, 10 μl of CCK8 detection reagent (Cellorlab, catalog number: CX001M, batch number: 2571100) was added to each well, and the cells were cultured in a 5% CO2 incubator at 37°C for 2-4 hours. The OD450 (chemiluminescence method) of the well plate was then read using a microplate reader.

[0312] Analysis revealed that the IC50 for the lubicatin group was 0.71 nM, while the IC50 for the lubicatin plus 6 μM IN10018 group was 0.30 nM. The IC50 for the group containing IN10018 was lower than that for the group without IN10018, indicating that the combined treatment group was more effective than either drug alone, as shown in Figure 1.

[0313] Example 2: Study of IN10018 and Rubicatin in Colon Cancer CT26 Cells

[0314] Mouse colon cancer CT26 cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI-1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) supplemented with 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, the culture medium was plated in 24-well plates. After 24 hours of cell growth, four groups were assigned: the first group served as the control group, receiving culture medium; the second group received IN10018 at a concentration of 5 μM; the third group received lupicatin (DC Chemicals, catalog number: DC12502, batch number: DC1250203) at a concentration of 0.00142 μM; and the fourth group received a combination of IN10018 (5 μM) and lupicatin (0.00142 μM). The drugs were mixed and incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0315] 48 hours after drug treatment, cells were observed under a microscope and photographed, and the images were saved. Cells were then collected for flow cytometry analysis and washed twice with flow cytometry buffer (PBS + 2% FBS). 0.5 μl of AF647 anti-calreticulin antibody (abcam, catalog number: ab196159, lot number: CR33676773) was added to each well, mixed thoroughly, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow cytometry buffer (PBS + 2% FBS). Cell apoptosis detection kit (Beyotime, catalog number: CL062L, lot number: 021921210811) was added, 195 μl of annexin V-FITC conjugate was added, and the cells were gently mixed. Then, 5 μl of annexin V-FITC antibody was added and gently mixed. 10 μl of PI dye was added, mixed gently, and the cells were incubated at room temperature in the dark for 15 minutes before analysis on a flow cytometer.

[0316] Microscopic observation of cells revealed poor cell status in the rubicatin monotherapy and dual-drug combination groups, with the dual-drug combination group showing the worst results, with more cell death. Cells in the control and IN10018 groups were in good condition (see Figure 2). Results showed that the CRT and Annexin V positivity rates in the dual-drug combination group were higher than those in the monotherapy group (see Figure 3).

[0317] Example 3: Synergistic effect of IN10018 and MRTX1133 in colon cancer CT26 cells

[0318] Mouse colon cancer CT26 cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI-1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) plus 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, they were plated in 96-well plates at 3,000 cells per well. After 24 hours of cell growth, medium containing MRTX1133 (Shanghai Chaolan Chemical Technology Center, catalog number: P2621928-55-8, batch number: 131005) was added. Ten drug concentrations were set up, starting with a 30 μM concentration, diluted fivefold, and ending with a control with zero drug concentration. Three wells were plated for each drug concentration. Simultaneously, another set of cells was established using the same drug concentrations as above. The difference was that 5 μM IN10018 was added to each well, the drugs were mixed, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 72 hours.

[0319] After 72 hours of drug exposure, cells were observed under a microscope. 10 μl of CCK8 detection reagent (Cellorlab, catalog number: CX001M, batch number: 2571100) was added to each well and incubated in a 37°C 5% CO2 incubator for 2-4 hours. The plate was then read using a microplate reader (chemiluminescence) at OD450. Analysis revealed that the IC50 for the MRTX1133 group was 0.28 μM; the IC50 for the MRTX1133 + 5 μM IN10018 group was 0.02 μM. The IC50 for the group containing IN10018 was significantly lower than that for the group without IN10018, indicating that the combination therapy was superior to the monotherapy group, as shown in Figure 4.

[0320] Example 4: Study of IN10018 and MRTX1133 in colon cancer CT26 cells

[0321] Mouse colon cancer CT26 cells (Institute of Cell Biology, Chinese Academy of Sciences) were cultured in RPMI-1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) plus 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, the culture medium was plated in 24-well plates. After 24 hours of cell growth, four groups were set up: the first group served as the control group, receiving culture medium; the second group received IN10018 at a concentration of 5 μM; the third group received MRTX1133 (Shanghai Chaolan Chemical Technology Center, catalog number: P2621928-55-8, batch number: 131005) at a concentration of 6 μM; and the fourth group received a combination of IN10018 (5 μM) and MRTX1133 (6 μM). The drugs were mixed and incubated in a 5% CO2 incubator at 37°C for 48 hours.

[0322] After 48 hours of drug treatment, cells were observed under a microscope and photographed, and the photographs were saved. Cells were then collected for flow cytometry analysis and washed twice with flow cytometry buffer (PBS + 2% FBS). 0.5 μl of AF647 anti-calreticulin antibody (abcam, catalog number: ab196159, batch number: CR33676773) was added to each well, mixed thoroughly, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow cytometry buffer (PBS + 2% FBS). A cell apoptosis detection kit (Beyotime, catalog number: CL062L, batch number: 021921210811) was used, 195 μl of annexin V-FITC conjugate was added, the cells were gently mixed, 5 μl of annexin V-FITC antibody was added, and the mixture was gently mixed. 10 μl of PI dye was added and the mixture was incubated at room temperature in the dark for 15 minutes before analysis on a flow cytometer.

[0323] Microscopic observation of cells revealed poor cell status in the MRTX1133 monotherapy group and the MRTX1133-drug combination group, with the MRTX1133-drug combination group showing the worst cell death, while the control group and IN10018 group showed good cell status (see Figure 5). The results showed that the CRT- and Annexin V-positive rates in the MRTX1133-drug combination group were higher than those in the monotherapy group (see Figure 6).

[0324] Example 5: Synergistic Effect of IN10018 and MRTX1133 in Lung Cancer KPL Cells

[0325] Mouse lung carcinoma KPL (Institute of Cell Biology, Chinese Academy of Sciences) was cultured in RPMI-1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, they were plated in 96-well plates at 3,000 cells / well. After 24 hours of cell growth, medium containing MRTX1133 (Shanghai Chaolan Chemical Technology Center, catalog number: P2621928-55-8, batch number: 131005) was added. Ten drug concentrations were set up, starting with a 30 μM concentration, diluted fivefold, and ending with a control with zero drug concentration. Three wells were plated for each drug concentration. Simultaneously, another set of cells was established using the same drug concentrations as above. The difference was that 10 μM IN10018 was added to each well, the drugs were mixed, and the cells were cultured at 37° C. in a 5% CO 2 incubator for 72 hours.

[0326] After 72 h of drug treatment, the cells were observed under a microscope, 10 μl of CCK8 detection reagent (Cellorlab, catalog number: CX001M, batch number: 2571100) was added to each well, and the cells were cultured in a 5% CO2 incubator at 37°C for 2-4 hours. The plate was then read using a microplate reader OD450 (chemiluminescence method).

[0327] Analysis revealed that the IC50 for the MRTX1133 group was 1.76 μM, while the IC50 for the MRTX1133 + 10 μM IN10018 group was 0.008 μM. The IC50 for the group containing IN10018 was significantly lower than that for the group without IN10018, indicating that the combined treatment with both drugs was more effective than either drug alone, as shown in Figure 7.

[0328] Example 6: Study of IN10018 and MRTX1133 in lung cancer KPL cells

[0329] Mouse lung carcinoma KPL (Institute of Cell Biology, Chinese Academy of Sciences) was cultured in RPMI-1640 (Shanghai Yuanpei, catalog number: L210KJ, batch number: F210916) + 10% FBS (Gibco, catalog number: 10099-141c, batch number: 2158737cp) and passaged twice. When cells were in good condition, the culture medium was plated in 24-well plates. After 24 hours of cell expansion, four groups were divided: the first group was a control group, which received culture medium; the second group was treated with IN10018 at a concentration of 10 μM; the third group was treated with MRTX1133 (Shanghai Chaolan Chemical Technology Center, catalog number: P2621928-55-8, batch number: 131005) at a concentration of 15 μM; and the fourth group was treated with a combination of IN10018 (10 μM) and MRTX1133 (15 μM). The drugs were mixed and incubated in a 5% CO2 incubator at 37°C for 48 hours.

[0330] After 48 hours of drug treatment, cells were observed under a microscope and photographed, and the photographs were saved. Cells were then collected for flow cytometry analysis and washed twice with flow cytometry buffer (PBS + 2% FBS). 0.5 μl of AF647 anti-calreticulin antibody (abcam, catalog number: ab196159, batch number: CR33676773) was added to each well, mixed thoroughly, and incubated at 4°C in the dark for 20 minutes. After 20 minutes, cells were washed twice with flow cytometry buffer (PBS + 2% FBS). A cell apoptosis detection kit (Beyotime, catalog number: CL062L, batch number: 021921210811) was used, 195 μl of annexin V-FITC conjugate was added, the cells were gently mixed, 5 μl of annexin V-FITC antibody was added, and the mixture was gently mixed. 10 μl of PI dye was added and the mixture was incubated at room temperature in the dark for 15 minutes before analysis on a flow cytometer.

[0331] Microscopic observation of cells revealed poor cell status in the MRTX1133 monotherapy group and the MRTX1133-drug combination group, with the MRTX1133-drug combination group showing the worst cell death, while the control group and IN10018 group showed good cell status (see Figure 8). The results showed that the CRT- and Annexin V-positive rates in the MRTX1133-drug combination group were higher than those in the monotherapy group (see Figure 9).

[0332] Example 7: Study on the Induction of Immunogenic Cell Death Targets by Crizotinib and IN10018 in Mouse Colon Cancer CT26 Cells

[0333] Compound information is shown in Table 1:

[0334] Table 1.

[0335] The main reagent information of the experiment is shown in Table 2:

[0336] Table 2.

[0337] The experimental design is shown in Table 3:

[0338] Table 3:

[0339] Cell culture:

[0340] CT26 cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Cells were routinely digested and passaged using trypsin two to three times a week. When cells reached 80%-90% confluence and were in the exponential growth phase, they were harvested and plated.

[0341] CT26 cells were trypsinized, harvested, and counted. Based on the count, the cells were diluted with RPMI-1640 + 10% FBS to a concentration of 50,000 cells / mL. The cells were then plated onto 24-well cell culture plates, with 1 mL of the cell suspension per well (i.e., 50,000 cells). After plating, the cells were cultured in a 37°C, 5% CO2 incubator.

[0342] Add the test compound:

[0343] 24 hours after plating, the test compound IN10018 and crizotinib were added to different wells, respectively. The groups and drug concentrations are shown in Table 3.

[0344] Collect cells for flow cytometry:

[0345] After 48 hours of drug treatment, the cells were photographed using a microscope, and then trypsinized and collected for flow cytometry staining.

[0346] After washing the cells twice with flow cytometry buffer (DPBS + 2% FBS), each group of cells was divided equally into two aliquots. 0.5 μL of AF647 Anti-Calreticulin Antibody was added to each well of one aliquot, mixed, and incubated at 4°C in the dark for 20 minutes. After washing once with flow cytometry buffer, 195 μL of Annexin-V-FITC conjugate was added. After gently mixing the cells, 5 μL of Annexin-V-FITC antibody was added. After further mixing, 10 μL of PI dye was added and mixed. The cells were incubated at room temperature in the dark for 15 minutes before flow cytometry analysis.

[0347] Data analysis: After the experiment, Flowjo (V10) software was used to analyze the cell positive rate.

[0348] Experimental results:

[0349] 48 hours after drug treatment, cells in each group were observed under a microscope. Cell death was evident in the crizotinib monotherapy group, with the most significant cell death in the combination therapy group. Cell viability was relatively good in the control and IN10018 groups. Flow cytometric analysis showed that the CRT and Annexin-V positivity rates in the combination therapy group were significantly higher than those in the monotherapy group. The relevant test results are shown in Figure 10.

[0350] Example 8: In vivo pharmacodynamic study of the test drug on a BALB / c mouse model of subcutaneous homografts of CT26-KRAS G12C colon cancer cells

[0351] Experimental Materials:

[0352] Species: Mouse; Strain: BALB / c mice; Age and weight: 6-8 weeks, 18-22 g; Gender: Female; Number: 49 (excluding remaining mice in each group); Supplier: Shanghai Lingchang Biotechnology Co., Ltd.

[0353] Upon arrival, animals were housed in the experimental environment for 3-7 days before experiments began. Animals were housed in an SPF-grade animal facility in IVC (independent ventilation system) cages (3-4 animals per cage). Each cage's animal information card indicated the number of animals in the cage, sex, strain, date of receipt, dosing regimen, study number, group, and start date of the experiment. All cages, bedding, and drinking water were sterilized before use. Cages, feed, and drinking water were changed twice weekly. The housing environment and lighting conditions were as follows: temperature: 20-26°C; humidity: 40-70%; photoperiod: 12 hours of light, 12 hours of dark (lights on 8:00 AM - lights off 8:00 PM); cages: made of polycarbonate, 300 mm x 180 mm x 150 mm. The litter was corn cobs, which were changed twice a week. The experimental animals were free to eat food (irradiation sterilized, dry granular food) throughout the experimental period. The experimental animals were free to drink sterilized water. Animal identification: The experimental animals were identified with ear tags.

[0354] The information of test and control substances is shown in Table 4.

[0355] Table 4:

[0356] Note: (INX0082 is a PD1 antibody)

[0357] Experimental methods and steps:

[0358] Mouse colon cancer CT26-KRAS G12C cells were cultured as monolayers in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin / amphotericin B at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged twice weekly using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0359] 0.1 mL (0.3 × 10 6 CT26-KRAS G12C cells were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 55 mm 3 The grouping information is shown in Table 5.

[0360] Table 5:

[0361] Note: 1. N: number of mice in each group;

[0362] 2. Dosing volume: 10 μl / g based on mouse body weight. If body weight loss exceeds 15%, the dosing regimen should be adjusted accordingly.

[0363] The preparation of the test substances is shown in Table 6.

[0364] Table 6: Preparation of test substances

[0365] NOTE: The drug should be gently mixed thoroughly before administration to animals.

[0366] Daily observation of experimental animals:

[0367] Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight change (measured three times per week), physical signs, or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0368] Tumor measurements and experimental parameters:

[0369] Tumor diameter was measured with a vernier caliper three times a week. Tumor volume was calculated using the formula: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0370] The anti-tumor efficacy of the compound was evaluated by TGI (%) or relative tumor growth rate T / C (%). Relative tumor growth rate T / C (%) = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : RTV of negative control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), V t is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0371] TGI (%) reflects the rate of tumor growth inhibition. TGI (%) = [1 - (average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0372] Statistical analysis:

[0373] Statistical analysis included the mean and standard error (SEM) of the tumor volume at each time point for each group (see Table 7 for specific data). Statistical analysis was performed based on the tumor volume data on day 19 after administration to evaluate the differences between the groups. Comparisons between two groups were analyzed using T-test, and comparisons between three or more groups were analyzed using one-way ANOVA. If there was a significant difference in the F value, the Games-Howell method was used for testing. If there was no significant difference in the F value, the Dunnet (2-sided) method was used for analysis. All data analyses were performed using SPSS 17.0. p<0.05 was considered to be significantly different.

[0374] Experimental results:

[0375] The body weight of the experimental animals is used as a reference indicator for indirectly measuring drug toxicity. In this model, all drug-treated groups showed no significant weight loss (as shown in Figure 11), and no mice became ill or died.

[0376] The effects of the test substances on body weight in a female BALB / c mouse model bearing subcutaneous homografts of mouse colon cancer CT26-KRAS G12C cells are shown in Figures 11 and 12 .

[0377] In this study, we evaluated the in vivo efficacy of the test compound alone and in combination in a mouse colon cancer CT26-KRAS G12C homograft tumor model. Nineteen days after the start of dosing, the average tumor volume of the tumor-bearing mice in the solvent control group reached 1746 mm 3 The average tumor volume of the D-1553 5 mg / kg group was 995 mm 3 (T / C was 57.0%, TGI was 44.4%, p value was 0.251), which showed a certain anti-tumor effect compared with the solvent control; the average tumor volume of the test substance IN10018, 25 mg / kg administration group was 1352 mm 3 (T / C was 74.7%, TGI was 23.4%, and p value was 0.938), and had no tumor inhibitory effect compared with the solvent control group; the average tumor volume of the INX0082, 10 mg / kg administration group was 920 mm 3 (T / C is 54.7%, TGI is 48.9%, p is 0.348), and it also has a certain anti-tumor effect compared with the solvent control group. The average tumor volume of the group treated with the test substance D-1553, 5 mg / kg and IN10018, 25 mg / kg was 650 mm 3 (T / C is 36.2%, TGI is 64.8%, p is 0.063), showing a better tumor inhibition effect than the solvent control group; the average tumor volume of the group receiving combined administration of D-1553, 5 mg / kg and INX0082, 10 mg / kg was 668 mm3 (T / C is 39.2%, TGI is 63.8%, p is 0.053), which also has a better anti-tumor effect than the solvent control group; the average tumor volume of the test substance D-1553, 5 mg / kg, IN10018, 25 mg / kg and INX0082, 10 mg / kg combined administration group was 412 mm 3 (T / C was 22.9%, TGI was 78.9%, p was 0.016), showing a significant tumor inhibition effect compared with the solvent control group, as shown in Table 7 and Figure 13.

[0378] Table 7:

[0379] Note: a. Mean ± SEM, n = 7.

[0380] bp (one-way ANOVA) values ​​were calculated based on tumor volume.

[0381] Example 9: In vivo anti-tumor efficacy study of the combination of MRTX1133, anti-mouse TIGIT antibody, and IN10018 in a BALB / c mouse subcutaneous homograft model of mouse colon cancer CT26 cells

[0382] Experimental Materials:

[0383] The information of experimental animals is shown in Table 8.

[0384] Table 8:

[0385] The rearing environment is shown in Table 9.

[0386] Table 9:

[0387] The test sample information is shown in Table 10.

[0388] Table 10:

[0389] The relevant excipient information is shown in Table 11.

[0390] Table 11:

[0391] Experimental methods and steps:

[0392] Mouse colorectal cancer cells CT26 (sourced from Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60043) were maintained and passaged by Ins Biotechnology (Nanjing) Co., Ltd. Cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged two to three times weekly using trypsin-EDTA. When cells reached the exponential growth phase and 80%-90% saturation, they were harvested, counted, and plated.

[0393] 0.1 mL contains 3×10 5 A cell suspension of 100 cells was subcutaneously inoculated on the right back of each mouse. 3 Around 3 d after 4 d (13 days after cell inoculation), the mice were randomly divided into groups according to tumor volume and given medication. The grouping information is shown in Table 12.

[0394] Table 12:

[0395] Note:

[0396] 1. N: number of mice in each group;

[0397] 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight drops by more than 15%, stop dosing the animal; resume dosing after the body weight returns to the initial value.

[0398] The preparation information of test substance and control solvent is shown in Table 13.

[0399] Table 13:

[0400] Daily observation of experimental animals:

[0401] Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on the animals' daily behaviors, such as activity, food and water intake (visual observation only), weight changes, physical signs, or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0402] Experiment termination:

[0403] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3 If the animal is seriously ill or in pain, it must be euthanized. If the animal has any of the following conditions, notify the veterinarian and euthanize it: obvious weight loss, weight loss greater than 20%; cannot eat and drink freely; the average tumor volume of the control group reaches 3,000mm 3The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0404] Tumor measurements and experimental parameters:

[0405] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured with a vernier caliper 2-3 times a week. The formula for calculating tumor volume is: V = 0.5 × a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0406] The tumor inhibition efficacy of the compound was evaluated using the TGI (%), which reflects the rate of tumor growth inhibition. The tumor growth inhibition rate (TGI) (%) was calculated using the following formula, referring to the tumor volume on the first day after grouping: TGI (%) = [1 - (average tumor volume of a given treatment group - average tumor volume of that treatment group at the start of treatment) / (average tumor volume of the solvent control group - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0407] Statistical analysis:

[0408] Statistical analysis was performed using Prism GraphPad software based on tumor volume at the end of the study. Multiple group comparisons were performed using two-way ANOVA and Fisher's LSD test. P < 0.05 was considered significant.

[0409] Experimental results:

[0410] In vivo efficacy study of the combination therapy between MRTX1133 and IN10018 in a subcutaneous homograft tumor model of colorectal cancer in CT-26BALB / c mice

[0411] After cell inoculation, tumor growth was observed every day. Groups were divided according to tumor volume on the 13th day after inoculation. The average tumor volume in the group was about 57 mm 3 Due to tumor burden, the control group was euthanized on the 27th day after inoculation, i.e., the 14th day after group administration; the other treatment groups were euthanized on the 29th day after inoculation, i.e., the 16th day after group administration, and the entire experiment was terminated.

[0412] On the 14th day after group administration, the tumor volume of the control group was 2799.4±687.0mm 3 The tumor volume of the MRTX1133 (5 mg / kg) monotherapy group was 1067.8 ± 302.1 mm 3 The tumor volume of the MRTX1133+IN10018 (5+25 mg / kg) combination group was 674.9±189.0 mm3 Comparing the tumor volume on day 14 with that of the control group, the tumor inhibition rate (TGI) of the MRTX1133 (5 mg / kg) monotherapy group was 63.2% (p<0.0001); the tumor inhibition rate (TGI) of the MRTX1133+IN10018 (5+25 mg / kg) combination therapy group was 77.5% (p<0.0001), both showing significant statistical differences (see Figure 14).

[0413] On the 14th day after group administration, the control group was euthanized due to excessive tumor size, and the other groups continued to be administered and observed until the 16th day. The tumor volume of the MRTX1133 (5 mg / kg) monotherapy group was 1672.1±548.1 mm 3 The tumor volume of the MRTX1133+IN10018 (5+25 mg / kg) combination group was 1013.5±351.8 mm 3 Statistical comparison between the two groups was performed, and the P value was p<0.0001, indicating a significant statistical difference, see Figure 14.

[0414] The evaluation of the tumor inhibition effect of each group is detailed in Table 14. The tumor volume of each dose group at different time periods is shown in Figure 14. Comprehensively considering the entire dosing cycle, the tumor volume of the MRTX1133+IN10018 (5+25 mg / kg) combination treatment group was smaller than that of the MRTX1133 (5 mg / kg) monotherapy group, and both were statistically significant. This indicates that the combined administration of MRTX1133+IN10018 (5+25 mg / kg) has a better effect in inhibiting tumor growth, and IN10018 25 mg / kg can significantly enhance the synergy of the MRTX1133+IN10018 (5+25 mg / kg) combination treatment.

[0415] Table 14: Evaluation of the tumor inhibition effect of the test substance on the mouse colon cancer CT26 cell BALB / c mouse transplant tumor model (based on the data on the 14th / 16th day after group administration)

[0416] Note:

[0417] 1. Calculated according to the number of days after group administration, the data are mean ± standard deviation (mean ± SD);

[0418] 2. TGI (%) = [1-(T 14 -T0) / (V 14 -V0)]×100%;

[0419] 3. ****: p < 0.0001, vs. control group, Two-way ANOVA;

[0420] 4. ****: p < 0.0001, vs. MRTX1133 + IN10018 (5 + 25 mg / kg) group, Two-way ANOVA;

[0421] In vivo efficacy study of the combination of MRTX1133, TIGIT, and IN10018 in a CT26 colorectal cancer subcutaneous homograft model in BALB / c mice

[0422] After cell inoculation, tumor growth was observed every day. As described above, the patients were grouped according to tumor volume on the 13th day after inoculation. The average tumor volume in the group was approximately 57 mm 3 Due to tumor burden, the control group was euthanized on the 27th day after inoculation, i.e., the 14th day after group administration; the other treatment groups were euthanized on the 29th day after inoculation, i.e., the 16th day after group administration, and the entire experiment was terminated.

[0423] On the 14th day after group administration, the tumor volume of the control group was 2799.4±687.0mm 3 The tumor volumes of the MRTX1133 (5 mg / kg) and TIGIT (3 mg / kg) monotherapy groups were 1067.8 ± 302.1 mm 3 and 976.8±640.4mm 3 The tumor volumes of the MRTX1133+IN10018 (5+25 mg / kg) and IN10018+TIGIT (25+3 mg / kg) combination groups were 674.9±189.0 mm 3 and 1315.6±870.2mm 3 The tumor volume of the MRTX1133+IN10018+TIGIT (5+25+3 mg / kg) triple-drug combination group was 535.5±288.9 mm 3 . Compared with the control group, the tumor inhibition rates (TGI) of the MRTX1133 (5 mg / kg) and TIGIT (3 mg / kg) single-drug groups were 63.2% (p<0.0001) and 66.5% (p<0.0001), respectively; the tumor inhibition rates (TGI) of the MRTX1133+IN10018 (5+25 mg / kg) and IN10018+TIGIT (25+3 mg / kg) combination groups were 77.5% (p<0.0001) and 54.1% (p<0.0001), respectively; and the tumor inhibition rate (TGI) of the MRTX1133+IN10018+TIGIT (5+25+3 mg / kg) triple-drug combination group was 82.5% (p<0.0001), all of which were statistically significant.

[0424] On the 14th day after group administration, the control group was euthanized due to excessive tumor size, and the other groups continued to be administrated and observed until the 16th day. The tumor volumes of the MRTX1133 (5 mg / kg) and TIGIT (3 mg / kg) monotherapy groups were 1672.1±548.1 mm, respectively. 3 and 1225.8±814.5mm 3 The tumor volumes of the MRTX1133+IN10018 (5+25 mg / kg) and IN10018+TIGIT (25+3 mg / kg) combination groups were 1013.5±351.8 mm 3 and 1857±1240.4mm 3 The tumor volume of the MRTX1133+IN10018+TIGIT (5+25+3 mg / kg) triple-drug combination group was 809.3±511.4 mm 3 Statistical analysis was performed to compare the comprehensive tumor volume with the MRTX1133+IN10018+TIGIT (5+25+3 mg / kg) triple-drug combination group. The P values ​​of the MRTX1133 (5 mg / kg) and TIGIT (3 mg / kg) single-drug groups were p<0.0001 and p=0.0339, respectively; the P values ​​of the MRTX1133+IN10018 (5+25 mg / kg) and IN10018+TIGIT (25+3 mg / kg) two-drug combination groups were p=0.2965 and p<0.0001, respectively, which were statistically significant compared with the combination treatment groups except MRTX1133+IN10018 (5+25 mg / kg).

[0425] The tumor inhibition evaluation of each group is detailed in Table 15. The tumor volumes of each dose group at different time periods are shown in Figure 15. Taking into account the entire dosing cycle, the tumor volume of the MRTX1133+IN10018+TIGIT (5+25+3 mg / kg) triple-drug combination treatment group was also one of the smallest groups.

[0426] Table 15: Evaluation of the tumor inhibition effect of the test substance on the mouse colon cancer CT26 cell BALB / c mouse transplant tumor model (based on the data on the 14th / 16th day after group administration)

[0427] Note:

[0428] 1. Calculated according to the number of days after group administration, the data are mean ± standard deviation (mean ± SD);

[0429] 2. TGI (%) = [1-(T 14 -T0) / (V 14 -V0)]×100%;

[0430] 3. ****: p < 0.0001, vs. control group, Two-way ANOVA;

[0431] 4. *: p < 0.05, ****: p < 0.0001, vs. MRTX1133 + IN10018 + TIGIT (5 + 25 + 3 mg / kg) group, Two-way ANOVA;

[0432] Study on the effect of combination therapy of MRTX1133, TIGIT and IN10018 on body weight changes and clinical status in the CT26 mouse colorectal cancer subcutaneous transplant model of BALB / c mice

[0433] The experiment was carried out according to the dosing regimen. During the experiment, the animals' activities such as eating and drinking were observed every day, and the weight of the animals was recorded 2 to 3 times a week. On the day of euthanasia of the animals in each group, that is, the 14th day after the group administration in the control group, and the 16th day after the group administration in the other groups, the weight of the control group changed from 19.4g on the day of group administration (Day 0) to 23.7g on the 14th day, with a weight change rate of 22.0%; the average weight of the MRTX1133 (5mg / kg) and TIGIT (3mg / kg) single-drug groups changed from 19.4g and 19.6g on the day of group administration (Day 0) to 22.3g and 23.1g on the 16th day, with a weight change rate of 15.1% and 18.2%, respectively; the weight of the MRTX1133+IN10018 (5+25mg / kg) and IN10018+TIGIT (25+3mg / kg) combination groups changed from 19.4g and 19.6g on the day of group administration (Day 0) to 22.3g and 23.1g on the 16th day, respectively, with a weight change rate of 15.1% and 18.2%, respectively. The body weight of the MRTX1133+IN10018+TIGIT (5+25+3 mg / kg) triple-drug combination group changed from 19.5 g on the day of group administration (Day 0) to 21.2 g on the 16th day, with a body weight change rate of 8.5%.

[0434] Overall, there was no significant weight loss in the animals in each group throughout the entire dosing cycle, and the animals were in good spirits, with normal feeding and motility. The weight data for each group are shown in Table 16, and the weight changes and change rates of each dose group at different time periods are shown in Figure 16. There was no significant weight loss in the animals in each group, and the animals were in good spirits, with normal feeding and motility, demonstrating tolerance to the combination treatment of MRTX1133 (5 mg / kg), TIGIT (3 mg / kg), and IN10018 (25 mg / kg).

[0435] Table 16: Evaluation of the body weight changes of the test substance in the BALB / c mouse transplant tumor model of mouse colon cancer CT26 cells (based on the data on the 14th / 16th day after group administration)

[0436] Note:

[0437] 1. Calculate the number of animals surviving on day 0 / day 14 (control group) or day 16 (other groups) according to the number of days after drug administration.

[0438] 2. Data are mean ± standard deviation (mean ± SD);

[0439] 3. The control group is calculated based on the data on the 14th day after group administration, and the other groups are calculated based on the data on the 16th day after group administration;

[0440] 4. Weight change rate = [1-(W 14 / 16 -W0) / W0]*100%;

[0441] Example 10: Study on the induction of immunogenic cell death targets by the test compounds Lurbinectedin and AMP945 on mouse breast cancer 4T1 cells in vitro

[0442] Experimental Materials:

[0443] 1) Drugs used in this experiment

[0444] Lurbinectedin was provided by DC Chemicals, Lot No.: DC12502

[0445] AMP945 provided by MCE, Lot No.:143253

[0446] 2) Antibodies used in this experiment

[0447] Restructuring Alexa 647 fluorescent anti-calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773). Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).

[0448] Experimental methods:

[0449] 4T1 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) were cultured in RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp) at 37°C and 5% CO2. Two to three times a week, cells were routinely digested and passaged using trypsin. When the cells were in the exponential growth phase and 80%-90% confluent, they were harvested and plated. After trypsinization, the 4T1 cells were harvested and counted. Based on the count results, the cells were diluted with RPMI 1640 + 10% FBS to a concentration of 50,000 cells / mL. The cells were then plated in 12-well cell culture plates, with 2 mL of cell suspension per well, i.e., 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell spreading, six groups were set up: the first group served as the control group, receiving culture medium; the second group received AMP945 at a concentration of 3 μM; the third group received AMP945 at a concentration of 6 μM; the fourth group received Lurbinectedin at a concentration of 0.002 μM; the fifth group received a combination of AMP945 (3 μM) and Lurbinectedin (0.002 μM); and the sixth group received a combination of AMP945 (6 μM) and Lurbinectedin (0.002 μM). The drugs were mixed and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0450] Experimental results

[0451] After 48 hours of drug treatment, cells were collected for flow analysis and washed twice with flow cytometry buffer (PBS + 2% FBS). 0.5 μl of AF647 anti-calreticulin antibody (abcam) was added to each well and mixed. Incubated at 4°C in the dark for 20 minutes, flow cytometry buffer was added, and 195 μl of Annexin-V-FITC conjugate was added using an annexin staining kit (Beyotime). After pipetting and mixing with the cells, 5 μl of Annexin-V-FITC antibody was added and gently mixed. Finally, 10 μl of PI dye was added and mixed. The mixture was incubated at room temperature in the dark for 15 minutes, and the samples were sent to a flow cytometer for signal measurement.

[0452] The results of flow cytometry analysis showed that the CRT positivity rate and Annexin-V positivity rate of the combination of the two drugs were significantly better than those of the single drug group and the control group, as shown in Figure 17.

[0453] Example 11: Study on the induction of immunogenic cell death targets by the test compounds Crizotinib and AMP945 on mouse breast cancer 4T1 cells in vitro

[0454] Experimental Materials:

[0455] 1) Drugs used in this experiment

[0456] Crizotinib was provided by MCE, Lot No.: 06049

[0457] AMP945 provided by MCE, Lot No.:143253

[0458] 2) Antibodies used in this experiment

[0459] Restructuring Alexa 647 fluorescent anti-calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773). Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).

[0460] Experimental methods:

[0461] 4T1 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60352) were cultured in RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp) at 37°C and 5% CO2. Two to three times a week, cells were routinely digested and passaged using trypsin. When the cells were in the exponential growth phase and 80%-90% confluent, they were harvested and plated. After trypsinization, the 4T1 cells were harvested and counted. Based on the count results, the cells were diluted in RPMI 1640 + 10% FBS to a concentration of 50,000 cells / mL. The cells were then plated in 12-well cell culture plates, with 2 mL of cell suspension per well, i.e., 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO2 incubator. After 24 hours of cell spreading, six groups were set up: Group 1, a control group, received culture medium; Group 2, AMP945 at a concentration of 3 μM; Group 3, AMP945 at a concentration of 6 μM; Group 4, crizotinib at a concentration of 3 μM; Group 5, a combination of AMP945 (3 μM) and crizotinib (3 μM); and Group 6, a combination of AMP945 (6 μM) and crizotinib (3 μM). The drugs were mixed and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0462] Experimental results

[0463] After 48 hours of drug treatment, cells were collected for flow analysis and washed twice with flow cytometry buffer (PBS + 2% FBS). 0.5 μl of AF647 anti-calreticulin antibody (abcam) was added to each well and mixed. Incubated at 4°C in the dark for 20 minutes, flow cytometry buffer was added, and 195 μl of Annexin-V-FITC binding solution was added using the Annexin staining kit (Beyotime). After pipetting and mixing with the cells, 5 μl of Annexin-V-FITC antibody was added and gently mixed. Finally, 10 μl of PI dye was added and mixed. The mixture was incubated at room temperature in the dark for 15 minutes, and the samples were sent to the flow cytometer for signal measurement.

[0464] The results of flow cytometry analysis showed that the CRT positivity rate and Annexin-V positivity rate of the combination of the two drugs were significantly better than those of the single drug group and the control group, as shown in Figure 18.

[0465] All references mentioned in this application are incorporated herein by reference in their entirety, just as if each reference were listed separately. It should be understood that after reading the disclosure of this application, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the claims appended hereto.

Claims

1. Use of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor in the preparation of a medicament for treating a tumor in a subject, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

2. A pharmaceutical combination product comprising a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor for treating a tumor in a subject, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

3. A method for treating tumors, comprising administering to a subject a therapeutically effective amount of a FAK inhibitor, a substance that induces immunogenic cell death, and an immune checkpoint inhibitor, wherein the substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

4. The use, pharmaceutical combination or method according to any one of claims 1 to 3, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows:

5. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

6. The use, pharmaceutical combination or method of claim 5, wherein the RNA polymerase II inhibitor is lurbinectedin, SEL-120 or a pharmaceutically acceptable salt thereof.

7. The use, pharmaceutical combination or method according to any one of claims 5 to 6, wherein the RNA polymerase II inhibitor is lurbinectedin.

8. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

9. The use, pharmaceutical combination product or method according to claim 8, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

10. The use, pharmaceutical combination or method according to any one of claims 8 to 9, wherein the ALK / ROS1 inhibitor is crizotinib.

11. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

12. The use, pharmaceutical combination or method according to claim 11, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

13. The use, pharmaceutical combination or method according to any one of claims 11 to 12, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

14. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

15. The use, pharmaceutical combination or method of claim 14, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000 or a pharmaceutically acceptable salt thereof.

16. The use, pharmaceutical combination or method according to any one of claims 14-15, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

17. The use, pharmaceutical combination product or method according to any one of claims 1 to 16, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor or a TIGIT antibody.

18. The use, pharmaceutical combination or method according to any one of claims 1 to 17, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, tislelizumab, nivolumab, toripalimab, atezolizumab, durvalumab, avelumab, atezolizumab, camrelizumab, sintilimab, cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005 or MX-10181.

19. The use, pharmaceutical combination, or method according to any one of claims 1 to 17, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

20. The use, pharmaceutical combination product or method according to any one of claims 1 to 17, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody 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.

21. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is lurbinectedin; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

22. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

23. The use, pharmaceutical combination or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

24. The use, pharmaceutical combination product or method according to any one of claims 1 to 4, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

25. The use, pharmaceutical combination or method according to any one of claims 1 to 24, wherein the FAK inhibitor, the substance that induces immunogenic cell death and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.

26. The use, pharmaceutical combination or method of any one of claims 1 to 25, wherein the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, Liposarcoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovarian cancer, salivary gland cancer, metastases caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), gastric cancer, melanoma or pancreatic cancer.

27. The use, pharmaceutical combination or method according to any one of claims 1 to 26, wherein the tumor is lung cancer, colon cancer (including colorectal cancer) or breast cancer.

28. A kit or pharmaceutically acceptable composition comprising: (a) FAK inhibitors; (b) substances that induce immunogenic cell death; and (c) immune checkpoint inhibitors, The substance that induces immunogenic cell death is an RNA polymerase II inhibitor, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor or a KRAS G12D inhibitor.

29. The kit or composition according to claim 28, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, wherein the structure of IN10018 is as follows:

30. The kit or composition according to any one of claims 28 to 29, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

31. The kit or composition according to claim 30, wherein the RNA polymerase II inhibitor is lurbinectedin, SEL-120 or a pharmaceutically acceptable salt thereof.

32. The kit or composition of any one of claims 30-31, wherein the inhibitor of RNA polymerase II is lurbinectedin.

33. The kit or composition according to any one of claims 28 to 29, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

34. The kit or composition of claim 33, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922, Lorlatinib), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

35. The kit or composition of any one of claims 33-34, wherein the ALK / ROS1 inhibitor is crizotinib or a pharmaceutically acceptable salt thereof.

36. The kit or composition of any one of claims 28-29, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

37. The kit or composition of claim 36, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036, or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510), or a pharmaceutically acceptable salt thereof.

38. The kit or composition of any one of claims 36-37, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

39. The kit or composition of any one of claims 28-29, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

40. The kit or composition of claim 39, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

41. The kit or composition of any one of claims 39-40, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

42. The kit or composition of any one of claims 28-41, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, a PD-1 / PD-L1 small molecule inhibitor, or a TIGIT antibody.

43. The kit or composition of any one of claims 28 to 42, wherein the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody, and further, the anti-PD-1 / PD-L1 antibody is pembrolizumab, Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, durvalumab, Avelumab, Atezolizumab, Camrelizumab, Sintilimab, Cemiplimab, envafolimab, BMS-936559, JS003, SHR-1316, GS-4224, AN-4005, or MX-10181.

44. The kit or composition of any one of claims 28 to 42, wherein the immune checkpoint inhibitor is a PD-1 / PD-L1 small molecule inhibitor, and further, the PD-1 / PD-L1 small molecule inhibitor is INCB-086550, lazertinib, IMH-010, CA-170, ABSK043, or RRx-001.

45. The kit or composition of any one of claims 28 to 42, wherein the immune checkpoint inhibitor is a TIGIT antibody, and further, the TIGIT antibody 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.

46. ​​The kit or composition of any one of claims 28-29, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is lurbinectedin; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

47. The kit or composition of any one of claims 28-29, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is crizotinib or a pharmaceutically acceptable salt thereof; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

48. The kit or composition of any one of claims 28-29, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, and the substance that induces immunogenic cell death is D-1553 or a pharmaceutically acceptable salt thereof; the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody or a PD-1 / PD-L1 small molecule inhibitor, in particular, the immune checkpoint inhibitor is an anti-PD-1 / PD-L1 antibody.

49. The kit or composition of any one of claims 28-29, wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the substance that induces immunogenic cell death is MRTX1133 or a pharmaceutically acceptable salt thereof; and the immune checkpoint inhibitor is a TIGIT antibody.

50. A kit or composition as claimed in any one of claims 28 to 49 for use as a medicament.

51. The kit or composition of claim 50, wherein the drug is used to treat a tumor, and the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer , ovarian cancer, salivary gland cancer, metastases caused by spindle cell cancer, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), gastric cancer, melanoma or pancreatic cancer.

52. The kit or composition of claim 51, wherein the tumor is lung cancer, colon cancer (including colorectal cancer), or breast cancer.

53. A FAK inhibitor for use in the treatment of tumors to enhance immunogenic cell death induced by a substance that induces immunogenic cell death, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II, an ALK / ROS1 inhibitor, a KRAS G12C inhibitor, or a KRAS G12D inhibitor.

54. The FAK inhibitor according to claim 53, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886, or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945, or a pharmaceutically acceptable salt thereof, further preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, and the structure of IN10018 is as follows: The FAK inhibitor according to claim 53 or 54, wherein the substance that induces immunogenic cell death is an inhibitor of RNA polymerase II.

56. The FAK inhibitor according to claim 55, wherein the RNA polymerase II inhibitor is lurbinectedin, SEL-120 or a pharmaceutically acceptable salt thereof, in particular lurbinectedin. The FAK inhibitor according to claim 53 or 54, wherein the substance that induces immunogenic cell death is an ALK / ROS1 inhibitor.

58. The FAK inhibitor of claim 57, wherein the ALK / ROS1 inhibitor is crizotinib, SIM-0201, XZP-3621, TQ-B3139, SAF-189s, Ceritinib, Lorlatinib (PF-06463922), Alectinib, Ensartinib, APG-2449, Brigatinib, TQ-B3101, Entrectinib, Repotrectinib or a pharmaceutically acceptable salt thereof, in particular crizotinib, Entrectinib or a pharmaceutically acceptable salt thereof.

59. The FAK inhibitor of any one of claims 57-58, wherein the ALK / ROS1 inhibitor is crizotinib.

60. The FAK inhibitor according to claim 53 or 54, wherein the substance that induces immunogenic cell death is a KRAS G12C inhibitor.

61. The FAK inhibitor of claim 60, wherein the KRAS G12C inhibitor is D-1553, ARS-3248, GF-105, JAB-21822, JDQ-443, LY-3537982, Sotorasib (Sotorasib / AMG510), Adagrasib (MRTX849), GDC-6036 or a pharmaceutically acceptable salt thereof, in particular D-1553, Sotorasib (Sotorasib / AMG510) or a pharmaceutically acceptable salt thereof.

62. The FAK inhibitor according to any one of claims 60-61, wherein the KRAS G12C inhibitor is D-1553 or a pharmaceutically acceptable salt thereof.

63. The FAK inhibitor according to claim 53 or 54, wherein the substance that induces immunogenic cell death is a KRAS G12D inhibitor.

64. The FAK inhibitor of claim 63, wherein the KRAS G12D inhibitor is MRTX1133, HRS-4642, JAB-22000, or a pharmaceutically acceptable salt thereof.

65. The FAK inhibitor of any one of claims 63-64, wherein the KRAS G12D inhibitor is MRTX1133 or a pharmaceutically acceptable salt thereof.

66. The FAK inhibitor of any one of claims 53 to 65, wherein the drug is used to treat a tumor, and the tumor is bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal carcinoma, neuroendocrine cancer, Secretory carcinoma, ovarian cancer, salivary gland cancer, metastatic tumor caused by spindle cell carcinoma, anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma or hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is breast cancer, ovarian cancer, colon cancer (including colorectal cancer), lung cancer (including small cell lung cancer and non-small cell lung cancer), gastric cancer, melanoma or pancreatic cancer.

67. The FAK inhibitor of claim 66, wherein the tumor is lung cancer or colon cancer (including colorectal cancer) or breast cancer.

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