Method for increasing concentration of macromolecular drug in tumor tissue

Abstract

According to the invention, IN10018 and a macromolecular drug are combined to treat tumors, and the macromolecular drug is a monoclonal antibody, a bispecific antibody or an antibody conjugate.

Description

Method for increasing the concentration of macromolecular drugs in tumor tissue

[0001] This application claims priority to Chinese application No. 202310343152.0 filed on March 31, 2023, Chinese application No. 202311276741.8 filed on September 28, 2023, and Chinese application No. 202410283445.9 filed on March 12, 2024. Field of the Invention

[0002] The present invention belongs to the field of medicinal chemistry and specifically relates to the combined use of focal adhesion kinase (FAK) inhibitor IN10018 and macromolecular drugs in treating tumors. Background Art

[0003] Malignant tumors (cancer) have become a major public health issue posing a serious threat to global health. According to the latest statistics, malignant tumor deaths account for 23.91% of all causes of death in China, and the incidence and mortality of malignant tumors have been steadily increasing over the past decade. Annual medical expenses related to malignant tumors in China exceed 220 billion RMB.

[0004] In recent years, the development of large-molecule drugs, primarily monoclonal antibodies, bispecific antibodies, and antibody conjugates, has steadily accelerated, with numerous large-molecule drugs entering the anti-cancer market. These drugs can precisely recognize tumor antigens and, through blocking antigen-mediated downstream reactions or by carrying small-molecule conjugates, precisely kill tumor cells, thereby achieving effective anti-tumor activity. Tumor tissue consists of tumor cells and the surrounding stroma. This stroma-rich tumor microenvironment harbors numerous mechanisms that hinder the diffusion (or penetration) of large-molecule drugs. These interstitial barriers, including fibroblasts and fibrotic collagen, which are in close contact with tumor cells, further hinder the effective delivery of anti-cancer drugs to tumor cells. Based on this mechanism, agents that inhibit tumor fibrosis or reduce the presence of fibrotic stroma may help increase the concentration of large-molecule drugs in tumor tissue. Therefore, developing drugs that inhibit tumor fibrosis or reduce the presence of fibrotic stroma may advance cancer treatment and hold significant clinical value.

[0005] Summary of the Invention

[0006] The inventors discovered that the focal adhesion kinase (FAK) inhibitor IN10018 helps increase the concentration of macromolecular drugs in tumor tissue. Regardless of the rationale, it is believed that activated FAK pathways significantly promote tumor fibrosis, and that the FAK inhibitor IN10018 can reduce FAK activity and significantly reduce tumor fibrosis, thereby facilitating the penetration of macromolecular drugs into the tumor microenvironment.

[0007] In one aspect, the present disclosure provides use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a drug for increasing the concentration of a macromolecular drug in tumor tissue, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. The structure of IN10018 is as follows:

[0008] In another aspect, the present disclosure provides a pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug for treating a tumor in a subject. The macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. The structure of IN10018 is as follows:

[0009] In another aspect, the present disclosure provides the use of IN10018 or a pharmaceutically acceptable salt thereof for increasing the concentration of a macromolecular drug in tumor tissue, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. The structure of IN10018 is as follows:

[0010] In another aspect, the present disclosure provides a method for treating tumors, comprising administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug to a subject in need thereof, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. The structure of IN10018 is as follows:

[0011] Another aspect of the present disclosure provides a kit or a pharmaceutically acceptable composition comprising:

[0012] (a) IN10018 or a pharmaceutically acceptable salt thereof; and

[0013] (b) macromolecular drugs, wherein the macromolecular drugs are monoclonal antibodies, bispecific antibodies or antibody conjugates;

[0014] The structure of IN10018 is as follows:

[0015] In another aspect, the present disclosure provides a pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody or an antibody conjugate, for treating a tumor in a subject.

[0016] In another aspect, the present disclosure provides the use of IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug in the preparation of a combination drug for treating tumors, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate.

[0017] In another aspect, the present disclosure provides the use of IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a drug for use in combination with a macromolecular drug for treating tumors, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate.

[0018] In another aspect, the present disclosure provides the use of a macromolecular drug in preparing a drug for use in combination with IN10018 or a pharmaceutically acceptable salt thereof for treating tumors, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate.

[0019] In another aspect, the present disclosure provides a kit comprising: IN10018 or a pharmaceutically acceptable salt thereof; and instructions, wherein the instructions indicate that IN10018 or a pharmaceutically acceptable salt thereof can be used for treating tumors in combination with a macromolecular drug, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate.

[0020] In another aspect, the present disclosure provides a kit comprising: a macromolecular drug; and instructions indicating that the macromolecular drug can be used in combination with IN10018 or a pharmaceutically acceptable salt thereof to treat tumors; the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate.

[0021] Optionally, the pharmaceutically acceptable salt of IN10018 is tartrate.

[0022] Optionally, the monoclonal antibody is racotumomab, rituximab, iodine [131I] metuximab (131I-Metuximab), JMT-103, necitumumab, alemtuzumab, elotuzumab, bevacizumab, ofatumumab, tocilizumab, atezolizumab, toripalimab, HX-008, carrelizumab, Camrelizumab, Ocrelizumab, Sugemalimab, Lenzilumab, Sintilimab, Vilobelimab, Margetuximab, Siltuximab, Mogamulizumab, Amivantamab, Cadonilimab, Inebilizumab, Iodine I 131 derlotuximabbiotin), Isatuximab, Serplulimab, Retifanlimab, Cetuximab, Adebrelimab, Tislelizumab, Penpulimab, Teprotumumab, Itolizumab, Dostarlimab, Denosumab umab), Obinutuzumab, Nimotuzumab, Teclistamab, Daratumumab, Dinutuximab, Tafasitamab, Socazolimab, Dupilumab, Prolgolimab, Blinatumomab, Geptaluzumab anolimab), panitumumab, canakinumab, ramucirumab, envafolimab, belimumab, leronlimab, ranibizumab, natalizumab, cosibelimab, zimberelimab, trastuzumab ), Catumaxomab, Durvalumab, avelumab, Ublituximab, Cemiplimab, Pembrolizumab, Glofitamab, Pertuzumab, Bermekimab, tremelimumab, ipilimumab, NacituzumabNaxitamab, infliximab, nivolumab, omburtamab, crizanlizumab, burosumab, talquetamab, besilesomab, alirocumab, arcitumomab, or biosimilars thereof; in particular, trastuzumab or a biosimilar thereof; preferably trastuzumab.

[0023] Biosimilars, also known as biosimilars, are biological drugs that are highly similar to approved original biological drugs in terms of quality, safety, and efficacy. Examples include bevacizumab biosimilars, adalimumab biosimilars, and rituximab biosimilars.

[0024] Optionally, the bispecific antibody is Teclistamab, Blinatumomab, Cadonilimab, Mosunetuzumab, Catumaxomab, Ublituximab, Amivantamab, Talquetamab, Epcoritamab, Glofitamab, Zenidatuzumab, Zanidatamab), elranatamab, tebotelimab, amivantamab, SI-B001, odronextamab, KN-026, KN-046, ivonescimab, SHR-1701, M7824, GEN-3009, navicixizumab, GB-261, CM-355, plamotamab, or their biosimilars.

[0025] Optionally, the antibody conjugate is Loncastuximab Tesirine, Ibritumomab Tiuxetan, Tisotumab Vedotin, Sacituzumab Govitecan, Enfortumab Vedotin, Inotuzumab Ozogamicin, Gemtuzumab Ozogamicin, Belantamab Mafodotin, Trastuzumab Emtansine, Moxetumomab Pasudotox, Polatuzumab Vedotin, Disitamab Vedotin, Brentuximab Vedotin, Trastuzumab deruxtecan, DS-8201), Trastuzumab Emtansine, Cetuximab Sarotalocan, Mirvetuximab Soravtansine, Trastuzumab Duocarmazine, ARX-788, KL-A264, Upifimab Rilsodotin, Tusamitamab Ravtansine, Naptumomab Estafenatox, Datopotamab Deruxtecan, Oportuzumab Monatox, SHR-A1811, Patritumab Deruxtecan, Telisotuzumab Vedotin), TrastuzumabDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, BMS-98 6148, SOT-102, ESG-401, CD117-ADC, LCB-14, W-0101, REGN-5093-M114, CX-2029, BDC-1001, DGN549-C (Pivekimab Sunirine), AVID-100, MRG-003, CAT-5001, HDP-101, MORAb-202 (Farletuzumab Ecteribulin), L-DOS47, Praluzatamab Ravtansine, BA3021 (Ozuriftamab Vedotin), DS-7300, BA3011 (Mecbotamab Vedotin), Camidanlumab Tesirine, Ladiratuzumab Vedotin, LMB-2, SAT-012, DEBIO-1562, PSMA-ADC, MRG-002, OBI-999, Coltuximab Ravtansine, KL-A166, DX-126-262, NBE-002, OXS-1550, Lorvotuzumab Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103ADC, TE-1218, TE-1112, T-PNU, BV-001, DS-1062, SKB264, Ab6000-Dxd or their biosimilars; in particular, ESG-401, DS-1062, SKB264, Sacituzumab Govitecan, Trastuzumab deruxtecan, DS-8201), Ab6000-Dxd or its biosimilars; preferably ESG-401, DS-1062, SKB264, Sacituzumab Govitecan / Trastuzumab deruxtecan (DS-8201), or Ab6000-Dxd; preferably ESG-401, Sacituzumab Govitecan / Trastuzumab deruxtecan, DS-8201, or Ab6000-Dxd.Govitecan), Trastuzumab deruxtecan (DS-8201), or Ab6000-Dxd.

[0026] Optionally, the macromolecular drug is an antibody conjugate.

[0027] Optionally, the antibody conjugate is Loncastuximab Tesirine, Ibritumomab Tiuxetan, Tisotumab Vedotin, Sacituzumab Govitecan, Enfortumab Vedotin, Inotuzumab Ozogamicin, Gemtuzumab Ozogamicin, Belantamab Mafodotin, Trastuzumab Emtansine, Moxetumomab Pasudotox, Polatuzumab Vedotin, Disitamab Vedotin, Brentuximab Vedotin, Trastuzumab deruxtecan, DS-8201), Trastuzumab Emtansine, Cetuximab Sarotalocan, Mirvetuximab Soravtansine, Trastuzumab Duocarmazine, ARX-788, KL-A264, Upifimab Rilsodotin, Tusamitamab Ravtansine, Naptumomab Estafenatox, Datopotamab Deruxtecan, Oportuzumab Monatox, SHR-A1811, Patritumab Deruxtecan, Telisotuzumab Vedotin), TrastuzumabDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, BMS-98 6148, SOT-102, ESG-401, CD117-ADC, LCB-14, W-0101, REGN-5093-M114, CX-2029, BDC-1001, DGN549-C (Pivekimab Sunirine), AVID-100, MRG-003, CAT-5001, HDP-101, MORAb-202 (Farletuzumab Ecteribulin), L-DOS47, Praluzatamab Ravtansine, BA3021 (Ozuriftamab Vedotin), DS-7300, BA3011 (Mecbotamab Vedotin), Camidanlumab Tesirine, Ladiratuzumab Vedotin, LMB-2, SAT-012, DEBIO-1562, PSMA-ADC, MRG-002, OBI-999, Coltuximab Ravtansine, KL-A166, DX-126-262, NBE-002, OXS-1550, Lorvotuzumab Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103ADC, TE-1218, TE-1112, T-PNU, BV-001, DS-1062, SKB264, or biosimilars thereof; in particular, ESG-401, DS-1062, SKB264, Sacituzumab Govitecan, Ab6000-Dxd or biosimilars thereof; preferably, ESG-401, DS-1062, SKB264, Sacituzumab Govitecan, Trastuzumab deruxtecan, DS-8201) or Ab6000-Dxd; preferably ESG-401, gosatuzumab (Sacituzumab Govitecan), deruxtecan (Trastuzumabderuxtecan, DS-8201) or Ab6000-Dxd.

[0028] Optionally, the antibody conjugate is a Trop-2 antibody conjugate.

[0029] Optionally, the Trop-2 antibody conjugate is ESG-401, DS-1062, SKB264, gosartan (Sacituzumab Govitecan), or a biosimilar thereof; preferably ESG-401, DS-1062, SKB264 or gosartan (Sacituzumab Govitecan); preferably ESG-401 or gosartan (Sacituzumab Govitecan).

[0030] Optionally, the IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug are administered to the subject simultaneously or sequentially.

[0031] Optionally, the above-mentioned use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition is for treating tumors.

[0032] Optionally, the tumor is selected from bladder cancer, breast cancer, cervical cancer, colon cancer (including colorectal cancer), esophageal cancer, esophageal squamous cell carcinoma, head and neck cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), melanoma, myeloma, rhabdomyosarcoma, inflammatory myofibroblastic tumor, neuroblastoma, pancreatic cancer, prostate cancer, kidney cancer, renal cell carcinoma, sarcoma (including osteosarcoma), skin cancer (including squamous cell carcinoma), gastric cancer, testicular cancer, thyroid cancer, uterine cancer, mesothelioma, bile duct cancer, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastatic tumor caused by spindle cell carcinoma , anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is gastric cancer, lung cancer, breast cancer, glioma, esophageal cancer, pancreatic cancer, head and neck cancer, colon cancer or ovarian cancer; more preferably, the tumor is gastric cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer or lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] Figure 1 shows panoramic scan thumbnails of frozen sections from Example 1. Images (thumbnail) were obtained by scanning the entire section using a digital pathology imaging panoramic scanning system. Blue represents DAPI-stained cell nuclei, and green represents GFP protein fluorescence from the administered drug, T-GFP. A and B, respectively, are panoramic scans of two animals in the control group; C and D, respectively, are panoramic scans of two animals in the IN10018+T-GFP group.

[0035] Figure 2 shows the percentage of T-GFP-positive cells in Example 1. It shows the percentage of tumor infiltration of the test substance T-GFP in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018.

[0036] Figure 3 shows the H-Score of T-GFP-positive cells in Example 1. It shows the H-Score of the tumor infiltration of the test substance T-GFP in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018.

[0037] Figure 4 shows the changes in body weight of different groups of MDA-MB-468 tumor-bearing mice after administration of Example 2. The data are expressed as mean + standard error (SEM).

[0038] Figure 5 shows the relative body weight changes of MDA-MB-468 tumor-bearing mice in different groups after administration of Example 2. The data are expressed as mean + standard error (SEM).

[0039] Figure 6 shows the changes in tumor volume in MDA-MB-468 tumor-bearing mice during the administration period of Example 2. The data are expressed as mean + standard error (SEM).

[0040] FIG7 shows thumbnails of panoramic scans of frozen sections in Example 3.

[0041] FIG8 shows the tumor growth curves of mice after administration of different test substances in Example 4. The data points represent the average tumor volume within the group, and the error bars represent the standard error (SEM).

[0042] FIG9 shows the binding of tumor cells to antibodies in each treatment group at the end point of the experiment in Example 5. The staining intensity represents the binding intensity of tumor cells to antibodies in each group.

[0043] FIG10 shows the data of Example 6 on the day of group administration 14 days after vaccination.

[0044] FIG11 shows the total fluorescence intensity of Example 6. The data points represent the total fluorescence intensity of each animal in the group, and the error bars represent the standard error (SEM).

[0045] FIG12 shows the fluorescence images of H-Cy5.5 injection in Example 6, wherein A and B represent the fluorescence images of the G1 control group, C and D represent the fluorescence images of the G2 IN10018 25 mg / kg group, and E represents the fluorescence image of the negative control group.

[0046] Figure 13 shows the mean fluorescence intensity curve of the tumor site of H-Cy5.5, the test substance in Example 6, in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice following administration of IN10018. The value at 0 h represents the mean fluorescence intensity of the negative control group. Data points represent the mean fluorescence intensity within each group, and error bars represent the standard error of the experiment (SEM).

[0047] Figure 14 shows fluorescence images of the test substance H-Cy5.5 in Example 6 48 hours after injection into a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice. A and B represent fluorescence images of the G1 control group, while C and D represent fluorescence images of the G2 IN10018 25 mg / kg group.

[0048] Figure 15 shows the tumor growth curve of the subcutaneous xenograft tumor model of BALB / c-nude mice inoculated with a mixture of NCI-N87 & NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts in Example 7 after administration. The data points represent the mean tumor volume within the group, and the error bars represent the standard error (SEM).

[0049] Figure 16 shows the body weight change curve of the BALB / c-nude mouse subcutaneous xenograft tumor model inoculated with a mixture of NCI-N87 & NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts after administration of the drug. The data points represent the mean body weight within the group, and the error bars represent the standard error (SEM).

[0050] Figure 17 shows the body weight change rate curve of Example 7 NCI-N87 & NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts inoculated subcutaneously in BALB / c-nude mice after administration of the xenograft tumor model. The data points represent the average body weight change rate within the group, and the error bars represent the standard error (SEM).

[0051] Figure 18 shows the percent (%) change in body weight for Example 8. Data points represent the percent group mean change in body weight, and error bars represent the standard error of the mean (SEM).

[0052] Figure 19 shows the tumor growth curves of Example 8. Data points represent group means, and error bars represent standard error of the mean (SEM).

[0053] FIG20 shows the tumor growth curve of Example 9.

[0054] FIG21 shows the tumor growth curve of Example 10.

[0055] FIG22 shows the tumor growth curve of Example 11.

[0056] FIG23 shows the tumor growth curve of Example 12. DETAILED DESCRIPTION

[0057] 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.

[0058] 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.

[0059] 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.

[0060] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0061] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as 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.

[0062] 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.

[0063] 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."

[0064] definition

[0065] The following terms and symbols used in this application have the meanings described below, unless the context indicates otherwise.

[0066] The "monoclonal antibody" used herein refers to monoclonal antibody drugs, i.e., monoclonal antibody drugs, which are generally biological preparations, are large molecular proteins, and their target is a specific protein on the cell membrane. In some embodiments, the monoclonal antibody is Racotumomab, Rituximab, Iodine [131I] Metuximab (131I-Metuximab), JMT-103, Necitumumab, Alemtuzumab, Elotuzumab, Bevacizumab, Ofatumumab, Tocilizumab, Atezolizumab, Toripalimab, HX-008, Carrelizumab, Camrelizumab, Ocrelizumab, Sugemalimab, Lenzilumab, Sintilimab, Vilobelimab, Margetuximab, Siltuximab, Mogamulizumab, Amivantamab, Cadonilimab, Inebilizumab, Iodine I 131derlotuximabbiotin), Isatuximab, Serplulimab, Retifanlimab, Cetuximab, Adebrelimab, Tislelizumab, Penpulimab, Teprotumumab, Itolizumab, Dostarlimab, Denosumab, Obinutuzumab, Nimotuzumab, Terituzumab Teclistamab, Daratumumab, dinutuximab, Tafasitamab, Socazolimab, Dupilumab, Prolgolimab, Blinatumomab, Geptanolimab, Panitumumab, Canakinumab, Ramucirumab, Envafolimab, Belimumab, LeliLeronlimab, Ranibizumab, Natalizumab, Cosibelimab, Zimberelimab, Trastuzumab, Catumaxomab, Durvalumab, Avelumab, Ublituximab, Cemiplimab, Pembrolizumab, Glofitamab, Pertuzumab, Bermekimab, Tesitumumab remelimumab), ipilimumab, naxitamab, infliximab, nivolumab, omburtamab, crizanlizumab, burosumab, talquetamab, besilesomab, alirocumab, arcitumomab, or a biosimilar thereof; in some embodiments, trastuzumab or a biosimilar thereof; in some embodiments, trastuzumab.

[0067] As used herein, "bispecific antibodies" (bsAb, Bispecific Antibody) refers to antibodies that have two specific antigen-binding sites. In some embodiments, the bispecific antibodies are teclistamab, blinatumomab, cadonilimab, mosunetuzumab, catumaxomab, ublituximab, amivantamab, talquetamab, epcoritamab, glofitamab, zenidatuzumab, tacs ... Zanidatamab, elranatamab, tebotelimab, amivantamab, SI-B001, odronextamab, KN-026, KN-046, ivonescimab, SHR-1701, M7824, GEN-3009, navicixizumab, GB-261, CM-355, plamotamab, or their biosimilars.

[0068] As used herein, an "antibody-drug conjugate" (ADC) refers to a drug in which a biologically active small molecule drug is linked to a monoclonal antibody via a chemical linker. The monoclonal antibody then acts as a carrier to deliver the small molecule drug to target cells. In some embodiments, the macromolecular drug is an antibody conjugate. For example, the antibody conjugates are Loncastuximab Tesirine, Ibritumomab Tiuxetan, Tisotumab Vedotin, Sacituzumab Govitecan, Enfortumab Vedotin, Inotuzumab Ozogamicin, Gemtuzumab Ozogamicin, Belantamab Mafodotin, Trastuzumab Emtansine, Moxetumomab Pasudotox, Polatuzumab Vedotin, Disitamab Vedotin, Brentuximab Vedotin, Trastuzumab deruxtecan, DS-8201), Trastuzumab Emtansine, Cetuximab Sarotalocan, Mirvetuximab Soravtansine, Trastuzumab Duocarmazine, ARX-788, KL-A264, Upifimab Rilsodotin, Tusamitamab Ravtansine, Naptumomab Estafenatox, Datopotamab Deruxtecan, Oportuzumab Monatox, SHR-A1811, Patritumab Deruxtecan, Telisotuzumab Vedotin), TrastuzumabDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, BMS-98 6148, SOT-102, ESG-401, CD117-ADC, LCB-14, W-0101, REGN-5093-M114, CX-2029, BDC-1001, DGN549-C (Pivekimab Sunirine), AVID-100, MRG-003, CAT-5001, HDP-101, MORAb-202 (Farletuzumab Ecteribulin), L-DOS47, Praluzatamab Ravtansine, BA3021 (Ozuriftamab Vedotin), DS-7300, BA3011 (Mecbotamab Vedotin), Camidanlumab Tesirine, Ladiratuzumab Vedotin, LMB-2, SAT-012, DEBIO-1562, PSMA-ADC, MRG-002, OBI-999, Coltuximab Ravtansine, KL-A166, DX-126-262, NBE-002, OXS-1550, Lorvotuzumab Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103ADC, TE-1218, TE-1112, T-PNU, BV-001, Ab6000-Dxd, or a biosimilar thereof; in some embodiments, ESG-401, Sacituzumab Govitecan, Trastuzumab deruxtecan (DS-8201), Ab6000-Dxd, or a biosimilar thereof; in some embodiments, ESG-401, Sacituzumab Govitecan, or Trastuzumab deruxtecan (DS-8201), Ab6000-Dxd, or a biosimilar thereof deruxtecan, DS-8201) or Ab6000-Dxd.

[0069] Trastuzumab is an anti-Her 2 monoclonal antibody.

[0070] ESG-401 is an antibody conjugate targeting Trop-2 jointly developed by Shanghai Shijian Biotechnology Co., Ltd. and Suzhou Lianning Biopharmaceutical Co., Ltd., disclosed in WO2021225892A1.

[0071] Sacituzumab Govitecan is an antibody conjugate targeting Trop-2 developed by Immunomedics, with the trade name Trodelvy.

[0072] 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.

[0073] 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.

[0074] "Simultaneous or sequential administration" in this application refers to the simultaneous or sequential administration of two or more drugs within a dosing cycle (e.g., within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours) or at certain time intervals. The modes of drug administration (e.g., oral, intravenous, intramuscular, or subcutaneous administration, etc.) may be the same or different, and the dosing frequency / cycle of the two or more drugs may be the same or different. When the treatment method, product, or use of the present disclosure involves two drugs, the two drugs may be administered simultaneously or separately at certain time intervals.

[0075] 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.

[0076] 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, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine cancer, ovarian cancer, salivary gland cancer, metastatic tumors caused by spindle cell carcinoma. , anaplastic large cell lymphoma, undifferentiated thyroid cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, glioma and hematological malignancies, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML); preferably, the tumor is lung cancer, breast cancer, glioma, esophageal cancer, pancreatic cancer, head and neck cancer, colon cancer or ovarian cancer; more preferably, the tumor is gastric cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer or lung cancer.

[0077] 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.

[0078] As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerable, and suitable for administration to a subject.

[0079] 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.

[0080] In addition, pharmaceutically acceptable acid addition salts can be prepared 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 readily identify various synthetic methods for preparing non-toxic pharmaceutically acceptable acid addition salts without undue experimentation. In some embodiments, the pharmaceutically acceptable salt of IN10018 is a tartrate salt.

[0081] 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.).

[0082] As used herein, the term "inhibit" refers to a decrease in the baseline activity of a biological activity or process.

[0083] As used herein, the term "kit" refers to a box for containing chemical reagents for detecting chemical components, drug residues, virus species, etc. The kit of the present invention may include (i) IN10018 or a pharmaceutically acceptable salt thereof and / or a macromolecular drug; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug can be used to treat tumors in a subject, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug can be used to treat tumors in a subject, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. In one embodiment, the kit includes (i) a macromolecular drug; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug can be used to treat tumors in a subject, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate. In one embodiment, the kit includes (i) IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug; and (ii) instructions indicating that IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug can be used to treat a tumor in a subject, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody conjugate.

[0084] The compound of the test kit can be contained in a separate container. Alternatively, two or more compounds are contained in the same container. For example, the test kit can include a first container, a second container and a package insert, wherein the first container includes at least one dose of a drug including IN10018 or a pharmaceutically acceptable salt thereof, the second container includes at least one dose of a macromolecular drug, the macromolecular drug being a monoclonal antibody, a bispecific antibody or an antibody conjugate, and the package insert includes instructions for using the drug to treat the tumor of the subject. The first container and the second container can include the same or different shapes (e.g., vials, syringes and bottles) and / or materials (e.g., plastic or glass). The test kit can also include other materials that can assist in administering the drug, such as diluents, filters, IV bags and lines, needles and syringes.

[0085] The precise amount of IN10018, or a pharmaceutically acceptable salt thereof, and the macromolecular drug administered to a subject will depend on various factors, such as the specific drug or compound, the drug formulation, the route of administration, the type of disease, the condition, the identity of the subject or host being treated, etc., but can nevertheless be routinely determined by one skilled in the art. For example, determining an effective amount will also depend on the extent, severity, and type of cell proliferation. A skilled artisan will be able to determine an appropriate dosage based on these and other factors.

[0086] IN10018 or its pharmaceutically acceptable salt and macromolecular drug can be administered by a suitable route such as oral, intravenous, intramuscular or subcutaneous administration.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] The amount of IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug 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.

[0092] In some embodiments, IN10018 or a pharmaceutically acceptable salt thereof is administered at a dose of 5 mg / day to 100 mg / day, eg, 20 mg / day, in adults, calculated as the free base.

[0093] The macromolecular drug is administered in a dosage range of 1-20 mg / kg per week in adults. In a specific embodiment, trastuzumab is administered in a dosage range of 1-5 mg / kg, for example, 2 mg / kg per week in adults. In a specific embodiment, ESG-401 is administered in a dosage range of 2-20 mg / kg, for example, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg per week in adults. In a specific embodiment, gosartuzumab is administered in a dosage range of 2-20 mg / kg, for example, 6 mg / kg or 10 mg / kg per week in adults.

[0094] 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.

[0095] Example

[0096] 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.

[0097] The experimental methods in the following examples without specifying specific conditions can be carried out according to conventional conditions of such reactions or according to conditions recommended by the manufacturers.

[0098] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial channels.

[0099] The abbreviations used in the examples have the following meanings:

[0100] Example 1: In vivo pharmacological study of the tumor invasion of green fluorescent protein-labeled trastuzumab (T-GFP) in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018

[0101] Purpose of the experiment:

[0102] The purpose of this study was to evaluate the effects of IN10018 before and after administration of a timed regimen in a subcutaneous xenograft model of human gastric cancer NCI-N87 cells in Balb / c-nude mice. Tumors treated with the test substance, trastuzumab labeled with green fluorescent protein (T-GFP), were frozen sectioned and stained with multiple immunofluorescence staining. Panoramic scanning was performed to observe the expression of various protein markers. Image analysis was performed using HALO pathology image analysis software to assess the extent of infiltration within the tumor.

[0103] Experimental design:

[0104] The groups and medication are shown in Table 1.

[0105] Table 1. Animal groups and dosing regimens for in vivo efficacy experiments

[0106] Note:

[0107] 1. N: number of mice in each group;

[0108] 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight decreases by more than 15%, stop dosing the animal; resume dosing when the body weight recovers to 10% of the decrease.

[0109] Experimental Materials:

[0110] Mice: 7-8 week old female BALB / c-nude mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Upon arrival, the animals were acclimated to the experimental environment for at least 3 days before the experiment began. The animals were housed in cages (5 per cage) in an SPF-grade animal room. All cages, bedding, and drinking water were sterilized before use. All experimental personnel were required to wear protective clothing and latex gloves when working in the animal room. Cages, feed, and drinking water were changed twice a week. The housing environment and lighting conditions were as follows:

[0111] Temperature: 20-26℃

[0112] Humidity: 40-70%

[0113] Photoperiod: 12 hours of light, 12 hours of no light

[0114] Cages: Made of polycarbonate, 300 mm × 180 mm × 150 mm. Bedding is corn cobs, changed twice a week.

[0115] Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.

[0116] Drinking water: Experimental animals can drink sterile water freely.

[0117] Cage identification: The animal information card for each cage should indicate the number of animals in the cage, sex, strain, receipt date, dosing regimen, experiment number, group, and experiment start date.

[0118] Animal identification: Experimental animals were identified with ear tags.

[0119] The test sample information is shown in Table 2.

[0120] Table 2:

[0121] The experimental reagent information is shown in Table 3.

[0122] Table 3:

[0123] The experimental equipment information is shown in Table 4.

[0124] Table 4:

[0125] Experimental methods and steps:

[0126] Cell culture:

[0127] Human gastric cancer NCI-N87 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60491) 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 were in the exponential growth phase and at 80%-90% saturation, they were harvested, counted, and plated.

[0128] Cell seeding and grouping:

[0129] 0.1 mL contains 5×10 6 A cell suspension of 100 cells in PBS:Matrigel = 1:1 was subcutaneously inoculated on the right back of each mouse. 3 Around 14 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 1.

[0130] The preparations of the test substances and control solvents are shown in Table 5.

[0131] Table 5:

[0132] Daily observation of experimental animals:

[0133] The design and any modifications to this experimental protocol were reviewed and approved by the IACUC. The use and welfare of experimental animals were carried out in accordance with AAALAC guidelines. Animal health and mortality were monitored daily. Routine examinations included observation of tumor growth and the effects of drug treatment on daily animal behavior, such as activity, food and water intake (visual observation only), weight change, physical signs, or other abnormalities. Group deaths and adverse reactions were recorded based on the number of animals in each group.

[0134] Experiment termination:

[0135] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0136] Tumor measurement and dissection:

[0137] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured with a vernier caliper three 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.

[0138] On the 28th day after continuous IN10018 treatment, that is, the 42nd day after vaccination, all animals were injected with T-GFP once through the tail vein. Six hours after the injection, all animals were euthanized, the tumors were removed, and immersed in liquid nitrogen. After about 10 minutes, the tumor samples were transferred to dry ice and immediately proceeded to the next step of research.

[0139] Frozen sections:

[0140] Tumor tissue quickly frozen in liquid nitrogen is first precipitated with 20%-30% sucrose solution, then embedded in OCT to create frozen blocks. Cryosections are prepared using a cryostat and mounted on glass slides with good adhesion. Microscopic examination of the sections ensures that the sections are intact, without obvious breakage or wrinkles. After preparation, the slides can be left at room temperature for a period of time before staining or stored in a refrigerator.

[0141] DAPI counterstaining and mounting:

[0142] Add DAPI working solution (about 100-150 μl) to the glass slide, immersing the tissue area. Then incubate at room temperature in a humidified chamber for 3-5 minutes, wash with 1×TBST (or PBS) for 5 minutes, then wash with distilled water for 3 minutes, and finally add fluorescent antifade mounting medium to seal the slide. Fix the coverslip with nail polish, and then use the instrument for panoramic scanning and subsequent data analysis.

[0143] Panoramic scanning and quantitative analysis:

[0144] A digital pathology image panoramic scanning system was used to scan the entire slice and obtain pathology image information of the entire slice. The scan results were quantitatively analyzed using the pathology image analysis software Halo. The HALO Highplex FL (Indica Labs; Albuquerque, NM) analysis module of the Halo software was used. During the analysis process, the parameters corresponding to the analysis module were adjusted according to the actual situation of the image. After determining the optimal parameters, all similar images were analyzed using the same set of analysis templates to avoid the influence of human subjective factors. Each time the information circled in different areas represents an analysis layer, it identifies the different staining signals of each cell in the analysis field of view. At the same time, after the quantitative analysis of a single layer of each image is completed, the overall information of all results will be statistically summarized and analyzed.

[0145] Analytical indicators:

[0146] GFP-positive cell ratio: For each defined cell type, the number of positive cells in the sample and the percentage of this type of cells in the total number of cells in the sample are counted.

[0147] H-Score: that is, histochemical score. By taking each staining channel as the unit, the analysis results include statistics on the overall number of positive cells in each channel of each sample and the number of cells in the three levels of weak, moderate, and positive. The corresponding positive cells are counted in the nucleus, cytoplasm, and cell membrane respectively, and finally the corresponding percentage of the total cells and the H-Score value of each channel are calculated. The value range is 0 to 300, and the calculation method is: H-Score = [1*(%Cells1+)+2*(%Cells2+)+3*(%Cells3+)], where %Cells1+, %Cells2+, and %Cells3+ are the percentages of cells with weak, moderate, and positive expression levels automatically identified by the software.

[0148] Experimental results:

[0149] Image scan results

[0150] A digital pathology image panoramic scanning system was used to scan the entire slide, obtaining pathology image information for the entire slide. The report displays thumbnails of each image, as shown in Figure 1.

[0151] Study on the proportion of tumor infiltration of the test substance T-GFP in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018

[0152] The experiment was conducted according to the dosing schedule. On day 28 after continuous IN10018 treatment (i.e., day 42 after inoculation), all animals received a single injection of T-GFP via the tail vein. Six hours after injection, all animals were euthanized, tumors excised, and frozen embedded blocks and sections were prepared. After DAPI staining, whole-slide scans were performed using a digital pathology image panoramic scanning system. Quantitative analysis of the scans was performed using the HALO Highplex FL analysis module (Indica Labs; Albuquerque, NM) of the Halo pathology image analysis software. During analysis, the analysis module parameters were adjusted based on the image's specific conditions. Once optimal parameters were determined, all similar images were analyzed using the same analysis template to minimize subjective influences. Each circled area represents an analysis layer. The layer name can be customized to identify the different staining signals of individual cells within the analysis field of view. After completing the quantitative analysis of each individual layer in each image, the overall results are summarized. For each defined cell type, the number of positive cells in the sample and the percentage of that cell type in the total sample are counted.

[0153] In the first animal of the control group, a total of 132,264 cells were detected, of which 7,532 were GFP-positive, representing a 5.69% GFP-positive percentage. In the second animal of the control group, a total of 235,747 cells were detected, of which 11,757 were GFP-positive, representing a 4.99% GFP-positive percentage. The average GFP-positive percentage for the entire control group was 5.34%. In the first animal of the IN10018+T-GFP group, a total of 104,126 cells were detected, of which 13,470 were GFP-positive, representing a 12.94% GFP-positive percentage. In the second animal of the IN10018+T-GFP group, a total of 182,491 cells were detected, of which 14,994 were GFP-positive, representing a 8.22% GFP-positive percentage. The average GFP-positive percentage for the entire IN10018+T-GFP group was 10.58%. See Table 6 and Figure 2.

[0154] Table 6: Evaluation of the percentage of tumor infiltration of the test substance T-GFP in the subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018

[0155] Note:

[0156] 1. GFP-positive cell percentage = GFP-positive cell number / total number of cells detected * 100%;

[0157] 2. The percentage of GFP-positive cells in each group is the average of the percentages of GFP-positive cells in the two animals in the same group.

[0158] H-Score study of the tumor invasion degree of the test substance T-GFP in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018

[0159] The experiment was conducted according to the experimental plan. After full-slide scanning using a digital pathology image panoramic scanning system, the HALO Highplex FL (Indica Labs; Albuquerque, NM) analysis module of the Halo software was used to count the total number of positive cells in each channel of each sample, as well as the number of cells in the weak, moderate, and positive grades, for each staining channel. The corresponding positive cells were counted in the nucleus, cytoplasm, and cell membrane, and the corresponding percentage of the total cells and the H-Score value of each channel were finally calculated.

[0160] The percentages of weakly positive, moderately positive and strongly positive cells detected in the first animal of the control group were 4.87%, 0.51% and 0.31% respectively, and the H-Score calculated by the formula was 6.83; the percentages of weakly positive, moderately positive and strongly positive cells detected in the second animal of the control group were 4.21%, 0.56% and 0.22% respectively, and the H-Score calculated by the formula was 5.98. The average H-Score of the entire control group was 6.40. In the first animal of the IN10018+T-GFP group, the percentages of weakly positive, moderately positive, and strongly positive cells were 9.83%, 1.66%, and 1.45%, respectively, resulting in an H-Score of 17.49. In the second animal of the IN10018+T-GFP group, the percentages of weakly positive, moderately positive, and strongly positive cells were 5.97%, 1.30%, and 0.95%, respectively, resulting in an H-Score of 11.41. The average H-Score for the entire IN10018+T-GFP group was 14.45. See Table 7 and Figure 3.

[0161] Table 7: H-Score evaluation of the tumor infiltration degree of the test substance T-GFP in the human gastric cancer NCI-N87 cell subcutaneous xenograft tumor model of Balb / c-nude mice after administration of IN10018

[0162] Note:

[0163] 1. Percentage of positive cells = number of GFP (weak, moderate, strong) positive cells / total number of cells detected * 100%;

[0164] 2. H-Score = [1*(%Cells 1+ )+2*(%Cells 2+ )+3*(%Cells 3+ )], where %Cells 1+ 、%Cells 2+ 、%Cells 3+ are the percentages of cells with weak, moderate, and positive expression levels automatically identified by the software;

[0165] 3. The average H-Score within a group is the average of the H-Scores of the two animals in the group;

[0166] Experimental Conclusion

[0167] In this study, we evaluated the tumor invasion of the test compound, T-GFP, in a subcutaneous xenograft model of human gastric cancer NCI-N87 cells in Balb / c-nude mice before and after administration of IN10018. The tumor invasion levels and corresponding H-Scores for each group 6 hours after T-GFP administration are shown in Table 7, Figures 1, 2, and 3.

[0168] Comparing the percentage of tumor infiltration and the corresponding H-Score in two mice in the blank control group, after 27 days of IN10018 administration, the IN10018+T-GFP group showed a higher percentage of GFP+ positive cells and a higher H-Score than the control group. These data suggest that a period of IN10018 administration can more effectively promote drug binding to human gastric cancer cells, potentially leading to better in vivo efficacy.

[0169] Example 2: Pharmacodynamic Study of IN10018 and ESG-401 in Balb / C Nude Mice Bearing MDA-MB-468 Tumor Model

[0170] The experimental design is shown in Table 8.

[0171] Table 8: Experimental design

[0172] Note:

[0173] an is the number of animals in each group;

[0174] b. Dosing volume: Adjust the dosage based on body weight to 10 mL / kg or 5 mL / kg. If body weight loss is >15%, the dosage may be adjusted based on body weight.

[0175] Experimental Materials

[0176] Mice: Female BALB / c-nude mice (weight: 19-23 g), 6-8 weeks old, were purchased from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning Sciences. Upon arrival, the animals were acclimated to the experimental environment for at least 3 days before the experiment began. The animals were housed in cages (3-4 per cage). All cages, bedding, and drinking water were sterilized before use. All experimental personnel were required to wear protective clothing and latex gloves when working in the animal room. Cages, feed, and drinking water were changed twice weekly. The housing environment and lighting conditions were as follows:

[0177] Temperature: 18-26℃.

[0178] Humidity: 40%-70%.

[0179] Cage: Made of polycarbonate. Dimensions: 300mm x 180mm x 150mm. Bedding: corn cob.

[0180] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, date of receipt, compound, study number, group number, and treatment start date.

[0181] Animal identification: Animals are marked with ear clipping numbers.

[0182] The test sample information is shown in Table 9.

[0183] Table 9:

[0184] The experimental reagent information is shown in Table 10.

[0185] Table 10:

[0186] The formulations of the compounds are shown in Table 11.

[0187] Table 11:

[0188] Experimental methods

[0189] MDA-MB-468 cells (Biyuntian) were cultured in vitro (37°C, 0% CO2) in L-15 (Gibco) medium supplemented with 10% FBS (Gibco) and 1% PS (Gibco). Cells were passaged 2-3 times per week. MDA-MB-468 cells were harvested in the exponential phase and resuspended in a suspension of PBS and Matrigel (PBS:Matrigel volume ratio of 1:1) at a cell density of 5 × 10 7 / mL, and a cell suspension was prepared for inoculation into mice. Each mouse was subcutaneously inoculated with 0.2 mL of MDA-MB-468 tumor cell suspension (10×106 When the average tumor volume of mice reached 160 mm 3 Around day 7 after cell inoculation, mice were randomly divided into groups according to tumor volume, and drug administration began on the same day of grouping. Specific drug administration information is shown in Table 8.

[0190] Clinical observation

[0191] All procedures related to animal handling, care, and treatment in the study were performed according to guidelines approved by the Institutional Animal Care and Use Committee (IACUC) under the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). During routine monitoring, animals were examined daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain / loss (measured every two days or twice a week), eye / hair changes, and any other abnormal effects specified in the protocol. Deaths and observed clinical signs were recorded based on the number of animals in each cage.

[0192] Tumor measurement

[0193] The primary endpoint of the experiment was to see whether tumor growth could be delayed or cured. 3 Or 3 weeks after drug administration, whichever is shorter, the experimental endpoint treatment will be performed.

[0194] Tumor size was measured twice a week using a vernier caliper. Tumor volume was expressed in mm 3 The calculation formula of tumor volume is: V = 0.5 × a × b 2 , where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used to calculate the relative tumor growth rate (T / C%). T / C (%) is calculated using the formula: T / C% = (T i -T0) / (V i -V0)×100%,T i is the average tumor volume of the treatment group on a certain day, T0 is the average tumor volume of the treatment group on the first day of treatment (before drug administration), V i To measure T i The average tumor volume of the control group on that day, V0 is the average tumor volume of the control group on the first day of treatment. The TGI (%) of each group was calculated using the formula: Tumor Growth Inhibition Rate (TGI) (%) = [100-T / C].

[0195] Sample collection

[0196] On day 45 after dosing, according to experimental requirements, the experiment ended for groups 2 through 4. At this point, the mice in group 1 were divided into a control group and a dosing group for additional experiments. The experiment ended on day 63, according to experimental requirements. The mice in the additional experiment were euthanized, and their tumors were harvested and stored in 10% formalin.

[0197] Statistical analysis

[0198] Experimental data are presented as mean and standard error of the mean (SEM), including tumor volume for each group at each time point. Statistical analysis of differences in tumor volume between groups was performed. P values ​​were calculated using GraphPad Prism 7.0 software.

[0199] T-test was used to analyze the difference in tumor volume of each group at each time point, and p<0.05 indicated a significant difference.

[0200] Experimental results

[0201] After 29 days of dosing, all groups were discontinued for observation, as required by the experiment. On day 45 after dosing, mice in Groups 2 through 4 were terminated, as required by the experiment. Specific grouping and dosing details are detailed in the dosing records in Table 8 of the Appendix.

[0202] The body weight and tumor volume data of mice in all groups during the experiment are as follows:

[0203] The body weight of MDA-MB-468 tumor-bearing mice was regularly monitored as an indirect measure of toxicity. Following administration, no significant decrease in the mean body weight of mice in each group was observed. Detailed changes in body weight and relative body weight of MDA-MB-468 tumor-bearing mice following administration are shown in Figures 4 and 5.

[0204] Tumor volume

[0205] The tumor growth curves during the experiment are shown in Figure 6. After the 45th day of the experiment, the average tumor volumes of the control group, ESG-401 group, IN10018 group, and ESG-401 + IN10018 combined group were 414.79 mm 3 、190.10mm 3 , 240.14mm 3 and 94.48mm 3 .

[0206] T / C (%), TGI (%) and p-value

[0207] The T / C and TGI data at different time points during drug administration are shown in Table 12. After 45 days of administration, compared with the control group, the T / C values ​​in the ESG-401, IN10018, and ESG-401 + IN10018 combined groups were 10.30%, 30.13%, and -28.00%, respectively; and the TGI values ​​were 89.70%, 69.87%, and 128.00%, respectively.

[0208] The significance analysis at different time points is shown in Table 13. After 45 days of administration, ESG-401 and IN10018 alone and in combination showed significant anti-tumor effects compared with the control group.

[0209] Table 12: Tumor T / C (%) and TGI (%) at different time points

[0210] Table 13: Significance analysis of tumor volume at different time points Note: T-test was used for statistical analysis, and the p-value was calculated based on the tumor volume and compared with the control group.

[0211] Experimental results

[0212] The purpose of this study was to evaluate the anti-tumor efficacy of ESG-401 and IN10018 alone and in combination. During the dosing period, no significant weight loss was observed in the mice in the dosing group, indicating that the mice tolerated the compounds well.

[0213] On the 45th day of administration, the average tumor volumes of the control group, ESG-401 group, IN10018 group, and ESG-401 + IN10018 combined group were 414.79 mm 3 、190.10mm 3 , 240.14mm 3 and 94.48mm 3 .

[0214] The T / C ratios of the ESG-401 group, IN10018 group, and ESG-401 + IN10018 combined group were 10.30%, 30.13%, and -28.00%, respectively; and the TGI ratios were 89.70%, 69.87%, and 128.00%, respectively.

[0215] Compared with the control group, ESG-401, IN10018 alone and the combination of the two drugs showed significant anti-tumor effects (p<0.05).

[0216] In summary, ESG-401, IN10018 alone, and their combination showed significant anti-tumor effects in Balb / C nude tumor-bearing mice of the MDA-MB-468 model.

[0217] Example 3: In vivo pharmacological study of the effect of ESG-401 on tumor invasion after administration of IN10018 in the MDA-MB-468 model

[0218] Reagent information is shown in Table 14.

[0219] Table 14

[0220] The experimental equipment information is shown in Table 15.

[0221] Table 15

[0222] Tissue paraffin embedding and sectioning experimental steps

[0223] Sample collection: Immerse the fresh mouse tumor collected in Example 2 in 10% neutral formalin for at least 24 hours. Do not freeze at 4°C. Remove the tumor from the fixative and trim the target area in a fume hood using a scalpel. Place the trimmed tissue and the corresponding label in a dehydration box and label the specimen bottle with the specimen code. Place the tissue in a container of water to briefly rinse the fixative off.

[0224] Dehydration: Place the embedding frame in the basket of the dehydrator and dehydrate using a gradient of alcohols: 75% alcohol for 2 hours, 85% alcohol for 1 hour, 90% alcohol for 1 hour, 95% alcohol for 40 minutes, absolute ethanol I for 30 minutes, absolute ethanol II for 30 minutes, benzene for 10 minutes, xylene I for 5 minutes, xylene II for 5 minutes, wax I for 1 hour, wax II for 1 hour, wax III for 1 hour.

[0225] Embedding: Embed the wax-soaked tissue in an embedding machine. First, place the melted wax in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding base mold and embedding frame according to the embedding surface requirements. Cool in a -20°C freezer. Once the wax solidifies, remove the wax block from the embedding frame and trim it.

[0226] Sectioning: Place the trimmed wax block on a paraffin microtome to slice 3 μm thick. Float the slices on a 42°C warm water slide to flatten the tissue. Remove the tissue with a glass slide and bake in a 60°C oven. Once the water is dried and the wax is melted, remove the slices and store at room temperature until ready for use.

[0227] Sirius red staining experimental steps

[0228] Dewax the paraffin sections to water: place the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and wash with distilled water.

[0229] Staining with Sirius Red Dye: Incubate with Sirius Red Dye for 20-30 minutes, then rinse with pure alcohol.

[0230] Dehydration and sealing: Place the slices in 95% alcohol II for 15 minutes, absolute ethanol I for 10 minutes, absolute ethanol II for 10 minutes, xylene I for 10 minutes, and xylene II for 10 minutes in sequence for dehydration and transparency. Take the slices out of the xylene and dry them slightly, then seal them with neutral gum.

[0231] Microscopic examination was performed using a scanner (3D histech, Pannoramic MIDI) for image acquisition and analysis. Thumbnails are shown in Figure 7.

[0232] Sirius staining results showed that the degree of fibrosis in the ESG-401 monotherapy group was similar to that in the control group, while tumor fibrosis was significantly suppressed in the IN10018 and ESG-401 combination group compared to the ESG-401 monotherapy group. Anti-human IgG secondary antibodies were able to recognize the binding of ESG-401 to tumor cells in vivo, demonstrating that the antibody and tumor cells were able to produce binding staining in the ESG-401 monotherapy group, while the IN10018 and ESG-401 combination group exhibited more pronounced antibody-tumor cell binding staining compared to the ESG-401 monotherapy group.

[0233] Example 4: In vivo pharmacodynamic study of IN10018 and gosartumomab (TRODELVY) in a BALB / c nude mouse model of pancreatic cancer PC-07-0067 subcutaneous xenograft tumors

[0234] Experimental design

[0235] PDX model establishment: The human pancreatic cancer model PC-07-0067 was initially established from a surgically resected clinical specimen, which was implanted into nude mice and designated as P0. P0 tumor tissue was implanted into the next generation, designated P1, and this number was repeated in nude mice. FP3 tumors were revived from P2 tumors, and the next generation from FP3 was designated FP4, and so on.

[0236] Animals: BALB / c nude mice, female, 6-8 weeks old, weighing 18-22 g. A total of 60 mice were required. These were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0237] Tumor inoculation: 20-30mm3 The PC-07-0067FP5 tumor tissue piece was subcutaneously inoculated on the right back of each mouse and the tumor was allowed to grow. When the average tumor volume reached about 100-150 mm 3 Randomization and drug administration began at 1:00 PM. The experimental groups and drug administration schedule are shown in the table below.

[0238] The animal experimental groups and dosing regimens are shown in Table 16.

[0239] Table 16:

[0240] Note:

[0241] 1. N: number of mice in each group;

[0242] 2. Dosing volume: 10 μL / g based on mouse body weight. If body weight loss exceeds 15%, discontinue dosing immediately and resume dosing when body weight loss returns to within 10%.

[0243] Animal husbandry: The experiment can only begin after the animals have been kept in the experimental environment for 3-7 days after arrival. The animals are kept in IVC (independent ventilation system) cages (5 per cage) in an SPF-level animal room. All cages, bedding and drinking water must be sterilized before use. All experimenters should wear protective clothing and latex gloves when operating in the animal room. The animal information card for each cage should indicate the number of animals in the cage, gender, strain, receipt date, dosing regimen, experimental number, group and start date of the experiment. Cages, feed and drinking water are changed twice a week. The breeding environment and lighting conditions are as follows: temperature: 20-26°C, humidity: 40-70%, light cycle: 12 hours of light and 12 hours of no light.

[0244] Feed ingredients: Feed meets the standards for laboratory animal food. Maximum contaminant levels are within controllable limits and are routinely inspected by the manufacturer. High-pressure sterilized drinking water is used.

[0245] Animal grouping: Before administration, animals were weighed and tumor volumes were measured, and the animals were randomly grouped according to tumor volume (randomized block design).

[0246] Observation: The design and any modifications to this experimental protocol must be reviewed and approved by the Institutional Animal Care Committee (IACUC). The use and welfare of experimental animals will be carried out in accordance with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals will be monitored daily. Routine examinations will include observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as activity, food and water intake, weight changes (measured twice weekly), physical signs, or other abnormalities. The number of deaths and adverse reactions within each group will be recorded based on the number of animals in each group.

[0247] Experimental indicators: The experimental indicators are used to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice a week with a vernier caliper. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0248] The tumor inhibition efficacy of a compound was evaluated using TGI (%) or tumor growth rate (T / C) (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) is calculated as follows: TGI (%) = [1 - (average tumor volume at the end of dosing in a given treatment group - average tumor volume at the start of dosing in that treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × 100%.

[0249] Tumor proliferation rate T / C (%): The calculation formula is as follows: T / C (%) = T i / V i × 100%. Where V i is the average tumor volume of the solvent control group at a certain measurement, T i It is the average tumor volume of the drug-treated group at the same measurement.

[0250] After the experiment, the tumor weight will be measured and T weight / C weight Percentage, T weight and C weight Represent the tumor weights of the drug-treated group and the solvent control group, respectively.

[0251] Termination of the experiment: 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:

[0252] Significant weight loss, with a weight loss of more than 20%; unable to eat or drink freely; the average tumor volume in the control group reached 2,000 mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0253] Data Analysis: T-tests were used for comparisons between two groups. One-way ANOVA was used for comparisons between three or more groups. If the F value showed a significant difference, multiple comparisons were performed after the ANOVA analysis. All data were analyzed using SPSS 17.0. A p value < 0.05 was considered significant.

[0254] The compounds to be tested are shown in Table 17.

[0255] Table 17:

[0256] The detailed scheme for preparing the medicine is shown in Table 18: Note: The tartrate salt of IN10018 was weighed in the experiment;

[0257] The drug needs to be mixed thoroughly and gently before administration to animals.

[0258] Experimental results

[0259] After 14 days of administration, all groups were observed after drug withdrawal according to experimental requirements. On the 14th day after administration, according to experimental requirements, the experiment was terminated and all experimental mice were sampled (for use in Example 5). Specific grouping and administration details are detailed in the administration records in Appendix Table 16.

[0260] The body weight and tumor volume data of mice in all groups during the experiment are as follows:

[0261] The body weight of PC-07-0067 tumor-bearing mice was regularly monitored as an indirect measure of toxicity. No significant decrease in the average body weight of mice in each group was observed after administration.

[0262] Tumor volume

[0263] The tumor growth curve during the experiment is shown in Figure 8. After the 14th day of the experiment, the average tumor volume of the control group, TRODELVY group and TRODELVY + IN10018 combined group was 1512 mm 3 , 423mm 3 and 104mm 3 .

[0264] T / C (%), TGI (%) and p-value

[0265] After 14 days of administration, the T / C of the TRODELVY group and the TRODELVY+IN10018 combined group were 27.95% and 6.86%, respectively; the TGI were 78.91% and 102.10%, respectively.

[0266] In summary, in NOD-SCID tumor-bearing mice of the PC-07-0067 model, the combination of TRODELVY and IN10018 showed a stronger anti-tumor effect than the TRODELVY monotherapy group.

[0267] Example 5: In vivo pharmacological study of TRODELVY on tumor invasion after administration of IN10018 in the PC-07-0067 model

[0268] The experimental methods were the same as in Example 3. The experimental tissue samples were from Example 4. A color thumbnail is shown in Figure 9. The anti-human IgG secondary antibody was able to recognize the binding of TRODELVY to tumor cells in vivo. The results showed that the antibody bound to tumor cells significantly in the TRODELVY monotherapy group, while the combination of IN10018 and TRODELVY showed even more significant antibody-tumor cell binding compared to the TRODELVY monotherapy group.

[0269] Example 6: In vivo pharmacological study of the tumor invasion of the test substance, fluorescein Cy5.5-labeled hantuyumab (H-Cy5.5), in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after administration of IN10018

[0270] The purpose of this study was to evaluate the extent of IN10018 infiltration within the tumor by fluorescence imaging of the tumor sites of mice administered with the test substance, fluorescein Cy5.5-labeled hantuyumab (H-Cy5.5), before and after administration of IN10018 for 7 days in a subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice.

[0271] Grouping and dosing are shown in Table 19.

[0272] Table 19: Animal groups and dosing regimens for in vivo efficacy experiments

[0273] Note:

[0274] 1. N: number of mice in each group;

[0275] 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight drops by more than 15%, stop dosing. Resume dosing when the body weight recovers to 10% of the decrease.

[0276] Experimental Materials:

[0277] Mice: Female BALB / c-nude mice, 6-8 weeks old, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Upon arrival, the animals were acclimated to the experimental environment for at least 3 days before the experiment began. The animals were housed in cages (5 per cage) in an SPF-grade animal room. All cages, bedding, and drinking water were sterilized before use. All experimental personnel were required to wear protective clothing and latex gloves when working in the animal room. Cages, feed, and drinking water were changed twice a week. The housing environment and lighting conditions were as follows:

[0278] Temperature: 20-26℃

[0279] Humidity: 40-70%

[0280] Photoperiod: 12 hours of light, 12 hours of no light

[0281] Cages: Made of polycarbonate, 300 mm × 180 mm × 150 mm. Bedding is corn cobs, changed twice a week.

[0282] Food: The experimental animals had free access to food (irradiated sterilized, dry pelleted food) throughout the experimental period.

[0283] Drinking water: Experimental animals can drink sterile water freely.

[0284] Cage identification: The animal information card for each cage should indicate the number of animals in the cage, sex, strain, receipt date, dosing regimen, experiment number, group, and experiment start date.

[0285] Animal identification: Experimental animals were identified with ear tags.

[0286] The test sample information is shown in Table 20 below.

[0287] The experimental reagent information is shown in Table 21.

[0288] Experimental methods and steps

[0289] Cell culture

[0290] Human gastric cancer cell line NCI-N87 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60491) was maintained and passaged by Ins Biotechnology (Nanjing) Co., Ltd. Cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged using trypsin-EDTA two to three times weekly. When cells were in the exponential growth phase and at 80%-90% saturation, they were harvested, counted, and plated.

[0291] Cell seeding and grouping

[0292] 0.2 mL containing 1×10 7 A PBS cell suspension of 100 cells was subcutaneously inoculated on the right back of each mouse. When the tumor volume reached 200.0 mm 3 Around 14 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 19.

[0293] Synthesis of H-Cy5.5

[0294] H-Cy5.5 was synthesized at the Institute of Biophysics, Chinese Academy of Sciences, Beijing. The trastuzumab concentration was 7.44 mg / ml, or 50.27 μM. The synthesized Cy5.5 concentration was 101 μM, resulting in a dye / protein labeling ratio of approximately 2.01. For in vivo mouse imaging, the recommended injection dose is 1–3 nmol Cy5.5 per animal. For this experiment, a dose of 3 nmol Cy5.5 per animal was used.

[0295] The preparation of the test substance and control solvent is shown in Table 22.

[0296] Table 22

[0297] Daily observation of experimental animals

[0298] The development of this experimental protocol and any modifications were evaluated and approved by the Institutional Animal Care Committee (IACUC) before implementation. The use and welfare of experimental animals will comply with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observing tumor growth and the effects of drug treatment on the animals' daily behavior, such as behavioral activity, food and water intake (visual inspection only), weight changes, physical signs, or other abnormalities. The number of deaths and side effects of animals within each group were recorded based on the number of animals in each group.

[0299] Experiment terminated

[0300] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3 If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0301] Tumor measurement

[0302] The experimental indicator is to examine whether tumor growth is inhibited, delayed or cured. Tumor diameter is measured with a vernier caliper three times a week.

[0303] 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.

[0304] Tumor imaging

[0305] On the seventh day after administration of IN10018, the G1 control group and the G2 IN10018 25 mg / kg group were injected with H-Cy5.5 via the tail vein, and the cells were then cytosed 6, 24, and 48 hours after the injection. The Lumina III small animal in vivo imaging system images animals and detects their fluorescence intensity.

[0306] Experimental indicators

[0307] Mouse fluorescence signal value: Perform in vivo imaging of mice, gate the entire torso, and collect the total fluorescence intensity (Total Radiant Efficiency) of the entire torso.

[0308] Average fluorescence signal value of the tumor site: The mouse was imaged in vivo, and the tumor site of the mouse was clearly gated under white light. The average fluorescence intensity (Avg Radiant Efficiency) of the tumor within the gate was collected.

[0309] Statistical analysis

[0310] Statistical analysis was performed using Prism Graphpad software at the end of the experiment. Overall comparisons of mean fluorescence intensity at different time periods were performed using two-way ANOVA and Fisher's LSD test, with P < 0.05 considered significant.

[0311] Experimental results

[0312] On the 14th day after tumor inoculation, 14 animals with appropriate tumor volumes were randomly divided into groups. The average tumor volume was approximately 200 mm 3 The day was defined as dosing day 0. Group G2 began to receive IN10018 25 mg / kg as planned. Figure 10 shows the data on the day of dosing 14 days after inoculation. The average tumor volume of the G1 control group was 200.7 ± 30.8 mm 3 The average tumor volume of the G2 IN10018 25 mg / kg group was 200.0 ± 33.4 mm 3 (mean ± SEM).

[0313] Study on the overall fluorescence intensity of the test substance H-Cy5.5 in the subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice 6 hours after injection

[0314] The experiment was carried out according to the dosing schedule. On the 7th day after continuous treatment with IN10018, that is, on the 21st day after inoculation, all animals were injected with 150ul of H-Cy5.5 through the tail vein. The animals were imaged using the Lumina III small animal in vivo imaging system, and the overall fluorescence intensity of their trunk was detected.

[0315] Six hours after H-Cy5.5 tail vein injection, mice in both groups were imaged in vivo, and the trunk area was gated to measure total radiant efficiency (Total Radiant Efficiency). Several animals not injected with H-Cy5.5 were also imaged as negative controls. The total radiant efficiency (Total Radiant Efficiency) was 2.86E+11±1.08E+11 in the G1 control group, 2.88E+11±1.09E+11 in the G2 IN10018 25mg / kg treatment group, and 2.04E+10±1.18E+10 in the negative control group. Comparing the total radiant efficiency data with the negative control group, the p-values ​​for the G1 control group and the G2 IN10018 25mg / kg treatment group were 0.0167 and 0.0167, respectively. The overall fluorescence intensity data comparing the G1 control group and the G2 IN10018 25 mg / kg treatment group showed a p value of 0.3176 (see Table 23 and Figures 11 and 12 for details).

[0316] Table 23: Evaluation of the overall fluorescence intensity of the test substance H-Cy5.5 in the subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice 6 hours after injection

[0317] Note:

[0318] 1.mean±SEM;

[0319] 2.*: p < 0.05, t-test, Mann-Whitney test, vs. negative control group;

[0320] 3. t-test, Mann-Whitney test, G1 control group vs. G2 IN10018 25 mg / kg group;

[0321] Study on the tumor infiltration degree of the test substance H-Cy5.5 in the subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice after 6 hours, 24 hours and 48 hours of injection

[0322] The experiment was carried out according to the experimental plan. On the 7th day after continuous treatment with IN10018, that is, on the 21st day after vaccination, all animals were injected with H-Cy5.5 through the tail vein. At 6 hours, 24 hours and 48 hours after the injection, the animals were injected with H-Cy5.5. The Lumina III small animal in vivo imaging system images animals, detects the average fluorescence intensity of their tumor sites, and clearly gates the tumor sites of mice under white light field of view, and collects the average fluorescence intensity (Avg Radiant Efficiency) of the tumor within the gate.

[0323] Six hours after H-Cy5.5 injection, the mean fluorescence intensity of the tumor site in the G1 control group was 3.31E+09±1.25E+09, while the mean fluorescence intensity of the tumor site in the G2IN10018 25 mg / kg treatment group was 4.02E+09±1.52E+09. Twenty-four hours after H-Cy5.5 injection, the mean fluorescence intensity of the tumor site in the G1 control group was 5.31E+09±2.01E+09, while the mean fluorescence intensity of the tumor site in the G2IN10018 25 mg / kg treatment group was 6.41E+09±2.42E+09. Forty-eight hours after H-Cy5.5 injection, the mean fluorescence intensity of the tumor site in the G1 control group was 6.61E+09±2.50E+09, while the mean fluorescence intensity of the tumor site in the G2IN10018 25 mg / kg treatment group was 8.04E+09±3.04E+09. Statistical analysis of the mean fluorescence intensity at each time point for the G1 control group and the G2 IN10018 25 mg / kg treatment group revealed a p-value of 0.0148 for both groups at 48 hours after injection (see Table 24 and Figures 13 and 14 for details).

[0324] Table 24: Evaluation of the mean fluorescence intensity of the tumor site of the test substance H-Cy5.5 in the subcutaneous xenograft tumor model of human gastric cancer NCI-N87 cells in Balb / c-nude mice 6h, 24h and 48h after injection

[0325] Note:

[0326] 1. The mean fluorescence intensity of the tumor site in each group at 6h, 24h and 48h, mean ± SEM;

[0327] 2.*: p < 0.05, G1 control group vs. G2 IN10018 25 mg / kg group, Two-way ANOVA;

[0328] Experimental Conclusion

[0329] In this study, we evaluated the tumor invasion of the test substance H-Cy5.5 in a subcutaneous xenograft model of human gastric cancer NCI-N87 cells in Balb / c-nude mice before and after administration of IN10018. Tumor invasion and corresponding fluorescence intensity were measured 6, 24, and 48 hours after H-Cy5.5 administration. The overall fluorescence intensity of the animals 6 hours after administration showed that the average overall fluorescence intensity was consistent across all groups of animals after the same dose of H-Cy5.5, indicating that the injection was successful. From the mean fluorescence intensity values ​​of the animal tumor sites at 6 hours, 24 hours, and 48 hours after drug administration, it can be seen that H-Cy5.5 in the tumor site tends to be enriched over time, and the mean fluorescence intensity values ​​of the G2 IN10018 25 mg / kg group are always higher than those of the G1 control group, and there is a statistical difference at 48 hours. The above data indicate that the administration of IN10018 can more effectively promote the binding of the drug to human gastric cancer NCI-N87 cells, which may result in better in vivo efficacy.

[0330] Example 7: In vivo anti-tumor efficacy study of Enhertu (trastuzumab) in combination with IN10018 in a subcutaneous xenograft tumor model of BALB / c-nude mice inoculated with a mixture of human gastric cancer NCI-N87 cells and mouse embryonic fibroblasts NIH-3T3 cells

[0331] Experimental design

[0332] Grouping and dosing are shown in Table 25

[0333] Table 25: Animal groups and dosing regimens for in vivo efficacy experiments

[0334] Note:

[0335] 1. N: number of mice in each group;

[0336] 2. Dosing volume: 10 mL / kg based on mouse body weight. If the body weight drops by more than 15%, stop dosing. Resume dosing when the body weight recovers to 10% of the decrease.

[0337] 3. Enhertu was administered starting on the 7th day after group administration and was only given twice, on the 7th day and 28th day after group administration;

[0338] 4. IN10018 was administered starting on the day of grouping;

[0339] The experimental animals, breeding environment and DPBS source were the same as those in Example 6. The test sample information is shown in Table 26.

[0340] Experimental methods and steps

[0341] Cell culture

[0342] Human gastric cancer cell line NCI-N87 (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60491) was 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.

[0343] Mouse embryonic fibroblast NIH-3T3 cells (source: Nanjing Kebai Biotechnology Co., Ltd., catalog number: CBP60317) were maintained and passaged by Ins Biotechnology (Nanjing) Co., Ltd. Cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% calf serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged two to three times weekly using trypsin-EDTA. When both cell lines were in the exponential growth phase and had a confluence of 80%-90%, cells were harvested, counted, and plated.

[0344] Cell seeding and grouping

[0345] For mice inoculated with a mixture of NCI-N87 and NIH-3T3 cells, 0.2 mL of 10 × 10 6 NCI-N87 cells and 5×10 5 A mixed cell suspension of NIH-3T3 cells was subcutaneously inoculated on the right back of each mouse. On day 8 after inoculation, the tumor volume of mice inoculated with mixed NCI-N87 and NIH-3T3 cells reached 154 mm 3 At around 37 ℃, the patients were randomly divided into groups according to the tumor volume and given drugs. The grouping information is shown in Table 25.

[0346] The preparation of test substances and control solvents is shown in Table 27.

[0347] Daily observation of experimental animals

[0348] The development of this experimental protocol and any modifications were evaluated and approved by the Institutional Animal Care Committee (IACUC) before implementation. The use and welfare of experimental animals will comply with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observing tumor growth and the effects of drug treatment on the animals' daily behavior, such as behavioral activity, food and water intake (visual inspection only), weight changes, physical signs, or other abnormalities. The number of deaths and side effects of animals within each group were recorded based on the number of animals in each group.

[0349] Experiment terminated

[0350] If the animal's health condition continues to deteriorate, or the tumor volume exceeds 3,000 mm 3 If the following conditions occur, notify the veterinarian and euthanize the animal: obvious emaciation, weight loss greater than 20%; inability to eat and drink freely; the average tumor volume of the control group reaches 2,000mm 3 The experiment was terminated. The animal developed the following clinical manifestations and continued to worsen: piloerection, arched back, pale ears, nose, eyes, or feet, rapid breathing, convulsions, continuous diarrhea, dehydration, slow movement, and vocalization.

[0351] Tumor measurements and experimental parameters

[0352] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured with a vernier caliper three 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.

[0353] 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 dosing group - average tumor volume of that dosing 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%.

[0354] Statistical analysis

[0355] 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.

[0356] Experimental results

[0357] After cell inoculation, tumor growth was observed every day. On the 8th day after inoculation, the patients were divided into groups according to tumor volume and given medication (marked as Day 0). The average tumor volume in the group was approximately 154 mm 3 All drugs except Enhertu (3 mg / kg) were administered starting on Day 0. Enhertu (3 mg / kg) was administered to all groups starting on Day 7 and twice, on Day 7 and Day 28. Due to tumor burden, all groups were euthanized on Day 43 after inoculation, which was 35 days after group administration, and the entire experiment was terminated.

[0358] On the 35th day after group administration, the tumor volume of the control group was 1523.7±173.3mm 3 The tumor volume of the Enhertu (3 mg / kg) treatment group was 1575.2 ± 547.4 mm 3 The tumor volume of the IN10018 (25 mg / kg) treatment group was 976.8 ± 171.2 mm 3 The tumor volume of the Enhertu+IN10018 (3+25 mg / kg) combination treatment group was 626±209.8 mm 3 ;

[0359] Comparing the comprehensive tumor volume with the control group, the tumor inhibition rates (TGI) of the Enhertu (3 mg / kg) and IN10018 (25 mg / kg) monotherapy groups were -3.8% (p=0.7634) and 40.0% (p=0.0015), respectively; the tumor inhibition rate (TGI) of the Enhertu + IN10018 (3 + 25 mg / kg) combination therapy group was 65.6% (p<0.0001). The p values ​​for the comparison of the comprehensive tumor volume-related monotherapy groups Enhertu (3 mg / kg), IN10018 (25 mg / kg), and Enhertu + IN10018 (3 + 25 mg / kg) combination therapy groups were p<0.0001 and p=0.0921, respectively. See Table 28 for details. The tumor volume of each dose group at different time periods is shown in Figure 15.

[0360] Table 28: Evaluation of the tumor inhibitory effect of the test substance on the BALB / c-nude mouse transplant tumor model of NCI-N87 & NIH-3T3 cells (based on the data on day 35 after group administration)

[0361] Note:

[0362] 1. Calculated according to the number of days after group administration, data are mean ± standard error (mean ± SEM);

[0363] 2. TGI (%) = [1-(T 35 -T0) / (V 35 -V0)]×100%;

[0364] 3. **: p < 0.01, ****: p < 0.0001, vs. control group, Two-way ANOVA, Fisher's LSD test;

[0365] 4.****: p<0.0001, vs.Enhertu+IN10018(3+25mg / kg), Two-way ANOVA, Fisher's LSD test;

[0366] The experiment was carried out according to the drug administration plan. During the experiment, the animals' activities such as eating and drinking were observed every day, and the animal weights were recorded three times a week. For the co-inoculated NCI-N87 human gastric cancer cells and NIH-3T3 mouse embryonic fibroblasts, after 35 days of dosing, the average body weight of the control group increased from 18.5g on Day 0 to 22.0g, with a body weight increase of 18.4%. The average body weight of the Enhertu (3mg / kg) and IN10018 (25mg / kg) monotherapy groups increased from 17.9g and 19.2g on Day 0 to 20.9g and 21.5g on Day 35, respectively, with body weight change rates of 16.9% and 11.6%, respectively. The average body weight of the Enhertu + IN10018 (3+25mg / kg) combination therapy group increased from 18.9g on Day 0 to 20.7g on Day 35, with a body weight change rate of 9.5%. Throughout the dosing period, the animals in each group showed no significant weight loss and remained in good condition. See Table 29 for details. The body weight changes and change rates of each dosage group at different time periods are shown in Figures 16 and 17.

[0367] Table 29: Evaluation of Body Weight Changes of the Test Substances in the BALB / c-nude Mouse Xenograft Model of NCI-N87 & NIH-3T3 Cells (Based on Data on Day 35 After Group Dosing)

[0368] Note:

[0369] 1. Calculate the number of animals surviving on day 0 / the number of animals surviving on day 35 according to the number of days after group administration.

[0370] 2. Data are mean ± standard error (mean ± SEM);

[0371] 3. Weight change rate = (W 35 -W0) / W0*100%;

[0372] Experimental Conclusion

[0373] In this study, we evaluated the in vivo efficacy of Enhertu in combination with IN10018 in a subcutaneous xenograft tumor model in BALB / c-nude mice inoculated with a mixture of NCI-N87 and NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts. Compared to the control group, both the IN10018 (25 mg / kg) monotherapy group and the Enhertu + IN10018 (3 + 25 mg / kg) combination therapy group demonstrated significant tumor growth inhibition, with statistically significant differences compared to the control group. Furthermore, the Enhertu + IN10018 (3 + 25 mg / kg) combination therapy group demonstrated smaller tumor volumes and superior tumor inhibition rates throughout the experimental period compared to the monotherapy groups. These data demonstrate that the combination of Enhertu and IN10018 offers superior therapeutic efficacy compared to monotherapy.

[0374] Overall, the animals showed good weight changes during the 35-day dosing cycle, and no abnormalities were found in their activity, water intake, and mental state throughout the dosing cycle, indicating that the animals tolerated Enhertu (3 mg / kg) and IN10018 (25 mg / kg) alone and in combination.

[0375] Example 8: Evaluation of the anti-tumor effect of IN10018 in the PC-07-0041 human pancreatic cancer PDX model in female BALB / c nude mice

[0376] Experimental design

[0377] Grouping and dosing are shown in the table

[0378] Table 30: Animal groups and dosing regimens for in vivo efficacy experiments

[0379] Note:

[0380] 1. N: number of mice in each group;

[0381] 2. Dosage: Adjust the dosage to 10 μL / g based on body weight;

[0382] 3. Initially, the dose was 1, but it was ineffective, so the dose was increased to 3.

[0383] Experimental animals: purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., other information is the same as Example 6.

[0384] The rearing environment and DPBS source were the same as those in Example 6.

[0385] The test sample information is shown in Table 9.

[0386] Experimental methods and steps

[0387] PDX model establishment:

[0388] Mice were implanted with PC-07-0041 human pancreatic cancer tumor cells, which were established from surgically resected patient tissue. Passage 0 (P0) was defined as the next generation from the P0 tumor implantation, and this number was repeated throughout the duration of the mouse implantation. FP4 tumor tissue was used in this study.

[0389] Cell seeding and grouping

[0390] Each mouse was implanted with a PC-07-0041FP4 tumor piece (approximately 30 mm) subcutaneously on the right side. 3 Treatment was started on day 19 after tumor implantation, when the average tumor size reached approximately 119 mm. 3 The animals were randomized into groups based on tumor volume using Excel-based randomization software. Each group consisted of 5 tumor-bearing mice. The test articles were administered to the mice according to the pre-determined protocol shown in the experimental design table.

[0391] The preparation of the test substance and control solvent is shown in Table 31.

[0392] Note: The tartrate salt of IN10018 was weighed in the experiment;

[0393] Daily observation of experimental animals

[0394] The development of this experimental protocol and any modifications were evaluated and approved by the Institutional Animal Care Committee (IACUC) before implementation. The use and welfare of experimental animals will comply with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health and mortality of the animals were monitored daily. Routine examinations included observing tumor growth and the effects of drug treatment on the animals' daily behavior, such as behavioral activity, food and water intake (visual inspection only), weight changes, physical signs, or other abnormalities. The number of deaths and side effects of animals within each group were recorded based on the number of animals in each group.

[0395] Tumor measurements and experimental parameters

[0396] The experimental indicator is to examine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured with a vernier caliper twice a week. The formula for calculating tumor volume is: V = 0.5 × a × b 2 , a and b represent the long and short diameters of the tumor, respectively. Tumor size was then used to calculate TGI (%) and T / C (%) values.

[0397] The TGI calculation formula for each group is: TGI (%) = [1-(Ti-T0) / (Vi-V0)] × 100%; Ti is the average tumor volume of the treatment group on a given day, T0 is the average tumor volume of the treatment group on the day of treatment initiation, Vi is the average tumor volume of the vector control group on the same day as Ti, and V0 is the average cancer volume of the vector group on the day of treatment initiation.

[0398] The T / C value (percentage) is an indication of antitumor effectiveness; T and C are the mean volumes of the treated and control groups, respectively, on a given day.

[0399] Tumor weights were measured at the end of the study. weight The value (percentage) is calculated using the formula: T / C weight % = T weight / C weight x 100%, where T weight and C weight are the mean tumor weights of the treatment and vehicle control groups, respectively.

[0400] Statistical analysis

[0401] Tumor volumes and weights were compared between groups using one-way analysis of variance. Because a significant F statistic (the ratio of the treatment variance to the error variance) was obtained, the Games-Howell test was used for comparisons between groups. All data were analyzed using SPSS 29.0, and p < 0.05 was considered statistically significant.

[0402] Experimental results

[0403] Animal body weights were monitored regularly as an indicator of toxicity. No mortality or morbidity was observed. None of the mice showed significant weight loss. Weight changes in the different treatment groups are shown in Figure 18.

[0404] Tumor growth inhibition by IN10018 and ESG-401 in the PC-07-0041 PDX model was calculated based on tumor volume measurements on day 28, as shown in Tables 32, 33 and Figure 19.

[0405] Table 32: Average tumor volume at different times Note: 1. Mean ± SEM

[0406] Table 33:

[0407] The tumor weights of tumor-bearing mice in different treatment groups are shown in Table 34.

[0408] Table 34:

[0409] In this study, the efficacy of IN10018 and ESG-401 in the PC-07-0041 human pancreatic xenograft model was evaluated in female BALB / c nude mice.

[0410] The average tumor size in control mice reached 944 mm on day 28 after the start of treatment. 3 Compared with the control group, IN10018+ESG-401 (25 mg / kg, QD + 1 / 3 mg / kg, BIW) treatment produced significant antitumor activity; the average tumor size was 197 mm 3 (T / C=20.83%, TGI=90.55%, p=0.007). IN10018 (25 mg / kg, QD) and ESG-401 (1 / 3 mg / kg, BIW) treatment did not produce significant antitumor activity; the average tumor size was 1252 mm 3 (T / C = 132.65%, TGI = -37.38%, p = 0.525) and 510 mm 3 (T / C=54.00%, TGI=52.60%, p=0.075) The results of tumor weight were basically consistent with those of tumor volume.

[0411] In this study, the test compounds IN10018 and ESG-401 were well tolerated by tumor-bearing mice at the dose levels indicated. No significant weight loss was observed in any treatment group.

[0412] Example 9: In vivo efficacy evaluation of the test drug IN10018 as a single agent or in combination with other drugs in the human rectal cancer xenograft model LD1-0038-361855

[0413] Experimental animals: NU / NU mice, female, weighing 18-21 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and housed in the same environment as in Example 6.

[0414] Human rectal cancer tumor tissue (case model number: LD1-0038-361855) was passaged to FP5+2 and used in this efficacy experiment.

[0415] The test sample information is shown in Table 26.

[0416] The human rectal cancer LD1-0038-361855 xenograft tumor was cut into approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) pieces and inoculated subcutaneously into NU / NU mice. The mice were observed and tumor growth was monitored. On day 23 of inoculation, the average tumor volume of the tumor-bearing mice was 156.69 mm. 3 The grouping and administration were carried out at 37 days, and the day of grouping and administration was defined as day 0. The specific grouping information is as shown in Table 35:

[0417] Table 35. Dosing and Grouping Note: N: number of animals; Dosage volume: adjusted according to the weight of tumor-bearing mice (0.2 ml / 20 g).

[0418] The main purpose is to detect the growth inhibitory effect or complete cure ability of the test drug on the human rectal cancer transplant tumor model LD1-0038-361855.

[0419] Tumor volume and tumor-bearing mouse weight measurement: Use vernier calipers to measure twice a week. The tumor volume is calculated as V = 0.5a × b 2 , a, b represent the long diameter and wide diameter of the tumor, respectively;

[0420] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : negative control group RTV). The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV = T Vi / T V0 , where T V0 is the average tumor volume measured at the time of group administration (i.e., d0), T Vi is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0421] Tumor growth inhibition rate TGI (%) = [1-(T ti -T t0 ) / (V ci -V c0 )]×100,T ti is the average tumor volume of the compound group after the start of administration, T t0 is the average tumor volume of the compound group at the first administration, V c0 is the average tumor volume of the vehicle control group at the first administration, V ci The mean tumor volume of the vehicle control group after the start of drug administration.

[0422] The body weight of all tumor-bearing mice was measured twice a week. The relative change ratio of the body weight of the mice after administration was calculated: RCBW (%) = (BW i –BW0) / BW0×100, BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the first drug administration.

[0423] At the end of the experiment, the tumor masses were weighed and photographed.

[0424] Data Analysis

[0425] All data are expressed as mean ± SEM, where SEM = SD / SQRT (n), and n = number of animals in each experimental group. One-way ANOVA was used to compare tumor volume differences between the treatment and control groups. All data were analyzed using GraphPad, and p < 0.05 was considered significant.

[0426] Experimental results

[0427] The results of tumor volume changes in each treatment group are shown in Figure 20 and Table 36.

[0428] On the 24th day after the start of administration, the average tumor volume of the tumor-bearing mice in the control group was 1379.41±179.25mm 3 The average tumor volumes of the tumor-bearing mice in the Enhertu, IN10018, and Enhertu+IN10018 groups were 361.30±113.01mm, respectively. 3 、1190.95±463.62mm 3 , and 151.79±85.19mm 3 The tumor volume growth inhibition rates (TGI) (%) were 83.27%, 15.52%, and 100.39%, respectively; and the relative tumor proliferation rates (T / C) (%) were 26.11%, 85.55%, and 10.98%, respectively. The experimental results demonstrated that under the experimental conditions, the Enhertu, IN10018, and Enhertu+IN10018 groups all exhibited statistically significant inhibitory effects on the in vivo rectal cancer xenograft model LD1-0038-361855.

[0429] At the end of the experiment (day 24), all tumor-bearing mice were euthanized, and the subcutaneous tumors were excised and weighed. The average tumor weights for the control group, the Enhertu group, the IN10018 group, and the Enhertu + IN10018 group were 1.420±0.164 g, 0.278±0.083 g, 1.078±0.439 g, and 0.101±0.058 g, respectively. Tumor weights generally corresponded to tumor volume results.

[0430] Table 37: Data Statistics

[0431] Example 10: In vivo efficacy evaluation of Enhertu and IN10018 as single agents or in combination in the human ovarian cancer xenograft model LD2-0032-200651

[0432] Experimental animals: NU / NU mice, female, weighing 18-21 g, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and housed in the same environment as in Example 6.

[0433] Human ovarian cancer (poorly differentiated adenocarcinoma) tumor tissue (model number LD2-0032-200651) was passaged to FP3+5 and used in this efficacy experiment.

[0434] The test sample information is shown in Table 26.

[0435] Human ovarian cancer LD2-0032-200651 xenografts were cut into approximately 3 mm × 3 mm × 3 mm (approximately 45-60 mg) pieces and inoculated subcutaneously into NU / NU mice. The mice were observed and tumor growth was monitored. On day 18 of inoculation, the average tumor volume of the tumor-bearing mice was 180.47 mm. 3 The grouping and administration were carried out at the same time, and the day of grouping and administration was defined as day 0. The specific grouping information is as shown in Table 38:

[0436] Table 38. Dosing and Grouping Note: N: number of animals; Dosage volume: adjusted according to the weight of tumor-bearing mice (0.2 ml / 20 g);

[0437] The first dose of Enhertu began on the third day after group assignment.

[0438] The main purpose is to detect the growth inhibitory effect or complete cure ability of the test drugs Enhertu and IN10018 as single drugs or in combination on the human ovarian cancer transplant tumor model LD2-0032-200651.

[0439] Tumor volume and tumor-bearing mouse weight measurement: Use vernier calipers to measure twice a week. The tumor volume is calculated as V = 0.5a × b 2 , a, b represent the long diameter and wide diameter of the tumor, respectively;

[0440] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : negative control group RTV). The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV = T Vi / T V0 , where T V0 is the average tumor volume measured at the time of group administration (i.e., d0), T Viis the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0441] Tumor growth inhibition rate TGI (%) = [1-(T ti -T t0 ) / (V ci -V c0 )]×100,T ti is the average tumor volume of the compound group after the start of administration, T t0 is the average tumor volume of the compound group at the first administration, V c0 is the average tumor volume of the vehicle control group at the first administration, V ci The mean tumor volume of the vehicle control group after the start of drug administration.

[0442] The body weight of all tumor-bearing mice was measured twice a week. The relative change ratio of the body weight of the mice after administration was calculated: RCBW (%) = (BW i –BW0) / BW0×100, BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the time of first drug administration. At the end of the experiment, the tumor mass was weighed and photographed.

[0443] Data Analysis

[0444] All data are expressed as mean ± SEM, where SEM = SD / SQRT (n), and n = number of animals in each experimental group. One-way ANOVA was used to compare tumor volume differences between the treatment and control groups. All data were analyzed using GraphPad, and p < 0.05 was considered significant.

[0445] Experimental results

[0446] The results of tumor volume changes in each treatment group are shown in Figure 21 and Table 39.

[0447] Table 39:

[0448] On the 21st day after the start of drug administration, the average tumor volume of the tumor-bearing mice in the control group was 2132.47±513.67mm 3 The average tumor volumes of the tumor-bearing mice in the Enhertu 3mg / kg QW*3 group, the IN10018 25mg / kg QD*21 group, and the IN10018 25mg / kg QD*21 combined with Enhertu 3mg / kg QW*3 group were 1294.64±285.36mm 3 、1289.97±128.06mm 3 and 700.63±129.27mm3 The tumor volume growth inhibition rates (TGI) (%) were 43.20%, 47.11%, and 76.00%, respectively; and the relative tumor proliferation rates (T / C) (%) were 61.08%, 58.04%, and 32.15%, respectively. The experimental results showed that under the experimental conditions, the test drug IN10018 25mg / kg QD*21 combined with Enhertu 3mg / kg QW*3 group exhibited a statistically significant inhibitory effect on tumor growth in the ovarian cancer xenograft model LD2-0032-200651. The tumor volume in the IN10018 25mg / kg QD*21 combined with Enhertu 3mg / kg QW*3 group was smaller than that in the single-drug groups, but did not show a statistically significant difference compared with the Enhertu 3mg / kg QW*3 single-drug group.

[0449] At the end of the experiment (day 21), all tumor-bearing mice were euthanized, and the subcutaneous tumors were excised and weighed. The average tumor weights for the control group, the Enhertu 3 mg / kg QW*3 group, the IN10018 25 mg / kg QD*21 group, and the IN10018 25 mg / kg QD*21 combined with Enhertu 3 mg / kg QW*3 group were 2.075±0.466 g, 1.212±0.271 g, 1.189±0.139 g, and 0.711±0.137 g, respectively. Tumor weights were generally consistent with tumor volume results.

[0450] The body weight of experimental animals can be used as an indirect indicator for monitoring drug toxicity. No mice in any of the drug-dosing groups showed significant weight loss after administration, indicating that tumor-bearing mice tolerated the drugs well at the experimental doses.

[0451] Example 11: Evaluation of the anti-tumor effects of Enhertu and IN10018 in female NOD-SCID mice in the LU-01-1626 human lung cancer patient-derived xenograft (PDX) model

[0452] Experimental animals: NOD SCID mice, female, weighing 18-20 g, purchased from Saiye Biotechnology Co., Ltd., and housed in the same environment as in Example 6.

[0453] Human lung cancer tumor tissue, passaged to FP5, was used in this efficacy experiment.

[0454] The test sample information is shown in Table 26.

[0455] Each mouse was implanted with a LU-01-1626FP5 tumor piece (approximately 30 mm) subcutaneously on the right side. 3Treatment was started on day 18 after tumor implantation, when the average tumor size reached approximately 109 mm. 3 The animals were randomly divided into groups according to tumor volume using Excel-based randomization software. Each group consisted of 4 tumor-bearing mice. The test articles were administered to the mice according to the protocol shown in Table 40 of the experimental design.

[0456] Table 40: Note: 1. N: animal number;

[0457] 2. Dosage volume: The dosage volume is adjusted based on 10 μL / g body weight.

[0458] The primary endpoint is to observe whether tumor growth can be delayed or whether the mice can be cured. Tumor volume and weight of tumor-bearing mice were measured twice a week using a vernier caliper. The tumor volume was calculated as V = 0.5a × b 2 , a, b represent the long diameter and wide diameter of the tumor, respectively;

[0459] Tumor growth inhibition rate TGI (%) = [1-(T ti -T t0 ) / (V ci -V c0) ]×100, T ti is the average tumor volume of the compound group after the start of administration, T t0 is the average tumor volume of the compound group at the first administration, V c0 is the average tumor volume of the vehicle control group at the first administration, V ci The mean tumor volume of the vehicle control group after the start of drug administration.

[0460] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV :RTV in treatment group; C RTV : negative control group RTV). The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV = T Vi / T V0 , where T V0 is the average tumor volume measured at the time of group administration (i.e., d0), T Vi is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0461] The body weight of all tumor-bearing mice was measured twice a week. The relative change ratio of the body weight of the mice after administration was calculated: RCBW (%) = (BWi –BW0) / BW0×100, BW i BW0 is the body weight after the start of drug administration, and BW1 is the body weight at the first drug administration.

[0462] Data Analysis

[0463] Summary statistics of tumor volume at each time point for each group are provided, including the mean and standard error of the mean (SEM).

[0464] The data obtained on day 31 after the start of treatment were statistically analyzed for differences in tumor volume among the groups.

[0465] One-way ANOVA was performed to compare tumor volumes between groups. Since no significant F statistic (ratio of treatment variance to error variance) was obtained, the Games-Howell test was used for comparisons between groups. All data were analyzed using SPSS 29.0, and p < 0.05 was considered statistically significant.

[0466] The results of tumor volume changes in each treatment group are shown in Figure 22 and Table 41.

[0467] Table 41: Note: 1. Mean ± SEM;

[0468] 2. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treatment group by the group mean tumor volume of the control group (T / C);

[0469] 3.TGI(%)=[1-(T31-T0) / (V31-V0)]×100;

[0470] 4. Calculate p-values ​​based on tumor size.

[0471] On the 31st day after the start of administration, the average tumor volume of the tumor-bearing mice in the control group was 1977±135mm 3 The average tumor volumes of the tumor-bearing mice in the Enhertu group, IN10018 group, and IN10018 combined with Enhertu group were 664±157mm 3 、1459±123mm 3 and 405±73mm 3 ; The tumor volume growth inhibition rate TGI (%) was 70.28%, 27.74% and 84.16% respectively; the relative tumor proliferation rate T / C (%) was 33.58%, 73.80% and 20.49% respectively.

[0472] The experimental results showed that the tumor inhibition effect of the combination of the two drugs was better than that of the single drug group.

[0473] Example 12: Evaluation of the anti-tumor effects of test drugs Ab6000-DXD and IN10018 on female BALB / c nude mice in the OVCAR-3 human ovarian cancer (CDX) model

[0474] Experimental animals: BALB / c nude mice, female, 6-8 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., and housed in the same environment as in Example 6.

[0475] OVCAR-3 human ovarian cancer cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. To establish subcutaneous tumors, OVCAR-3 human ovarian cancer cells were harvested by trypsin digestion, centrifuged, washed, and suspended in ice-cold PBS + 5% FCS and growth factor-reduced Matrigel (1:1) at a cell concentration of 1 × 10 7 cells / ml. Then 200 μl of cell suspension containing OVCAR-3 cells was subcutaneously injected into the right flank of nude mice (one site per mouse). When the tumor volume reached 217 mm 3 On day 31 after cell injection, mice were randomly assigned to treatment groups and blank control groups. Group information is shown in Table 42.

[0476] Table 42:

[0477] Tumor diameters were measured twice a week (Monday and Thursday) using a caliper. The volume of each tumor [in mm] was calculated according to the formula 3 [unit], "tumor volume = length x diameter 2 x0.5". To monitor the side effects of treatment, mice were examined daily for abnormalities and their body weights were measured twice a week (Monday and Thursday). Animals were sacrificed at the end of the study; for ethical reasons, animals were not sacrificed if tumor necrosis or tumor size exceeded 2000 mm during the study. 3 The animals will be killed early.

[0478] IN 10018 information is shown in Table 26.

[0479] The full-length sequence of Ab6000 is as follows:

[0480] Heavy chain sequence: VH-CH (Human IgG1 Mutation):

[0481] Light chain sequence: VL-CL (Human Kappa):

[0482] Preparation of Ab6000-DXD ADC drug conjugates

[0483] An appropriate amount of reducing agent (TCEP·HCl, Thermo, CAS No.: 51805-45-9) was added to reduce the antibody (Ab6000) to expose the sulfhydryl groups between the antibody chains. The small molecule Deruxtecan (MCE, CAS No.: 1599440-13-7) was dissolved in DMSO and prepared into a 10 mM solution.

[0484] The dissolved solution was added at a molar ratio of 15:1 between the antibody and the linker-payload, and the antibody and the linker+payload were coupled together through a Michael addition reaction between the thiol group and the maleimide group on the linker.

[0485] The mixture after coupling was buffer exchanged to remove residual small molecules. The purity and DAR (drug / antibody ratio) value of the antibody conjugate after the reaction were determined by HPLC-SEC (size exclusion chromatography) and HPLC-HIC (hydrophobic chromatography), respectively. The coupling results are shown in Table 43.

[0486] Table 43: ADC drug conjugate preparation

[0487] The tumor volumes of each drug-treated group are shown in Figure 23. On day 14 after drug administration, the tumor volume of the control group was 616 mm 3 The tumor volume of the ab6000-DXD monotherapy group was 425 mm 3 The tumor volume of the IN10018 monotherapy group was 510 mm 3 The tumor volume of the ab6000-DXD and IN10018 combination group was 344 mm 3 The data showed that the tumor inhibition effect of the combination of the two drugs was better than that of the single drug group.

[0488] 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 IN10018 or a pharmaceutically acceptable salt thereof in the preparation of a drug for increasing the concentration of a macromolecular drug in tumor tissue, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody or an antibody conjugate, and the structure of IN10018 is as follows:

2. A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug for treating a tumor in a subject, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody or an antibody conjugate, and the structure of IN10018 is as follows:

3. Use of IN10018 or a pharmaceutically acceptable salt thereof in increasing the concentration of a macromolecular drug in tumor tissue, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody or an antibody conjugate, and the structure of IN10018 is as follows:

4. A method for treating a tumor, comprising administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and a macromolecular drug to a subject in need thereof, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody or an antibody conjugate, and the structure of IN10018 is as follows:

5. A kit or a pharmaceutically acceptable composition comprising: (a) IN10018 or a pharmaceutically acceptable salt thereof; and (b) macromolecular drugs, which are monoclonal antibodies, bispecific antibodies or antibody conjugates; The structure of IN10018 is as follows:

6. The use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable salt of IN10018 is tartrate.

7. The use, pharmaceutical combination, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 6, wherein the monoclonal antibody is racotumomab, rituximab, iodine [131I] metuximab (131I-Metuximab), JMT-103, necitumumab, alemtuzumab, elotuzumab, bevacizumab, ofatumumab, tocilizumab, atezolizumab, toripalimab alimab), HX-008, Camrelizumab, Ocrelizumab, Sugemalimab, Lenzilumab, Sintilimab, Vilobelimab, Margetuximab, Siltuximab, Mogamulizumab, Amivantamab, Cadonilimab, Inebilizumab, Iodine I 131 derlotuximabbiotin), Isatuximab, Serplulimab, Retifanlimab, Cetuximab, Adebrelimab, Tislelizumab, Penpulimab, Teprotumumab, Itolizumab, Dostarlimab, Denosumab, Obinutuzumab, Nimotuzumab (Nimotuzumab), Teclistamab, Daratumumab, dinutuximab, Tafasitamab, Socazolimab, Dupilumab, Prolgolimab, Blinatumomab, Geptanolimab, Panitumumab, Canakinumab, Ramucirumab, envafolimab, Belimumab, Leronlimab, Ranibizumab, Natalizumab, Cosibelimab, Zimberelimab, Trastuzumab, Catumaxomab, Durvalumab, avelumab, Ublituximab, Cemiplimab, Pabo Pembrolizumab, Glofitamab, Pertuzumab, Bermekimab, tremelimumab, ipilimumab, Naxitamab, infliximab, nivolumab, Omburtamab, Crizanlizumab, burosumab, Talquetamab, Besokomab(Besilesomab), alirocumab, arcitumomab, or biosimilars thereof; in particular, trastuzumab or a biosimilar thereof; preferably, trastuzumab.

8. The use, pharmaceutical combination, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 6, wherein the bispecific antibody is teclistamab, blinatumomab, cadonilimab, mosunetuzumab, catumaxomab, ulituximab, amivantamab, talquetamab, epcoritamab, gefituzumab (Glofitamab), Zanidatamab, Elranatamab, Tebotelimab, Amivantamab, SI-B001, Odronextamab, KN-026, KN-046, Ivonescimab, SHR-1701, M7824, GEN-3009, Navicixizumab, GB-261, CM-355, Plamotamab, or their biosimilars.

9. The use, pharmaceutical combination, method, kit or pharmaceutically acceptable composition of any one of claims 1 to 6, wherein the antibody conjugate is Loncastuximab Tesirine, Ibritumomab Tiuxetan, Tisotumab Vedotin, Sacituzumab Govitecan, Enfortumab Vedotin, Inotuzumab Ozogamicin, Gemtuzumab Ozogamicin, Belantamab Mafodotin, Trastuzumab Emtansine, Moxetumomab Pasudotox, Polatuzumab Vedotin, Disitamab Vedotin, Brentuximab Vedotin, Trastuzumab deruxtecan (DS-8201), Trastuzumab Emtansine, Cetuximab Sarotalocan, Mirvetuximab Soravtansine, Trastuzumab Duocarmazine, ARX-788, KL-A264, Upifitamab Rilsodotin, Tusamitamab Ravtansine, Naptumomab Estafenatox, Datopotamab Deruxtecan, Oportuzumab Monatox), SHR-A1811, Patritumab Deruxtecan, Telisotuzumab vedotin, TrastuzumabDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, BMS-98 6148, SOT-102, ESG-401, CD117-ADC, LCB-14, W-0101, REGN-5093-M114, CX-2029, BDC-1001, DGN549-C (Pivekimab Sunirine), AVID-100, MRG-003, CAT-5001, HDP-101, MORAb-202 (Farletuzumab Ecteribulin), L-DOS47, Praluzatamab Ravtansine, BA3021 (Ozuriftamab Vedotin), DS-7300, BA3011 (Mecbotamab Vedotin), Camidanlumab Tesirine, Ladiratuzumab Vedotin, LMB-2, SAT-012, DEBIO-1562, PSMA-ADC, MRG-002, OBI-999, Coltuximab Ravtansine, KL-A166, DX-126-262, NBE-002, OXS-1550, Lorvotuzumab Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103ADC, TE-1218, TE-1112, T-PNU, BV-001, DS-1062, SKB264, Ab6000-Dxd or their biosimilars; especially ESG-401, DS-1062, SKB264, Sacituzumab Govitecan, Ab6000-Dxd or their biosimilars; preferably ESG-401, DS-1062, SKB264, Sacituzumab Govitecan, Ab6000-Dxd or their biosimilars deruxtecan, DS-8201) or Ab6000-Dxd.

10. The use, pharmaceutical combination, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 6, wherein the macromolecular drug is an antibody conjugate, especially an antibody conjugate of Trop-2, preferably ESG-401, DS-1062, SKB264 or Sacituzumab Govitecan.

11. The use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 10, wherein the IN10018 or a pharmaceutically acceptable salt thereof and the macromolecular drug are administered to the subject simultaneously or sequentially.

12. The use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 11, for treating a tumor.

13. The use, pharmaceutical combination, method, kit or pharmaceutically acceptable composition of claim 12, wherein the tumor is selected from 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, ovariectomy, ovarian cancer ... Preferably, the tumor is gastric cancer, lung cancer, breast cancer, glioma, esophageal cancer, pancreatic cancer, head and neck cancer, colon cancer or ovarian cancer; more preferably, the tumor is gastric cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer or lung cancer.

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