Methods for increasing the concentration of high molecular weight drugs in tumor tissue

Abstract

This disclosure relates to the treatment of tumors using IN10018 in combination with a macromolecular drug, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

Description

[Technical Field]

[0001] This application claims priority to China Patent Application No. 202310343152.0 filed on March 31, 2023, priority to China Patent Application No. 202311276741.8 filed on September 28, 2023, and priority to China Patent Application No. 202410283445.9 filed on March 12, 2024.

[0002] This invention belongs to the field of drug chemistry. Specifically, this invention relates to the treatment of tumors using a combination of the focal adhesion kinase (FAK) inhibitor IN10018 and a high molecular weight drug. [Background technology]

[0003] Malignant tumors (cancer) have become one of the major public health problems threatening human health worldwide. As recent statistics show, deaths from malignant tumors account for 23.91% of all deaths in China, and both the incidence and mortality rates of malignant tumors have been on a continuous upward trend over the past few decades, with annual medical costs for malignant tumors in China exceeding 220 billion yuan.

[0004] In recent years, the research and development process of macromolecular drugs, primarily monoclonal antibodies, bispecific antibodies, and antibody-drug conjugates, has gradually accelerated, and many macromolecular drugs have been introduced to the antitumor therapy market. Macromolecular drugs can kill tumor cells by accurately identifying tumor antigens and inhibiting the cascade reaction between the antigen and downstream, or by carrying small molecule conjugates, thereby expanding their effective antitumor activity through this unique mechanism. Tumor tissue includes tumor cells and their surrounding stroma, and various mechanisms exist in this stroma-rich tumor microenvironment that inhibit the diffusion (penetration) of macromolecular drugs. These stroma barriers include fibroblasts and fibrous collagen that are in close contact with tumor cells, further inhibiting the effective action of antitumor drugs on tumor cells. Based on these mechanisms, reagents that can suppress fibrous proliferation of tumors or reduce fibrous stroma may contribute to increasing the concentration of macromolecular drugs in tumor tissue. As can be seen, the development of drugs that can suppress fibrous proliferation of tumors or reduce fibrous stroma has the potential to advance tumor treatment and has significant clinical value. [Overview of the Initiative]

[0005] The inventors have found that the focal adhesion kinase (FAK) inhibitor IN10018 contributes to increasing the concentration of high molecular weight drugs in tumor tissue. Aside from the limitations of the reason, it is thought that the activated FAK pathway can significantly promote tumor fibrosis, and that the FAK inhibitor IN10018 reduces FAK activity, significantly reduces fibrous proliferation of tumors, and contributes to the penetration of high molecular weight drugs into the tumor microenvironment.

[0006] In one aspect of the present disclosure, the use of IN10018 or a pharmaceutically acceptable salt thereof in the manufacture of a drug for increasing the concentration of a polymer drug in tumor tissue, wherein the polymer drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows: [ka]

[0007] A pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and a polymeric drug, relating to another aspect of this disclosure, is for the treatment of tumors in a subject, wherein the polymeric drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows: [ka]

[0008] In another aspect of the present disclosure, the 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-drug conjugate, and the structure of IN10018 is as follows: [ka]

[0009] A method for treating a tumor according to another aspect of the present disclosure comprises administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and a polymeric drug to a subject in need of treatment, wherein the polymeric drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows. [ka]

[0010] Kits or pharmaceutically acceptable compositions relating to other embodiments of this disclosure are: (a) IN10018 or a pharmaceutically acceptable salt thereof, (b) comprising a monoclonal antibody, a bispecific antibody, or a macromolecular drug which is an antibody-drug conjugate, The structure of IN10018 is as follows: [ka]

[0011] In another aspect of this disclosure, a pharmaceutical combination product of IN10018 or a pharmaceutically acceptable salt thereof and a polymeric drug, wherein the polymeric drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and is intended for the treatment of a tumor in a subject.

[0012] In another aspect of this disclosure, the use of IN10018 or a pharmaceutically acceptable salt thereof and a polymeric drug in the manufacture of a combination drug for treating tumors, wherein the polymeric drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

[0013] In another aspect of the present disclosure, the use of IN10018 or a pharmaceutically acceptable salt thereof in the manufacture of a combination drug for treating tumors in combination with a polymer drug, wherein the polymer drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

[0014] In another aspect of the present disclosure, in the use of a polymer drug in the manufacture of a combination drug for treating tumors in combination with IN10018 or a pharmaceutically acceptable salt thereof, the polymer drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

[0015] A kit relating to another aspect of the present disclosure comprises IN10018 or a pharmaceutically acceptable salt thereof, and a statement indicating that the IN10018 or a pharmaceutically acceptable salt thereof can be used in combination with a macromolecular drug to treat tumors, wherein the macromolecular drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

[0016] A kit relating to another aspect of the present disclosure comprises a polymer drug and a statement indicating that the polymer drug can be used in combination with IN10018 or a pharmaceutically acceptable salt thereof to treat a tumor, wherein the polymer drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

[0017] Selectively, the pharmaceutically acceptable salts of IN10018 are tartrates.

[0018] Selectively, the monoclonal antibodies include lacotumomab, rituximab, iodine[131I]metuximab, JMT-103, necitumumab, alemtuzumab, elotuzumab, bevacizumab, ofatumumab, tocilizumab, atezolizumab, tripalimab, HX-008, and camrelizumab. Camrelizumab, Ocrelizumab, Sugemalimab, Lenzilumab, Sintilimab, Vilobelimab, Margetuximab, Siltuximab, Mogamulizumab, Amivantamab, Cadonilimab, Inebilizumab, Iodine [131I]derlotuximab biotin biotin), Isatuximab, Serplulimab, Retifanlimab, Cetuximab, Adebrelimab, Tislelizumab, Penpulimab, Teprotumumab, Itolizumab, Dostarlimab, Denosma Denosumab, Obinutuzumab, Nimotuzumab, Teclistamab, Daratumumab, Dinutuximab, Tafasitamab, Socazolimab, Dupilumab, Prolgolimab, Blinatumomab,Geptanolimab, Panitumumab, Canakinumab, Ramucirumab, Envafolimab, Belimumab, Leronlimab, Ranibizumab, Natalizumab, Kosiberimab Cosibelimab, Zimberelimab, Trastuzumab, Catumaxomab, Durvalumab, Avelumab, Ublituximab, Cemiplimab, Pembrolizumab, Grofitamab (G The biosimilars are lofitamab, pertuzumab, bermekimab, tremelimumab, ipilimumab, naxitamab, infliximab, nivolumab, omburtamab, crizanlizumab, burosumab, talquetamab, besilesomab, alirocumab, arcitumomab, or their biosimilars, and more particularly trastuzumab or its biosimilar, preferably trastuzumab.

[0019] Biosimilars, also known as biosimilars, are biopharmaceuticals that are highly similar to approved original biopharmaceuticals in terms of quality, safety, and efficacy. Examples include bevacizumab biosimilar, adalimumab biosimilar, and rituximab biosimilar.

[0020] Selectively, the bispecific antibodies include tecristamab, blinatumomab, cadonilimab, mosunetuzumab, catumaxomab, ublituximab, amivantamab, talquetamab, epcoritamab, grofitamab, and zanidatamab (Z This includes anidatamab, elranatamab, tebotelimbab, amivantamab, SI-B001, odronextamab, KN-026, KN-046, Ivonescimab, SHR-1701, M7824, GEN-3009, navicixizumab, GB-261, CM-355, plamotamab, or their biosimilars.

[0021] Selectively, the antibody-drug conjugates include loncastuximab tesirine, ibritumomab tiuxetan, tisotumab vedotin, sacituzumab govitecan, enfortumab vedotin, inotuzumab ozogamicin, gemtuzumab ozogamicin, belantamab mafodotin, trastuzumab emtansine, moxetumomab pasudotox, and polatuzumab vedotin. 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 Estafenatox), Datopotamab Deruxtecan, Oportuzumab Monatox, SHR-A1811, Patritumab Deruxtecan, Telisotuzumab vedotin, Trastuzumab DuocalmazineDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, B MS-986148, 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 Meltansine Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103 ADC, 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 deruxtecan (DS-8201), Ab6000-Dxd or their biosimilars, preferably ESG-401, DS-1062, SKB264, sacituzumab govitecanThe drug is Govitecan / trastuzumab deruxtecan (DS-8201) or Ab6000-Dxd, preferably ESG-401, sacituzumab govitecan, trastuzumab deruxtecan (DS-8201), or Ab6000-Dxd.

[0022] Selectively, the polymer drug is an antibody-drug conjugate.

[0023] Selectively, the antibody-drug conjugates include loncastuximab tesirine, ibritumomab tiuxetan, tisotumab vedotin, sacituzumab govitecan, enfortumab vedotin, inotuzumab ozogamicin, gemtuzumab ozogamicin, belantamab mafodotin, trastuzumab emtansine, moxetumomab pasudotox, and polatuzumab vedotin. 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 Estafenatox), Datopotamab Deruxtecan, Oportuzumab Monatox, SHR-A1811, Patritumab Deruxtecan, Telisotuzumab vedotin, Trastuzumab DuocalmazineDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, B MS-986148, 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 Meltansine Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103 ADC, TE-1218, TE-1112, T-PNU, BV-001, DS-1062, SKB264, or their biosimilars, particularly ESG-401, DS-1062, SKB264, Sacituzumab Govitecan, Ab6000-Dxd, or their biosimilars, preferably ESG-401, DS-1062, SKB264, Sacituzumab Govitecan Govitecan, Trastuzumab deruxtecanThe active ingredient is deruxtecan (DS-8201) or Ab6000-Dxd, preferably ESG-401, sacituzumab govitecan, trastuzumab deruxtecan (DS-8201), or Ab6000-Dxd.

[0024] Selectively, the antibody-drug conjugate is a Trop-2 antibody-drug conjugate.

[0025] Selectively, the antibody-drug conjugate of Trop-2 is ESG-401, DS-1062, SKB264, sacituzumab govitecan, or a biosimilar thereof, preferably ESG-401, DS-1062, SKB264, or sacituzumab govitecan, and preferably ESG-401 or sacituzumab govitecan.

[0026] Selectively, IN10018 or a pharmaceutically acceptable salt thereof and the polymer drug are administered to the subject simultaneously or sequentially.

[0027] Selectively, the above uses, pharmaceutical combination products, methods, kits, or pharmaceutically acceptable compositions are used to treat tumors.

[0028] Selectively, the tumors include 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), malignant 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, cholangiocarcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine tumor, ovarian cancer, salivary gland cancer, metastatic tumors from spindle cell carcinoma, and anaplastic large cell lymphocytes. The tumors are selected from, for example, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematopoietic malignancies, such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), and preferably the tumor is gastric cancer, lung cancer, breast cancer, glioma, esophageal cancer, pancreatic cancer, head and neck cancer, colon cancer, or ovarian cancer, and more preferably the tumor is gastric cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer, or lung cancer.

[0029] To more clearly illustrate the technical means in the embodiments of this disclosure, the drawings of the embodiments will be briefly described below. However, it is clear that the drawings in the following description relate only to some embodiments of this disclosure and do not limit the invention. [Brief explanation of the drawing]

[0030] [Figure 1] The image shows panoramic scan thumbnails of frozen sections from Example 1. The images (thumbnails) were obtained by performing a full slide scan of the entire section using a digital pathology image panoramic scanning system. In the blue areas, the cell nuclei are DAPI-stained, and in the green areas, the GFP protein fluorescence of the administered drug T-GFP itself is represented. A and B are panoramic scan images of two animals from the control group, respectively, while C and D are panoramic scan images of two animals from the IN10018+T-GFP group, respectively. [Figure 2]This shows the percentage of T-GFP-positive cells in Example 1. This shows the percentage of tumor invasion after administration of the test substance T-GFP to a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model. [Figure 3] The H-Score scores of T-GFP-positive cells from Example 1 are shown. The H-Score scores for the degree of tumor invasion after administration of IN10018 to a Balb / c-nude mouse subcutaneous tumor xenograft model of human gastric cancer NCI-N87 cells with the test substance T-GFP are shown. [Figure 4] The changes in body weight of MDA-MB-468 tumor-bearing mice in different groups after administration of Example 2 are shown. Data are expressed as mean + standard error (SEM). [Figure 5] The changes in relative body weight of MDA-MB-468 tumor-bearing mice in different groups after administration of Example 2 are shown. Data are expressed as mean + standard error (SEM). [Figure 6] The changes in tumor volume in MDA-MB-468-bearing mice during the administration period of Example 2 are shown. Data are expressed as mean + standard error (SEM). [Figure 7] The panoramic scan thumbnails of the frozen sections from Example 3 are shown. [Figure 8] The tumor growth curves of mice after administration of different test substances in Example 4 are shown, with data points representing the group-average tumor volume and the error line representing the standard error (SEM). [Figure 9] The binding status of tumor cells to antibodies in each treatment group at the end of the experiment in Example 5 is shown, and the staining intensity represents the binding strength between tumor cells and antibodies within each group. [Figure 10] The data from the day of group administration 14 days after vaccination in Example 6 are shown. [Figure 11] The total fluorescence intensity in Example 6 is shown, with data points representing the total fluorescence intensity of each animal in the group, and the error line representing the standard error (SEM). [Figure 12]The fluorescence imaging images of the H-Cy5.5 injection in Example 6 are shown. A and B show the fluorescence images of the G1 control group, C and D show the fluorescence images of the G2 IN10018 25 mg / kg group, and E shows the fluorescence image of the negative control group. [Figure 13] The graph shows the mean fluorescence intensity curve of the tumor site after administration of IN10018 to a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model using the test substance H-Cy5.5 in Example 6. The value at 0h represents the mean fluorescence intensity of the negative control group. The data points represent the group mean fluorescence intensity values, and the error line represents the standard error (SEM). [Figure 14] The images show fluorescence imaging 48 hours after injection of the test substance H-Cy5.5 in a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model in Example 6. A and B show fluorescence images of the G1 control group, and C and D show fluorescence images of the G2 IN10018 25 mg / kg group. [Figure 15] The tumor growth curves after administration of a BALB / c-nude mouse subcutaneous tumor xenograft model in which NCI-N87&NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts were co-inoculated in Example 7 are shown, with data points representing the group-average tumor volume and the error line representing the standard error (SEM). [Figure 16] The graph shows the weight change curve after administration of a BALB / c-nude mouse subcutaneous tumor xenograft model in which NCI-N87&NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts were co-inoculated in Example 7. The data points represent the group mean weight, and the error line represents the standard error (SEM). [Figure 17] The graph shows the rate of weight change after administration of a BALB / c-nude mouse subcutaneous tumor xenograft model in which NCI-N87&NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts were co-inoculated in Example 7. The data points represent the group mean rate of weight change, and the error line represents the standard error (SEM). [Figure 18] The percentage change in body weight for Example 8 is shown. The data points represent the group mean change in percentage of body weight, and the error bars represent the standard error (SEM) of the mean. [Figure 19]The tumor growth curve for Example 8 is shown. The data points represent the group mean, and the error bars represent the standard error (SEM) of the mean. [Figure 20] The tumor growth curve for Example 9 is shown. [Figure 21] The tumor growth curve for Example 10 is shown. [Figure 22] The tumor growth curve for Example 11 is shown. [Figure 23] The tumor growth curve for Example 12 is shown. [Modes for carrying out the invention]

[0031] To further clarify the purpose, technical means, and advantages of the embodiments of this disclosure, the technical means of the embodiments of this disclosure will be described clearly and completely below with reference to the drawings of the embodiments. Clearly, the embodiments described are some, but not all, embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the embodiments described are all within the scope of the protection of the present invention.

[0032] The present invention can be implemented in other specific forms without departing from the fundamental attributes of the invention. It should be understood that, to the extent that they do not contradict each other, any one and all embodiments of the present invention can be combined with the technical features of any one other embodiment or more other embodiments to obtain other embodiments. The present invention includes other embodiments obtained from such combinations.

[0033] All publications and patents referenced in this disclosure are incorporated herein by reference. Any uses or terms used in any publication or patent incorporated by reference shall be governed by the uses and terms of this disclosure if they conflict with the uses or terms used herein.

[0034] The section titles used herein are for structural purposes only and should not be interpreted as limiting the subject matter.

[0035] Unless otherwise specified, all technical and scientific terms used herein have their general meanings within the field to which the subject matter for protection pertains. If a term has multiple definitions, the definition provided herein shall prevail.

[0036] All numerical values ​​of quantitative nature, such as dosages, described herein and in the claims should be understood to be modified in all cases by the term “approximately,” unless otherwise specified in the examples or elsewhere. Furthermore, it should be understood that any numerical range given herein is intended to include all sub-regions within that range and any combination of the endpoints of that range or sub-range.

[0037] Similar words used in this disclosure, such as “include,” “contain,” or “incorporate,” are intended to mean that the element preceding the word covers the elements and their equivalents listed after the word, and do not exclude any elements not described. The terms “contain” or “include” as used herein may be open, semi-closed, or closed. In other words, the terms above also include “substantially consisting of” or “consisting of.”

[0038] definition The following terms and symbols used in this application have the following meanings unless otherwise specified in the context.

[0039] As used herein, "monoclonal antibody" refers to a monoclonal antibody drug, i.e., a monoclonal antibody drug, which is generally a biopharmaceutical, a large molecule protein, and whose target of action is a specific protein on the cell membrane. In some embodiments, the monoclonal antibodies are lacotumomab, rituximab, iodine[131I]metuximab, JMT-103, necitumumab, alemtuzumab, elotuzumab, bevacizumab, ofatumumab, tocilizumab, atezolizumab, toripalimab, HX-008, and Camre. Camrelizumab, Ocrelizumab, Sugemalimab, Lenzilumab, Sintilimab, Vilobelimab, Margetuximab, Siltuximab, Mogamulizumab, Amivantamab, Cadonilimab, Inebilizumab, Iodine[131I]delrotuximab biotin I 131 derlotuximab biotin), Isatuximab, Serplulimab, Retifanlimab, Cetuximab, Adebrelimab, Tislelizumab, Penpulimab, Teprotumumab, Itolizumab, Dostarlimab, Denosumab, Obinutuzumab, Nimotuzumab, Teclistamab, Daratumumab,Dinutuximab, Tafasitamab, Socazolimab, Dupilumab, Prolgolimab, Blinatumomab, Geptanolimab, Panitumumab, Canakinumab, Ramucirumab, Envafol imab), belimumab, leronlimab, ranibizumab, natalizumab, cosibelimab, zimberelimab, trastuzumab, catumaxomab, durvalumab, avelumab, ublituximab ), cemiplimab, pembrolizumab, grofitamab, pertuzumab, bermekimab, tremelimumab, ipilimumab, naxitamab, infliximab, nivolumab, omburtamab, Crizanlizumab, burosumab, talquetamab, besilesomab, alirocumab, arcitumomab, or their biosimilars; in some embodiments, trastuzumab or its biosimilars; and in some embodiments, trastuzumab.

[0040] As used herein, "bispecific antibody" (bsAb) refers to an antibody having two specific antigen-binding sites. In some embodiments, the bispecific antibody is tecristamab, blinatumomab, cadonilimab, mosunetuzumab, catumaxomab, ublituximab, amivantamab, talquetamab, epcoritamab, grofitamab, or zanidatama. These include Zanidatamab, Elranatamab, Tebotelimbab, Amivantamab, SI-B001, Odronextamab, KN-026, KN-046, Ivonescimab, SHR-1701, M7824, GEN-3009, Navicixizumab, GB-261, CM-355, Plamotamab, or their biosimilars.

[0041] As used herein, "antibody-drug conjugate" (ADC) refers to a drug that uses a monoclonal antibody as a carrier to deliver a small molecule drug with biological activity to target cells, by linking the small molecule drug to a monoclonal antibody via a chemical chain. In some embodiments, the macromolecule drug is an antibody-drug conjugate. For example, the above antibody-drug conjugates include loncastuximab tesirine, ibritumomab tiuxetan, tisotumab vedotin, sacituzumab govitecan, enfortumab vedotin, inotuzumab ozogamicin, gemtuzumab ozogamicin, belantamab mafodotin, trastuzumab emtansine, moxetumomab pasudotox, and polatuzumab vedotin. 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 Estafenatox, Datopotamab deluxtecanDeruxtecan), Oportuzumab Monatox, SHR-A1811, Patritumab Deruxtecan, Telisotuzumab vedotin, Trastuzumab Duocalmazine Duocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, B MS-986148, 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 Meltansine Mertansine), TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103 ADC, TE-1218, TE-1112, T-PNU, BV-001, Ab6000-Dxd, or their biosimilars, and in some embodiments, ESG-401, Sacituzumab GovitecanThe active ingredient is Govitecan, trastuzumab deruxtecan (DS-8201), Ab6000-Dxd, or a biosimilar thereof, and in some embodiments, it is ESG-401, sacituzumab Govitecan, trastuzumab deruxtecan (DS-8201), or Ab6000-Dxd.

[0042] Trastuzumab is a monoclonal antibody against Her 2.

[0043] ESG-401 is an antibody-drug conjugate against Trop-2 jointly developed by Shanghai Shijian Biotechnology Co., Ltd. and Suzhou Liannang Biopharmaceutical Co., Ltd., and is disclosed in WO2021225892A1.

[0044] Sacituzumab Govitecan is an antibody-drug conjugate against Trop-2 developed by Immunomedics, and its trade name is Trodelvy.

[0045] As used herein, “combination drug” or “combination drug product” may refer to a fixed combination of a single dose unit form (for example, in which the active ingredients of all drugs are present in a single dosage form) or a kit of combination drug products, or a combination of one drug and instructions indicating that the drug can be used in combination with one or more other drugs.

[0046] As used herein, “combination therapy” or “combination drug” means treating a disease by using one drug in combination with one or more other drugs, and includes not only combinations of one drug with one or more other drugs, but also combinations of one drug with instructions indicating that the drug can be used in combination with one or more other drugs.

[0047] "Administered simultaneously or sequentially" in this application means administering two or more drugs simultaneously or at regular time intervals within a single administration cycle (e.g., within 4 weeks, 3 weeks, 2 weeks, 1 week, or 24 hours), where the method of drug administration (e.g., oral, intravenous, intramuscular, or subcutaneous) may be the same or different, and the frequency / cycle of administration of the two or more drugs may be the same or different. If the therapeutic method, product, or use of this disclosure involves two drugs, the two drugs may be administered simultaneously or individually at regular time intervals.

[0048] As used herein, the term “treatment” means administering one or more drug substances to a subject having a disease or symptoms of the disease to cure, alleviate, reduce, modify, treat, improve, enhance, or influence the disease or symptoms of the disease. In some embodiments, the disease is a tumor or cancer.

[0049] As used herein, the term "tumor" refers to an abnormal lesion formed by the clonal abnormal proliferation of cells in a local tissue that has lost normal growth regulation at the genetic level under the action of various oncogenic factors. The tumors mentioned above include 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), malignant 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, cholangiocarcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine tumor, ovarian cancer, salivary gland cancer, metastatic tumors from spindle cell carcinoma, anaplastic large cell lymphoma, and thyroid cancer. Undifferentiated carcinoma, non-Hodgkin lymphoma, Hodgkin lymphoma, glioma, and hematopoietic malignancies, including, but not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). Preferably, the tumors are lung cancer, breast cancer, glioma, esophageal cancer, pancreatic cancer, head and neck cancer, colon cancer, or ovarian cancer. More preferably, the tumors are gastric cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer, or lung cancer.

[0050] As used herein, the terms “subject” or “subject” refer to mammals and non-mammals. Mammals refer to any member of the mammalian species and include, but are not limited to, humans; non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats and pigs; domestic animals such as rabbits, dogs and cats; and laboratory animals such as rodents such as rats, mice and guinea pigs. Examples of non-mammals include, but are not limited to, birds. The term “subject” is not limited to a specific age or sex. In some embodiments, the subject is human.

[0051] As used herein, the term "pharmaceutically acceptable" means non-toxic, biologically tolerable, and suitable for administration to a subject.

[0052] As used herein, the term “pharmaceutically acceptable salt” refers to an acid addition salt that is non-toxic, biologically tolerable, and suitable for administration to a subject, including acid addition salts formed with inorganic acids such as hydrochlorides, hydrobroms, carbonates, bicarbonates, phosphates, sulfates, sulfites, and nitrates, as well as formates, acetates, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfons, p-toluenesulfons, 2-hydroxyethanesulfons, benzoates, salicylates, stearates, and salts of the formula HOOC-(CH2) n This includes, but is not limited to, acid addition salts formed with organic acids, such as salts formed with alkanedicarboxylic acids of -COOH (where n is 0 to 4 in the formula).

[0053] Furthermore, pharmaceutically acceptable acid addition salts may be produced by dissolving a free base in a suitable solvent and treating the solution with an acid, following the usual procedure for preparing acid addition salts from basic compounds. Those skilled in the art can determine various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable acid addition salts without excessive experimentation. In some embodiments, the pharmaceutically acceptable salt of IN10018 is a tartrate.

[0054] As used herein, “pharmaceutically acceptable composition” means that the formulation, along with other components and / or the subject receiving treatment with it, must be chemically and / or toxicologically compatible. As used herein, “therapeutic dose” means an amount sufficient to produce a beneficial therapeutic effect on the subject. The therapeutic dose 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., administration method, pharmacokinetics of the compound, disease severity and course, subject's medical history, subject's health status, subject's responsiveness to the drug, etc.).

[0055] As used herein, the term "inhibition" refers to a reduction in the baseline activity of a biological activity or process.

[0056] As used herein, the term “kit” refers to a box for containing chemical reagents for detecting chemical components, residual drugs, types of viruses, etc. The kit of the present invention comprises (i) IN10018 or a pharmaceutically acceptable salt thereof and / or a polymeric drug, and (ii) instructions indicating that a tumor can be treated in a subject using IN10018 or a pharmaceutically acceptable salt thereof and the polymeric drug, wherein the polymeric drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate. In one embodiment, the kit comprises (i) IN10018 or a pharmaceutically acceptable salt thereof, and (ii) instructions indicating that a tumor can be treated in a subject using IN10018 or a pharmaceutically acceptable salt thereof and the polymeric drug, wherein the polymeric drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate. In one embodiment, the kit comprises (i) a high molecular weight drug and (ii) instructions indicating that a tumor can be treated in a subject using IN10018 or a pharmaceutically acceptable salt thereof and the high molecular weight drug, wherein the high molecular weight drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate.

[0057] The compounds in the kit may be contained in separate containers. Selectively, two or more compounds may be contained in the same container. For example, the kit may include a first container, a second container, and a package insert, wherein the first container contains at least one dose of a drug comprising IN10018 or a pharmaceutically acceptable salt thereof, the second container contains at least one dose of a high molecular weight drug, the high molecular weight drug being a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the package insert includes instructions for using the drug to treat a target tumor. The first and second containers may be the same or different shapes (e.g., vials, syringes, and bottles) and / or materials (e.g., plastic or glass). The kit may further include other materials that help administer the drug, such as diluents, filters, IV bags and pipelines, needles, and syringes.

[0058] The precise amount of IN10018 or its pharmaceutically acceptable salts and high molecular weight drugs administered to a subject depends on various factors such as the drug or compound, drug formulation, route of administration, type of disease, symptoms, and the status of the subject or host being treated, but can still usually be determined by a person skilled in the art. For example, the determination of an effective dose also depends on the degree, severity, and type of cell proliferation. A person skilled in the art can determine an appropriate dose in accordance with these and other factors.

[0059] IN10018 or its pharmaceutically acceptable salts and high molecular weight drugs may be administered by an appropriate method, such as oral, intravenous, intramuscular, or subcutaneous administration.

[0060] For example, when administered orally, the drug may be administered orally with a pharmaceutically acceptable carrier such as an inert diluent or an absorbable food carrier. These may be encapsulated in hard or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into the patient's food. For example, the drug may be combined with one or more excipients and may be used in the form of ingestible tablets, oral tablets, lozenges, capsules, elixirs, suspensions, syrups, or wafers. Tablets, lozenges, pills, capsules, etc., may further contain adhesives such as tragacanth gum, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, fructose, lactose, or aspartame; or flavorings.

[0061] For example, when administered intravenously or intraperitoneally as an infusion or injection, the drug solution can be prepared in water and optionally mixed with a non-toxic surfactant.

[0062] Exemplary drug dosage forms for injection or infusion include sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient suitable for immediate preparation of sterile injection or infusion solutions or dispersions. In all cases, the final dosage form must be sterile, fluid, and stable under manufacturing and storage conditions.

[0063] A sterile injection solution may be prepared by incorporating the required amount of drug, along with the various other necessary components, into a suitable solvent, and then sterilizing by filtration. In the case of sterile powders for preparing a sterile injection solution, preferred preparation methods are vacuum drying and freeze-drying techniques, which can produce powders of any desired components present after prior sterile filtration, in addition to the powder of the active ingredient.

[0064] The amount of IN10018 or its pharmaceutically acceptable salts and high molecular weight drugs required for treatment can vary depending not only on the specific reagent selected, but also on the route of administration, the nature of the disease being treated, and the patient's age and condition, and can ultimately be determined by the attending physician or clinician. However, generally, the dose is in the range of approximately 0.1 to 50 mg / kg body weight per day.

[0065] In some embodiments, IN10018 or a pharmaceutically acceptable salt thereof is administered to adults at a dose of 5 mg / day to 100 mg / day, for example, 20 mg / day, where the dose is calculated based on the free base.

[0066] The above-mentioned high molecular weight drugs are administered to adults in a dose range of 1 to 20 mg / kg per week. In a specific embodiment, trastuzumab is administered to adults in a dose of 1 to 5 mg / kg per week, for example, 2 mg / kg. In a specific embodiment, ESG-401 is administered to adults in a dose of 2 to 20 mg / kg per week, 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. In a specific embodiment, sacituzumab govitecan is administered to adults in a dose of 2 to 20 mg / kg per week, for example, 6 mg / kg or 10 mg / kg.

[0067] Technical and scientific terms used herein that are not specifically defined have meanings that are generally understood by those skilled in the art. [Examples]

[0068] To further illustrate the present invention, the following examples are provided. It should be understood that these examples are merely illustrative and not intended to limit the scope of the present invention.

[0069] In the following examples, experimental methods for which specific conditions are not explicitly stated may be carried out under normal conditions or conditions recommended by the manufacturer.

[0070] Unless otherwise specified, the experimental materials and reagents used in the following examples are all commercially available.

[0071] The meanings of the abbreviations used in the examples are as follows:

[0072] [Table 1]

[0073] Example 1: In vivo pharmacological study of tumor invasion after administration of IN10018 to a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model using trastuzumab (T-GFP) labeled with green fluorescent protein.

[0074] Experimental objective: The purpose of this experiment is to evaluate the degree of invasion within the tumor by administering trastuzumab (T-GFP), labeled with the test substance green fluorescent protein, before and after administering IN10018 for a certain period of time to a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model. Frozen sections were prepared, stained with multiplex immunofluorescence, and panoramic scans were performed. The expression of each protein marker was observed, and image analysis was performed using HALO pathology image analysis software.

[0075] Experimental design: Table 1 shows the groupings and administration status.

[0076] [Table 2]

[0077] Experimental materials: Mice: Female BALB / c-nude mice aged 7-8 weeks were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. After purchasing the animals, they were allowed to adapt to the experimental environment for at least 3 days before the experiment began. The animals were housed in cages in the SPF housing room (5 animals / cage). All cages, bedding, and drinking water must be sterilized before use. All researchers should wear protective clothing and latex gloves when working in the housing room. Cages, feed, and drinking water were changed twice a week. The housing environment and light exposure conditions were as follows: Temperature: 20~26℃ Humidity: 40~70% Light irradiation cycle: 12 hours of light irradiation followed by 12 hours of no light irradiation. Cage: Made of polycarbonate, with dimensions of 300mm x 180mm x 150mm. Corn cob is used as bedding and is replaced twice a week. Food: Experimental animals are free to consume (radiation-sterilized, dried particulate food) throughout the entire experimental phase. Drinking water: Laboratory animals are free to drink sterile water. Cage Labels: Each cage's animal information card should clearly indicate the number of animals in the cage, sex, breed, date of receipt, administration scheme, experiment number, group, and experiment start date. Animal labeling: Laboratory animals are marked with ear tags.

[0078] Table 2 shows information about the test substance.

[0079] [Table 3]

[0080] Table 3 shows information about the experimental reagents.

[0081] [Table 4]

[0082] Table 4 shows information about the experimental equipment.

[0083] [Table 5]

[0084] Experimental method and steps:

[0085] Cell culture: Human gastric cancer NCI-N87 (derived from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60491) cells were cultured in vitro in a monolayer. The culture conditions were RPMI-1640 medium with 10% fetal bovine serum added, cultured at 37°C in a 5% CO2 incubator. The cells were subcultured with standard digestion using pancreatin-EDTA 2-3 times per week. When the cells were in the exponential growth phase and the saturation reached 80%-90%, the cells were harvested, counted, and then inoculated.

[0086] Cell inoculation and grouping: 5 x 10 6 A cell suspension containing 100 cells in a PBS:Matrigel=1:1 mixture was subcutaneously inoculated at 0.1 mL into the posterior dorsal right side of each mouse. The tumor volume was approximately 125.6 mm². 3 If this occurs (14 days after cell inoculation), the patients are randomly divided into groups based on tumor volume and administered accordingly. The group division information is shown in Table 1.

[0087] Table 5 shows the preparation information for the test substance and the control solvent.

[0088] [Table 6]

[0089] Daily observations of laboratory animals: The development and any modifications of this experimental scheme were both evaluated and approved by IACUC. The use and care of experimental animals were carried out in accordance with AAALAC regulations. The health status and mortality of the animals were monitored daily, and periodic examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake (visual only), weight changes, external signs, or other abnormal conditions. The number of animal deaths and adverse events within each group were recorded based on the number of animals in each group.

[0090] Experiment stopped: If the animal's health condition is persistently deteriorating, or if the tumor volume reaches 3000 mm³ 3 Euthanasia is necessary if the animal exceeds a certain threshold or is suffering from a serious illness or pain. Euthanasia should be performed in the following cases, informing the veterinarian: The animal is clearly emaciated, with a weight loss greater than 20%, unable to freely access food and water, and the average tumor volume of the control group is 2000 mm². 3 The experiment was stopped. The animals exhibited clinical symptoms such as bristly fur, hunched posture, pale ears, noses, eyes, or feet, shortness of breath, seizures, continuous diarrhea, dehydration, sluggishness, and vocalizations, and their condition steadily worsened.

[0091] Tumor measurement and dissection: The experimental indicators were used to consider whether tumor growth was suppressed, delayed, or cured. Tumor diameter was measured using calipers, three times per week. The formula for calculating tumor volume was V = 0.5 × a × b 2 Here, a and b represent the long and short diameters of the tumor, respectively.

[0092] On day 28 of continuous treatment with IN10018, i.e., day 42 after inoculation, each animal received a single tail vein injection of T-GFP. Six hours after injection, all animals were euthanized, the tumors were removed and immersed in liquid nitrogen. After approximately 10 minutes, the tumor samples were transferred to dry ice and immediately subjected to further research.

[0093] Frozen section: For tumor tissue rapidly frozen with liquid nitrogen, a sedimentation flow is first completed according to a 20%-30%-30% (sucrose solution) flow, and then OCT embedding is performed to create a frozen-embedded block. Frozen sections are prepared using a cryomicrotome, and the sections are attached to a highly adhesive glass slide. When observed under a microscope, if the section is complete, clearly undamaged, and wrinkle-free, the preparation of the frozen section is complete. The glass slide is then left to stand at room temperature for a specific time and stained directly, or stored in a low-temperature refrigerator.

[0094] DAPI counterstaining and mounting: It is preferable to drop approximately 100-150 μl of DAPI working solution onto a glass slide and immerse the tissue area. Next, incubate in a wet box at room temperature for 3-5 minutes, wash with 1×TBST (or PBS) for 5 minutes, wash with distilled water for 3 minutes, and finally mount by dropping an antifluorescent quenching mounting medium. The coverslip is then fixed with nail polish, and a panoramic scan and subsequent data analysis are performed using an instrument.

[0095] Panoramic scan and quantitative analysis: A full slide scan was performed on the entire section using a digital pathology image panoramic scanning system to obtain pathology image information for the entire section. Quantitative analysis of the scan results was performed 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, each parameter corresponding to the analysis module was adjusted according to the actual conditions of the image. After determining the optimal parameters, all similar images were analyzed using the same analysis template to avoid the influence of artificial subjective factors. The information displayed for each different region represents only one analysis layer, identifying the different staining signal conditions of each cell within the analysis field. Simultaneously, once the quantitative analysis of a single layer of each image is completed, the overall information of all results is statistically compiled and analyzed.

[0096] Analysis metrics: Percentage of GFP-positive cells: For each defined cell type, the number of positive cells in the sample and the percentage of the total number of cells of that type are statistically recorded.

[0097] The H-Score is a histochemical score. It is calculated by analyzing each staining channel as a unit, statistically counting the total number of positive cells for each channel in each sample, as well as the number of cells at three levels: weakly positive, moderately positive, and positive. The corresponding positive cell status is then statistically calculated for the three regions: the cell nucleus, cytoplasm, and cell membrane. Finally, the corresponding percentage of the total cells and the H-Score value for each channel are calculated. The value ranges from 0 to 300, and the calculation formula is H-Score = [1 × (%Cells1+) + 2 × (%Cells2+) + 3 × (%Cells3+)], where %Cells1+, %Cells2+, and %Cells3+ are the percentages of cells at the three expression levels (weakly positive, moderately positive, and positive), as automatically identified by the software.

[0098] Experimental results: Image scan results A full slide scan was performed on the entire point using a digital pathology image panoramic scanning system to obtain pathology image information for the entire section. Figure 1 shows thumbnails of each image displayed in the report.

[0099] Study on the percentage of tumor invasion after administration of the test substance T-GFP to IN10018 in a Balb / c-nude mouse subcutaneous tumor xenograft model of human gastric cancer NCI-N87 cells.

[0100] The experiment was conducted according to the administration scheme. On day 28 of treatment with continuous administration of IN10018, i.e., day 42 after inoculation, all animals received a single tail vein injection of T-GFP. Six hours after injection, all animals were euthanized, the tumors were dissected, frozen-embedded blocks were prepared, frozen sections were made, and after DAPI staining, all slides were scanned using a digital pathology image panoramic scanning system. Quantitative analysis of the scan results was performed using the HALO Highplex FL (Indica Labs, Albuquerque, NM) analysis module of the pathology image analysis software Halo. During the analysis process, each parameter corresponding to the analysis module was adjusted according to the actual conditions of the image. After determining the optimal parameters, all similar images were analyzed using the same analysis template to avoid the influence of artificial subjective factors. Information displayed for different regions each time represents only one analysis layer. Layer names may be customized to identify the different staining signal conditions of each cell within the analysis field. Once the quantitative analysis of a single layer of each image was completed, the overall information of all results was statistically summarized. For each defined cell type, the number of positive cells in the sample and the percentage of cells of that type that accounted for the total number of cells in the sample were statistically analyzed.

[0101] In the first animal of the control group, the total number of cells detected was 132,264, of which 7,532 were GFP-positive, representing a GFP-positive cell ratio of 5.69%. In the second animal of the control group, the total number of cells detected was 235,747, of which 11,757 were GFP-positive, representing a GFP-positive cell ratio of 4.99%. The average positive cell ratio for the entire control group was 5.34%. In the first animal of the IN10018+T-GFP group, the total number of cells detected was 104,126, of which 13,470 were GFP-positive, representing a GFP-positive cell ratio of 12.94%. In the second animal of the IN10018+T-GFP group, the total number of cells detected was 182,491, of which 14,994 were GFP-positive, representing a GFP-positive cell ratio of 8.22%. The average positive cell ratio for the entire IN10018+T-GFP group was 10.58%. This is shown in Table 6 and Figure 2.

[0102] [Table 7]

[0103] Study of the degree of tumor invasion H-Score score after administration of the test substance T-GFP to a Balb / c-nude mouse subcutaneous tumor xenograft model of human gastric cancer NCI-N87 cells.

[0104] The experiment was conducted according to the experimental scheme. After performing a full slide scan 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 statistically analyze the total number of positive cells for each channel in each sample, as well as the number at three levels: weakly positive, moderately positive, and positive. The corresponding positive cell status was then statistically analyzed for the three regions: the cell nucleus, cytoplasm, and cell membrane. Finally, the corresponding percentage of the total cells and the H-Score value for each channel were calculated.

[0105] In the first animal of the control group, the percentages of weakly positive, moderately positive, and strongly positive cells detected were 4.87%, 0.51%, and 0.31%, respectively, and the H-Score calculated using the formula was 6.83. In the second animal of the control group, the percentages of weakly positive, moderately positive, and strongly positive cells detected were 4.21%, 0.56%, and 0.22%, respectively, and the H-Score calculated using the formula was 5.98. The average H-Score for 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 detected were 9.83%, 1.66%, and 1.45%, respectively, and the H-Score calculated using the formula was 17.49. In the second animal of the IN10018+T-GFP group, the percentages of weakly positive, moderately positive, and strongly positive cells detected were 5.97%, 1.30%, and 0.95%, respectively, and the H-Score calculated using the formula was 11.41. The average H-Score for the entire IN10018+T-GFP group was 14.45. These figures are shown in Table 7 and Figure 3.

[0106] [Table 8]

[0107] Experimental conclusion In this experiment, the inventors evaluated the degree of tumor invasion before and after administration of IN10018 in a xenograft model of human gastric cancer NCI-N87 cells and Balb / c-nude mice with subcutaneous tumors, using the test substance T-GFP. The degree of tumor invasion and the corresponding H-Score score 6 hours after T-GFP administration for each group are shown in Table 7, Figures 1, 2, and 3.

[0108] Compared to the percentage of tumor infiltration and corresponding H-Score scores in two mice in the blank control group, the percentage of GFP+-positive cells and corresponding H-Score scores in the IN10018+T-GFP group were both higher than in the control group after administration of IN10018 for a certain period (27 days). These data suggest that after administration of IN10018 for a certain period, the binding of the drug to human gastric cancer cells can be more effectively promoted, potentially leading to better efficacy in the body.

[0109] Example 2: Pharmacodynamic studies of IN10018 and ESG-401 in Balb / C nude mice of the tumor-bearing MDA-MB-468 model.

[0110] The experimental design is shown in Table 8.

[0111] [Table 9]

[0112] Experimental materials Mice: Female BALB / c-nude mice (weight: 19-23 grams), 6-8 weeks old, were purchased from the Laboratory Animal Management Department of the Shanghai Institute of Family Planning. After purchasing the animals, they were adapted to the experimental environment for at least 3 days before the experiment began. The animals were housed in cages (3-4 animals / cage). All cages, bedding, and drinking water must be sterilized before use. All researchers should wear protective clothing and latex gloves when working in the animal room. Cages, feed, and drinking water were changed twice a week. The rearing environment and lighting conditions were as follows: Temperature: 18~26℃. Humidity: 40%~70%. Cage: Made of polycarbonate. Dimensions: 300mm x 180mm x 150mm. Bedding is corn cob. Cage identification: Each cage's identification label includes the number of animals, sex, date and time of receipt, compound, study number, group number, and treatment start date. Animal identification: Animals were numbered using the ear-punch method.

[0113] The information of the test substance is shown in Table 9.

[0114] [Table 10]

[0115] The information of the experimental reagents is shown in Table 10.

[0116] [Table 11]

[0117] The preparation of the compound is shown in Table 11.

[0118] [Table 12]

[0119] Experimental method MDA-MB-468 cells (Biyuntian) were cultured in vitro in L-15 (Gibco) medium supplemented with 10% FBS (Gibco) and 1% P.S. (Gibco) (37 °C, 0% CO2). Subculture was performed 2 - 7 3 times a week. Logarithmic-phase MDA-MB-468 cells were collected and resuspended in a suspension of PBS and Matrigel (the volume ratio of PBS to Matrigel is 1:1) to prepare a cell suspension with a cell concentration of 5×10 6 / mL, which was used for mouse inoculation. 0.2 mL of the MDA-MB-468 tumor cell suspension (10×10 3 cells) was subcutaneously inoculated into the right back of each mouse. When the average tumor volume of the mice reached about 160 mm

[0120] Clinical observation All procedures related to the treatment, care, and study of animals were carried out under the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), in accordance with guidelines approved by the Animal Experimentation Committee (IACUC). During routine monitoring, any effects of tumor growth and treatment on the normal behavior of the animals, such as activity, food and water consumption (observation only), weight gain / loss (weight measured twice every two days or once a week), and any other abnormal effects specified in the agreement, such as eye / hair, were examined daily. Deaths and observed clinical signs were recorded based on the number of animals in each mouse cage.

[0121] Tumor measurement The main objective of the experiment is to observe whether tumor growth is delayed or whether remission occurs. The average tumor volume in the control group of mice was 2000 mm³. 3 If the time limit was exceeded, or if it occurred within 3 weeks after administration, the experiment was terminated based on the shorter of the two time intervals.

[0122] The size of the tumor was measured using calipers, twice a week, and the tumor volume was recorded in mm². 3 The formula for calculating tumor volume is V = 0.5 × a × b 2 Here, a and b are the long and short diameters of the tumor, respectively. Next, the tumor size is used to calculate the relative tumor growth rate T / C (%). The formula for calculating T / C (%) is T / C% = (T i -T0) / (V i -V0) × 100%, and T i V is the mean tumor volume on a specific day in the treatment group, T0 is the mean tumor volume on the first day of treatment (before administration) in the treatment group, and V i is, T i V0 is the mean tumor volume of the control group on the day of measurement, and V0 is the mean tumor volume of the control group on the first day of treatment. The TGI (%) for each group was calculated using the formula TGI (%) = [100 - T / C].

[0123] Sample collection On day 45 after administration, the experiments with mice in groups 2 and 4 were terminated according to the experimental requirements. In this case, mice in group 1 were divided into a control group and a treatment group, and additional experiments were continued. Based on the experimental requirements, the experiments with mice that underwent additional experiments were terminated on day 63. Subsequently, the mice that underwent additional experiments were euthanized, their tumors were collected, and preserved in 10% formalin.

[0124] statistical analysis Experimental data are presented as mean values ​​and standard error (SEM) of the mean values, including tumor volume for each group at each time point. The differences in tumor volume between groups were statistically analyzed. P-values ​​were calculated using Graphpad Prism 7.0 software.

[0125] A difference analysis was performed on the tumor volume of each group at each time point using a T-test, and a p<0.05 indicates a significant difference.

[0126] Experimental results 29 days after administration, administration was stopped for all groups and observation was conducted, based on experimental requirements. On the 45th day after administration, the experiment for mice in groups 2 to 4 was terminated, based on experimental requirements. For details on the specific group divisions and administration status, please refer to the administration records in attached Table 8.

[0127] The data for body weight and tumor volume of mice in all groups during the experimental period are as follows:

[0128] Body weight of MDA-MB-468-bearing mice was measured periodically as an indirect indicator of toxicity. No significant decrease in the average body weight of mice in each group was observed after administration. Detailed changes in body weight and relative body weight after administration of MDA-MB-468-bearing mice are shown in Figures 4 and 5.

[0129] Tumor volume The tumor growth curves during the experimental period are shown in Figure 6. On day 45 after the experiment, the mean tumor volume of the control group, ESG-401 group, IN10018 group, and ESG-401 + IN10018 combination therapy group was 414.79 mm², respectively. 3, 190.10mm 3 , 240.14mm 3 and 94.48mm 3 That is the case.

[0130] T / C (%) and TGI (%) and p-value Table 12 shows T / C and TGI data at different time points during the administration period. At 45 days after administration, compared to the control group, the T / C ratios were 10.30%, 30.13%, and -28.00% for the ESG-401 group, 30.13% for the IN10018 group, and -28.00% for the ESG-401 + IN10018 combination therapy group, respectively, and the TGI ratios were 89.70%, 69.87%, and 128.00% for the ESG-401 group, IN10018 group, and -28.00% for the IN10018 group.

[0131] Table 13 shows significance analyses at different time points. At 45 days after administration, ESG-401 monotherapy, IN10018 monotherapy, and combination therapy of both all showed significant antitumor effects compared to the control group.

[0132] [Table 13]

[0133] [Table 14]

[0134] Experimental results The purpose of this experiment was to evaluate the antitumor efficacy of ESG-401 monotherapy, IN10018 monotherapy, and combination therapy of both compounds. During the administration period, no significant decrease in body weight was observed in the treated mice, indicating that the mice possessed high tolerance to the compounds.

[0135] On day 45 after administration, the mean tumor volume in the control group, ESG-401 group, IN10018 group, and ESG-401 + IN10018 combination therapy group was 414.79 mm², respectively. 3 , 190.10mm 3 , 240.14mm 3 and 94.48mm 3 That is the case.

[0136] The T / C ratios for the ESG-401 group, IN10018 group, and ESG-401 + IN10018 combination therapy group were 10.30%, 30.13%, and -28.00%, respectively, and the TGI ratios were 89.70%, 69.87%, and 128.00%, respectively.

[0137] Compared to the control group, ESG-401 monotherapy, IN10018 monotherapy, and combination therapy of both all showed significant antitumor effects (p<0.05).

[0138] Based on the above, in Balb / C nude tumor-bearing mice of the MDA-MB-468 model, administration of ESG-401 monotherapy, administration of IN10018 monotherapy, and administration of both in combination all showed significant antitumor effects.

[0139] Example 3: In vivo pharmacological study of tumor invasion after administration of IN10018 in the MDA-MB-468 model of ESG-401.

[0140] Reagent information is shown in Table 14.

[0141] [Table 15]

[0142] Table 15 shows information about the experimental equipment.

[0143] [Table 16]

[0144] Experimental steps for paraffin-embedded tissue sections Material Collection: Fresh mouse tumors collected in Example 2 were immersed in 10% neutral formalin for at least 24 hours. Do not freeze in a refrigerator at 4°C. Remove the tumors from the fixative, trim the target tissue with a scalpel in a fume hood, place the trimmed tissue and corresponding label in a dehydration cassette, and attach a specimen code to the specimen bottle. Lightly rinse the fixative from the tissue in a container of water.

[0145] Dehydration: The embedding blocks were placed in the dehydration basket of a dehydrator and sequentially dehydrated with alcohols of varying concentrations. 75% alcohol 2h - 85% alcohol 1h - 90% alcohol 1h - 95% alcohol 40min - Anhydrous ethanol I 30min - Anhydrous ethanol II 30min - Alcohol benzene 10min - Xylene I 5min - Xylene II 5min - Paraffin I 1h - Paraffin II 1h - Paraffin III 1h.

[0146] Embedding: Tissue immersed in paraffin was embedded in an embedding apparatus. First, molten paraffin was placed in an embedding block, and before the paraffin solidified, the tissue was removed from the dehydration cassette and placed in the embedding mold and embedding block according to the requirements of the embedding surface. After cooling on a -20°C cooling plate and the paraffin solidified, the paraffin block was removed from the embedding block and trimmed.

[0147] Sections: Trimmed paraffin blocks were placed in a paraffin microtome, and sections were cut to a thickness of 3 μm. The sections were floated in 42°C warm water in a spreading machine to spread the tissue, placed on glass slides, and dried in a 60°C oven. After the moisture had completely dried and the paraffin had melted, the sections were removed and stored at room temperature for use.

[0148] Experimental steps for picrosilius red staining Deparaffinization and immersion of paraffin sections: The sections were sequentially immersed 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, and 70% alcohol for 5 minutes, and then washed with distilled water.

[0149] Immersion and staining with picrosilius red staining solution: The specimens were incubated in picrosilius red staining solution for 20-30 minutes and then washed with pure alcohol.

[0150] Dehydration and mounting: The sections were dehydrated and cleared by sequentially placing them in 95% alcohol II for 15 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, xylene I for 10 minutes, and xylene II for 10 minutes. The sections were then removed from the xylene, allowed to dry slightly, and mounted in neutral balsam.

[0151] The samples were examined under a microscope, and images were acquired and analyzed using a scanner (3D histech, Panoramic MIDI). Thumbnails are shown in Figure 7.

[0152] As can be seen from the picrosilius red staining results, the degree of fibrous proliferation in the ESG-401 monotherapy group was similar to that of the control group, but tumor fibrosis was significantly suppressed in the IN10018 and ESG-401 combination group compared to the ESG-401 monotherapy group. The anti-human IgG secondary antibody can distinguish the binding action of ESG-401 in the body to tumor cells, and as can be seen from the results, in the ESG-401 monotherapy group, antibody-tumor cell binding staining was generated, while the IN10018 and ESG-401 combination group had a more pronounced antibody-tumor cell binding staining effect compared to the ESG-401 monotherapy group.

[0153] Example 4: In vivo pharmacodynamic studies of IN10018 and sacituzumab govitecan (TRODELVY) in a pancreatic cancer PC-07-0067 subcutaneous tumor xenograft BALB / c nude mouse model.

[0154] Experimental Design Establishment of the PDX Model: The establishment of the human pancreatic cancer PC-07-0067 model involves defining the P0 generation as the first generation of clinical samples obtained by surgical excision and transplanted into nude mice. The next generation of tumor tissue obtained by transplanting the P0 generation is called the P1 generation, and this is used by analogy to continue transplanting into nude mice. The FP3 tumor is obtained by resuscitation of the P2 generation, and the next generation generated by the FP3 generation is defined as FP4, and this is used by analogy.

[0155] Animals: BALB / c nude mice, female, 6-8 weeks old, weighing 18-22 grams. A total of 60 mice were needed. Provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0156] Tumor inoculation: 20-30mm 3 PC-07-0067 FP5 tumor tissue blocks were subcutaneously inoculated into the posterior dorsal right side of each mouse, and tumor growth was allowed to occur until the average tumor volume reached approximately 100-150 mm². 3 If this occurred, random group assignment and administration were initiated. The experimental group assignment and administration scheme are shown in the table below.

[0157] Table 16 shows the animal experiment groupings and administration scheme.

[0158] [Table 17]

[0159] Animal Rearing: After purchasing the animals, they were reared in the experimental environment for 3-7 days before the experiment began. The animals were reared in an SPF rearing room in IVC (Individual Ventilation System) cages (5 animals per cage). All cages, bedding, and drinking water must be sterilized before use. All researchers should wear protective clothing and latex gloves when working in the rearing room. The animal information card in each cage should clearly indicate the number of animals in the cage, sex, breed, date of receipt, administration scheme, experiment number, group, and experiment start date. Cages, feed, and drinking water were changed twice a week. The rearing environment and light exposure conditions were as follows: Temperature: 20-26°C, Humidity: 40-70%, Light exposure cycle: 12 hours of light exposure followed by 12 hours of no light exposure.

[0160] Feed composition: Feed conforms to laboratory animal food testing standards. The maximum levels of contaminants are within controllable limits and are subject to regular testing by the manufacturer. Drinking water is autoclaved.

[0161] Animal grouping: Animals were weighed and tumor volume measured before administration. Based on tumor volume, they were randomly assigned to groups (random grouping design).

[0162] Observations: The development and any modifications of this experimental scheme are only permissible after evaluation and approval by the Animal Care Committee (IACUC). The use and welfare of experimental animals were carried out in accordance with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of the animals were monitored daily, and periodic examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake, weight changes (weight measured twice a week), external signs, or other abnormal conditions. The number of animal deaths and adverse events within each group were recorded based on the number of animals in each group.

[0163] Experimental Indicators: Experimental indicators were used to consider whether tumor growth was suppressed, delayed, or cured. Tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume was V = 0.5a × b 2Here, a and b represent the long and short diameters of the tumor, respectively.

[0164] The tumor inhibitory effect of the compounds was evaluated by TGI (%) or tumor growth rate T / C (%). TGI (%) reflects the tumor growth inhibition rate. TGI (%) calculation: TGI (%) = [1 - (average tumor volume at the end of administration for a treatment group - average tumor volume at the start of administration for that treatment group) / (average tumor volume at the end of treatment for the solvent control group - average tumor volume at the start of treatment for the solvent control group)] × 100%.

[0165] The formula for calculating tumor growth rate T / C (%) is as follows: T / C (%) = T i / V i ×100%. V i This is the mean tumor volume of the solvent control group at a specific measurement, and T i This represents the average tumor volume of the treatment group during the same measurement cycle.

[0166] After the experiment is completed, the tumor weight is detected, T weight / C weight Calculate the percentage, T weight and C weight The values ​​represent the tumor weight of the treatment group and the solvent control group, respectively.

[0167] Experiment stopped if the animal's health condition continues to deteriorate or the tumor volume reaches 3000 mm³. 3 If the animal exceeds a certain threshold, or if it has a serious illness or is in pain, euthanasia may be necessary. In the following situations, notify a veterinarian and have the animal euthanized:

[0168] They were clearly underweight, lost more than 20% of their body weight, were unable to freely consume food and water, and the average tumor volume in the control group was 2000 mm². 3 The experiment was stopped. The animals exhibited clinical symptoms such as bristly fur, hunched posture, pale ears, noses, eyes, or feet, shortness of breath, seizures, continuous diarrhea, dehydration, sluggishness, and vocalizations, and their condition steadily worsened.

[0169] Data Analysis: T-tests were used for comparisons between two groups. For comparisons between three or more groups, one-way ANOVA was used. If there was a significant difference in the F-score, multiple comparisons should be performed after the ANOVA analysis. All data were analyzed using SPSS 17.0. A p-value of <0.05 was considered statistically significant.

[0170] The test compounds are shown in Table 17.

[0171] [Table 18]

[0172] Table 18 shows a detailed scheme for drug preparation.

[0173] [Table 19]

[0174] Experimental results Fourteen days after administration, administration was stopped for all groups based on experimental requirements, and observation was conducted. On the 14th day after administration, the experiment was terminated based on experimental requirements, and materials were collected from all experimental mice (used in Example 5). For details on specific group divisions and administration status, please refer to the administration records in Table 16 attached.

[0175] The data for body weight and tumor volume of mice in all groups during the experimental period are as follows:

[0176] Body weight of PC-07-0067 tumor-bearing mice was measured periodically as an indirect indicator of toxicity. No significant decrease in the average body weight of mice in any group was observed after administration.

[0177] Tumor volume The tumor growth curves during the experimental period are shown in Figure 8. On day 14 after the experiment, the mean tumor volume of the control group, the TRODELVY group, and the TRODELVY + IN10018 combination therapy group was 1512 mm², respectively. 3 , 423mm 3and 104 mm 3 It is as follows.

[0178] T / C (%), TGI (%), and p value 14 days after administration, in the TRODELVY group and the combined administration group of TRODELVY + IN10018, the T / C values were 27.95% and 6.86% respectively, and the TGI values were 78.91% and 102.10% respectively.

[0179] From the above, in the NOD-SCID tumor-bearing mice of the PC-07-0067 model, the combined administration of TRODELVY and IN10018 showed a higher antitumor effect compared to the TRODELVY monotherapy group.

[0180] Example 5 In vivo pharmacological study of the degree of tumor infiltration after administration of IN10018 in the PC-07-0067 model of TRODELVY

[0181] The experimental method was the same as that in Example 3. The experimental tissue samples were from Example 4. The color thumbnails are shown in Figure 9. The anti-human IgG secondary antibody can identify the binding effect between TRODELVY in the body and tumor cells. As can be seen from the results, in the TRODELVY monotherapy group, the binding between the antibody and tumor cells was significant, and the combined group of IN10018 and TRODELVY had a more significant antibody-tumor cell binding effect compared to the TRODELVY monotherapy group.

[0182] Example 6 In vivo pharmacological study of the degree of tumor infiltration after administration of IN10018 in a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model of the test substance labeled with the fluorescent dye Cy5.5-trastuzumab (H-Cy5.5)

[0183] The purpose of this experiment was to perform fluorescence imaging on the tumor sites of mice administered with trastuzumab labeled with the fluorescent dye Cy5.5 (H-Cy5.5) of the test substance before and after administration of IN10018 for a certain period (7 days) in a human gastric cancer NCI-N87 cell Balb / c-nude mouse subcutaneous tumor xenograft model, and to evaluate the degree of infiltration inside the tumor.

[0184] Table 19 shows the group assignments and treatment regimens.

[0185] [Table 20]

[0186] Experimental materials: Mice: Female BALB / c-nude mice aged 6-8 weeks were purchased from Shanghai Lingchang Biotechnology Co., Ltd. After purchasing the animals, they were adapted to the experimental environment for at least 3 days before the experiment began. The animals were housed in cages in the SPF housing room (5 animals / cage). All cages, bedding, and drinking water must be sterilized before use. All researchers should wear protective clothing and latex gloves when working in the housing room. Cages, feed, and drinking water were changed twice a week. The housing environment and light exposure conditions were as follows: Temperature: 20~26℃ Humidity: 40~70% Light irradiation cycle: Irradiate with light for 12 hours, then do not irradiate with light for 12 hours. Cage: Made of polycarbonate, with dimensions of 300mm x 180mm x 150mm. Corn cob is used as bedding and is replaced twice a week. Food: Experimental animals are free to consume (radiation-sterilized, dried particulate food) throughout the entire experimental phase. Drinking water: Laboratory animals are free to drink sterile water. Cage Labels: Each cage's animal information card should clearly indicate the number of animals in the cage, sex, breed, date of receipt, administration scheme, experiment number, group, and experiment start date. Animal labeling: Laboratory animals are marked with ear tags.

[0187] Table 20 shows information about the test substance.

[0188] [Table 21] Table 21 shows information about the experimental reagents.

[0189]

Table 22

[0190] Experimental methods and steps Cell culture Human gastric cancer NCI-N87 (derived from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60491) is maintained in passage by YingShi Biotechnology (Nanjing) Co., Ltd. The cells are cultured in monolayer in vitro, and the culture conditions are to add 10% fetal bovine serum to RPMI-1640 medium and culture in a 5% CO2 incubator at 37°C. Subculture was performed by normal digestion using pancreatin-EDTA 2-3 times a week. When the cells were in the exponential growth phase and the saturation was 80%-90%, the cells were collected, counted, and then inoculated.

[0191] Cell inoculation and grouping 1×10 7 A PBS cell suspension containing cells was subcutaneously inoculated at 0.2 mL on the right back of each mouse. When the tumor volume reached about 200.0 mm 3 (on the 14th day after cell inoculation), based on the tumor volume, random grouping was performed for administration, and the grouping information is shown in Table 19.

[0192] Synthesis of H-Cy5.5 H-Cy5.5 was synthesized at the Institute of Biophysics, Chinese Academy of Sciences, Beijing. The concentration of trastuzumab was 7.44 mg / ml, that is, 50.27 uM, the concentration of Cy5.5 after synthesis was 101 uM, and the labeling ratio Dye / Protein was about 2.01. Based on mouse bioimaging, it was proposed to inject 1-3 nmol of Cy5.5 per animal. In this experiment, it was selected to inject 3 nmol of Cy5.5 per animal.

[0193] The preparation information of the test substance and the control solvent is shown in Table 22.

[0194]

Table 23

[0195] Daily observation of laboratory animals The establishment and any modifications to this experimental scheme are only permissible after evaluation and approval by the Animal Care Committee (IACUC). The use and welfare of experimental animals were carried out in accordance with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of the animals were monitored daily, and periodic examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake (visual only), weight changes, external signs, or other abnormal conditions. The number of animal deaths and adverse events within each group were recorded based on the number of animals in each group.

[0196] Experiment stopped If the animal's health condition is persistently deteriorating, or if the tumor volume reaches 3000 mm³ 3 Euthanasia is necessary if the animal exceeds a certain threshold or is suffering from a serious illness or pain. Euthanasia should be performed in the following cases, informing the veterinarian: The animal is clearly emaciated, with a weight loss greater than 20%, unable to freely access food and water, and the average tumor volume of the control group is 2000 mm². 3 The experiment was stopped. The animals exhibited clinical symptoms such as bristly fur, hunched posture, pale ears, noses, eyes, or feet, shortness of breath, seizures, continuous diarrhea, dehydration, sluggishness, and vocalizations, and their condition steadily worsened.

[0197] Tumor measurement The experimental indicators were used to consider whether tumor growth was suppressed, delayed, or cured. Tumor diameter was measured using calipers, three times per week. The formula for calculating tumor volume was V = 0.5 × a × b 2 Here, a and b represent the long and short diameters of the tumor, respectively.

[0198] Tumor imaging On day 7 after IN10018 administration, H-Cy5.5 was administered via tail vein injection to the G1 control group and the G2 IN10018 25 mg / kg group. At 6, 24, and 48 hours after injection, the animals were imaged using the IVIS® Lumina III small animal in vivo imaging system, and the fluorescence intensity was detected.

[0199] Experimental indicators Fluorescence signal values ​​in mice: In vivo imaging was performed on mice, the entire torso was gated, and the total radiant efficiency of the entire torso was collected.

[0200] Average fluorescence signal value of tumor sites: In vivo imaging was performed on mice, and the tumor sites of the mice were clearly gated in a white light field. The average fluorescence intensity (Avg Radiant Efficiency) of the tumors within the gate was collected.

[0201] statistical analysis Statistical analysis was performed using Prism Graphpad software based on tumor volume at the end of the study. For a comprehensive comparison of average fluorescence intensity over different periods, analysis was performed using two-way ANOVA and Fisher's LSD test, and a statistically significant difference was considered to exist if P<0.05.

[0202] Experimental results Fourteen days after tumor inoculation, 14 animals with appropriate tumor volumes were selected and randomly divided into groups, with an average tumor volume of approximately 200 mm³. 3 The day of administration was defined as day 0, and IN10018 was administered to group G2 at a dose of 25 mg / kg according to the plan. Figure 10 shows the data for the day of group division administration, 14 days after vaccination. The mean tumor volume of the G1 control group was 200.7 ± 30.8 mm 3 The mean tumor volume in the G2 IN10018 25 mg / kg group was 200.0 ± 33.4 mm². 3 (mean ± SEM)

[0203] Study of total fluorescence intensity 6 hours after injection of the test substance H-Cy5.5 into a Balb / c-nude mouse subcutaneous tumor xenograft model of human gastric cancer NCI-N87 cells. The experiment was conducted according to the administration scheme. On the 7th day after continuous treatment with IN10018, i.e., the 21st day after inoculation, each animal received a single tail vein injection of 150 µl of H-Cy5.5. Six hours after injection, the animals were imaged using the IVIS® Lumina III small animal in vivo imaging system, and the total fluorescence intensity of the torso region was detected.

[0204] In vivo imaging was performed on two groups of mice 6 hours after tail vein injection of H-Cy5.5. The torso region of the mice was gated, and the total radiant efficiency (TFE) was detected. Animals not injected with H-Cy5.5 were selected as negative controls and imaged. The TFE of the G1 control group was 2.86E+11±1.08E+11, the TFE of the G2 IN10018 25 mg / kg treatment group was 2.88E+11±1.09E+11, and the TFE of the negative control animals was 2.04E+10±1.18E+10. The TFE data were integrated and compared with the negative control group, and the p-values ​​for the G1 control group and the G2 IN10018 25 mg / kg treatment group were p=0.0167 and p=0.0167, respectively. Integrating the total fluorescence intensity data, the G1 control group showed a p-value of p=0.3176 compared to the G2 IN10018 25 mg / kg treatment group. For details, see Table 23 and Figures 11 and 12.

[0205] [Table 24]

[0206] Study of tumor invasion 6, 24, and 48 hours after injection of the test substance H-Cy5.5 in a Balb / c-nude mouse subcutaneous tumor xenograft model of human gastric cancer NCI-N87 cells.

[0207] The experiment was conducted according to the experimental scheme. On the 7th day after continuous treatment with IN10018, i.e., the 21st day after inoculation, all animals received a single tail vein injection of H-Cy5.5. At 6, 24, and 48 hours after injection, the animals were imaged using the IVIS® Lumina III small animal in vivo imaging system, and the average fluorescence intensity of the tumor site was detected. Clear gating was performed on the tumor site of the mice in a white light field, and the average fluorescence intensity (Avg Radiant Efficiency) of the tumor within the gate was collected.

[0208] 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, and the mean fluorescence intensity of the tumor site in the G2 IN10018 25mg / 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, and the mean fluorescence intensity of the tumor site in the G2 IN10018 25mg / kg treatment group was 6.41E+09±2.42E+09. 48 hours after H-Cy5.5 injection, the mean fluorescence intensity of tumor sites in the G1 control group was 6.61E+09±2.50E+09, and the mean fluorescence intensity of tumor sites in the G2 IN10018 25 mg / kg treatment group was 8.04E+09±3.04E+09. Statistical analysis was performed by integrating the mean fluorescence intensity data at each time point for the G1 control group and the G2 IN10018 25 mg / kg treatment group. The p-value for the two groups at 48 hours after injection was p=0.0148. See Table 24 and Figures 13 and 14 for details.

[0209] [Table 25]

[0210] Experimental conclusion In this experiment, the inventors evaluated the degree of tumor invasion before and after administration of IN10018 to a xenograft model of human gastric cancer NCI-N87 cells in Balb / c-nude mice with subcutaneous tumors, using the test substance H-Cy5.5. The degree of tumor invasion and corresponding fluorescence intensity 6, 24, and 48 hours after H-Cy5.5 administration for each group are shown. As can be seen from the total fluorescence intensity of the animals 6 hours after administration, the fact that the average total fluorescence intensity was the same for each group after injecting the same dose of H-Cy5.5 into the animals indicates that there were no problems with this injection. As can be seen from the average fluorescence intensity values ​​of the tumor sites in animals 6, 24, and 48 hours after administration, H-Cy5.5 in the tumor sites tends to concentrate over time. The average fluorescence intensity values ​​of the G2 IN10018 25 mg / kg group must all be higher than those of the G1 control group, and a statistically significant difference exists at 48 hours. These data suggest that administering IN10018 can more effectively promote the binding of the drug to human gastric cancer NCI-N87 cells, potentially leading to better in vivo efficacy.

[0211] Example 7: Study of the in vivo antitumor drug efficacy after administering Enhertu (trastuzumab deruxtecan) in combination with IN10018 to a BALB / c-nude mouse subcutaneous tumor xenograft model in which human gastric cancer NCI-N87 cells and mouse embryonic fibroblast cells NIH-3T3 cells were co-inoculated.

[0212] Experimental Design Table 25 shows the group assignments and treatment regimens.

[0213] [Table 26]

[0214] The animal rearing environment and DPBS source were the same as in Example 6, and information on the test substance is shown in Table 26.

[0215] [Table 27]

[0216] Experimental Method and Steps cell culture Human gastric cancer NCI-N87 (derived from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60491) is subcultured by Yingshi Biotechnology (Nanjing) Co., Ltd. Cells were cultured in vitro in a monolayer culture under the following conditions: RPMI-1640 medium with 10% fetal bovine serum added, cultured at 37°C in a 5% CO2 incubator. Subculture was performed using standard digestion treatment with pancreatin-EDTA 2-3 times per week.

[0217] Mouse embryonic fibroblasts (NIH-3T3 cells, derived from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60317) were passaged by Yingshi Biotechnology (Nanjing) Co., Ltd. The cells were cultured in vitro in a monolayer under the following conditions: RPMI-1640 medium with 10% calf serum, cultured at 37°C in a 5% CO2 incubator. The cells were passaged using standard digestion with pancreatin-EDTA 2-3 times per week. When both cell types were in the exponential growth phase and confluence reached 80%-90%, the cells were harvested, counted, and inoculated.

[0218] Cell inoculation and grouping Mice inoculated with a mixture of NCI-N87 and NIH-3T3 cells were subjected to a 10 × 10 6 NCI-N87 cells and 5 × 10 5 A mixed cell suspension containing 100 NIH-3T3 cells was subcutaneously inoculated into the posterior dorsal right side of each mouse at a dose of 0.2 mL. Eight days after inoculation, the tumor volume of mice inoculated with the mixed NCI-N87 and NIH-3T3 cells was approximately 154 mm². 3 In such cases, the patients were randomly divided into groups based on tumor volume and administered accordingly. The group divisions are shown in Table 25.

[0219] Table 27 shows the preparation information for the test substance and the control solvent.

[0220] [Table 28]

[0221] Daily observation of laboratory animals The establishment and any modifications to this experimental scheme are only permissible after evaluation and approval by the Animal Care Committee (IACUC). The use and welfare of experimental animals were carried out in accordance with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of the animals were monitored daily, and periodic examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake (visual only), weight changes, external signs, or other abnormal conditions. The number of animal deaths and adverse events within each group were recorded based on the number of animals in each group.

[0222] Experiment stopped If the animal's health condition is persistently deteriorating, or if the tumor volume reaches 3000 mm³ 3 Euthanasia is necessary if the animal exceeds a certain threshold or is suffering from a serious illness or pain. Euthanasia should be performed in the following cases, informing the veterinarian: The animal is clearly emaciated, with a weight loss greater than 20%, unable to freely access food and water, and the average tumor volume of the control group is 2000 mm². 3 The experiment was stopped. The animals exhibited clinical symptoms such as bristly fur, hunched posture, pale ears, noses, eyes, or feet, shortness of breath, seizures, continuous diarrhea, dehydration, sluggishness, and vocalizations, and their condition steadily worsened.

[0223] Tumor measurement and experimental indicators The experimental indicators were used to consider whether tumor growth was suppressed, delayed, or cured. Tumor diameter was measured using calipers, three times per week. The formula for calculating tumor volume was V = 0.5 × a × b 2 Here, a and b represent the long and short diameters of the tumor, respectively.

[0224] The tumor inhibitory effect of the compounds was evaluated by TGI (%), which reflects the tumor growth inhibition rate. The tumor growth inhibition rate TGI (%) was calculated based on the following formula, referring to the tumor volume on day 1 after group division: TGI (%) = [1 - (average tumor volume of a certain treatment group - average tumor volume of that treatment group at the start of treatment) / (average tumor volume of the solvent control group - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0225] statistical analysis Statistical analysis was performed using Prism Graphpad software based on tumor volume at the end of the study. Multiple groups were compared and analyzed using two-way ANOVA and Fisher's LSD test. A statistically significant difference was considered to exist if P < 0.05.

[0226] Experimental results After inoculation of cells, tumor growth was observed daily. On the 8th day after inoculation, patients were divided into groups based on tumor volume and administered (indicated as Day 0). The average tumor volume of the assigned groups was approximately 154 mm². 3 In this experiment, other drugs besides Enhertu (3 mg / kg) were administered on Day 0, and Enhertu (3 mg / kg) was administered to all groups starting on Day 7, for a total of two doses, on Day 7 and Day 28, respectively. Based on the tumor burden, all groups were euthanized on Day 43 after inoculation, i.e., Day 35 after group administration, and the experiment was terminated.

[0227] 35 days after group administration, the tumor volume in the control group was 1523.7 ± 173.3 mm². 3 The tumor volume in the Enhertu (3 mg / kg) treatment group was 1575.2 ± 547.4 mm². 3 The tumor volume in the IN10018 (25 mg / kg) treatment group was 976.8 ± 171.2 mm². 3 The tumor volume in the Enhertu + IN10018 (3 + 25 mg / kg) dual therapy group was 626 ± 209.8 mm². 3 And, When tumor volume was aggregated and compared with the control group, the tumor inhibition rate (TGI) for the Enhertu (3 mg / kg) monotherapy group and the IN10018 (25 mg / kg) monotherapy group was -3.8% (p=0.7634) and 40.0% (p=0.0015), respectively. The tumor inhibition rate (TGI) for the Enhertu + IN10018 (3 + 25 mg / kg) dual therapy group was 65.6% (p<0.0001). When tumor volume was aggregated and compared with the related monotherapy groups Enhertu (3 mg / kg), IN10018 (25 mg / kg), and the Enhertu + IN10018 (3 + 25 mg / kg) dual therapy group, the p-values ​​were p<0.0001 and p=0.0921, respectively. See Table 28 for details. Figure 15 shows the tumor volume at different time ranges for each dose group.

[0228] [Table 29]

[0229] The experiment was conducted according to the administration scheme, and the animals' activities, such as food and water intake, were observed daily during the experiment. The animals' body weight was recorded three times a week. In the group inoculated with a mixture of NCI-N87 human gastric cancer cells and NIH-3T3 mouse embryonic fibroblasts, 35 days after group administration, the mean body weight of the control group increased from 18.5g on Day 0 (the day of group administration) to 22.0g, with a weight gain rate of 18.4%. The mean body weight of the Enhertu (3mg / kg) monotherapy group and the IN10018 (25mg / kg) monotherapy group increased from 17.9g and 19.2g on Day 0 (the day of group administration) to 20.9g and 21.5g on Day 35, respectively, with weight change rates of 16.9% and 11.6%, respectively. The mean body weight of the Enhertu + IN10018 (3+25mg / kg) dual therapy group increased from 18.9g on Day 0 (the day of group administration) to 20.7g on Day 35, with a weight change rate of 9.5%. Throughout the entire administration cycle, animals in each group showed no significant decrease in body weight and remained in good condition. See Table 29 for details. Figures 16 and 17 show the changes in body weight and the rate of change over different time ranges for each dose group.

[0230] [Table 30]

[0231] Experimental conclusion In this experiment, the inventors evaluated the in vivo efficacy of the test substance Enhertu after administering it in combination with IN10018 to a BALB / c-nude mouse subcutaneous tumor xenograft model that had been inoculated with a mixture of NCI-N87&NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts. In experimental animal populations inoculated with a mixture of NCI-N87 & NIH-3T3 human gastric cancer cells and mouse embryonic fibroblasts, both the IN10018 (25 mg / kg) monotherapy group and the Enhertu + IN10018 (3 + 25 mg / kg) dual therapy group showed significant tumor growth inhibition compared to the control group, demonstrating statistically significant differences. Furthermore, the Enhertu + IN10018 (3 + 25 mg / kg) dual therapy group exhibited smaller tumor volume and superior tumor inhibition rates throughout all experimental cycles compared to the monotherapy group. These data indicate that the combination of Enhertu and IN10018 achieves higher therapeutic efficacy compared to monotherapy.

[0232] Integrating all administration cycles, the animals showed favorable weight changes during the 35-day continuous administration cycle, and no abnormalities were observed in activity, food and water intake, or mental status throughout the entire administration cycle, indicating that the animals tolerated treatment with Enhertu (3 mg / kg), IN10018 (25 mg / kg) monotherapy and combination therapy.

[0233] Example 8 Evaluation of the antitumor effect of IN10018 in a female BALB / c nude mouse PC-07-0041 human pancreatic cancer PDX model.

[0234] Experimental Design The groupings and dosages are shown in the table.

[0235] [Table 31]

[0236] Experimental animals: Purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., and other information is the same as in Example 6.

[0237] The rearing environment and DPBS source are the same as in Example 6.

[0238] Table 9 shows information about the test substance.

[0239] Experimental Method and Steps Establishment of the PDX model: Mice were transplanted with PC-07-0041 human pancreatic cancer tumor cells, which were obtained from patient tissue surgically removed. Generation 0 was defined as (P0). The next passage after P0 tumor transplantation was defined as passage 1 (P1), and thus similarity was observed over the continuous transplantation period in mice. FP4 tumor tissue was used in this study.

[0240] Cell inoculation and grouping To induce tumor formation, a PC-07-0041 FP4 tumor fragment (approximately 30 mm) was placed on the right side of each mouse. 3 A subcutaneous transplant was performed. Treatment was started 19 days after tumor transplantation, with an average tumor size of approximately 119 mm. 3 The animals were randomly assigned to groups based on tumor volume using Excel-based randomization software. Each group consisted of five tumor-bearing mice. The test substance was administered to the mice according to the predetermined scheme shown in the experimental design sheet.

[0241] The preparation of the test substance and control solvent is shown in Table 31.

[0242] [Table 32]

[0243] Daily observation of laboratory animals The establishment and any modifications to this experimental scheme are only permissible after evaluation and approval by the Animal Care Committee (IACUC). The use and welfare of experimental animals were carried out in accordance with the rules of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of the animals were monitored daily, and periodic examinations included observing the effects of tumor growth and drug treatment on the animals' daily behavior, such as behavior, food and water intake (visual only), weight changes, external signs, or other abnormal conditions. The number of animal deaths and adverse events within each group were recorded based on the number of animals in each group.

[0244] Tumor measurement and experimental indicators The experimental indicators were used to consider whether tumor growth was suppressed, delayed, or cured. Tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume was V = 0.5 × a × b 2 Here, a and b represent the long and short diameters of the tumor, respectively. Next, the tumor size is used to calculate the TGI (%) and T / C (%) values.

[0245] The TGI calculation formula for each group is TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100%, where Ti is the average tumor volume of the treatment group on a specific day, T0 is the average tumor volume of the treatment group on the initiation day, Vi is the average tumor volume of the carrier control group on the day Ti is measured, and V0 is the average tumor volume of the carrier group on the initiation day.

[0246] The T / C ratio (percentage) is an indicator of antitumor efficacy, where T and C represent the average volumes of the treatment group and the control group, respectively, on a specific day.

[0247] Tumor weight will be measured at the end of the study. T / C weight The value (percentage) is from the formula T / C weight %=T weight / C weight It is calculated as ×100%, T weight and C weight These figures represent the average tumor weights for the treatment group and the carrier-control group, respectively.

[0248] statistical analysis Tumor volume and tumor weight were compared between each group using one-way ANOVA. A significant F-statistic (ratio of treatment variance to error variance) was obtained, and the groups were compared using the Games-Howell test. All data were analyzed using SPSS 29.0, and a p-value of <0.05 was considered statistically significant.

[0249] Experimental results Animal body weight was measured regularly as an indicator of toxicity. There were no deaths or illnesses. No significant weight loss was observed in any of the mice. Figure 18 shows the weight changes in the different treatment groups.

[0250] Based on the tumor volume measurements on day 28, the inhibition of tumor growth by IN10018 and ESG-401 in the PC-07-0041 PDX model was calculated. See Tables 32, 33, and Figure 19 for details.

[0251] [Table 33]

[0252] [Table 34]

[0253] Table 34 shows the tumor weights of tumor-bearing mice in different treatment groups.

[0254] [Table 35]

[0255] This study evaluated the therapeutic effects of IN10018 and ESG-401 in a PC-07-0041 human pancreas xenograft model in female BALB / c nude mice.

[0256] The average tumor size in the control group mice was 944 mm on day 28 after the start of treatment. 3Compared to the control group, treatment with IN10018 + ESG-401 (25 mg / kg, QD+1 / 3 mg / kg, BIW) showed significant antitumor activity, with an average tumor size of 197 mm. 3 (T / C = 20.83%, TGI = 90.55%, p = 0.007). Treatment with IN10018 (25 mg / kg, QD) and ESG-401 (1 / 3 mg / kg, BIW) did not show significant antitumor activity, and the mean tumor size was 1252 mm, respectively. 3 (T / C=132.65%, TGI=-37.38%, p=0.525), and 510mm 3 (T / C = 54.00%, TGI = 52.60%, p = 0.075). The tumor weight results were essentially in agreement with the tumor volume results.

[0257] In this study, the test compounds IN10018 and ESG-401 showed high resistance in tumor-bearing mice at dose levels. No significant weight loss was observed in any of the treatment groups.

[0258] Example 9 Evaluation of the efficacy of the test drug IN10018 when administered as monotherapy or in combination with other drugs in the human rectal cancer xenograft model LD1-0038-361855.

[0259] Experimental animals: NU / NU mice, female, weighing 18-21g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the rearing environment was the same as in Example 6.

[0260] Human-derived rectal cancer tumor tissue (case model number: LD1-0038-361855) was passaged up to the FP5+2 generation and used in this drug efficacy experiment.

[0261] Table 26 shows information about the test substance.

[0262] Tumor blocks from human-derived rectal cancer LD1-0038-361855, transplanted into the body, were excised into tumor tissue pieces approximately 3mm x 3mm x 3mm (approximately 45-60mg) in size and subcutaneously inoculated into NU / NU mice. The mice were observed after inoculation to monitor tumor growth, and the average tumor volume of the tumor-bearing mice was 156.69mm² on day 23 after inoculation. 3 If this occurs, group-based administration will be performed, with the day of group-based administration defined as day 0. Specific group-based information is shown in Table 35 below.

[0263] [Table 36]

[0264] The primary objective is to detect the growth inhibitory effect or complete cure effect of the test drug in the human-derived rectal cancer in vivo transplant tumor model LD1-0038-361855.

[0265] Measurement of tumor volume and body weight of tumor-bearing mice: Measured twice a week using calipers. The formula for calculating tumor volume is V = 0.5a × b 2 Here, a and b represent the longest diameter and widest diameter of the tumor, respectively.

[0266] The formula for calculating the relative tumor growth rate T / C (%) is as follows: T / C% = T RTV / C RTV ×100%(T RTV :Treatment group RTV, C RTV (RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated using the formula: RTV = T Vi / T V0 And in the formula, T V0 This is the average tumor volume measured at the time of group administration (i.e., d0), and T Vi This is the average tumor volume at a specific measurement, and T RTV and C RTV For this purpose, data from the same day was used.

[0267] Tumor growth inhibition rate TGI(%)=[1-(T ti -T t0) / (V ci -V c0 )]×100, and T ti is the average tumor volume after the start of administration of the compound group, and T t0 is the average tumor volume at the first administration of the compound group, and V c0 is the average tumor volume at the first administration of the vehicle control group, and V ci is the average tumor volume after the start of administration of the vehicle control group.

[0268] The body weights of all tumor-bearing mice were measured twice a week. At the same time, the relative change rate of the body weight of the mice after administration was calculated: RCBW(%) = (BW i -BW0) / BW0×100, where BW i is the body weight after the start of administration, and BW0 is the body weight at the first administration.

[0269] At the end of the experiment, the tumor blocks were weighed and photographed.

[0270] Data analysis All data are presented as mean ± standard error of the mean (SEM), SEM = SD / SQRT(n), where n = the number of animals in the experimental group. One-Way ANOVA test was used to compare the differences between the tumor volumes of the treatment groups and the control group. All data were analyzed by GraphPad, and a significant difference was determined if p < 0.05.

[0271] Experimental results The results of the changes in tumor volume of each treatment group are shown in Figure 20 and Table 36.

[0272] 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 ± 179.25 mm 3 , and the tumor-bearing mice in the test drug Enhertu group, IN10018 group, and Enhertu + IN10018 group had average tumor volumes of 361.30 ± 113.01 mm 3 , 1190.95 ± 463.62 mm 3 , and 151.79 ± 85.19 mm 3The tumor volume growth inhibition rates (TGI) were 83.27%, 15.52%, and 100.39%, respectively, and the relative tumor growth rates (T / C) were 26.11%, 85.55%, and 10.98%, respectively. As can be seen from the experimental results, under the experimental conditions, the Enhertu group, the IN10018 group, and the Enhertu + IN10018 group all showed a statistically significant tumor growth inhibition effect in the rectal cancer in vivo transplant tumor model LD1-0038-361855.

[0273] On the final day of the experiment (day 24), all tumor-bearing mice were euthanized. Subcutaneous tumor blocks were removed and weighed. The average weights of the tumor blocks in the control group, the Enhertu group, the IN10018 group, and the Enhertu+IN10018 group were 1.420±0.164g, 0.278±0.083g, 1.078±0.439g, and 0.101±0.058g, respectively. The tumor weight results were in good agreement with the tumor volume results.

[0274] [Table 37]

[0275] Example 10 Evaluation of the efficacy of the test drugs Enhertu and IN10018 when administered as monotherapy or in combination in the human ovarian cancer xenograft tumor model LD2-0032-200651.

[0276] Experimental animals: NU / NU mice, female, weighing 18-21g, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the rearing environment was the same as in Example 6.

[0277] Human-derived ovarian cancer (poorly differentiated adenocarcinoma and differentiated adenocarcinoma) tumor tissue (model number LD2-0032-200651) was passaged up to FP3+5 generations and used in this drug efficacy experiment.

[0278] Table 26 shows information about the test substance.

[0279] Tumor blocks from human-derived ovarian cancer LD2-0032-200651 in vivo transplanted tumors were excised into tumor tissue pieces approximately 3mm x 3mm x 3mm (approximately 45-60mg) in size and subcutaneously inoculated into NU / NU mice. The mice were observed after inoculation to monitor tumor growth, and the average tumor volume of the tumor-bearing mice was 180.47mm² on day 18 after inoculation. 3 If this occurs, group-based administration will be performed, with the day of group-based administration defined as day 0. Specific group-based information is shown in Table 38 below.

[0280] [Table 38]

[0281] The primary objective is to detect the growth inhibitory effect or complete cure effect of the test drugs Enhertu and IN10018 when administered as monotherapy or in combination in the human-derived ovarian cancer in vivo transplant tumor model LD2-0032-200651.

[0282] Measurement of tumor volume and body weight of tumor-bearing mice: Measured twice a week using calipers. The formula for calculating tumor volume is V = 0.5a × b 2 Here, a and b represent the longest diameter and widest diameter of the tumor, respectively.

[0283] The formula for calculating the relative tumor growth rate T / C (%) is as follows: T / C% = T RTV / C RTV ×100%(T RTV :Treatment group RTV, C RTV (RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated using the formula: RTV = T Vi / T V0 And in the formula, T V0 This is the average tumor volume measured at the time of group administration (i.e., d0), and T Vi This is the average tumor volume at a specific measurement, and T RTV and C RTV For this purpose, data from the same day was used.

[0284] Tumor growth inhibition rate TGI(%)=[1-(T ti -T t0 ) / (V ci -V c0 )] × 100, T ti This is the average tumor volume after the start of administration of the compound group, and T t0 V is the average tumor volume at the time of the first administration of the compound group. c0 V is the mean tumor volume at the time of the first dose in the solvent control group. ci This represents the mean tumor volume after the start of administration in the solvent control group.

[0285] The body weight of all tumor-bearing mice was measured twice a week. Simultaneously, the relative percentage change in body weight of mice after administration was calculated: RCBW(%) = (BW i -BW0) / BW0×100, where BW i BW0 represents the body weight after the start of administration, while BW0 represents the body weight at the time of the first dose. At the end of the experiment, the tumor block was weighed and photographed.

[0286] Data Analysis All data are presented as mean ± standard error (SEM), where SEM = SD / SQRT(n), and n = number of animals in the experimental group. One-way ANOVA was used to compare the tumor volume of the treatment group with that of the control group. All data were analyzed using GraphPad, and a p < 0.05 result was considered statistically significant.

[0287] Experimental results The results of tumor volume changes in each treatment group are shown in Figure 21 and Table 39.

[0288] [Table 39]

[0289] On day 21 after the start of administration, the mean tumor volume of tumor-bearing mice in the control group was 2132.47 ± 513.67 mm². 3The tumor-bearing mice in the Enhertu 3 mg / kg QW x 3 group, the IN10018 25 mg / kg QD x 21 group, and the IN10018 25 mg / kg QD x 21 group combined with Enhertu 3 mg / kg QW x 3 group had average tumor volumes of 1294.64 ± 285.36 mm², respectively. 3 , 1289.97±128.06mm 3 , and 700.63±129.27mm 3 The tumor volume growth inhibition rates (TGI) were 43.20%, 47.11%, and 76.00%, respectively, and the relative tumor growth rates (T / C) were 61.08%, 58.04%, and 32.15%, respectively. As can be seen from the experimental results, under the experimental conditions, the combination group of the test drug IN10018 25 mg / kg QD×21 and Enhertu 3 mg / kg QW×3 showed a statistically significant tumor growth inhibition effect in the ovarian cancer in vivo transplant tumor model LD2-0032-200651. The tumor volume of the combination group of IN10018 25 mg / kg QD×21 and Enhertu 3 mg / kg QW×3 was smaller than the tumor volume of each monotherapy group, but it did not show a statistically significant difference compared to the Enhertu 3 mg / kg QW×3 monotherapy group.

[0290] On the final day of the experiment (day 21), all tumor-bearing mice were euthanized, and the subcutaneous tumor blocks from the tumor-bearing mice were removed and weighed. The mean weights of the tumor blocks for the control group, the Enhertu 3 mg / kg QW × 3 group, the IN10018 25 mg / kg QD × 21 group, and the group receiving IN10018 25 mg / kg QD × 21 and 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. The tumor weight results were essentially consistent with the tumor volume results.

[0291] The body weight of experimental animals can be used as an indirect indicator for measuring drug toxicity. No significant decrease in body weight occurred in the mice after administration in each treatment group, indicating that tumor-bearing mice possess high tolerance to each drug at the experimental dose.

[0292] Example 11 Evaluation of the antitumor effects of the test drugs Enhertu and IN10018 in female NOD-SCID mice in a patient-derived xenograft (PDX) model of human lung cancer LU-01-1626.

[0293] Experimental animals: NOD SCID mice, female, weighing 18-20g, purchased from Sai-Ye Biotechnology Co., Ltd., and reared in the same environment as in Example 6.

[0294] Human-derived lung cancer tumor tissue was passaged up to the FP5 generation and used in this drug efficacy experiment.

[0295] Table 26 shows information about the test substance.

[0296] To promote tumor growth, a LU-01-1626 FP5 tumor fragment (approximately 30 mm) was placed on the right side of each mouse. 3 A subcutaneous transplant was performed. Treatment was started 18 days after tumor transplantation, with an average tumor size of approximately 109 mm. 3 The animals were randomly assigned to groups based on tumor volume using Excel-based randomization software. Each group consisted of four tumor-bearing mice. The test substance was administered to the mice according to the scheme shown in experimental design sheet 40.

[0297] [Table 40]

[0298] The main objective is to observe whether tumor growth can be delayed or whether the mice can be cured. Measurement of tumor volume and body weight of tumor-bearing mice: Measured twice a week with calipers, and the formula for calculating tumor volume is V = 0.5a × b 2 Here, a and b represent the longest diameter and widest diameter of the tumor, respectively.

[0299] Tumor growth inhibition rate TGI(%)=[1-(T ti -T t0 ) / (V ci -V c0)] × 100, T ti This is the average tumor volume after the start of administration of the compound group, and T t0 V is the average tumor volume at the time of the first administration of the compound group. c0 V is the mean tumor volume at the time of the first dose in the solvent control group. ci This represents the mean tumor volume after the start of administration in the solvent control group.

[0300] The formula for calculating the relative tumor growth rate T / C (%) is as follows: T / C% = T RTV / C RTV ×100%(T RTV :Treatment group RTV, C RTV (RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated using the formula: RTV = T Vi / T V0 And in the formula, T V0 This is the average tumor volume measured at the time of group administration (i.e., d0), and T Vi This is the average tumor volume at a specific measurement, and T RTV and C RTV For this purpose, data from the same day was used.

[0301] The body weight of all tumor-bearing mice was measured twice a week. Simultaneously, the relative percentage change in body weight of mice after administration was calculated: RCBW(%) = (BW i -BW0) / BW0×100, where BW i BW0 is the body weight after the start of administration, and BW0 is the body weight at the time of the first dose.

[0302] Data Analysis We provided integrated statistical data (including mean and standard error of mean (SEM)) of tumor volume for each group at each time point.

[0303] Using data obtained on day 31 after the start of treatment, a statistical analysis of tumor volume differences between the groups was performed.

[0304] A one-way ANOVA was performed to compare tumor volumes between groups. Since a significant F-statistic (ratio of treatment variance to error variance) was not obtained, the Games-Howell test was used to compare the groups. All data were analyzed using SPSS 29.0, and a p-value of <0.05 was considered statistically significant.

[0305] The results of tumor volume changes in each treatment group are shown in Figure 22 and Table 41.

[0306] [Table 41]

[0307] On day 31 after the start of administration, the average tumor volume of tumor-bearing mice in the control group was 1977 ± 135 mm². 3 The tumor-bearing mice in the Enhertu group, the IN10018 group, and the IN10018 and Enhertu combination group each had an average tumor volume of 664 ± 157 mm². 3 , 1459±123mm 3 and 405±73mm 3 The tumor volume growth inhibition rates (TGI) were 70.28%, 27.74%, and 84.16%, respectively, and the relative tumor growth rates (T / C) were 33.58%, 73.80%, and 20.49%, respectively.

[0308] As the experimental results show, the tumor-inhibiting effect of the two-drug combination group was superior to that of the monotherapy group.

[0309] Example 12 Evaluation of the antitumor effects of the test drugs Ab6000-DXD and IN10018 in female BALB / c nude mice in an OVCAR-3 human ovarian cancer (CDX) model.

[0310] Experimental animals: BALB / c nude mice, female, 6-8 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the rearing environment was the same as in Example 6.

[0311] 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 collected by trypsin treatment, centrifuged, washed, and suspended in cold PBS + 5% FCS and growth factor-reduced Matrigel (1:1) to a cell concentration of 1 × 10⁶. 7 The cell count was set to cells / ml. A 200 μl cell suspension containing OVCAR-3 cells was subcutaneously injected into the right flank of nude mice (one site per mouse). The tumor volume was 217 mm². 3 If this occurred (31 days after cell injection), the mice were randomly assigned to either the treatment group or the blank control group. The group assignment information is shown in Table 42.

[0312] [Table 42]

[0313] The tumor diameter was measured twice a week (Monday and Thursday) using calipers. The volume of each tumor [mm²] 3 [Units are] "Tumor volume = length × diameter" 2 The calculation was performed according to the formula "x0.5". To monitor for side effects of the treatment, the mice were examined daily for abnormal conditions and their body weight was measured twice a week (Monday and Thursday). Animals were killed at the end of the study, and during the study period, for ethical reasons, if tumor necrosis occurred or the tumor size reached 2000 mm. 3 Animals exceeding a certain age were killed early.

[0314] Table 26 shows the information for IN10018.

[0315] The full-length sequence of Ab6000 is as follows:

[0316] Heavy chain sequence: VH-CH (Human IgG1 Mutation): EVQLVQSGAEVKKPGASVKVSCKASGYTFTRNFMHWVRQAPGQGLEWMGWIYPGDGETEYAQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARGVYGGFAGGYFDFWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0317] Light chain sequence: VL-CL (Human Kappa): DIQMTQSPSSLSASVGDRVTITCKASQNIYKNLAWYQQKPGKAPKLLIYDANTLQTGVPSRFSGSGSGSDFTLTISSLQPEDFATYFCQQYYSGWAFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0318] Preparation of Ab6000-DXD ADC drug conjugate An appropriate amount of reducing agent (TCEP HCl, Thermo, CAS No.: 51805-45-9) was added to reduce the antibody (Ab6000), exposing the sulfhydryl groups between antibody chains. Low molecular weight deruxtecan (MCE, CAS No.: 1599440-13-7) was then dissolved in DMSO to prepare a 10 mM solution.

[0319] The above solution, after dissolution, was added in a molar ratio of antibody to linker-payload of 15:1. A Michael addition reaction was carried out with the maleimide group on the linker via the sulfhydryl group, thereby binding the antibody to the linker and payload.

[0320] The bound mixture was subjected to buffer substitution to remove residual small molecules, and the purity and DAR (drug / antibody ratio) values ​​of the antibody-drug conjugate were measured by HPLC-SEC (size exclusion chromatography) and HPLC-HIC (hydrophobic chromatography), respectively. The binding results are shown in Table 43.

[0321] [Table 43]

[0322] The tumor volume for each treatment group is shown in Figure 23. On day 14 after administration, the tumor volume of the control group was 616 mm². 3 The tumor volume in the ab6000-DXD monotherapy group was 425 mm². 3 The tumor volume in the IN10018 monotherapy group was 510 mm². 3 The tumor volume in the group receiving the two-drug combination therapy of ab6000-DXD and IN10018 was 344 mm². 3 As the data shows, the tumor-inhibiting effect of the two-drug combination group was superior to that of the monotherapy group.

[0323] All references cited herein are incorporated herein by reference in their entirety, as each reference is listed individually. Those skilled in the art, after reading the disclosure of this invention, will understand that various changes or modifications can be made to the invention, and these equivalents are also limited to the scope of the claims appended to this application.

Claims

1. Use of IN10018 or a pharmaceutically acceptable salt thereof in the manufacture of a drug for increasing the concentration of a high molecular weight drug in tumor tissue, wherein the high molecular weight drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows: 【Chemistry 1】

2. A pharmaceutical combination product comprising IN10018 or a pharmaceutically acceptable salt thereof and a high molecular weight drug, for the treatment of a tumor in a subject, wherein the high molecular weight drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows. 【Chemistry 2】

3. Use of IN10018 or a pharmaceutically acceptable salt thereof in increasing the concentration of a high molecular weight drug in tumor tissue, wherein the high molecular weight drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows: 【Transformation 3】

4. A method for treating a tumor, comprising administering a therapeutically effective amount of IN10018 or a pharmaceutically acceptable salt thereof and a high molecular weight drug to a subject in need of treatment, wherein the high molecular weight drug is a monoclonal antibody, a bispecific antibody, or an antibody-drug conjugate, and the structure of IN10018 is as follows. 【Chemistry 4】

5. (a) IN10018 or a pharmaceutically acceptable salt thereof, (b) comprising a monoclonal antibody, a bispecific antibody, or a high molecular weight drug which is an antibody-drug conjugate, The structure of IN10018 is as follows: a kit or pharmaceutically acceptable composition. 【Transformation 5】

6. The pharmaceutically acceptable salt of IN10018 is a tartrate salt, as described in any one of claims 1 to 5, for use, pharmaceutical combination product, method, kit, or pharmaceutically acceptable composition.

7. The monoclonal antibodies mentioned above are lacotumomab, rituximab, iodine[131I]metuximab, JMT-103, necitumumab, alemtuzumab, elotuzumab, bevacizumab, ofatumumab, tocilizumab, atezolizumab, tripalimab, HX-008, and camrelizumab (C amrelizumab, ocrelizumab, sugemalimab, lenzilumab, cintilimab, vilobelimab, margetuximab, siltuximab, mogamulizumab, amivantamab, cadnilimab, inebilizumab, iodine[131I]delrotuximab biotin I 131 derlotuximab biotin), isatuximab, serplulimab, retifanlimab, cetuximab, adebrelimab, tislerizumab, penplimab, teprotumumab, itolizumab, dostarlimab, denosum Denosumab, Obinutuzumab, Nimotuzumab, Teclistamab, Daratumumab, Dinutuximab, Tafasitamab, Socazolimab, Dupilumab, Prolgolimab, Blinatumomab,Geptanolimab, Panitumumab, Canakinumab, Ramucirumab, Envafolimab, Belimumab, Leronlimab, Ranibizumab, Natalizumab, Cosibelimb Zimberelimab, Trastuzumab, Catumaxomab, Durvalumab, Avelumab, Ublituximab, Cemiprimab, Pembrolizumab, Glofitamab, Pertuzumab mab), bermekimab, tremelimumab, ipilimumab, naxitamab, infliximab, nivolumab, omburtamab, crizanlizumab, burosumab, talquetamab a) besilesomab, alirocumab, arcitumomab, or a biosimilar thereof, particularly trastuzumab or a biosimilar thereof, preferably trastuzumab, is the use, pharmaceutical combination product, method, kit, or pharmaceutically acceptable composition according to any one of claims 1 to 6.

8. The aforementioned bispecific antibodies are teclistamab, blinatumomab, cadnilimab, mosunetuzumab, catumaxomab, ublituximab, amivantamab, toalquetamab, epcoritamab, globitamab, zanidatamab, and elranatamab. A use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 6, which is teboterimab, amivantamab, SI-B001, odronextamab, KN-026, KN-046, ivonescimab, SHR-1701, M7824, GEN-3009, navicixizumab, GB-261, CM-355, plamotamab, or a biosimilar thereof.

9. The aforementioned antibody-drug conjugates include loncastuximab tesirine, ibritumomab tiuxetan, tisotumab vedotin, sacituzumab govitecan, enfortumab vedotin, inotuzumab ozogamicin, gemtuzumab ozogamicin, and belantamab mahodotin. Mafodotin, trastuzumab emtansine, moxetumomab pasudotox, polatuzumab vedotin, disitamab vedotin, brentuximab vedotin, trastuzumab deruxtecan (DS-8201), trastuzumab emtansine, cetuximab sarotalocan Sarotalocan, Mirbetuximab soravtansine, Trastuzumab duocarmazine, ARX-788, KL-A264, Upfitamab rillsodotin, Tusamitamab ravtansine, Naptumomab estafenatox, Datopotamab deruxtecan, Oportuzumab monatox Monatox, SHR-A1811, Patritumab Deruxtecan, Terisotuzumab Vedotin, Trastuzumab DuocalmazineDuocarmazine), MK-2140, TAA-013, BIO-106, DB-1305, MRG-004A, AZD-8205, ADCT-602, FOR-46, TPX-4589, B MS-986148, 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, Rorbotuzumab Mertansine, TAC-001, MT-8633, TRPH-222, TAK-164, STRO-001, CX-2043, TBL-0306M, HDP-103, DAN-311, PRO-1102, GM-103 ADC, TE-1218, TE-1112, T-PNU, BV-001, DS-1062, SKB264, Ab6000-DXd, or biosimilars thereof, particularly ESG-401, DS-1062, SKB264, sacituzumab govitecan, Ab6000-DXd, or biosimilars thereof, preferably ESG-401, DS-1062, SKB264, sacituzumab govitecan / trastuzumab deruxtecanA use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 6, wherein the compound is deluxtecan (DS-8201) or Ab6000-DXd.

10. The use, pharmaceutical combination product, method, kit or pharmaceutically acceptable composition according to any one of claims 1 to 6, wherein the polymer drug is an antibody-drug conjugate, particularly a Top-2 antibody-drug conjugate, 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 polymer drug are administered to the subject simultaneously or sequentially.

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

13. The aforementioned tumors include 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), malignant 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, cholangiocarcinoma, leiomyosarcoma, liposarcoma, nasopharyngeal cancer, neuroendocrine tumor, ovarian cancer, salivary gland cancer, metastatic tumors from spindle cell carcinoma, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, non-Hodgkin lymphoma, and Hodgkin's lymphoma. Lymphoma, glioma, and hematopoietic malignancies, selected from, for example, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), and 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, and more preferably the tumor is gastric cancer, breast cancer, pancreatic cancer, colon cancer, ovarian cancer, or lung cancer, the use, pharmaceutical combination product, method, kit, or pharmaceutically acceptable composition according to claim 12.

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