WO2023104151A1 - 治疗肿瘤的药物组合及用途 - Google Patents

治疗肿瘤的药物组合及用途 Download PDF

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WO2023104151A1
WO2023104151A1 PCT/CN2022/137545 CN2022137545W WO2023104151A1 WO 2023104151 A1 WO2023104151 A1 WO 2023104151A1 CN 2022137545 W CN2022137545 W CN 2022137545W WO 2023104151 A1 WO2023104151 A1 WO 2023104151A1
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cancer
inhibitor
doxorubicin
fak
immune checkpoint
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French (fr)
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张宝袁
刘学彬
王在琪
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应世生物科技(南京)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the invention belongs to the field of medicinal chemistry. Specifically, the present invention relates to the combined use of focal adhesion kinase (Focal Adhesion Kinase, FAK) inhibitors and other drugs to treat tumors.
  • focal adhesion kinase Fecal Adhesion Kinase, FAK
  • Immunogenic cell death is based on the additive effect of programmed cell death.
  • stress signals include endoplasmic reticulum stress (ER stress) and reactive oxygen species stress (oxidative stress).
  • ER stress endoplasmic reticulum stress
  • oxidative stress reactive oxygen species stress
  • DAMPs damage-associated molecular patterns
  • This kind of DAMPs will be specifically recognized by pattern recognition receptors on antigen-presenting cells (APCs) in the body, induce APCs to mature, differentiate and activate, and present them to immune cells such as effector T cells step by step, thereby Make immune cells generate antigen memory, and when tumor cells of the same origin are found again, immune cells will specifically recognize and kill tumor cells.
  • the new tumor-specific immune response initiated by ICD can increase the sensitivity to immune checkpoint inhibitors (Immuno-checkpoint inhibitors, ICIs), so as to enhance the effect of immune checkpoint inhibitors, and can produce immune Memory-lasting antitumor responses.
  • ICD immune checkpoint inhibitors
  • FAK also known as protein tyrosine kinase 2 (PTK2), is a non-receptor tyrosine kinase and is a key component of the focal adhesion complex. FAK plays an important role in mediating integrin and growth factor signaling to regulate tumor cell invasion, proliferation and survival.
  • PTK2 protein tyrosine kinase 2
  • chemotherapeutic drugs have been observed to trigger ICD in cancer cells, and ICD is understood to contribute to the antitumor effects of this therapy.
  • ICDs stimulate the immune system to attack cancer, especially in combination with anti-PD1 or PD-L1, resulting in long-lasting antitumor effects.
  • PLD doxorubicin hydrochloride liposome injection (doxil)
  • doxil doxorubicin hydrochloride liposome injection
  • Western Arizona Regional Medical Center explored Anti-PD1 (pembrolizumab) combined with chemotherapy (including PLD) to treat tumors, but the clinical effect did not meet expectations, and the clinical trial has been terminated. Therefore, it is still necessary to further search for drugs that can enhance the induction of ICD by chemotherapy drugs, improve the response rate of tumor treatment and the possibility of long-term benefits.
  • One aspect of the present disclosure provides the use of FAK inhibitors, anthracycline chemotherapeutics and immune checkpoint inhibitors in the preparation of drugs for treating tumors in subjects, wherein the FAK inhibitors, anthracycline chemotherapeutics and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.
  • Yet another aspect of the present disclosure provides a drug combination product of a FAK inhibitor, an anthracycline chemotherapeutic, and an immune checkpoint inhibitor for treating a tumor in a subject, wherein the FAK inhibitor, the anthracycline chemotherapeutic and the immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.
  • One aspect of the present disclosure provides a method for treating tumors, the method comprising simultaneously or sequentially administering therapeutically effective doses of FAK inhibitors, anthracycline chemotherapeutics, and immune checkpoint inhibitors to a subject in need.
  • kits or a pharmaceutically acceptable composition comprising: (a) a FAK inhibitor; (b) an anthracycline chemotherapeutic; and (c) an immune checkpoint inhibitor.
  • Another aspect of the present disclosure provides a use of a FAK inhibitor and anthracycline chemotherapeutics in the preparation of a drug for treating tumors, wherein the FAK inhibitor is used to enhance the immunogenicity induced by the anthracycline chemotherapeutics cell death.
  • Yet another aspect of the present disclosure provides FAK inhibitors for use in enhancing anthracycline chemotherapeutic-induced immunogenic cell death in the treatment of tumors.
  • Yet another aspect of the present disclosure provides a method of treating tumors, the method comprising simultaneously or sequentially administering a therapeutically effective amount of a FAK inhibitor and anthracycline chemotherapeutics to a subject in need, wherein the FAK inhibitor is used to enhance the Immunogenic cell death induced by anthracycline chemotherapeutics.
  • Yet another aspect of the present disclosure provides the use of FAK inhibitors, anthracycline chemotherapeutics and immune checkpoint inhibitors in the preparation of drugs for combined treatment of tumors.
  • Yet another aspect of the present disclosure provides a use of a FAK inhibitor in the preparation of a drug in combination with anthracycline chemotherapeutics and immune checkpoint inhibitors for treating tumors.
  • Yet another aspect of the present disclosure provides the use of anthracycline chemotherapeutics in the preparation of a combination drug for treating tumors with FAK inhibitors and immune checkpoint inhibitors.
  • Yet another aspect of the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a drug in combination with a FAK inhibitor and anthracycline chemotherapeutics for treating tumors.
  • Yet another aspect of the present disclosure provides the use of FAK inhibitors, anthracycline chemotherapeutics and immune checkpoint inhibitors in the preparation of drugs for combined treatment of tumors.
  • Yet another aspect of the present disclosure provides a use of a FAK inhibitor in the preparation of a drug for treating tumors in combination with anthracycline chemotherapeutics and immune checkpoint inhibitors.
  • Yet another aspect of the present disclosure provides the use of anthracycline chemotherapeutics in the preparation of a drug for treating tumors in combination with FAK inhibitors and immune checkpoint inhibitors.
  • Yet another aspect of the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a drug for treating tumors in combination with a FAK inhibitor and anthracycline chemotherapy drugs.
  • kits which includes: a FAK inhibitor; and instructions indicating that the FAK inhibitor can be used in combination with anthracycline chemotherapeutics and immune checkpoint inhibitors to treat tumors.
  • kits which includes: anthracycline chemotherapeutics; and instructions, the instructions indicating that the anthracycline chemotherapeutics can be used in combination with FAK inhibitors and immune checkpoint inhibitors to treat tumors.
  • kits which includes: an immune checkpoint inhibitor; and instructions, which indicate that the immune checkpoint inhibitor can be used in combination with FAK inhibitors and anthracycline chemotherapy drugs to treat tumors.
  • Another aspect of the present disclosure provides a method of treating a tumor, the method comprising administering a therapeutically effective amount of a FAK inhibitor and an anthracycline chemotherapeutic to a subject in need thereof.
  • Yet another aspect of the present disclosure provides a pharmaceutical combination of a FAK inhibitor and an anthracycline chemotherapeutic for use in treating a tumor in a subject in need thereof.
  • Another aspect of the present disclosure provides the use of a FAK inhibitor and anthracycline chemotherapeutics in the preparation of a combined drug for treating tumors.
  • Yet another aspect of the present disclosure provides the use of the FAK inhibitor in the preparation of a drug in combination with anthracycline chemotherapeutics for treating tumors.
  • Another aspect of the present disclosure provides the use of anthracycline chemotherapeutics in the preparation of a combination drug for treating tumors with FAK inhibitors.
  • Yet another aspect of the present disclosure provides the use of a FAK inhibitor and anthracycline chemotherapeutics in the preparation of a drug for combined treatment of tumors.
  • Yet another aspect of the present disclosure provides the use of a FAK inhibitor in the preparation of a drug for treating tumors in combination with anthracycline chemotherapeutics.
  • Yet another aspect of the present disclosure provides the use of anthracycline chemotherapeutics in the preparation of a drug for treating tumors in combination with FAK inhibitors.
  • kits which includes: a FAK inhibitor; and an instruction indicating that the FAK inhibitor can be used in combination with anthracycline chemotherapeutics to treat tumors.
  • kits which includes: an anthracycline chemotherapeutic drug; and an instruction indicating that the anthracycline chemotherapeutic drug can be used in combination with a FAK inhibitor to treat tumors.
  • One aspect of the present disclosure provides the use of FAK inhibitor, doxorubicin and immune checkpoint inhibitor in the preparation of a medicament for treating tumor in a subject, wherein the FAK inhibitor, the doxorubicin and the immune checkpoint inhibitor Checkpoint inhibitors are administered to the subject simultaneously or sequentially.
  • Yet another aspect of the present disclosure provides a pharmaceutical combination product of a FAK inhibitor, doxorubicin and an immune checkpoint inhibitor for treating a tumor in a subject, wherein the FAK inhibitor, the doxorubicin and the immune Checkpoint inhibitors are administered to the subject simultaneously or sequentially.
  • One aspect of the present disclosure provides a method for treating tumors, the method comprising simultaneously or sequentially administering therapeutically effective amounts of FAK inhibitors, doxorubicin and immune checkpoint inhibitors to a subject in need.
  • kits or a pharmaceutically acceptable composition comprising: (a) a FAK inhibitor; (b) doxorubicin; and (c) an immune checkpoint inhibitor.
  • Yet another aspect of the present disclosure provides a use of a FAK inhibitor and doxorubicin in the preparation of a medicament for treating tumors, wherein the FAK inhibitor is used to enhance the immunogenic cell death induced by the doxorubicin.
  • Yet another aspect of the present disclosure provides FAK inhibitors for use in enhancing doxorubicin-induced immunogenic cell death in the treatment of tumors.
  • Yet another aspect of the present disclosure provides a method of treating tumors, the method comprising simultaneously or sequentially administering a therapeutically effective amount of a FAK inhibitor and doxorubicin to a subject in need, wherein the FAK inhibitor is used to enhance the doxorubicin Mycin-induced immunogenic cell death.
  • Yet another aspect of the present disclosure provides the use of FAK inhibitor, doxorubicin and immune checkpoint inhibitor in the preparation of a drug for combined treatment of tumors.
  • Yet another aspect of the present disclosure provides a use of a FAK inhibitor in the preparation of a drug in combination with doxorubicin and an immune checkpoint inhibitor for treating tumors.
  • Another aspect of the present disclosure provides the use of doxorubicin in the preparation of a combination drug for treating tumors with FAK inhibitors and immune checkpoint inhibitors.
  • Yet another aspect of the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a drug in combination with a FAK inhibitor and doxorubicin for treating tumors.
  • Yet another aspect of the present disclosure provides the use of FAK inhibitor, doxorubicin and immune checkpoint inhibitor in the preparation of a drug for combined treatment of tumors.
  • Yet another aspect of the present disclosure provides a use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with doxorubicin and an immune checkpoint inhibitor.
  • Yet another aspect of the present disclosure provides the use of doxorubicin in the preparation of a medicament for treating tumors in combination with FAK inhibitors and immune checkpoint inhibitors.
  • Yet another aspect of the present disclosure provides the use of an immune checkpoint inhibitor in the preparation of a medicament for treating tumors in combination with a FAK inhibitor and doxorubicin.
  • kits comprising: a FAK inhibitor; and an instruction indicating that the FAK inhibitor can be used in combination with doxorubicin and an immune checkpoint inhibitor to treat tumors.
  • kits which includes: doxorubicin; and an instruction indicating that the doxorubicin can be used in combination with FAK inhibitors and immune checkpoint inhibitors to treat tumors.
  • kits which includes: an immune checkpoint inhibitor; and an instruction indicating that the immune checkpoint inhibitor can be used in combination with a FAK inhibitor and doxorubicin to treat tumors.
  • Another aspect of the present disclosure provides a method of treating a tumor, the method comprising administering a therapeutically effective amount of a FAK inhibitor and doxorubicin to a subject in need thereof.
  • Yet another aspect of the present disclosure provides a pharmaceutical combination product of a FAK inhibitor and doxorubicin for use in treating a tumor in a subject in need thereof.
  • Yet another aspect of the present disclosure provides the use of a FAK inhibitor and doxorubicin in the preparation of a combined drug for treating tumors.
  • Another aspect of the present disclosure provides the use of the FAK inhibitor in the preparation of a drug in combination with doxorubicin for treating tumors.
  • Another aspect of the present disclosure provides the use of doxorubicin in the preparation of a combination drug for treating tumors with FAK inhibitors.
  • Yet another aspect of the present disclosure provides the use of a FAK inhibitor and doxorubicin in the preparation of a medicament for combined treatment of tumors.
  • Yet another aspect of the present disclosure provides the use of a FAK inhibitor in the preparation of a medicament for treating tumors in combination with doxorubicin.
  • Yet another aspect of the present disclosure provides the use of doxorubicin in the preparation of a medicament for treating tumors in combination with a FAK inhibitor.
  • kits which includes: a FAK inhibitor; and an instruction indicating that the FAK inhibitor can be used in combination with doxorubicin to treat tumors.
  • kits which includes: doxorubicin; and an instruction indicating that the doxorubicin can be used in combination with a FAK inhibitor to treat tumors.
  • the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable salt thereof, preferably IN10018, Defactinib, AMP945 or a pharmaceutically acceptable salt thereof
  • An acceptable salt, more preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, the IN10018 structure is as follows:
  • the Defactinib is also known as Defatinib, and its CAS number is 1345713-71-4; the CAS number of the GSK2256098 is 1224887-10-8.
  • the CAS number of PF-00562271 is 717907-75-0; the CAS number of VS-4718 is 1061353-68-1; the APG-2449 is developed by Ascentage Pharma; the CAS number of AMP945 is 1393653- 34-3.
  • the anthracycline chemotherapeutic drug is doxorubicin, epirubicin, or daunorubicin, preferably doxorubicin.
  • the immune checkpoint inhibitor is anti-PD-1/PD-L1 antibody, PD-1/PD-L1 small molecule inhibitor or TIGIT inhibitor.
  • the immune checkpoint inhibitor is an anti-PD-1/PD-L1 antibody
  • the anti-PD-1/PD-L1 antibody is pembrolizumab, tislelizumab Tislelizumab, Nivolumab, Toripalimab, Atezolizumab, Durvalumab, Avelumab, Cardiac Camrelizumab, Sintilimab, Cemiplimab, Envafolimab, BMS-936559, JS003, SHR-1316, GS-4224 , AN-4005 or MX-10181, especially toripalimab.
  • the immune checkpoint inhibitor is a PD-1/PD-L1 small molecule inhibitor
  • the PD-1/PD-L1 small molecule inhibitor is INCB-086550, Lazertinib ), IMMH-010, CA-170, ABSK043 or RRx-001.
  • the immune checkpoint inhibitor is a TIGIT inhibitor
  • the TIGIT inhibitor is Ociperlimab (Ociperlimab/BGB-A1217), Vibostolimab (Vibostolimab), domvanalimab (AB154) , Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA-TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036, or IBI-321.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid Leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma Tumor, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymus Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma, Uveal Melanoma, Skin Cancer, Germ Cell Cancer carcinoma of the nasopharynx,
  • the tumor is acute myeloid leukemia, melanoma, thyroid cancer, colorectal cancer, esophageal cancer, hepatocellular carcinoma, ovarian cancer, fibrosarcoma, gastric cancer, non-small cell lung cancer or cholangiocarcinoma; further ovarian cancer or colorectal cancer.
  • Figure 1 shows the effect of FAKsiRNA on the knockdown of FAK and phosphorylated FAKY397.
  • Figure 2 shows the synergistic killing effect of 3 ⁇ M doxorubicin and 50nM FAKsiRNA knockout on ovarian cancer cell SK-OV-3.
  • Figure 3 shows the immunofluorescence staining of DAMPs after 3 ⁇ M doxorubicin combined with 50nM FAKsiRNA for 48 hours.
  • Figure 4 shows the 72-hour killing curve and IC50 value of IN10018 and doxorubicin on ovarian cancer cell SK-OV-3.
  • Figure 5 shows the synergistic effect of doxorubicin combined with 3 ⁇ M IN10018 for 48 hours on ovarian cancer cell line SK-OV-3 at different doses.
  • Figure 6 shows the results of staining with Annexin V kit after 48 hours of combined administration of 0.3 or 1.0 ⁇ M doxorubicin and 3 ⁇ M IN10018.
  • Figure 7 shows the results of immunofluorescence staining of DAMPs after 48 hours of combined administration of 0.3 ⁇ M doxorubicin and 3 ⁇ M IN10018.
  • Figure 8 shows the results of immunofluorescence staining of DAMPs after 48 hours of combined administration of 0.3 ⁇ M Daunorubicin or Epirubicin and 3 ⁇ M IN10018.
  • Figure 9 shows the synergistic killing effect of doxorubicin and 3 ⁇ M Defactinib combined with different doses for 72 hours on ovarian cancer cell SK-OV-3.
  • Figure 10 shows the results of immunofluorescent staining of DAMPs after 48 hours of combined administration of 1 ⁇ M doxorubicin and 3 ⁇ M Defactinib.
  • Figure 11 shows the white light micrographs of the cells taken after 48 hours of incubation with the drug.
  • Figure 12 shows the percentage of CRT-positive and Annexin V-positive CT26 cells after incubation with IN10018 and doxorubicin for 48 hours, wherein Figure 12a shows the percentage of CRT-positive CT26 cells after incubation with drugs for 48 hours, and Figure 12b shows the percentage of CT26 cells incubated with drugs The percentage of Annexin V positive CT26 cells after 48 hours.
  • Figure 17 shows the concentration of IFN- ⁇ protein detected in serum obtained from orbital blood collection after 7 days administration of CT26 mouse colon cancer cell line BABL/c mouse subcutaneous allograft tumor model.
  • Figure 18 shows the concentration of Granzyme B protein detected in serum obtained from orbital blood collection after 7 days of administration of CT26 mouse colon cancer cell line BABL/c mouse subcutaneous allograft tumor model.
  • Figure 19 shows the percentage of CRT-positive and Annexin V-positive 4T1 cells after 48 hours of incubation with doxorubicin and AMP945, wherein Figure 19a shows the percentage of CRT-positive 4T1 cells after incubation with drugs for 48 hours, and Figure 19b shows the percentage of cells incubated with drugs The percentage of Annexin V positive 4T1 cells after 48 hours.
  • Figure 20 shows the percentage of CRT-positive and Annexin V-positive CT26 cells after 48 hours of incubation with doxorubicin and AMP945, wherein Figure 20a shows the percentage of CRT-positive CT26 cells after 48 hours of incubation with drugs, and Figure 20b shows the percentage of CT26 cells incubated with drugs The percentage of Annexin V positive CT26 cells after 48 hours.
  • 21 depicts a waterfall diagram of the best change (%) in the total diameter of target lesions relative to baseline in the clinical study of IN10018 combined with PLD and anti-PD-1 in the treatment of ovarian cancer.
  • FAK inhibitor refers to a potent inhibitor of FAK, suitable for mammals, especially humans.
  • the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable salt thereof, and the structure of IN10018 is as follows:
  • the Defactinib is also known as Defatinib, and its CAS number is 1345713-71-4; the CAS number of the GSK2256098 is 1224887-10-8.
  • the CAS number of PF-00562271 is 717907-75-0; the CAS number of VS-4718 is 1061353-68-1; the APG-2449 is developed by Ascentage Pharma; the CAS number of AMP945 is 1393653- 34-3.
  • the FAK inhibitor is preferably IN10018, Defactinib, AMP945 or a pharmaceutically acceptable salt thereof, in some preferred embodiments, the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, Especially IN10018 tartrate.
  • anthracycline refers to a class of chemotherapeutic drugs derived from Streptomyces persei var. ciliary.
  • the anthracycline is doxorubicin, epirubicin, daunorubicin or aclarmycin, etc.
  • the anthracycline is doxorubicin.
  • doxorubicin used herein is doxorubicin (Doxorubicin), it can be understood that although the technical method of the present invention only mentions doxorubicin, in fact doxorubicin or its different salts or preparations are all protected .
  • PLD refers to doxorubicin hydrochloride liposome injection (doxil), which is doxorubicin hydrochloride liposome injection, and its active ingredient is doxorubicin.
  • immune checkpoint inhibitor refers to drugs that can improve the activity of the immune system by regulating immune checkpoint pathways (such as PD-1, TIGIT, CTLA-4, LAG-3, TIM-3, etc.).
  • the immune checkpoint inhibitor is an antagonist of the PD-1/PD-L1 (programmed cell death protein 1) pathway (also referred to as a "PD-1 inhibitor”) or a TIGIT inhibitor.
  • PD-1 inhibitors are also referred to in this disclosure as PD-1/PD-L1 inhibitors.
  • the PD-1/PD-L1 inhibitors described in the treatment methods, medicines and uses of the present disclosure are anti-PD-1/PD-L1 antibodies, including but not limited to pembrolizumab (Keytruda/Keytruda /K drug), Tislelizumab (Tislelizumab/Bizlezumab), Nivolumab (Nivolumab), Toripalimab (Toripalimab/Tuoyi), Durvalumab (Infineon/Du Valirumab/durvalumab), avelumab (Avelumab/Bavencio), atezolizumab (MPDL3280A/Atezolizumab/Tecentriq/T drug), BMS-936559 (fully human anti-PD-L1 IgG4 monoclonal antibody), GS-4224, AN-4005 or MX-10181, especially toripalimab.
  • pembrolizumab Keytruda/Keytrud
  • the PD-1 inhibitor is toripalimab.
  • a PD-1 inhibitor is used to treat a human subject.
  • PD-1 is human PD-1.
  • PD-1/PD-L1 inhibitors also include PD-1/PD-L1 small molecule inhibitors, such as INCB-086550, Lazertinib, IMMH-010, CA-170, ABSK043 or RRx-001.
  • TIGIT also known as WUCAM, Vstm3, VSIG9 is a receptor of the Ig superfamily and a new type of immune checkpoint after PD-1/PD-L1.
  • the immune checkpoint inhibitor is a TIGIT inhibitor (such as TIGIT antibody), including but not limited to Ociperlimab (Ociperlimab/BGB-A1217), vitamin Vibostolimab, domvanalimab (AB154), Tiragolumab, Belrestotug, Etigilimab, ONO-4686, JS-006, AZD-2936, HLX-301, SEA -TGT, M-6223, IBI-939, COM-902, AB-308, AGEN-1777, AK-127, BAT-6021, BAT-6005, ASP-8374, PM-1022, BMS-986207, HB0036 or IBI -321.
  • TIGIT inhibitors are used to treat human subjects.
  • antibodies herein include double antibodies.
  • “Pharmaceutical combination” or “pharmaceutical combination product” as used herein may refer to either a fixed combination in the form of a dosage unit (for example, all pharmaceutical active ingredients are present in one dosage form) or a kit for products administered in combination, or refers to the combination of a drug with instructions indicating that the drug may be used in combination with one or more other drugs.
  • combination therapy or “combination drug” refers to the combination of one drug and one or more other drugs to treat diseases, including the combination of one drug and one or more other drugs It also includes the combination of a drug and instructions indicating that the drug can be used in combination with one or more other drugs.
  • “Simultaneous or sequential administration” in this application refers to two or more drugs within one administration cycle (for example, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week or within 24 hours) at the same time or at a certain time interval
  • Sequential administration the way of drug administration (such as oral, intravenous, intramuscular or subcutaneous administration, etc.) may be the same or different, and the frequency/period of administration of two or more drugs may be the same or different.
  • the treatment method, product or use of the present disclosure involves two drugs, the two drugs can be administered separately at the same time or with a certain time interval.
  • the three drugs can be administered at the same time point, or two drugs can be administered at one time point and the remaining one drug can be administered at another time point, or all Each of the three drugs was administered at different time points.
  • the PD-1/PD-L1 inhibitor is administered intravenously (eg, as an intravenous infusion) or subcutaneously or orally.
  • the PD-1/PD-L1 inhibitor is administered as an intravenous infusion.
  • immune checkpoint inhibitors to treat cancer depends on the presence of tumor antigen-specific T cells within tumor tissue. This requires tumor tissue to express antigens that distinguish itself from their non-transformed counterparts, for example, through novel protein products called neoantigens. Tumor neoantigen burden is strongly associated with immunogenicity and sensitivity (for example, to checkpoint inhibitor therapy), implying that poorly immunogenic tumors should be largely resistant to these agents. Therapies designed to release tumor antigens that can be taken up by APCs, such as those that induce immunogenic cell death (ICD), may promote effective antitumor immunity, especially when further combined with checkpoint inhibitors.
  • ICD immunogenic cell death
  • treating refers to the administration of one or more drug substances to a subject suffering from a disease or symptoms of said disease for the purpose of curing, alleviating, alleviating, altering, curing, ameliorating, modifying or affecting said A disease or a symptom of said disease.
  • the disease is a tumor or cancer.
  • tumor refers to the abnormal lesion formed by the abnormal proliferation of cells in local tissues due to the loss of normal regulation of their growth at the gene level under the action of various tumorigenic factors.
  • examples include, but are not limited to: Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid Leukemia, Chronic Myeloid Leukemia, Thyroid Cancer, Glioma, Colorectal Cancer, Ovarian Cancer, Bladder Cancer, Prostate Cancer, Breast Cancer, Liposarcoma, Fibrosarcoma, Rhabdomyosarcoma, Leiomyosarcoma, Angiosarcoma, Neuroblastoma Tumor, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroes
  • subject or “subject” as used herein refers to both mammals and non-mammals.
  • Mammal means any member of the class Mammalia, which includes, but is not limited to: humans; non-human primates such as chimpanzees and other ape and monkey species; farm animals such as cattle, horses, sheep, goats and pigs; Domestic animals such as rabbits, dogs and cats; laboratory animals including rodents such as rats, mice and guinea pigs; etc. Examples of non-mammals include, but are not limited to, birds and the like.
  • the term "subject” does not define a specific age or gender. In some embodiments, the subject is a human.
  • pharmaceutically acceptable means non-toxic, biologically tolerable, suitable for administration to a subject.
  • the term "pharmaceutically acceptable salt” refers to a non-toxic, biologically tolerable acid addition salt suitable for administration to a subject, including but not limited to: acid addition salts formed with inorganic acids , such as hydrochloride, hydrobromide, carbonate, bicarbonate, phosphate, sulfate, sulfite, nitrate, etc.; and acid addition salts formed with organic acids, such as formate, ethyl salt, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate , benzoate, salicylate, stearate and salts formed with alkanedicarboxylic acids of formula HOOC-(CH 2 ) n -COOH (wherein n is 0-4), and the like.
  • inorganic acids such as hydrochloride, hydrobromide, carbonate, bi
  • pharmaceutically acceptable acid addition salts can be obtained by dissolving the free base in a suitable solvent and treating the solution with an acid according to conventional procedures for preparing acid addition salts from basic compounds.
  • Those skilled in the art can identify without undue experimentation the various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable acid addition salts.
  • composition means that it must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation, and/or compatible with the subject to be treated therewith.
  • therapeutically effective amount refers to an amount generally sufficient to produce a beneficial therapeutic effect on a subject. Routine influencing factors (e.g., mode of administration, pharmacokinetics of the compound, severity and course of the disease, subject's medical history, subject's health status, subject's The degree of response to the drug, etc.) to determine the therapeutically effective amount of the present invention.
  • Routine influencing factors e.g., mode of administration, pharmacokinetics of the compound, severity and course of the disease, subject's medical history, subject's health status, subject's The degree of response to the drug, etc.
  • inhibitor refers to a reduction in the baseline activity of a biological activity or process.
  • kit refers to a box used to contain chemical reagents for detection of chemical components, drug residues, virus types, etc.
  • the kit of the present invention may include (i) one, two or three of FAK inhibitors, anthracycline chemotherapeutics (such as doxorubicin) and immune checkpoint inhibitors; and (ii) instructions, The instructions state that FAK inhibitors, anthracycline chemotherapeutics, and immune checkpoint inhibitors may be used to treat tumors in the subject.
  • the kit includes (i) a FAK inhibitor; and (ii) instructions indicating that the FAK inhibitor, anthracycline chemotherapeutics, and immune checkpoint inhibitors can be used to treat a tumor in a subject .
  • the kit includes (i) an anthracycline chemotherapeutic; and (ii) instructions indicating that a FAK inhibitor, an anthracycline chemotherapeutic, and an immune checkpoint inhibitor can be used to treat in a subject Treat tumors.
  • the kit includes (i) an immune checkpoint inhibitor; and (ii) instructions stating that a FAK inhibitor, an anthracycline chemotherapeutic, and an immune checkpoint inhibitor can be used to treat in a subject Treat tumors.
  • the kit includes (i) a FAK inhibitor, an anthracycline chemotherapeutic, and an immune checkpoint inhibitor; and (ii) instructions stating that the FAK inhibitor, anthracycline chemotherapeutic, and and immune checkpoint inhibitors to treat tumors in a subject.
  • a kit includes (i) a FAK inhibitor; and (ii) instructions indicating that the FAK inhibitor and an anthracycline chemotherapeutic agent can be used to treat a tumor in a subject.
  • the kit includes (i) an anthracycline chemotherapeutic; and (ii) instructions indicating that the anthracycline chemotherapeutic and the FAK inhibitor can be used to treat a tumor in a subject.
  • kits can include a first container, a second container, a third container, and a package insert, wherein the first container includes at least one dose of a drug comprising a FAK inhibitor and the second container includes at least one dose of an anthracycline chemotherapeutic agent (eg, doxorubicin), the third container includes at least one dose of a drug that is an immune checkpoint inhibitor, and the package insert includes instructions for using the drug to treat the subject's tumor.
  • an anthracycline chemotherapeutic agent eg, doxorubicin
  • the third container includes at least one dose of a drug that is an immune checkpoint inhibitor
  • the package insert includes instructions for using the drug to treat the subject's tumor.
  • the first container, second container, and third container may comprise the same or different shapes (eg, vials, syringes, and bottles) and/or materials (eg, plastic or glass).
  • the kit may also include other materials that may facilitate administration of the drug, such as diluents, filters, IV bags and tubing, needles, and syringes.
  • the precise amount of FAK inhibitor, anthracycline, or immune checkpoint inhibitor administered to a subject will depend on various factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease, condition, The identity of the subject or host to be treated, etc., however, can be routinely determined by one skilled in the art. For example, determining an effective amount also depends on the degree, severity and type of cell proliferation. A skilled artisan will be able to determine appropriate dosages based on these and other factors.
  • FAK inhibitors, anthracyclines, or immune checkpoint inhibitors can be administered in a suitable manner such as oral, intravenous, intramuscular or subcutaneous administration.
  • the drug when administered orally, can be orally administered together with a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be mixed directly with the patient's food.
  • a pharmaceutically acceptable carrier such as an inert diluent or an absorbable edible carrier. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be mixed directly with the patient's food.
  • the medicament may be combined with one or more excipients and administered in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups or wafers and the like. Tablets, lozenges, pills, capsules, etc.
  • binders such as tragacanth, acacia, cornstarch or gelatin
  • excipients such as dicalcium phosphate
  • disintegrants such as cornstarch, potato starch , alginic acid, etc.
  • lubricants such as magnesium stearate
  • sweeteners such as sucrose, fructose, lactose or aspartame; or flavoring agents.
  • solutions of the drug can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Exemplary pharmaceutical dosage forms for injection or infusion include: sterile aqueous solutions, dispersions, or sterile powders containing the active ingredient suitable for the extemporaneous preparation of sterile injectable or infusion solutions or dispersion.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • Sterile injectable solutions can be prepared by incorporating the drug in the required amount in an appropriate solvent with various other ingredients enumerated above as required, followed by filtered sterilization.
  • the preferred methods of preparation may be vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any other desired ingredient previously sterile filtered.
  • the amount of FAK inhibitor, anthracycline, or immune checkpoint inhibitor required for treatment may vary not only with the particular agent chosen, but also with the route of administration, the nature of the disease being treated, and the age and age of the patient. Conditions vary and can ultimately be at the discretion of the attending physician or clinician. In general, however, dosages will range from about 0.1 to about 50 mg/kg body weight per day.
  • the FAK inhibitor is administered in a dosage range of 5 mg/day to 300 mg/day in an adult.
  • IN10018 or a pharmaceutically acceptable salt thereof is administered at a dose of 5 mg/day to 100 mg/day in an adult, the dose being calculated as free base.
  • the anthracyclines are administered in a dosage range of 1-40 mg/ m2 per week in adults.
  • doxorubicin or a pharmaceutically acceptable salt thereof is administered at a dose of 1-40 mg/m 2 per week in adults, and the dose is calculated as doxorubicin.
  • the immune checkpoint inhibitor is administered at a dosage of 2-10 mg/kg or 50-1200 mg in adults for each administration, once every 2 to 3 weeks. In a specific embodiment, the immune checkpoint inhibitor is administered at a dose of 3-10 mg/kg or 100-1200 mg in adults for each administration, and administered once every 2 to 3 weeks.
  • the present disclosure also discloses the following:
  • FAK inhibitors, anthracyclines and immune checkpoint inhibitors for use in a method of treating tumors in a subject, wherein the FAK inhibitors, the anthracyclines and the immune checkpoint inhibitors are controlled by Simultaneously or sequentially administered to the subject.
  • the compound for use according to embodiment 1, wherein the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or its pharmaceutically acceptable Acceptable salts are preferably IN10018, Defactinib, AMP945 or a pharmaceutically acceptable salt thereof, more preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, the IN10018 structure is as follows:
  • the immune checkpoint inhibitor is anti-PD-1/PD-L1 antibody or PD-1/PD-L1 small molecule inhibitor, especially toripalimab.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, liver Cell carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer , liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, thymus cancer, pancreatic cancer, endometrial cancer Carcinoma, cervical cancer, melanoma, glare melanoma, skin cancer, germ cell carcinoma,
  • kits or pharmaceutically acceptable composition comprising:
  • the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable Salt, preferably IN10018, Defactinib, AMP945 or a pharmaceutically acceptable salt thereof, more preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, the IN10018 structure is as follows:
  • kits or composition according to embodiment 7 or 8, wherein the anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the immune checkpoint inhibitor is an anti-PD-1/PD-L1 antibody, a PD-1/PD-L1 small molecule inhibitor Or a TIGIT inhibitor.
  • kits or composition according to any one of embodiments 7-10 wherein the FAK inhibitor is IN10018 or a pharmaceutically acceptable salt thereof, the anthracycline is doxorubicin, and the Immune checkpoint inhibitors are PD-1/PD-L1 inhibitors, especially toripalimab.
  • kit or composition according to any one of embodiments 7-11 for use as a medicament for use as a medicament.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myeloid hyperplasia Dysfunction syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma Sarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, thymus cancer, pancreatic cancer, endometrial cancer, cervical cancer, melanoma, Grave melanoma, skin cancer, germ cell cancer, na
  • a method of treating a tumor in a subject comprising simultaneously or sequentially administering to the subject the compounds of the kit or composition according to any one of embodiments 7-12.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, myelodysplastic syndrome, Acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma , Angiosarcoma, Neuroblastoma, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymic Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma, Grave Melanoma cancer, skin cancer, germ cell cancer, nasopharyn
  • a FAK inhibitor for use in enhancing anthracycline-induced immunogenic cell death.
  • the anthracycline is adriamycin, epirubicin, daunorubicin, preferably adriamycin.
  • a method of treating a tumor in a subject comprising simultaneously or sequentially administering to the subject a therapeutically effective amount of a FAK inhibitor, an anthracycline, and an immune checkpoint inhibitor.
  • anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, Myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma , Rhabdomyosarcoma, Leiomyosarcoma, Angiosarcoma, Neuroblastoma, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymic Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma melanoma, skin cancer, germ cell carcinoma, nas
  • the FAK inhibitor is IN10018, defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable Acceptable salts are preferably IN10018, Defactinib, AMP945 or a pharmaceutically acceptable salt thereof, more preferably IN10018 or a pharmaceutically acceptable salt thereof, especially IN10018 tartrate, the IN10018 structure is as follows:
  • anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the immune checkpoint inhibitor is anti-PD-1/PD-L1 antibody or PD-1/PD-L1 small molecule inhibitor.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, liver Cell carcinoma, cholangiocarcinoma, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer , liposarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, head and neck cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, thymus cancer, pancreatic cancer, endometrial cancer Carcinoma, cervical cancer, melanoma, glare melanoma, skin cancer, germ cell
  • a method of treating tumors by increasing immunogenic cell death in a subject comprising simultaneously or sequentially administering to the subject a therapeutically effective amount of a FAK inhibitor, an anthracycline, and an immune checkpoint inhibitor agent.
  • anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the immune checkpoint inhibitor is an anti-PD-1/PD-L1 antibody, a PD-1/PD-L1 small molecule inhibitor, or a TIGIT inhibitor .
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, Myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma , Rhabdomyosarcoma, Leiomyosarcoma, Angiosarcoma, Neuroblastoma, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymic Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma melanoma, skin cancer, germ cell carcinoma, na
  • FAK inhibitor for the manufacture of a medicament for treating a tumor in a subject, wherein the FAK inhibitor, anthracycline and immune checkpoint inhibitor are administered to the subject simultaneously or sequentially.
  • anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, Myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma , Rhabdomyosarcoma, Leiomyosarcoma, Angiosarcoma, Neuroblastoma, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymic Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma melanoma, skin cancer, germ cell carcinoma, n
  • anthracycline for the manufacture of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, said anthracycline and an immune checkpoint inhibitor are administered to said subject simultaneously or sequentially.
  • anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the FAK inhibitor is IN10018, Defactinib, GSK2256098, PF-00562271, VS-4718, APG-2449, AMP945, AMP886 or a pharmaceutically acceptable salt thereof
  • the structure of said IN10018 is as follows:
  • the immune checkpoint inhibitor is an anti-PD-1/PD-L1 antibody, a PD-1/PD-L1 small molecule inhibitor or a TIGIT inhibitor .
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, Myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma , Rhabdomyosarcoma, Leiomyosarcoma, Angiosarcoma, Neuroblastoma, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymic Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma melanoma, skin cancer, germ cell carcinoma, n
  • an immune checkpoint inhibitor for the manufacture of a medicament for treating a tumor in a subject, wherein a FAK inhibitor, an anthracycline and said immune checkpoint inhibitor are administered to said subject simultaneously or sequentially.
  • anthracycline is doxorubicin, epirubicin, daunorubicin, preferably doxorubicin.
  • the immune checkpoint inhibitor is an anti-PD-1/PD-L1 antibody, a PD-1/PD-L1 small molecule inhibitor or a TIGIT inhibitor .
  • the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma, Myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroid cancer, glioma, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma , Rhabdomyosarcoma, Leiomyosarcoma, Angiosarcoma, Neuroblastoma, Renal Cell Carcinoma, Head and Neck Cancer, Gastric Cancer, Esophageal Cancer, Gastroesophageal Junction Cancer, Thymic Cancer, Pancreatic Cancer, Endometrial Cancer, Cervical Cancer, Melanoma melanoma, skin cancer, germ cell carcinoma, n
  • SK-OV-3 cells were used as the research object, and 50nM FAKsiRNA was transfected with Lipofectamine 3000. After the experiment, the protein was harvested to detect the expression of FAK and phosphorylated FAKY397.
  • FAKsiRNA is provided by Gemma Gene, and its sequence is shown in the table below.
  • Doxorubicin was provided by MCE.
  • Alexa 488 fluorescent Anti-HMGB1 antibody (Abcam, ab195010). recombinant Alexa 647 fluorescent Anti-Calreticulin antibody (Abcam, ab196159). Annexin A1 rabbit antibody (Cell signaling technology, 32934S). Anti-rabbit IgG(H+L), F(ab')2 Fragment(Alexa 488 Conjugate) (Cell signaling technology, 4412). FAK antibody (CST, 3285S). Phospho FAKY397 antibody (Thermo fisher, 44-624G). HRP-labeled Alpha tubulin ( ⁇ -tubulin) antibody (Proteintech, HRP-66031)
  • SK-OV-3 cells were transfected with 50nM FAKsiRNA. Lipofectamine 3000 was used as transfection reagent. Downstream co-use and ICD biomarker experiments can be performed 24 hours after transfection. 72 hours after transfection, the protein of SK-OV-3 cells was collected and western blot (Western blot) was performed to detect the amount of phosphorylated FAKY397, FAK and Alpha tubulin. The transfection effect is based on the protein knockout status, as shown in Figure 1. FAKsiRNA knocked out phosphorylated FAKY397 and total FAK protein significantly.
  • FAK knockout can significantly enhance the cell killing effect of doxorubicin on ovarian cancer cell SK-OV-3.
  • ICD markers ICD markers
  • knockout of FAK could significantly enhance the exposure and release of DAMPs markers Calreticulin and Annexin A1, suggesting that knockout of FAK could effectively enhance the immunogenic cell death of ovarian cancer cells.
  • Example 2 IN10018 enhances the immunogenic cell death of ovarian cancer cells against doxorubicin
  • IN10018 was synthesized according to the method in patent WO2010058032.
  • Doxorubicin was provided by MCE.
  • Alexa 488 fluorescent Anti-HMGB1 antibody (Abcam, ab195010). recombinant Alexa 647 fluorescent Anti-Calreticulin antibody (Abcam, ab196159). Annexin A1 rabbit antibody (Cell signaling technology, 32934S). Anti-rabbit IgG(H+L), F(ab')2Fragment(Alexa 488 Conjugate) (Cell signaling technology, 4412).
  • Annexin V Apoptosis Kit (Abbkine, KTA0002).
  • CCK8 cell viability reagent (Cellor lab, CX001).
  • Microplate reader (Molecular devices M3). Fluorescence microscope (Olympus U-HGLGPS). Flow cytometer (BD LSRFortessa X-20)
  • 96-well cell plate was laid with 5000 SK-OV-3 cells/well. After 24 hours, the IC50 value of doxorubicin was used as the initial point to set 3 sub-doses and 2 sub-doses on the left and right sides respectively. Doxorubicin was combined with 3 ⁇ M IN10018. After 72 hours, the cell viability was detected by CCK8 method. Use Graphpad 8.0 for graph drawing. The results showed that IN10018 could significantly enhance the cell killing effect of different doses of doxorubicin on ovarian cancer cell SK-OV-3 (see Figure 5).
  • SK-OV-3 cells were treated with 0.3 ⁇ M or 1 ⁇ M doxorubicin combined with 3 ⁇ M IN10018, and the cells were harvested 48 hours later and stained with Annexin V kit and detected by flow cytometry.
  • the Q2 quadrant shows the late apoptosis
  • the Q3 quadrant shows the early apoptosis. The results showed that compared with the single drug group, early and late apoptosis was significantly enhanced in the combination group (see Figure 6).
  • SK-OV-3 cells were treated with 0.3 ⁇ M doxorubicin and 3 ⁇ M IN10018, and fluorescent staining was performed 48 hours later using antibodies against Calreticulin, HMGB1 and Annexin A1.
  • the results showed that compared with the single drug group, the release and exposure of Calreticulin, HMGB1 and Annexin A1 were significantly enhanced (see Figure 7).
  • IN10018 can significantly enhance the cell killing effect of doxorubicin on ovarian cancer cell SK-OV-3. This effect is related to its enhancement of early and late apoptosis. In the experiment of detecting ICD markers, it was found that IN10018 can significantly enhance the exposure and release of DAMPs markers Calreticulin, HMGB1 and Annexin A1, suggesting that IN10018 can effectively enhance the immunogenic cell death of ovarian cancer cells.
  • Example 3 IN10018 enhances ovarian cancer cell death against daunorubicin (daunorubicin) and epirubicin (epirubicin) immunogenic cell death
  • IN10018 was synthesized according to the method in patent WO2010058032. Daunorubicin is provided by MCE. Epirubicin is provided by MCE.
  • Example 4 Defactinib enhances immunogenic cell death of ovarian cancer cells against doxorubicin
  • Defactinib combined with doxorubicin can effectively induce the ICD effect of ovarian cancer cell line SK-OV-3
  • Defactinib is provided by DC chemicels.
  • Doxorubicin was provided by MCE.
  • Alexa 488 fluorescent Anti-HMGB1 antibody (Abcam, ab195010). recombinant Alexa 647 fluorescent Anti-Calreticulin antibody (Abcam, ab196159). Annexin A1 rabbit antibody (Cell signaling technology, 32934S). Anti-rabbit IgG(H+L), F(ab')2 Fragment(Alexa 488 Conjugate) (Cell signaling technology, 4412).
  • Microplate reader (Molecular devices M3). Fluorescence microscope (Olympus U-HGLGPS).
  • 96-well cell plate was laid with 5000 SK-OV-3 cells/well. After 24 hours, the IC50 value of doxorubicin was used as the initial point to set 3 sub-doses and 2 sub-doses on the left and right sides respectively. Doxorubicin was administered in combination with 3 ⁇ M Defactinib. After 72 hours, the cell viability was detected by CCK8 method. Use Graphpad 8.0 for graph drawing. The results showed that Defactinib could significantly enhance the cell killing effect of different doses of doxorubicin on ovarian cancer cell SK-OV-3 (see Figure 9). Student's T-test was used in statistical research, *** represents the P value is less than 0.005, **** represents the P value is less than 0.001.
  • Defactinib can significantly enhance the cell killing effect of doxorubicin on ovarian cancer cell SK-OV-3. In the experiment of detecting ICD markers, it was found that Defactinib can significantly enhance the exposure and release of DAMPs markers Calreticulin, HMGB1 and Annexin A1, suggesting that Defactinib can effectively enhance the immunogenic cell death of ovarian cancer cells.
  • Example 5 Pharmacodynamic study of IN10018 and doxorubicin in colon cancer CT26 cell line
  • Doxorubicin is provided by MCE, Cat No.:HY-15142, Lot No.:97451
  • Alexa 647 fluorescent Anti-Calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773).
  • Annexin-Modulation Assay Kit (Beyotime, Cat No.: C1062L, Lot No.: 021921210811).
  • CT26 cells were cultured with RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp), passaged twice, and the cells When in good condition, place the culture plate in a 24-well plate. After 24 hours of cell spreading, four groups were set up, the first group was the control group, the medium was added, the second group was IN10018, the concentration was 5 ⁇ M, the third group was doxorubicin (doxorubicin, MCE), the concentration was 1uM, The fourth group is the combination of IN10018 (5 ⁇ M) and doxorubicin (1 ⁇ M). Drugs were mixed and incubated for 48 h at 37 °C in a 5% CO2 incubator.
  • Example 6 In vivo anti-tumor drug efficacy study of IN10018 in colon cancer CT26 cell BALB/c mouse subcutaneous allograft tumor model
  • mice 6-8 week-old female BALB/c mice were purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • the animal information card for each cage should indicate the number of animals in the cage, sex, strain, date of receipt, dosing regimen, experiment number, group and date of start of the experiment. Cages, feed and drinking water were changed twice a week.
  • the feeding environment and light conditions are as follows:
  • Photoperiod 12 hours light, 12 hours no light.
  • Cage made of polycarbonate, volume 300mm ⁇ 180mm ⁇ 150mm.
  • the bedding is corn cobs, which are changed twice a week.
  • Drinking water Experimental animals can freely drink sterilized water.
  • Animal identification Experimental animals were identified with ear tags.
  • Colorectal cancer cell CT26 (sourced from the Cell Bank of the Chinese Academy of Sciences, item number: TCM37) was maintained and passaged by Huiyuan Biotechnology (Shanghai) Co., Ltd.
  • the cells were cultured in a single layer in vitro, and the culture conditions were RPMI-1640 medium plus 10% fetal bovine serum, and cultured in a 5% CO 2 incubator at 37°C. Passage with routine digestion with trypsin-EDTA two to three times a week. When the cells are in the exponential growth phase and the saturation is 80%-85%, the cells are harvested, counted and inoculated.
  • mice The number of mice in each group.
  • Dosing volume 10 mL/kg based on the body weight of the mouse. If the body weight drops by more than 15%, the animal stops the administration; when the body weight recovers to a 10% reduction, the administration is resumed.
  • Tumor diameters were measured with a vernier caliper three times a week.
  • TGI (%) [1-(average tumor volume of a certain administration group-average tumor volume at the beginning of the administration group)/(average tumor volume of the solvent control group-average tumor volume at the beginning of treatment of the solvent control group)] ⁇ 100%.
  • the in vivo efficacy of the test substance IN10018 and/or PD-L1 antibody combined with the positive drug Doxil in the CT26 mouse colorectal cancer cell BALB/c mouse subcutaneous allograft tumor model After the cells were inoculated, the tumor growth was observed every day. On the 9th day after the inoculation, the tumors were grouped according to the tumor volume. The average tumor volume of the enrolled groups was about 38 mm 3 . Control (G1) mice were euthanized on day 28 and PD-L1 antibody group (G3) #790 mice were euthanized on day 32 due to tumor burden. The whole experiment ended on the 35th day.
  • the tumor volume of the control group was 2176.53 ⁇ 491.44 mm 3 .
  • IN10018+Doxil (12.5+1.5mg/kg), PD-L1 antibody (2.5mg/kg), IN10018+Doxil+PD-L1 antibody (12.5+1.5+2.5mg/kg) and Doxil+PD-L1 antibody (1.5 +2.5mg/kg) the tumor volumes of each treatment group were 340.51 ⁇ 89.1mm 3 , 807.22 ⁇ 257.97mm 3 , 88.95 ⁇ 33.38mm 3 and 295.53 ⁇ 215.08mm 3 , see Table 4 for details.
  • the tumor volumes of each dose group at different time periods are shown in FIG. 13 .
  • the experiment was carried out according to the dosing regimen. During the experiment, the animals were observed for eating and drinking activities every day, and the animal weight was recorded 3 times a week. The animal weight curve is shown in Figure 14. During the whole administration period, the body weight of animals in each group had no significant decrease and was in good condition.
  • Example 7 In vivo anti-tumor efficacy study of Defactinib in colon cancer CT26 cell BALB/c mouse subcutaneous allograft tumor model
  • mice 6-8 week-old female BALB/c mice were purchased from Beijing Weitong Lihua Experimental Animal Co., Ltd. The experiments were started after the animals arrived and were acclimated to the experimental environment. The animals were kept in IVC (independent ventilation system) cages (5 per cage) in an SPF animal room. All cages, bedding and drinking water should be sterilized before use. All experimenters should wear protective clothing and latex gloves when operating in the animal room. The animal information card for each cage should indicate the number of animals in the cage, sex, strain, date of receipt, dosing regimen, experiment number, group and date of start of the experiment. Cages, feed and drinking water were changed twice a week. The feeding environment and light conditions are as follows:
  • Photoperiod 12 hours light, 12 hours no light.
  • Cage made of polycarbonate, volume 300mm ⁇ 180mm ⁇ 150mm.
  • the bedding is corn cobs, which are changed twice a week.
  • Drinking water Experimental animals can freely drink sterilized water.
  • Animal identification Experimental animals were identified with ear tags.
  • Colorectal cancer cell CT26 (sourced from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60043) was maintained and passaged by Yingshi Biotechnology (Nanjing) Co., Ltd.
  • the cells were cultured in a single layer in vitro, and the culture conditions were 10% fetal bovine serum added to RPMI-1640 medium, and cultured in a 5% CO2 incubator at 37°C. Passage with routine digestion with trypsin-EDTA two to three times a week. When the cells are in the exponential growth phase and the saturation is 80%-90%, the cells are harvested, counted and inoculated.
  • mice The number of mice in each group.
  • Dosing volume 10 mL/kg based on the body weight of the mouse. If the body weight drops by more than 15%, the animal stops the administration; when the body weight recovers to a 10% reduction, the administration is resumed.
  • Tumor diameters were measured with a vernier caliper three times a week.
  • TGI (%) [1-(average tumor volume of a certain administration group-average tumor volume at the beginning of the administration group)/(average tumor volume of the solvent control group-average tumor volume at the beginning of treatment of the solvent control group)] ⁇ 100%.
  • the in vivo efficacy of the test substance Defactinib and/or PD-L1 antibody combined with the positive drug Doxil in the CT26 mouse colorectal cancer cell BALB/c mouse subcutaneous allograft tumor model After the cells were inoculated, the tumor growth was observed every day. On the 9th day after the inoculation, the tumors were grouped according to the tumor volume, and the average tumor volume of the group was about 81 mm 3 . Due to tumor burden, all enrolled mice were euthanized on the 24th day after inoculation, that is, the 15th day after group administration, and the entire experiment was terminated.
  • the tumor volume of the control group was 3920.2 ⁇ 1697.5 mm 3 .
  • Defactinib+Doxil 25+1.5mg/kg
  • Doxil single drug 1.5mg/kg
  • PD-L1 antibody 10mg/kg
  • Doxil+PD-L1 antibody 1.5+10mg/kg
  • Defactinib+Doxil+ The tumor volumes of PD-L1 antibody (25+1.5+10mg/kg) treatment groups were 2942.2 ⁇ 1491.2mm 3 , 3130.9 ⁇ 1446.2mm 3 , 3645.2 ⁇ 1914.8mm 3 , 2964.6 ⁇ 1653.2mm 3 and 1546.5 ⁇ 1237.9mm 3 respectively , see Table 8 for details.
  • the comprehensive tumor volume was compared with the Defactinib+Doxil+PD-L1 antibody (25+1.5+10mg/kg) group for statistical analysis.
  • the tumor volumes of each dose group at different time periods are shown in FIG. 15 .
  • the experiment was carried out according to the dosing regimen. During the experiment, the animals were observed for eating and drinking activities every day, and the animal weight was recorded 3 times a week. The animal weight curve is shown in Figure 16. During the whole administration period, the body weight of animals in each group had no significant decrease and was in good condition.
  • the Defactinib+Doxil (25+1.5mg/kg) and Defactinib+Doxil+PD-L1 antibody (25+1.5+10mg/kg) groups had significant tumor growth inhibitory effects, compared with the control group There are statistical differences.
  • Defactinib+Doxil+PD-L1 antibody (25+1.5+10mg/kg) group was compared with Defactinib+Doxil (25+1.5mg/kg), Doxil single drug (1.5mg/kg), PD-L1 antibody (10mg/kg ) and Doxil+PD-L1 antibody (1.5+10mg/kg) group, the tumors in each treatment group were smaller, and compared with these single-drug groups and two-drug combination groups, there were statistical differences. The combination group and the two-drug combination group have a better effect of inhibiting tumor growth.
  • Example 8 ELISA detection of IFN- ⁇ and Granzyme B in the serum after the treatment of colon cancer CT26 cell BALB/c mouse subcutaneous allograft tumor model treated by the combined treatment of test substance Doxil and IN10018
  • test items are shown in Table 9.
  • mice from Shanghai Lingchang Biotechnology Co., Ltd.
  • the grouping information is shown in Table 11.
  • Dosing volume 10mL/kg based on the body weight of the mouse. If the body weight drops by more than 15%, the animal stops the administration; when the body weight recovers to a 10% reduction, the administration is resumed
  • the animals in the 4 groups were subjected to orbital blood collection, and the blood was placed in a collection tube without anticoagulant, and allowed to agglutinate naturally at room temperature for 30 to 60 minutes, and centrifuged at 1000 rpm at 4°C until the blood coagulated 10 minutes. Transfer the upper layer of serum to a new collection tube and store it in a negative 80°C refrigerator.
  • the incubation time needs to be extended appropriately. At this time, it can be incubated until the standard product has a very significant color change. If the sample concentration is high enough, a significant color change will also occur; add 100 ⁇ l/well of stop solution, and measure A450 immediately after mixing. value.
  • the concentration of IFN- ⁇ protein in each group was 363.10 ⁇ 4.06pg/ml for the blank control, 367.24 ⁇ 7.34pg/ml for IN10018, 359.13 ⁇ 5.71pg/ml for Doxil, 387.00 ⁇ 4.04pg/ml for Doxil+IN10018, the comprehensive protein concentration and Doxil+IN10018
  • the Doxil+IN10018 two-drug combination group had higher protein expression than the Doxil single-drug group.
  • Statistical analysis showed that the P value was p ⁇ 0.001, see Figure 17 for details.
  • the Granzyme B protein concentration in each group was blank control 34.06 ⁇ 47.49pg/ml, IN10018 50.48 ⁇ 53.08pg/ml, Doxil 17.24 ⁇ 30.80pg/ml, Doxil+IN10018 72.21 ⁇ 35.40pg/ml, the comprehensive protein concentration was compared with Doxil+IN10018 In comparison, the Doxil+IN10018 two-drug combination group had higher protein expression than the Doxil single-drug group. Statistical analysis showed that the P value was p ⁇ 0.05, see Figure 18 for details.
  • each group was compared with the Doxil+IN10018 group.
  • the Doxil+IN10018 two-drug combination group had higher protein expression than the Doxil single-drug group.
  • Statistical analysis showed that the P value was p ⁇ 0.001.
  • each group was compared with the Doxil+IN10018 group.
  • the Doxil+IN10018 two-drug combination group had higher protein expression than the Doxil single-drug group.
  • Statistical analysis showed that the P value was p ⁇ 0.05.
  • Example 9 Study on Induction of Immunogenic Cell Death Targets of Mouse Breast Cancer 4T1 Cells in Vitro by Tested Compounds Doxorubicin and AMP945
  • Doxorubicin is provided by MCE, Lot No.: 97451
  • AMP945 is provided by MCE, Lot No.: 143253
  • Alexa 647 fluorescent Anti-Calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773).
  • Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).
  • 4T1 cells (from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60352) were treated with RPMI1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c, Lot No.: 2158737cp) cultured at 37°C, 5% CO 2 .
  • Routine digestion passaging with trypsin two to three times a week. When the cells were in the exponential growth phase and reached 80%-90% confluency, the cells were harvested and plated. 4T1 cells were digested with trypsin and collected and counted.
  • the cells were diluted with RPMI1640+10% FBS to a concentration of 50,000 cells per milliliter, and then plated on a 12-well cell culture plate. Spread 2ml of cell suspension, that is, 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO 2 incubator.
  • the first group was the control group, and the medium was added
  • the second group was AMP945, the concentration was 3 ⁇ M
  • the third group was AMP945, the concentration was 6 ⁇ M
  • the fourth group was Adriamycin (Adriamycin (Adriamycin) Mycin, MCE)
  • the concentration is 0.5uM
  • the fifth group is the combination of AMP945 (3 ⁇ M) and doxorubicin (0.5 ⁇ M)
  • the sixth group is the combination of AMP945 (6 ⁇ M) and doxorubicin (0.5 ⁇ M).
  • Drugs were mixed and incubated for 48 h at 37 °C in a 5% CO2 incubator.
  • the cells were collected for flow analysis, and the cells were washed twice with flow buffer (PBS+2% FBS) and 0.5 ⁇ l AF647, and anti-calreticulin antibody (abcam) was added to each well and mixed. Incubate at 4°C and avoid light, add flow buffer after incubation for 20min, use annexin staining kit (Beyotime), add 195 ⁇ l Annexin-V-FITC conjugate, mix with cells, add 5 ⁇ l Annexin - V-FITC antibody, mixed gently, and finally added 10 ⁇ l PI dye to mix, incubated at room temperature in the dark for 15 minutes, and sent the sample to the flow cytometer for signal determination.
  • flow buffer PBS+2% FBS
  • abcam anti-calreticulin antibody
  • Example 10 Study on Induction of Immunogenic Cell Death Targets of Mouse Colorectal Cancer CT26 Cells in Vitro by Test Compounds Doxorubicin and AMP945
  • Doxorubicin is provided by MCE, Lot No.: 97451
  • AMP945 is provided by MCE, Lot No.: 143253
  • Alexa 647 fluorescent Anti-Calreticulin antibody (Abcam, Cat No.: ab196159, Lot No.: CR33676773).
  • Annexin V-apoptosis detection kit (Beyotime, Cat No.: C1062L, Lot No.: 122221220706).
  • CT26 cells (from Nanjing Kebai Biotechnology Co., Ltd., product number: CBP60043) were treated with RPMI 1640 (Shanghai Yuanpei, Cat No.: L210KJ, Lot No.: F210916) + 10% FBS (Gibco, Cat No.: 10099-141c , Lot No.: 2158737cp) cultured at 37°C, 5% CO 2 . Routine digestion passaging with trypsin two to three times a week. When the cells were in the exponential growth phase and reached 80%-90% confluency, the cells were harvested and plated. CT26 cells were digested with trypsin and collected and counted.
  • the cells were diluted with RPMI1640+10% FBS to a concentration of 50,000 cells per milliliter, and then plated on a 12-well cell culture plate. Spread 2ml of cell suspension, that is, 100,000 cells. After plating, the cells were cultured in a 37°C, 5% CO 2 incubator.
  • the first group is the control group, add medium
  • the second group is AMP945, the concentration is 0.75 ⁇ M
  • the third group is AMP945, the concentration is 1.5 ⁇ M
  • the fourth group is doxorubicin (Adriamycin, MCE)
  • the concentration is 0.5uM
  • the fifth group is AMP945 (0.75 ⁇ M) combined with adriamycin (0.5 ⁇ M)
  • the sixth group is AMP945 (1.5 ⁇ M) and adriamycin (0.5 ⁇ M) joint use.
  • Drugs were mixed and incubated for 48 h at 37 °C in a 5% CO2 incubator.
  • the cells were collected for flow analysis, and the cells were washed twice with flow buffer (PBS + 2% FBS) and 0.5 ⁇ l AF647, and anti-calreticulin antibody (abcam) was added to each well and mixed. Incubate at 4°C and avoid light, add flow buffer after incubation for 20min, use annexin staining kit (Beyotime), add 195 ⁇ l Annexin-V-FITC conjugate, mix with cells, add 5 ⁇ l Annexin - V-FITC antibody, mixed gently, and finally added 10 ⁇ l PI dye to mix, incubated at room temperature in the dark for 15 minutes, and sent the sample to the flow cytometer for signal determination.
  • flow buffer PBS + 2% FBS
  • abcam anti-calreticulin antibody
  • Example 11 Clinical study of IN10018 combined with PLD and anti-PD-1 in the treatment of ovarian cancer
  • IN10018 in combination with pegylated liposomal doxorubicin (PLD) and anti-PD-1 antibody for the treatment of platinum-resistant recurrent ovarian cancer is currently being investigated in a Phase Ib/II clinical study (IN10018-010) in China Evaluate.
  • the IN10018 used its tartrate
  • the PLD used a commercially available doxorubicin hydrochloride liposome injection (common name)
  • the anti-PD-1 antibody research used a commercially available special Ripleyzumab.
  • Study subjects will receive IN10018 100mg orally once daily (QD) combined with PLD 40mg/ m2 every 4 weeks (Q4W) and toripalimab 3mg/kg every 2 weeks (Q2W) intravenous infusion medicine.
  • QD Quality of Service
  • Q2W toripalimab 3mg/kg every 2 weeks
  • a treatment cycle is 28 days.
  • Table 12 Summary of baseline characteristics of 26 patients with platinum-resistant recurrent ovarian cancer in the ongoing study
  • CT computed tomography
  • MRI contrast-enhanced magnetic resonance imaging
  • MRI is the test of choice for brain imaging, and other imaging techniques, such as PET/CT and bone scans, may be used if needed.
  • Imaging studies during the screening period must be performed within 28 days prior to study treatment assignment. During the study period, the subjects will receive tumor imaging examinations every 8 weeks (56 ⁇ 7 days), until the investigators evaluate the subjects to find the disease progression confirmed by imaging, start a new anti-tumor treatment, withdraw from the study or die ( whichever occurs first).
  • Efficacy indicators include objective response rate (ORR), disease control rate (DCR), duration of response (DOR), progression-free survival (PFS) and overall survival (OS).
  • ORR objective response rate
  • DCR disease control rate
  • DOR duration of response
  • PFS progression-free survival
  • OS overall survival
  • Three-drug combination (IN10018+PLD+anti-PD-1) therapy has a total of 13 evaluable patients in the study of platinum-resistant recurrent ovarian cancer, most of which only have the results of the first tumor imaging evaluation, so only the results of the first tumor evaluation are presented efficacy data.
  • partial response (PR) was observed in 7 cases at the first tumor assessment, ORR was 53.8%% (7/13), DCR was 100% (13/13), 83.5% (11/13 ) patients had shrinkage of tumor target lesions.
  • Table 13 Summary of anti-tumor efficacy of two-drug and three-drug combination
  • Table 14 The curative effect list of the first tumor evaluation of the combination of three drugs
  • the best change of the total diameter of target lesions relative to the baseline (the total diameter of the smallest target lesions after treatment - the total diameter of target lesions at baseline)/total diameter of target lesions at baseline x 100%.
  • the safety analysis shows that the safety of the three-drug combination (IN10018+PLD+anti-PD-1) treatment is generally equivalent to that of the two-drug combination (IN10018+PLD) treatment, and the safety characteristics are comparable to the historical data of the respective drugs, and no adverse effects caused by the combination were observed Incidents have increased significantly.
  • a summary of the safety of the two-drug and three-drug combinations is shown in Table 15 below.

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Abstract

涉及FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂联合治疗肿瘤。还涉及FAK抑制剂和蒽环类化疗药联合治疗肿瘤。

Description

治疗肿瘤的药物组合及用途 技术领域
本发明属于药物化学领域。具体地，本发明涉及粘着斑激酶(Focal Adhesion Kinase,FAK)抑制剂与其它药物联用治疗肿瘤。
背景技术
肿瘤是威胁人们健康的第二大杀手。免疫原性细胞死亡(Immunogenic cell death，ICD)是基于细胞程序性死亡的叠加效应。癌细胞接触化疗或者靶向治疗类药物的同时可能激活细胞内部的压力信号，这一类压力信号包含内质网压力(ER stress)和活性氧压力(oxidative stress)。在压力信号的作用下细胞会先尝试修复压力，如果压力造成的损伤超出了修复的能力，细胞会启动程序性死亡过程。在这个过程中往往伴随着一类叫做损伤相关分子(Damage associated molecular patterns，DAMPs)的释放。这类DAMPs会特异性的被机体内抗原呈递细胞(Antigen-presenting cells，APC)上的模式识别受体识别，诱导APC成熟、分化和激活，并逐级呈递给效应T细胞等免疫细胞，从而使免疫细胞产生抗原记忆，再次发现相同来源的肿瘤细胞时免疫细胞就会特异性识别并杀伤肿瘤细胞。ICD启动的新的针对肿瘤的特异性免疫反应可增加对免疫检查点抑制剂(Immuno-check point inhibitor，ICI)的敏感，以此增效免疫检查点类抑制剂的作用，并可产生具有免疫记忆持久的抗肿瘤的反应。
FAK，又称为蛋白酪氨酸激酶2(PTK2)，是一种非受体酪氨酸激酶，并且是粘着斑复合体的关键组分。FAK在介导整合素和生长因子信号以调节肿瘤细胞的侵袭、增殖和存活方面发挥着重要作用。
已经观察到一些化疗药物可以触发癌细胞的ICD，并且ICD被理解有助于该疗法的抗肿瘤作用。重要地，ICD刺激免疫系统攻击癌症，尤其是与抗 -PD1或PD-L1联合，可产生持久的抗肿瘤效果。并且文献报道PLD(盐酸多柔比星脂质体注射液(doxil))能够诱发ICD，但是临床试验无论联合使用抗PD-1或PD-L1，均在临床上没有观察显著的临床效果。Western Arizona Regional Medical Center探索了Anti-PD1(pembrolizumab)联合化疗(其中包括PLD)治疗肿瘤，但临床效果并没有达到预期，临床试验已经终止。因此仍然需要进一步寻找可以增强化疗药物诱导ICD的药物，提高肿瘤治疗的应答率及长期获益的可能性。
发明内容
本公开一方面提供了FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂在制备用于在对象中治疗肿瘤的药物中的用途，其中所述FAK抑制剂、所述蒽环类化疗药和所述免疫检查点抑制剂被同时或依次施用于所述对象。
本公开又一方面提供了FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂的药物组合产品，其用于在对象中治疗肿瘤，其中所述FAK抑制剂、所述蒽环类化疗药和所述免疫检查点抑制剂被同时或依次施用于所述对象。
本公开一方面提供了一种治疗肿瘤的方法，该方法包括向有需要的对象同时或依次施用治疗有效量的FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂。
本公开又一方面提供了一种试剂盒或药学上可接受的组合物，其包括：(a)FAK抑制剂；(b)蒽环类化疗药；和(c)免疫检查点抑制剂。
本公开又一方面提供了FAK抑制剂和蒽环类化疗药在制备用于在治疗肿瘤的药物中的用途，其中所述FAK抑制剂用于增强所述蒽环类化疗药诱导的免疫原性细胞死亡。
本公开又一方面提供了FAK抑制剂，其在治疗肿瘤中用于增强蒽环类化疗药诱导的免疫原性细胞死亡。
本公开又一方面提供了一种治疗肿瘤的方法，该方法包括向有需要的对 象同时或依次施用治疗有效量的FAK抑制剂和蒽环类化疗药，其中所述FAK抑制剂用于增强所述蒽环类化疗药诱导的免疫原性细胞死亡。
本公开又一方面提供了FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂在制备用于联合治疗肿瘤的药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与蒽环类化疗药和免疫检查点抑制剂治疗肿瘤的联用药物中的用途。
本公开又一方面提供了蒽环类化疗药在制备用于与FAK抑制剂和免疫检查点抑制剂治疗肿瘤的联用药物中的用途。
本公开又一方面提供了免疫检查点抑制剂在制备用于与FAK抑制剂和蒽环类化疗药治疗肿瘤的联用药物中的用途。
本公开又一方面提供了FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂在制备用于联合治疗肿瘤的药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与蒽环类化疗药和免疫检查点抑制剂联合治疗肿瘤的药物中的用途。
本公开又一方面提供了蒽环类化疗药在制备用于与FAK抑制剂和免疫检查点抑制剂联合治疗肿瘤的药物中的用途。
本公开又一方面提供了免疫检查点抑制剂在制备用于与FAK抑制剂和蒽环类化疗药联合治疗肿瘤的药物中的用途。
本公开又一方面提供了一种试剂盒，其包括：FAK抑制剂；和说明书，该说明书指出该FAK抑制剂可用于与蒽环类化疗药和免疫检查点抑制剂联合治疗肿瘤。
本公开又一方面提供了一种试剂盒，其包括：蒽环类化疗药；和说明书，该说明书指出该蒽环类化疗药可用于与FAK抑制剂和免疫检查点抑制剂联合治疗肿瘤。
本公开又一方面提供了一种试剂盒，其包括：免疫检查点抑制剂；和说明书，该说明书指出该免疫检查点抑制剂可用于与FAK抑制剂和蒽环类化 疗药联合治疗肿瘤。
本公开另一方面提供了一种治疗肿瘤的方法，该方法包括向有需要的对象施用治疗有效量的FAK抑制剂和蒽环类化疗药。
本公开又一方面提供了FAK抑制剂和蒽环类化疗药的药物组合产品，其用于在有需要的对象中治疗肿瘤。
本公开又一方面提供了FAK抑制剂和蒽环类化疗药在制备用于治疗肿瘤的联用药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与蒽环类化疗药治疗肿瘤的联用药物中的用途。
本公开又一方面提供了蒽环类化疗药在制备用于与FAK抑制剂治疗肿瘤的联用药物中的用途。
本公开又一方面提供了FAK抑制剂和蒽环类化疗药在制备用于联合治疗肿瘤的药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与蒽环类化疗药联合治疗肿瘤的药物中的用途。
本公开又一方面提供了蒽环类化疗药在制备用于与FAK抑制剂联合治疗肿瘤的药物中的用途。
本公开又一方面提供了一种试剂盒，其包括：FAK抑制剂；和说明书，该说明书指出该FAK抑制剂可用于与蒽环类化疗药联合治疗肿瘤。
本公开又一方面提供了一种试剂盒，其包括：蒽环类化疗药；和说明书，该说明书指出该蒽环类化疗药可用于与FAK抑制剂联合治疗肿瘤。
本公开一方面提供了FAK抑制剂、阿霉素和免疫检查点抑制剂在制备用于在对象中治疗肿瘤的药物中的用途，其中所述FAK抑制剂、所述阿霉素和所述免疫检查点抑制剂被同时或依次施用于所述对象。
本公开又一方面提供了FAK抑制剂、阿霉素和免疫检查点抑制剂的药物组合产品，其用于在对象中治疗肿瘤，其中所述FAK抑制剂、所述阿霉 素和所述免疫检查点抑制剂被同时或依次施用于所述对象。
本公开一方面提供了一种治疗肿瘤的方法，该方法包括向有需要的对象同时或依次施用治疗有效量的FAK抑制剂、阿霉素和免疫检查点抑制剂。
本公开又一方面提供了一种试剂盒或药学上可接受的组合物，其包括：(a)FAK抑制剂；(b)阿霉素；和(c)免疫检查点抑制剂。
本公开又一方面提供了FAK抑制剂和阿霉素在制备用于在治疗肿瘤的药物中的用途，其中所述FAK抑制剂用于增强所述阿霉素诱导的免疫原性细胞死亡。
本公开又一方面提供了FAK抑制剂，其在治疗肿瘤中用于增强阿霉素诱导的免疫原性细胞死亡。
本公开又一方面提供了一种治疗肿瘤的方法，该方法包括向有需要的对象同时或依次施用治疗有效量的FAK抑制剂和阿霉素，其中所述FAK抑制剂用于增强所述阿霉素诱导的免疫原性细胞死亡。
本公开又一方面提供了FAK抑制剂、阿霉素和免疫检查点抑制剂在制备用于联合治疗肿瘤的药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与阿霉素和免疫检查点抑制剂治疗肿瘤的联用药物中的用途。
本公开又一方面提供了阿霉素在制备用于与FAK抑制剂和免疫检查点抑制剂治疗肿瘤的联用药物中的用途。
本公开又一方面提供了免疫检查点抑制剂在制备用于与FAK抑制剂和阿霉素治疗肿瘤的联用药物中的用途。
本公开又一方面提供了FAK抑制剂、阿霉素和免疫检查点抑制剂在制备用于联合治疗肿瘤的药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与阿霉素和免疫检查点抑制剂联合治疗肿瘤的药物中的用途。
本公开又一方面提供了阿霉素在制备用于与FAK抑制剂和免疫检查点 抑制剂联合治疗肿瘤的药物中的用途。
本公开又一方面提供了免疫检查点抑制剂在制备用于与FAK抑制剂和阿霉素联合治疗肿瘤的药物中的用途。
本公开又一方面提供了一种试剂盒，其包括：FAK抑制剂；和说明书，该说明书指出该FAK抑制剂可用于与阿霉素和免疫检查点抑制剂联合治疗肿瘤。
本公开又一方面提供了一种试剂盒，其包括：阿霉素；和说明书，该说明书指出该阿霉素可用于与FAK抑制剂和免疫检查点抑制剂联合治疗肿瘤。
本公开又一方面提供了一种试剂盒，其包括：免疫检查点抑制剂；和说明书，该说明书指出该免疫检查点抑制剂可用于与FAK抑制剂和阿霉素联合治疗肿瘤。
本公开另一方面提供了一种治疗肿瘤的方法，该方法包括向有需要的对象施用治疗有效量的FAK抑制剂和阿霉素。
本公开又一方面提供了FAK抑制剂和阿霉素的药物组合产品，其用于在有需要的对象中治疗肿瘤。
本公开又一方面提供了FAK抑制剂和阿霉素在制备用于治疗肿瘤的联用药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与阿霉素治疗肿瘤的联用药物中的用途。
本公开又一方面提供了阿霉素在制备用于与FAK抑制剂治疗肿瘤的联用药物中的用途。
本公开又一方面提供了FAK抑制剂和阿霉素在制备用于联合治疗肿瘤的药物中的用途。
本公开又一方面提供了FAK抑制剂在制备用于与阿霉素联合治疗肿瘤的药物中的用途。
本公开又一方面提供了阿霉素在制备用于与FAK抑制剂联合治疗肿瘤 的药物中的用途。
本公开又一方面提供了一种试剂盒，其包括：FAK抑制剂；和说明书，该说明书指出该FAK抑制剂可用于与阿霉素联合治疗肿瘤。
本公开又一方面提供了一种试剂盒，其包括：阿霉素；和说明书，该说明书指出该阿霉素可用于与FAK抑制剂联合治疗肿瘤。
可选的，所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000001
所述Defactinib也称为地法替尼，CAS号为1345713-71-4；所述GSK2256098的CAS号为1224887-10-8。所述PF-00562271的CAS号为717907-75-0；所述VS-4718的CAS号为1061353-68-1；所述APG-2449为亚盛医药研发；所述AMP945的CAS号为1393653-34-3。
可选的，所述蒽环类化疗药为阿霉素、表阿霉素、或柔红霉素，优选为阿霉素。
可选的，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
可选的，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体，例如，所述抗PD-1/PD-L1抗体为帕博利珠单抗(pembrolizumab)、替雷利珠单抗(Tislelizumab)、尼伏单抗(Nivolumab)、特瑞普利单抗(Toripalimab)、阿替利珠单抗(Atezolizumab)、度伐单抗(durvalumab)、阿维单抗(Avelumab)、卡瑞 利珠单抗(Camrelizumab)、信迪利单抗(Sintilimab)、西米普利单抗(Cemiplimab)、恩沃利单抗(envafolimab)、BMS-936559、JS003、SHR-1316、GS-4224、AN-4005或者MX-10181，尤其是特瑞普利单抗。
可选的，所述免疫检查点抑制剂为PD-1/PD-L1小分子抑制剂，例如，所述PD-1/PD-L1小分子抑制剂为INCB-086550、拉泽替尼(Lazertinib)、IMMH-010、CA-170、ABSK043或者RRx-001。
可选的，所述免疫检查点抑制剂为TIGIT抑制剂，例如，所述TIGIT抑制剂为欧司珀利单抗(Ociperlimab/BGB-A1217)、维博利单抗(Vibostolimab)、domvanalimab(AB154)、替瑞利尤单抗(Tiragolumab)、Belrestotug、艾替利单抗(Etigilimab)、ONO-4686、JS-006、AZD-2936、HLX-301、SEA-TGT、M-6223、IBI-939、COM-902、AB-308、AGEN-1777、AK-127、BAT-6021、BAT-6005、ASP-8374、PM-1022、BMS-986207、HB0036或IBI-321。
可选的，所述肿瘤为霍奇金淋巴瘤、非霍奇金淋巴瘤、非小细胞肺癌、小细胞肺癌、肝细胞癌、胆管癌、骨髓增生异常综合征、急性淋巴细胞白血病、急性髓系白血病、慢性髓系白血病、甲状腺癌、神经胶质瘤、结直肠癌、卵巢癌、膀胱癌、前列腺癌、乳腺癌、脂肪肉瘤、纤维肉瘤、横纹肌肉瘤、平滑肌肉瘤、血管肉瘤、神经母细胞瘤、肾细胞癌、头颈部癌、胃癌、食管癌、胃食管结合部癌、胸腺癌、胰腺癌、子宫内膜癌、宫颈癌、黑色素瘤、葡萄球膜黑色素瘤、皮肤癌、生殖细胞癌、鼻咽癌、口咽癌、或喉癌。例如，所述肿瘤为急性髓系白血病、黑色素瘤、甲状腺癌、结直肠癌、食道癌、肝细胞癌、卵巢癌、纤维肉瘤、胃癌、非小细胞肺癌或胆管癌；进一步的为卵巢癌或结直肠癌。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本公开的一些实施例， 而非对本发明的限制。
图1显示了FAKsiRNA对FAK及磷酸化FAKY397敲除的效果。
图2显示了3μM阿霉素与50nM FAKsiRNA敲除合用对卵巢癌细胞SK-OV-3的杀伤协同作用。
图3显示了3μM阿霉素与50nM FAKsiRNA合用48小时后进行DAMPs的免疫荧光染色。
图4显示了IN10018及阿霉素对卵巢癌细胞SK-OV-3的72小时杀伤曲线及IC50值。
图5显示了不同剂量下阿霉素与3μM IN10018合用48小时对卵巢癌细胞SK-OV-3的杀伤协同作用。
图6显示了0.3或1.0μM阿霉素与3μM IN10018合用48小时后采用Annexin V试剂盒染色的结果。
图7显示了0.3μM阿霉素与3μM IN10018合用48小时后进行DAMPs的免疫荧光染色结果。
图8显示了0.3μM Daunorubicin或Epirubicin与3μM IN10018合用48小时后进行DAMPs的免疫荧光染色结果。
图9显示了不同剂量下阿霉素与3μM Defactinib合用72小时对卵巢癌细胞SK-OV-3的杀伤协同作用。
图10显示了1μM阿霉素与3μM Defactinib合用48小时后进行DAMPs的免疫荧光染色结果。
图11显示了细胞与药物孵育48小时后，拍摄细胞的白光显微镜照片。
图12显示了与IN10018和阿霉素孵育48小时后CRT阳性和Annexin V阳性CT26细胞的百分比，其中图12a显示了与药物孵育48小时后CRT阳性CT26细胞的百分比，图12b显示了与药物孵育48小时后Annexin V阳性CT26细胞的百分比。
图13显示了小鼠结直肠癌CT26的BALB/c小鼠同种移植瘤模型中荷瘤 鼠在给予不同受试物后的肿瘤生长的变化。数据点代表组内平均值和标准误,n＝8。
图14显示了给予不同剂量药物后小鼠体重变化情况及给予不同剂量药物后小鼠体重变化。数据点代表组内平均值±标准误差，n＝8。
图15显示了小鼠结直肠癌CT26的BALB/c小鼠同种移植瘤模型中荷瘤鼠在给予不同受试物后的肿瘤生长的变化。数据点代表组内平均值和标准误,n＝8。
图16显示了给予不同剂量药物后小鼠体重变化情况及给予不同剂量药物后小鼠体重变化。数据点代表组内平均值±标准误差，n＝8。
图17显示了CT26小鼠结肠癌细胞BABL/c小鼠皮下同种移植瘤模型给药7天后，眼眶采血分离血清检测的IFN-γ蛋白浓度。
图18显示了CT26小鼠结肠癌细胞BABL/c小鼠皮下同种移植瘤模型给药7天后，眼眶采血分离血清检测的Granzyme B蛋白浓度。
图19显示了与阿霉素和AMP945孵育48小时后CRT阳性和Annexin V阳性4T1细胞的百分比，其中图19a显示了与药物孵育48小时后CRT阳性4T1细胞的百分比，图19b显示了与药物孵育48小时后Annexin V阳性4T1细胞的百分比。
图20显示了与阿霉素和AMP945孵育48小时后CRT阳性和Annexin V阳性CT26细胞的百分比，其中图20a显示了与药物孵育48小时后CRT阳性CT26细胞的百分比，图20b显示了与药物孵育48小时后Annexin V阳性CT26细胞的百分比。
图21描绘了IN10018联合PLD和抗PD-1治疗卵巢癌的临床研究中靶病灶总径相对于基线最佳改变(％)的瀑布图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公 开实施例的附图，对本公开实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于所描述的本公开的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明可在不偏离本发明基本属性的情况下以其它具体形式来实施。应该理解的是，在不冲突的前提下，本发明的任一和所有实施方案都可与任一其它实施方案或多个其它实施方案中的技术特征进行组合以得到另外的实施方案。本发明包括这样的组合得到的另外的实施方案。
本公开中提及的所有出版物和专利在此通过引用以它们的全部内容纳入本公开。如果通过引用纳入的任何出版物和专利中使用的用途或术语与本公开中使用的用途或术语冲突，那么以本公开的用途和术语为准。
本文所用的章节标题仅用于组织文章的目的，而不应被解释为对所述主题的限制。
除非另有规定，本文使用的所有技术术语和科学术语具有要求保护主题所属领域的通常含义。倘若对于某术语存在多个定义，则以本文定义为准。
除了在工作实施例中或另外指出之外，在说明书和权利要求中陈述的定量性质例如剂量的所有数字应理解为在所有情况中被术语“约”修饰。还应理解的是，本申请列举的任何数字范围意在包括该范围内的所有的子范围和该范围或子范围的各个端点的任何组合。
本公开中使用的“包括”、“含有”或者“包含”等类似的词语意指出现该词前面的要素涵盖出现在该词后面列举的要素及其等同，而不排除未记载的要素。本文所用的术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…组成”、或“由…组成”。
定义
本申请中所用的下列术语和符号具有如下所述的含义，其所处的上下文 中另有说明的除外。本文所用的术语“FAK抑制剂”是指FAK的有效抑制剂，可适于哺乳动物，特别是人。在一些实施方案中，所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000002
所述Defactinib也称为地法替尼，CAS号为1345713-71-4；所述GSK2256098的CAS号为1224887-10-8。所述PF-00562271的CAS号为717907-75-0；所述VS-4718的CAS号为1061353-68-1；所述APG-2449为亚盛医药研发；所述AMP945的CAS号为1393653-34-3。在一些实施方案中，所述FAK抑制剂优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，在一些优选实施方案中，所述FAK抑制剂为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐。
本文所用的术语“蒽环类药物”是指一类来源于波赛链霉菌青灰变种的化疗药物。在一些实施方案中，蒽环类药物为阿霉素、表阿霉素、柔红霉素或阿克拉霉素等。在一些优选实施方案中，蒽环类药物为阿霉素。
本文所用的术语“阿霉素”是多柔比星(Doxorubicin)，可以理解为虽然本发明的技术方法仅提及阿霉素，但其实多柔比星或其不同的盐或制剂均被保护。例如PLD，它是指盐酸多柔比星脂质体注射液(doxil)，是盐酸阿霉素脂质体注射剂，其有效成分为阿霉素。
本文所用的术语“免疫检查点抑制剂”是指能够通过调控免疫检查点通路(例如PD-1、TIGIT、CTLA-4、LAG-3、TIM-3等)来提高免疫系统活性的药物。在一些实施方案中，免疫检查点抑制剂为PD-1/PD-L1(程序性细胞死亡蛋白1)通路的拮抗剂(也称为“PD-1抑制剂”)或TIGIT抑制剂。PD-1抑 制剂在本公开中也称为PD-1/PD-L1抑制剂。例如，在本公开的治疗方法、药物和用途中所述PD-1/PD-L1抑制剂为抗PD-1/PD-L1抗体，包括但不限于帕博利珠单抗(可瑞达/Keytruda/K药)、替雷利珠单抗(Tislelizumab/百泽安)、尼伏单抗(Nivolumab)、特瑞普利单抗(Toripalimab/拓益)、度伐单抗(英飞凡/度伐鲁单抗/durvalumab)、阿维单抗(Avelumab/Bavencio)、阿替利珠单抗(MPDL3280A/Atezolizumab/Tecentriq/T药)、BMS-936559(全人源抗PD-L1的IgG4单克隆抗体)、GS-4224、AN-4005或者MX-10181，尤其是特瑞普利单抗。在一些优选的实施方案中，PD-1抑制剂为特瑞普利单抗。在一些实施方案中，PD-1抑制剂用于治疗人对象。在一些实施方案中，PD-1为人PD-1。PD-1/PD-L1抑制剂也包括PD-1/PD-L1小分子抑制剂，例如INCB-086550、拉泽替尼(Lazertinib)、IMMH-010、CA-170、ABSK043或者RRx-001。TIGIT(也称为WUCAM、Vstm3、VSIG9)是Ig超家族的一种受体，是继PD-1/PD-L1之后的新型免疫检查点。例如，在本公开的治疗方法、药物和用途中，所述免疫检查点抑制剂为TIGIT抑制剂(例如TIGIT抗体)，包括但不限于欧司珀利单抗(Ociperlimab/BGB-A1217)、维博利单抗(Vibostolimab)、domvanalimab(AB154)、替瑞利尤单抗(Tiragolumab)、Belrestotug、艾替利单抗(Etigilimab)、ONO-4686、JS-006、AZD-2936、HLX-301、SEA-TGT、M-6223、IBI-939、COM-902、AB-308、AGEN-1777、AK-127、BAT-6021、BAT-6005、ASP-8374、PM-1022、BMS-986207、HB0036或IBI-321。在一些实施方案中，TIGIT抑制剂用于治疗人对象。为了避免歧义，本文中抗体均包括双抗。
本文所用的“药物组合”或者“药物组合产品”既可以指采用一个剂量单位形式的固定组合(例如所有药物活性成分以一种剂型存在)或者成套药盒以组合施用的产品的情形，也可以指一种药物与指示该药物可与另外的一种或多种药物联合使用的说明书的组合情形。
本文所用的“联合治疗”或者“联用药物”是指一种药物与另外的一种或多种药物联合使用来治疗疾病，既包括一种药物与另外的一种或多种药物的组 合的情形，也包括一种药物与指示该药物可与另外的一种或多种药物联合使用的说明书的组合情形。
“同时或依次施用”在本申请中是指一个给药周期内(例如4周内、3周内、2周内、1周内或24小时以内)两种以上的药物同时或以一定时间间隔先后施用，药物施用的方式(例如口服、静脉、肌肉或皮下施用等)可以相同或不同，两种以上的药物的给药频率/周期可以相同或不同。当本公开的治疗方法、产品或用途涉及两种药物时，两种药物可同时或以一定时间间隔分别单独施用。当本公开的治疗方法、产品或用途涉及三种药物时，三种药物可以在同一时间点施用，或者两种药物在一个时间点施用而剩下一种药物在另一个时间点施用，或者所有三种药物各自在不同时间点施用。
在一些实施方案中，PD-1/PD-L1抑制剂被静脉(例如，作为静脉输注)或皮下施用或口服。优选地，PD-1/PD-L1抑制剂以静脉输注施用。
免疫检查点抑制剂治疗癌症的能力取决于肿瘤组织内肿瘤抗原特异性T细胞的存在。这要求肿瘤组织表达将自身与其非转化的对应物区分开来的抗原，例如，通过被称为新抗原(neoantigen)的新型蛋白产物。肿瘤新抗原负荷与免疫原性和敏感性(例如，对检查点抑制剂疗法的敏感性)密切相关，这意味着免疫原性较差的肿瘤应该在很大程度上对这些药物耐药。用于释放可被APC摄取的肿瘤抗原的疗法，如诱导免疫原性细胞死亡(ICD)的那些，可能会促进有效的抗肿瘤免疫，特别是当进一步与检查点抑制剂联合时。
本文所用的术语“治疗”是指给患有疾病或者具有所述疾病的症状的对象施用一种或多种药物物质，用以治愈、缓解、减轻、改变、医治、改善、改进或影响所述疾病或者所述疾病的症状。在一些实施方案中，所述疾病是肿瘤或者癌症。
本文所用的术语“肿瘤”是指机体在各种致瘤因素的作用下，局部组织的细胞在基因水平上失去对其生长的正常调控，从而导致其克隆型异常增生而形成的异常病变。实施例包括，但不限于：霍奇金淋巴瘤，非霍奇金淋巴 瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
本文所用的术语“对象”或“受试者”是指哺乳动物和非哺乳动物。哺乳动物是指哺乳类的任何成员，其包括但不限于：人；非人灵长类动物，如黑猩猩及其它猿类和猴类物种；农场动物，如牛、马、绵羊、山羊和猪；家畜，如兔、狗和猫；实验室动物，包括啮齿类动物，如大鼠、小鼠和豚鼠；等等。非哺乳动物的例子包括但不限于鸟等。术语“对象”并不限定特定的年龄或性别。在一些实施方案中，对象是人。
本文所用的术语“药学上可接受的”指的是无毒的、生物学上可耐受的，适合给对象施用的。
本文所用的术语“药学上可接受的盐”指的是无毒的、生物学上可耐受的适合给对象施用的酸加成盐，包括但不限于：与无机酸形成的酸加成盐，例如盐酸盐、氢溴酸盐、碳酸盐、碳酸氢盐、磷酸盐、硫酸盐、亚硫酸盐、硝酸盐等；以及与有机酸形成的酸加成盐，例如甲酸盐、乙酸盐、苹果酸盐、马来酸盐、富马酸盐、酒石酸盐、琥珀酸盐、柠檬酸盐、乳酸盐、甲磺酸盐、对甲苯磺酸盐、2-羟基乙磺酸盐、苯甲酸盐、水杨酸盐、硬脂酸盐和与式HOOC-(CH 2) n-COOH(其中n是0-4)的链烷二羧酸形成的盐等。
此外，药学上可接受的酸加成盐可以按照由碱性化合物制备酸加成盐的常规操作通过将游离碱溶于合适的溶剂并且用酸处理该溶液来得到。本领域 技术人员无需过多实验即可确定各种可用来制备无毒的药学上可接受的酸加成盐的合成方法。
本文所用的术语“药学上可接受的组合物”是指必须在化学和/或毒理学上与包括制剂的其他成分相容，和/或与接受其治疗的对象相容。本文所用的术语“治疗有效量”是指通常足以对对象产生有益治疗效果的量。可以通过常规方法(例如建模、剂量递增研究或临床试验)结合常规影响因素(例如给药方式、化合物的药代动力学、疾病的严重程度和病程、对象的病史、对象的健康状况、对象对药物的响应程度等)来确定本发明的治疗有效量。
本文所用的术语“抑制”是指生物活动或过程的基线活性的降低。
本文所用的术语“试剂盒”是指用于盛放检测化学成分、药物残留、病毒种类等化学试剂的盒子。本发明所述试剂盒可以是包括(i)FAK抑制剂、蒽环类化疗药(例如阿霉素)和免疫检查点抑制剂中的一种、二种或三种；以及(ii)说明书，所述说明书指出可使用FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂来在对象中治疗肿瘤。在一种实施方案中，试剂盒包括(i)FAK抑制剂；以及(ii)说明书，所述说明书指出可使用FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂来在对象中治疗肿瘤。在一种实施方案中，试剂盒包括(i)蒽环类化疗药；以及(ii)说明书，所述说明书指出可使用FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂来在对象中治疗肿瘤。在一种实施方案中，试剂盒包括(i)免疫检查点抑制剂；以及(ii)说明书，所述说明书指出可使用FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂来在对象中治疗肿瘤。在一种实施方案中，试剂盒包括(i)FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂；以及(ii)说明书，所述说明书指出可使用FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂来在对象中治疗肿瘤。在一种实施方案中，试剂盒包括(i)FAK抑制剂；以及(ii)说明书，所述说明书指出可使用FAK抑制剂和蒽环类化疗药来在对象中治疗肿瘤。在一种实施方案中，试剂盒包括(i)蒽环类化疗药；以及(ii)说明书，所述说明书指出可使用蒽环类化疗药和FAK抑制剂在对象中 治疗肿瘤。
试剂盒的化合物可以包含在分开的容器中。可选地，两种或更多种化合物包含在同一容器中。例如，试剂盒可以包括第一容器、第二容器、第三容器和包装插页，其中第一容器包括至少一个剂量的包括FAK抑制剂的药物，第二容器包括至少一个剂量的蒽环类化疗药(例如阿霉素)，第三容器包括至少一个剂量的免疫检查点抑制剂的药物，且所述包装插页包括使用药物治疗对象的肿瘤的说明。第一容器、第二容器和第三容器可以包含相同或不同形状(例如，小瓶、注射器和瓶)和/或材料(例如，塑料或玻璃)。试剂盒还可以包括可以有助于施用药物的其他材料，如稀释剂、过滤器、IV袋和管线、针和注射器。
给予受试者的FAK抑制剂、蒽环类药物、或免疫检查点抑制剂的精确量将取决于各种因素，例如给定的药物或化合物，药物制剂，给药途径，疾病类型，病症，所治疗的受试者或宿主的身份等，但是仍然可以由本领域技术人员常规确定。例如，确定有效量还取决于细胞增殖的程度，严重性和类型。技术人员将能够根据这些和其他因素确定合适的剂量。
FAK抑制剂、蒽环类药物、或免疫检查点抑制剂可选择合适的方式例如口服、静脉、肌肉或皮下施用给药。
例如，口服给药时，可以将药物与药学上可接受的载体例如惰性稀释剂或可吸收的食用载体一起口服给药。它们可以封装在硬壳或软壳明胶胶囊中，可以压制成片剂，或者可以直接与患者的食物混合。例如，药物可以与一种或多种赋形剂组合，并以可摄取的片剂，口腔片剂，锭剂，胶囊，酏剂，悬浮液，糖浆或糯米纸囊剂等形式使用。片剂，锭剂，丸剂，胶囊剂等可进一步包括：粘合剂，例如黄芪胶，阿拉伯胶，玉米淀粉或明胶；赋形剂，如磷酸二钙；崩解剂，例如玉米淀粉，马铃薯淀粉，海藻酸等；润滑剂，例如硬脂酸镁；或甜味剂，例如蔗糖，果糖，乳糖或阿斯巴甜；或调味剂。
例如，输注或注射静脉内或腹膜内给药时，药物的溶液可以在水中制备， 任选地与无毒的表面活性剂混合。
用于注射或输注的示例性药物剂型包括：无菌水溶液，分散液，或包含活性成分的无菌粉末，该无菌粉末适合于临时制备无菌注射或输注溶液或分散液。无论如何，最终剂型在生产和储存条件下均应无菌，流动且稳定。
无菌注射溶液可以通过将所需量的药物与所需的上述各种其他成分掺入适当的溶剂中，然后过滤灭菌来制备。对于用于制备无菌注射溶液的无菌粉末，优选的制备方法可以是真空干燥和冷冻干燥技术，其可以产生活性成分加上先前无菌过滤后存在的任何其他所需成分的粉末。
用于治疗所需的FAK抑制剂、蒽环类药物、或免疫检查点抑制剂的量不仅可以随所选择的特定试剂而变化，还可以随给药途径，所治疗疾病的性质以及患者的年龄和状况而变化，并且最终可以由主治医师或临床医生自行决定。然而，一般而言，剂量可以在每天约0.1至约50mg/kg体重的范围内。
所述FAK抑制剂以成年人中5mg/天-300mg/天的剂量范围施用。在一种具体的实施方案中，IN10018或其药学上可接受盐以成年人中5mg/天-100mg/天的剂量施用，所述剂量以游离碱计。
所述蒽环类药物以成年人中每周1-40mg/m 2的剂量范围施用。在一种具体的实施方式中，阿霉素或其药学上可接受盐以成年人中每周1-40mg/m 2的剂量施用，所述剂量以阿霉素计。
所述免疫检查点抑制剂每次给药以成年人中2-10mg/kg或者50-1200mg的剂量施用，每2周到3周给药一次。在一种具体的实施方式中，所述免疫检查点抑制剂每次给药以成年人中3-10mg/kg或者100-1200mg的剂量施用，每2周到3周给药一次。
本文所用的未具体定义的技术和科学术语具有本发明所属领域的技术人员通常理解的含义。
在一些实施方案中，本公开还公开了以下：
1.FAK抑制剂、蒽环类药物和免疫检查点抑制剂，其用于在对象中治疗 肿瘤的方法，其中所述FAK抑制剂、所述蒽环类药物和所述免疫检查点抑制剂被同时或依次施用于所述对象。
2.如实施方案1所述的用于所述用途的化合物，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000003
3.如实施方案1或2所述的用于所述用途的化合物，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
4.如实施方案1-3任一项所述的用于所述用途的化合物，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
5.如实施方案1-4任一项所述的用于所述用途的化合物，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
6.如实施方案1-5任一项所述的用于所述用途的化合物，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸 腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
7.一种试剂盒或药学上可接受的组合物，其包括：
(a)FAK抑制剂；
(b)蒽环类药物；和
(c)免疫检查点抑制剂。
8.如实施方案7所述的试剂盒或组合物，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000004
9.如实施方案7或8所述的试剂盒或组合物，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
10.如实施方案7-9任一项所述的试剂盒或组合物，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
11.如实施方案7-10任一项所述的试剂盒或组合物，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为PD-1/PD-L1抑制剂，尤其是特瑞普利单抗。
12.如实施方案7-11任一项所述的试剂盒或组合物，其用作药物。
13.如实施方案7-12任一项所述的试剂盒或组合物，其用于治疗肿瘤。
14.如实施方案13所述的试剂盒或组合物，所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
15.一种在对象中治疗肿瘤的方法，其中所述方法包括将根据实施方案7-12任一项所述的试剂盒或组合物的化合物同时或依次施用至所述对象。
16.如实施方案15所述的方法，所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
17.FAK抑制剂，其用于增强蒽环类药物诱导的免疫原性细胞死亡。
18.如实施方案17所述用途的FAK抑制剂，所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
19.如实施方案17或18所述用途的FAK抑制剂，其为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000005
20.如实施方案17-19任一项所述用途的FAK抑制剂，其中所述FAK抑制剂和所述蒽环类药物同时或依次施用。
21.一种在对象中治疗肿瘤的方法，其中所述方法包括向所述对象同时或依次施用治疗有效量的FAK抑制剂、蒽环类药物和免疫检查点抑制剂。
22.如实施方案21所述的方法，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000006
23.如实施方案21或22所述的方法，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
24.如实施方案21-23任一项所述的方法，其中所述免疫检查点抑制剂 为PD-1/PD-L1抑制剂。
25.如实施方案21-24任一项所述的方法，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为PD-1/PD-L1抑制剂，尤其是特瑞普利单抗。
26.如实施方案21-25任一项所述的方法，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
27.FAK抑制剂、蒽环类药物和免疫检查点抑制剂，其用于通过在对象中增加免疫原性细胞死亡来治疗肿瘤的方法，其中所述FAK抑制剂、所述蒽环类药物和所述免疫检查点抑制剂被同时或依次施用于所述对象。
28.如实施方案27所述的用于所述用途的化合物，其中所述FAK抑制剂为IN10018、defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000007
29.如实施方案27或28所述的方法，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
30.如实施方案27-29任一项所述的用于所述用途的化合物，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
31.如实施方案27-30任一项所述的用于所述用途的化合物，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂。
32.如实施方案27-31任一项所述的用于所述用途的化合物，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
33.一种通过在对象中增加免疫原性细胞死亡来治疗肿瘤的方法，其中所述方法包括向所述对象同时或依次施用治疗有效量的FAK抑制剂、蒽环类药物和免疫检查点抑制剂。
34.如实施方案33所述的方法，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000008
35.如实施方案33或34所述的方法，其中蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
36.如实施方案33-35任一项所述的方法，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
37.如实施方案33-36任一项所述的方法，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
38.如实施方案33-37任一项所述的方法，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
39.FAK抑制剂在制备用于在对象中治疗肿瘤的药物中的用途，其中将所述FAK抑制剂、蒽环类药物和免疫检查点抑制剂同时或依次施用于所述对象。
40.如实施方案39所述的用途，其中所述FAK抑制剂为IN10018、 Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000009
41.如实施方案39或40所述的用途，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
42.如实施方案39-41任一项所述的用途，其中所述免疫检查点抑制剂为PD-1/PD-L1抑制剂。
43.如实施方案39-42任一项所述的用途，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为PD-1/PD-L1抑制剂，尤其是特瑞普利单抗。
44.如实施方案39-43任一项所述的用途，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
45.蒽环类药物在制备用于在对象中治疗肿瘤的药物中的用途，其中将 FAK抑制剂，所述蒽环类药物和免疫检查点抑制剂同时或依次施用于所述对象。
46.如实施方案45所述的用途，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
47.如实施方案45或46所述的用途，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000010
48.如实施方案45-47任一项所述的用途，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
49.如实施方案45-48任一项所述的用途，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
50.如实施方案45-49任一项所述的用途，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖 细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
51.免疫检查点抑制剂在制备用于在对象中治疗肿瘤的药物中的用途，其中将FAK抑制剂、蒽环类药物和所述免疫检查点抑制剂同时或依次施用于所述对象。
52.FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂在制备用于治疗肿瘤的联用药物中的用途。
53.FAK抑制剂在制备用于与蒽环类化疗药和免疫检查点抑制剂治疗肿瘤的联用药物中的用途。
54.蒽环类化疗药在制备用于与FAK抑制剂和免疫检查点抑制剂治疗肿瘤的联用药物中的用途。
55.免疫检查点抑制剂在制备用于与FAK抑制剂和蒽环类化疗药治疗肿瘤的联用药物中的用途。
56.FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂在制备用于联合治疗肿瘤的药物中的用途。
57.FAK抑制剂在制备用于与蒽环类化疗药和免疫检查点抑制剂联合治疗肿瘤的药物中的用途。
58.蒽环类化疗药在制备用于与FAK抑制剂和免疫检查点抑制剂联合治疗肿瘤的药物中的用途。
59.免疫检查点抑制剂在制备用于与FAK抑制剂和蒽环类化疗药联合治疗肿瘤的药物中的用途。
60.如实施方案51-59任一项所述的用途，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受 的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
Figure PCTCN2022137545-appb-000011
61.如实施方案51-60任一项所述的用途，其中所述蒽环类药物为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
62.如实施方案51-61任一项所述的用途，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
63.如实施方案51-62任一项所述的用途，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类药物为阿霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
64.如实施方案51-63任一项所述的用途，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结直肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结直肠癌。
实施例
提供下面的实施例以进一步阐述本发明。应理解，这些实施例仅用于举 例说明本发明，而不用于限制本发明的范围。
下列实施例中未注明具体条件的实验方法均可以按照这类反应的常规条件进行或者按照制造厂商所建议的条件进行。
以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例中所用的缩写含义如下：
Figure PCTCN2022137545-appb-000012
Figure PCTCN2022137545-appb-000013
实施例1：FAK敲除增强卵巢癌细胞针对阿霉素免疫原性细胞死亡的研究
实验方案：
1)使用FAKsiRNA进行FAK敲除并检测敲除效率
使用SK-OV-3细胞作为研究对象，应用Lipofectamine 3000转染50nM FAKsiRNA，实验结束后收取蛋白进行FAK及磷酸化FAKY397表达检测。
2)FAK敲除联合阿霉素产生协同肿瘤杀伤作用
使用体外实验检测50nM FAKsiRNA敲除作用及3μM阿霉素在卵巢癌细胞SK-OV-3的细胞杀伤作用。48小时后对细胞死亡进行拍照。
3)FAK敲除联合阿霉素能够有效诱导卵巢癌细胞SK-OV-3的ICD作用
体外实验检测FAK敲除与阿霉素48小时合用后是否能有效提升细胞释放ICD的主要靶标Calreticulin(钙网蛋白)及Annexin A1(膜联蛋白A1)。
实验材料：
本实验所用药物：
FAKsiRNA由吉玛基因提供，序列如下表所示。阿霉素由MCE提供。
FAKsiRNA：
Figure PCTCN2022137545-appb-000014
本实验所用抗体：
重组Alexa
Figure PCTCN2022137545-appb-000015
488荧光Anti-HMGB1抗体(Abcam，ab195010)。重组Alexa
Figure PCTCN2022137545-appb-000016
647荧光Anti-Calreticulin抗体(Abcam，ab196159)。Annexin A1 rabbit antibody(Cell signaling technology，32934S)。Anti-rabbit IgG(H+L),F(ab')2 Fragment(Alexa
Figure PCTCN2022137545-appb-000017
488 Conjugate)(Cell signaling technology，4412)。FAK抗体(CST，3285S)。Phospho FAKY397抗体(Thermo fisher，44-624G)。HRP标记Alpha tubulin(α-微管蛋白)抗体(Proteintech，HRP-66031)
实验仪器：
荧光显微镜(Olympus U-HGLGPS)。化学发光成像仪(BIORAD chemidoc touch)
实验结果：
1)使用FAKsiRNA进行FAK敲除并检测敲除效率
使用50nM FAKsiRNA转染SK-OV-3细胞。转染试剂使用Lipofectamine 3000。转染后24小时即可进行下游合用及ICD生物标志物实验。转染后72小时对SK-OV-3细胞蛋白进行收集并进行western blot(蛋白印迹)检测磷酸化FAKY397,FAK及Alpha tubulin的量。转染效果以蛋白敲除情况作为标准，如图1所示。FAKsiRNA对于磷酸化FAKY397及总FAK蛋白的敲除作用非常显著。
2)FAK敲除联合阿霉素产生协同肿瘤杀伤作用
50nM FAKsiRNA转染SK-OV-3细胞24小时后加入3μM阿霉素。共 同培养48小时后对细胞进行拍照并发现FAK敲除联合阿霉素产生非常显著的肿瘤细胞协同杀伤作用。结果如图2所示。
3)PLD与FAK敲除合用显著增强SK-OV-3的ICD靶标Calreticulin，Annexin A1的释放及暴露
3μM阿霉素及50nM FAKsiRNA合用治疗SK-OV-3细胞，48小时后使用Hoechst 33342，Calreticulin，Annexin A1抗体进行荧光染色。结果如图3所示，显示合用组与单药组比较，Calreticulin及Annexin A1的释放及暴露均被显著的增强了。
实验结论：
FAK敲除能够显著增强阿霉素对卵巢癌细胞SK-OV-3的细胞杀伤作用。检测ICD标志物的实验中发现，FAK敲除能够显著增强DAMPs标志物Calreticulin及Annexin A1的暴露及释放，提示FAK敲除能够有效增强卵巢癌细胞产生免疫原性细胞死亡。
实施例2：IN10018增强卵巢癌细胞针对阿霉素免疫原性细胞死亡的研究
实验方案：
1)IN10018联合阿霉素产生协同肿瘤杀伤作用
使用体外实验检测IN10018及阿霉素在卵巢癌细胞SK-OV-3的细胞杀伤曲线并确定IC50值。探索不同剂量阿霉素与3μM IN10018合用的细胞毒作用。采用Annexin V法检测药物单用与合用细胞凋亡反应。学生T检验被用于统计学研究中，****意为P值小于0.001。
2)IN10018联合阿霉素能够有效诱导卵巢癌细胞SK-OV-3的ICD作用
体外实验检测IN10018与阿霉素48小时合用后是否能有效提升细胞释放ICD的主要靶标Calreticulin，HMGB1及Annexin A1。
实验材料：
1)本实验所用药物
IN10018按照专利WO2010058032中的方法合成。阿霉素由MCE提供。
2)本实验所用抗体
重组Alexa
Figure PCTCN2022137545-appb-000018
488荧光Anti-HMGB1抗体(Abcam，ab195010)。重组Alexa
Figure PCTCN2022137545-appb-000019
647荧光Anti-Calreticulin抗体(Abcam，ab196159)。Annexin A1rabbit antibody(Cell signaling technology，32934S)。Anti-rabbit IgG(H+L),F(ab')2Fragment(Alexa
Figure PCTCN2022137545-appb-000020
488Conjugate)(Cell signaling technology，4412)。
3)本实验所用试剂盒
Annexin V细胞凋亡试剂盒(Abbkine，KTA0002)。CCK8细胞活力试剂(Cellor lab，CX001)。
实验仪器：
酶标仪(Molecular devices M3)。荧光显微镜(Olympus U-HGLGPS)。流式细胞仪(BD LSRFortessa X-20)
实验结果：
1)IN10018与阿霉素单药对SK-OV-3的IC50曲线
以5000个SK-OV-3细胞/孔铺设96孔细胞板，24小时后加入最高浓度为20μM的药物以3倍浓度梯度稀释共设9个浓度，最低浓度为0.3nM。药物与细胞共同孵育72小时后每孔加入10μL CCK8溶液，于37度5％二氧化碳孵箱孵育2小时后，使用酶标仪设定吸光度波长为450nm进行读值。读取的数值与DMSO对照组进行比较并使用Graphpad 8.0进行绘图。IN10018的IC50为10(μM)，阿霉素的IC50为0.3(μM)，结果见图4。
2)不同剂量下阿霉素与3μM IN10018合用对SK-OV-3协同杀伤作用
以5000个SK-OV-3细胞/孔铺设96孔细胞板，24小时后以阿霉素的IC50 值作为初始点在其左侧及右侧各设置3个及2个亚剂量，这些剂量下阿霉素与3μM IN10018进行合用。72小时后使用CCK8法检测细胞活力。使用Graphpad 8.0进行图形绘制。结果显示IN10018能够显著增强不同剂量的阿霉素对卵巢癌细胞SK-OV-3的细胞杀伤作用(见图5)。
3)阿霉素与IN10018合用显著增强SK-OV-3的早期及晚期凋亡
使用0.3μM或1μM阿霉素与3μM IN10018联合治疗SK-OV-3细胞，48小时后收取细胞使用Annexin V试剂盒进行细胞染色并使用流式细胞仪进行检测。其中Q2象限显示的是晚期凋亡，Q3象限显示的是早期凋亡。结果显示合用组与单药组比较，早期及晚期凋亡被显著的增强了(见图6)。
4)阿霉素与IN10018合用显著增强SK-OV-3的ICD靶点Calreticulin、HMGB1及Annexin A1的释放及暴露
0.3μM阿霉素及3μM IN10018合用治疗SK-OV-3细胞，48小时后使用Calreticulin、HMGB1及Annexin A1抗体进行荧光染色。结果显示合用组与单药组比较，Calreticulin、HMGB1及Annexin A1的释放及暴露均被显著的增强了(见图7)。
实验结论：
IN10018能够显著增强阿霉素对卵巢癌细胞SK-OV-3的细胞杀伤作用。该作用与其增强早期及晚期凋亡有关。检测ICD标志物的实验中发现，IN10018能够显著增强DAMPs标志物Calreticulin、HMGB1及Annexin A1的暴露及释放，提示IN10018能够有效增强卵巢癌细胞产生免疫原性细胞死亡。
实施例3：IN10018增强卵巢癌细胞针对daunorubicin(柔红霉素)和epirubicin(表阿霉素)免疫原性细胞死亡的研究
实验方案：
IN10018联合Daunorubicin或Epirubicin能够有效诱导卵巢癌细胞SK-OV-3的ICD作用
体外实验检测IN10018与Daunorubicin或Epirubicin 48小时合用后是否能有效提升细胞释放ICD的主要靶标Calreticulin。
实验材料：
1)本实验所用药物
IN10018按照专利WO2010058032中的方法合成。Daunorubicin由MCE提供。Epirubicin由MCE提供。
2)本实验所用抗体
荧光Anti-Calreticulin抗体(Abcam，ab196159)。
实验仪器：
荧光显微镜(Olympus U-HGLGPS)。
实验结果：
Daunorubicin或Epirubicin与IN10018合用显著增强SK-OV-3的ICD靶点Calreticulin的释放及暴露。
0.3μM Daunorubicin或Epirubicin及3μM IN10018合用治疗SK-OV-3细胞，48小时后使用Calreticulin抗体进行荧光染色。结果显示合用组与单药组比较，Calreticulin的释放及暴露均被显著的增强了(见图8)。
实验结论：
检测ICD标志物的实验中发现，IN10018能够显著增强DAMPs标志物Calreticulin的暴露及释放，提示IN10018能够有效增强Daunorubicin或Epirubicin产生免疫原性细胞死亡。
实施例4：Defactinib增强卵巢癌细胞针对阿霉素免疫原性细胞死亡的研究
实验方案：
1)Defactinib联合阿霉素产生协同肿瘤杀伤作用
探索不同剂量阿霉素与3μM Defactinib合用的细胞毒作用。
2)Defactinib联合阿霉素能够有效诱导卵巢癌细胞SK-OV-3的ICD作用
体外实验检测Defactinib与阿霉素48小时合用后是否能有效提升细胞释放ICD的主要靶标Calreticulin，HMGB1及Annexin A1。
实验材料：
1)本实验所用药物
Defactinib由DC chemicels提供。阿霉素由MCE提供。
2)本实验所用抗体
重组Alexa
Figure PCTCN2022137545-appb-000021
488荧光Anti-HMGB1抗体(Abcam，ab195010)。重组Alexa
Figure PCTCN2022137545-appb-000022
647荧光Anti-Calreticulin抗体(Abcam，ab196159)。Annexin A1 rabbit antibody(Cell signaling technology，32934S)。Anti-rabbit IgG(H+L),F(ab')2 Fragment(Alexa
Figure PCTCN2022137545-appb-000023
488 Conjugate)(Cell signaling technology，4412)。
3)本实验所用试剂盒
CCK8细胞活力试剂(Cellor lab，CX001)。
实验仪器：
酶标仪(Molecular devices M3)。荧光显微镜(Olympus U-HGLGPS)。
实验结果：
1)不同剂量下阿霉素与3μM Defactinib合用对SK-OV-3协同杀伤作用
以5000个SK-OV-3细胞/孔铺设96孔细胞板，24小时后以阿霉素的IC50 值作为初始点在其左侧及右侧各设置3个及2个亚剂量，这些剂量下阿霉素与3μM Defactinib进行合用。72小时后使用CCK8法检测细胞活力。使用Graphpad 8.0进行图形绘制。结果显示Defactinib能够显著增强不同剂量的阿霉素对卵巢癌细胞SK-OV-3的细胞杀伤作用(见图9)。学生T检验被用于统计学研究中，***代表P值小于0.005，****代表P值小于0.001。
2)阿霉素与Defactinib合用显著增强SK-OV-3的ICD靶点Calreticulin，HMGB1及Annexin A1的释放及暴露1μM阿霉素及3μM Defactinib合用治疗SK-OV-3细胞，48小时后使用Calreticulin，HMGB1及Annexin A1抗体进行荧光染色。结果显示合用组与单药组比较，Calreticulin，HMGB1及Annexin A1的释放及暴露均被显著的增强了(见图10)。
实验结论：
Defactinib能够显著增强阿霉素对卵巢癌细胞SK-OV-3的细胞杀伤作用。检测ICD标志物的实验中发现，Defactinib能够显著增强DAMPs标志物Calreticulin，HMGB1及Annexin A1的暴露及释放，提示Defactinib能够有效增强卵巢癌细胞产生免疫原性细胞死亡。
实施例5：IN10018和阿霉素在结肠癌CT26细胞系中的药效研究
实验材料：
1)本实验所用药物
阿霉素由MCE提供,Cat No.:HY-15142,Lot No.:97451
2)本实验所用抗体
重组Alexa
Figure PCTCN2022137545-appb-000024
647荧光Anti-Calreticulin抗体(Abcam，Cat No.:ab196159,Lot No.:CR33676773)。Annexin-Modulation Assay Kit(Beyotime,Cat No.:C1062L,Lot No.:021921210811)。
实验方法：
CT26细胞用RPMI 1640(上海元培,Cat No.:L210KJ,Lot No.:F210916)+10％FBS(Gibco,Cat No.:10099-141c,Lot No.:2158737cp)培养，传代2次，细胞状态良好时，将培养板放置在24孔板中。细胞铺展24小时后，设立四组，第一组为对照组，添加培养基，第二组为IN10018，浓度为5μM，第三组为阿霉素(阿霉素，MCE)，浓度为1uM，第四组为IN10018(5μM)与阿霉素(1μM)联用。将药物混合，并在37℃下在5％CO 2培养箱中培养48小时。
实验结果
药物作用48h后，用显微镜观察细胞并拍照确认细胞杀伤效果。然后收集细胞进行流动分析，并使用流动缓冲液(PBS+2％FBS)和0.5μl AF647清洗细胞两次，每个孔中加入抗钙网蛋白抗体(abcam)，混合。在4℃且避光的条件下孵育，孵育20min后加入流动缓冲液，使用膜联蛋白染色试剂盒(Beyotime)，加入195μl Annexin-V-FITC结合物，与细胞进行吹打混合后，加入5μl Annexin-V-FITC抗体，轻轻混合，最后加入10μl PI染料混合，室温避光孵育15min，将样品送至流式细胞仪上进行信号测定。
显微镜下观察细胞，阿霉素单药组和两药联用组具有细胞杀伤作用，两药联用组细胞杀伤作用最为显著，而对照组和IN10018组细胞状态较好，见图11。流式细胞仪分析结果显示两药合用组的CRT阳性率和Annexin-V阳性率显著优于单药组及对照组，见图12a和12b。
实施例6：IN10018在结肠癌CT26细胞BALB/c小鼠皮下同种移植瘤模型中的体内抗肿瘤药效研究
试验材料：
小鼠：6-8周龄的雌性BALB/c小鼠购自上海斯莱克实验动物有限责任公司。
动物到达后在实验环境适应性饲养后开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼4只)。所有笼具、垫料及饮水在使用前均需灭菌。所有实验人员在动物房操作时应穿着防护服和乳胶手套。每笼动物信息卡应注明笼内动物数目，性别，品系，接收日期，给药方案，实验编号，组别以及实验开始日期。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下：
温度：20-26℃。湿度：40-70％。
光照周期：12小时光照，12小时无光照。
笼具：以聚碳酸酯制成，体积300mm×180mm×150mm。垫料为玉米芯，每周更换两次。
食物：实验动物在整个实验阶段中可自由进食(辐照灭菌，干颗粒状食物)。
饮水：实验动物可自由饮用灭菌水。
动物标识：实验动物以耳标进行标识。
化合物信息见表1
表1.化合物信息
Figure PCTCN2022137545-appb-000025
结直肠癌细胞CT26(来源中国科学院细胞库，货号：TCM37)由辉源生物科技(上海)有限公司保种维持传代。细胞体外单层培养，培养条件为RPMI-1640培养基中加10％胎牛血清，37℃5％CO 2培养箱中培养。一周两到三次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期，饱和度为80％-85％时，收取细胞，计数后接种。
细胞接种及分组
将0.1mL含有2×10 5个细胞的细胞悬液皮下接种于每只小鼠的右后背。当肿瘤体积达到～38mm 3时(细胞接种后第9天)，根据肿瘤体积进行随机分组给药，分组信息见表2。
表2.受试物对CT26小鼠移植瘤模型的给药方案
Figure PCTCN2022137545-appb-000026
注：1.每组小鼠数目；
2.给药体积：根据小鼠体重10mL/kg，如果体重下降超过15％，动物停止给药；待体重恢复至降低10％，再恢复给药。
受试物的配制
详见表3
表3.受试物配置方法
Figure PCTCN2022137545-appb-000027
实验动物日常观察
本实验方案的拟定及任何修改均通过了辉源生物IACUC的评估核准。 实验动物的使用及福利遵照AAALAC的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量(仅目测)，体重变化，外观体征或其它不正常情况，并每日对动物进行护理以及人工辅助排尿。基于各组动物数量记录了组内动物死亡数和副作用。
实验终止
若动物健康状况持续恶化，或瘤体积超过2,000mm 3，或有严重疾病，或疼痛，须处以安乐死。有以下情况者，通知兽医并处以安乐死：明显消瘦，体重降低大于20％；不能自由取食和饮水；对照组瘤体积平均值达到2,000mm 3，实验终止。动物出现以下临床表现且持续恶化：立毛，弓背，耳、鼻、眼或足色发白，呼吸仓促，抽搐，连续腹泻，脱水，行动迟缓，发声。
肿瘤测量和实验指标
用游标卡尺测量肿瘤直径，每周测量3次。肿瘤体积的计算公式为：V＝0.5×a×b 2，a和b分别表示肿瘤的长径和短径。
参照分组后第一天的肿瘤体积，根据以下公式计算肿瘤生长抑制率TGI(％)。TGI(％)＝[1-(某给药组的平均瘤体积-该给药组开始时平均瘤体积)/(溶剂对照组的平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]×100％。
统计分析
统计分析基于试验结束时肿瘤体积和肿瘤重量运用SPSS 24软件进行分析。多组间比较，采用one-way ANOVA检验，如方差齐性(F>0.05)，应用Tukey检验进行分析；如方差不齐(F<0.05)，应用Games-Howell检验进行分析。P<0.05认为有显著性差异。
实验结果
受试物IN10018和/或PD-L1抗体与阳性药Doxil联用在CT26小鼠结直肠癌细胞BALB/c小鼠皮下同种移植瘤模型中的体内药效。细胞接种后，每天观察肿瘤生长情况，接种后第9天根据肿瘤体积进行分组，入组平均肿瘤体积约为38mm 3。由于肿瘤负荷，对照组(G1)小鼠在第28天安乐死，PD-L1抗体组(G3)#790小鼠在第32天安乐死。整个实验在第35天结束。
分组给药后第28天，对照组的肿瘤体积为2176.53±491.44mm 3。IN10018+Doxil(12.5+1.5mg/kg)、PD-L1抗体(2.5mg/kg)、IN10018+Doxil+PD-L1抗体(12.5+1.5+2.5mg/kg)和Doxil+PD-L1抗体(1.5+2.5mg/kg)各治疗组的肿瘤体积分别为340.51±89.1mm 3,807.22±257.97mm 3,88.95±33.38mm 3和295.53±215.08mm 3，详见表4。综合肿瘤体积进行比较，IN10018+Doxil(12.5+1.5mg/kg)、PD-L1抗体(2.5mg/kg)、IN10018+Doxil+PD-L1抗体(12.5+1.5+2.5mg/kg)和Doxil+PD-L1抗体(1.5+2.5mg/kg)抑瘤率TGI分别为85.91％(p＝0.041),64.05％(p＝0.171),97.66％(p＝0.022)and 88.00％(p＝0.038)，详见表4。各剂量组在不同时间段的肿瘤体积如图13所示。
表4受试物对小鼠结肠癌CT26细胞的BALB/c小鼠移植瘤模型的抑瘤效果评价(基于第28天数据)
组别 第1天肿瘤体积(mm 3) 1 第25天肿瘤体积(mm 3) TGI(％)
对照组 38.86±1.17 2176.53±491.44 /
IN10018+Doxil 39.38±1.27 340.51±89.1 85.91
PD-L1抗体 38.74±1.4 807.22±257.97 64.05
IN10018+Doxil+PD-L1抗体 38.94±1.44 88.95±33.38 97.66
Doxil+PD-L1抗体 38.99±1.28 295.53±215.08 88.00
注：数据为平均值±标准误差。
实验按照给药方案进行，实验过程中，每天观察动物摄食饮水等活动，每周记录3次动物体重，动物体重曲线见图14。在整个给药周期中，各组组动物体重均无明显下降且状态良好。
结论
与空白对照组相比，IN10018+Doxil(12.5+1.5mg/kg)、PD-L1抗体(2.5mg/kg)、IN10018+Doxil+PD-L1抗体(12.5+1.5+2.5mg/kg)和Doxil+PD-L1抗体(1.5+2.5mg/kg)各治疗组均有明显肿瘤生长抑制作用，抑瘤率TGI均大于60％。IN10018+Doxil+PD-L1抗体(12.5+1.5+2.5mg/kg)的肿瘤抑制效果最好。
实施例7：Defactinib在结肠癌CT26细胞BALB/c小鼠皮下同种移植瘤模型中的体内抗肿瘤药效研究
试验材料：
小鼠：6-8周龄的雌性BALB/c小鼠购自北京维通利华实验动物有限责任公司。动物到达后在实验环境适应性饲养后开始实验。动物在SPF级动物房以IVC(独立送风系统)笼具饲养(每笼5只)。所有笼具、垫料及饮水在使用前均需灭菌。所有实验人员在动物房操作时应穿着防护服和乳胶手套。每笼动物信息卡应注明笼内动物数目，性别，品系，接收日期，给药方案，实验编号，组别以及实验开始日期。笼具、饲料及饮水每周更换两次。饲养环境及光照情况如下：
温度：20-26℃。湿度：40-70％。
光照周期：12小时光照，12小时无光照。
笼具：以聚碳酸酯制成，体积300mm×180mm×150mm。垫料为玉米芯，每周更换两次。
食物：实验动物在整个实验阶段中可自由进食(辐照灭菌，干颗粒状食物)。
饮水：实验动物可自由饮用灭菌水。
动物标识：实验动物以耳标进行标识。
化合物信息见表5
表5.化合物信息
Figure PCTCN2022137545-appb-000028
结直肠癌细胞CT26(来源南京科佰生物科技有限公司，货号：CBP60043)由应世生物科技(南京)有限公司维持传代。细胞体外单层培养，培养条件为RPMI-1640培养基中加10％胎牛血清，37℃5％CO2培养箱中培养。一周两到三次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期，饱和度为80％-90％时，收取细胞，计数后接种。
细胞接种及分组
将0.1mL含有3×10 5个细胞的细胞悬液皮下接种于每只小鼠的右后背。当肿瘤体积达到～81mm 3时(细胞接种后第9天)，根据肿瘤体积进行随机分组给药，分组信息见表6。
表6.受试物对CT26小鼠移植瘤模型的给药方案
Figure PCTCN2022137545-appb-000029
注：1.每组小鼠数目；
2.给药体积：根据小鼠体重10mL/kg，如果体重下降超过15％，动物停止给药；待体重恢复至降低10％，再恢复给药。
受试物的配制
详见表7
表7.受试物配置方法
Figure PCTCN2022137545-appb-000030
实验动物日常观察
本实验方案的拟定及任何修改均通过了云桥生物IACUC的评估核准。实验动物的使用及福利遵照AAALAC的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量(仅目测)，体重变化，外观体征或其它不正常情况。基于各组动物数量记录了组内动物死亡数和副作用。
实验终止
若动物健康状况持续恶化，或瘤体积超过4,000mm 3，或有严重疾病，或疼痛，须处以安乐死。有以下情况者，通知兽医并处以安乐死：明显消瘦，体重降低大于20％；不能自由取食和饮水；对照组瘤体积平均值达到4,000mm 3，实验终止。动物出现以下临床表现且持续恶化：立毛，弓背，耳、鼻、眼或足色发白，呼吸仓促，抽搐，连续腹泻，脱水，行动迟缓，发声。
肿瘤测量和实验指标
用游标卡尺测量肿瘤直径，每周测量3次。肿瘤体积的计算公式为：V＝0.5×a×b 2，a和b分别表示肿瘤的长径和短径。
参照分组后第一天的肿瘤体积，根据以下公式计算肿瘤生长抑制率TGI(％)。TGI(％)＝[1-(某给药组的平均瘤体积-该给药组开始时平均瘤体积)/(溶剂对照组的平均瘤体积-溶剂对照组开始治疗时平均瘤体积)]×100％。
统计分析
统计分析基于试验结束时肿瘤体积和肿瘤重量运用Prism Graphpad软件进行分析。多组间比较，采用Two-way ANOVA检验进行分析。P<0.05认为有显著性差异。
实验结果
受试物Defactinib和/或PD-L1抗体与阳性药Doxil联用在CT26小鼠结直肠癌细胞BALB/c小鼠皮下同种移植瘤模型中的体内药效。细胞接种后，每天观察肿瘤生长情况，接种后第9天根据肿瘤体积进行分组，入组平均肿瘤体积约为81mm 3。由于肿瘤负荷，所有入组小鼠在接种后的第24天，即分组给药之后的第15天安乐死，整个实验结束。
分组给药后第15天，对照组的肿瘤体积为3920.2±1697.5mm 3。Defactinib+Doxil(25+1.5mg/kg)、Doxil单药(1.5mg/kg)、PD-L1抗体(10mg/kg)、Doxil+PD-L1抗体(1.5+10mg/kg)和Defactinib+Doxil+PD-L1抗体(25+1.5+10mg/kg)各治疗组的肿瘤体积分别为2942.2±1491.2mm 3,3130.9±1446.2mm 3，3645.2±1914.8mm 3，2964.6±1653.2mm 3和1546.5±1237.9mm 3，详见表8。综合肿瘤体积与对照组进行比较，Defactinib+Doxil(25+1.5mg/kg)、Doxil单药(1.5mg/kg)、PD-L1抗体(10mg/kg)、Doxil+PD-L1抗体(1.5+10mg/kg)和Defactinib+Doxil+PD-L1抗体(25+1.5+10mg/kg)抑瘤率TGI分别为25.49％(p＝0.0470)，20.55％(p＝0.1513)，7.17％ (p＝0.9267)，24.89％(p＝0.0547)和61.83％(p<0.0001)。综合肿瘤体积与Defactinib+Doxil+PD-L1抗体(25+1.5+10mg/kg)组相比较，进行统计学分析，对照组，Defactinib+Doxil(25+1.5mg/kg)、Doxil单药(1.5mg/kg)、PD-L1抗体(10mg/kg)和Doxil+PD-L1抗体(1.5+10mg/kg)组的P值分别为p<0.0001，p＝0.0016，p＝0.0002，p<0.0001和p＝0.0013，详见表8。各剂量组在不同时间段的肿瘤体积如图15所示。
表8受试物对小鼠结肠癌CT26细胞的BALB/c小鼠移植瘤模型的抑瘤效果评价(基于分组给药后第15天数据)
Figure PCTCN2022137545-appb-000031
注：1.按照分组给药后的天数来计算，数据为平均值±标准误差。
2.*：p<0.05，****：p<0.0001，vs.对照组，Two-way ANOVA。
3.**：p<0.01，***：p<0.001，****：p<0.0001，vs.Defactinib+Doxil+PD-L1抗体(25+1.5+10mg/kg)组，Two-way ANOVA。
实验按照给药方案进行，实验过程中，每天观察动物摄食饮水等活动，每周记录3次动物体重，动物体重曲线见图16。在整个给药周期中，各组组动物体重均无明显下降且状态良好。
结论
与空白对照组相比，Defactinib+Doxil(25+1.5mg/kg)和Defactinib+Doxil+PD-L1抗体(25+1.5+10mg/kg)组均有明显肿瘤生长抑制作用，和对照组相比都有统计学差异。Defactinib+Doxil+PD-L1抗体 (25+1.5+10mg/kg)组相对于Defactinib+Doxil(25+1.5mg/kg)、Doxil单药(1.5mg/kg)、PD-L1抗体(10mg/kg)和Doxil+PD-L1抗体(1.5+10mg/kg)组各治疗组的肿瘤更小，且和这些单药组以及两药联用组相比均具有统计学差异，其相对于各单药组和两药联用组，具有更好的抑制肿瘤生长的效果。
实施例8：受试物Doxil和IN10018的配伍治疗在结肠癌CT26细胞BALB/c小鼠皮下同种移植瘤模型治疗后血清中IFN-γ和Granzyme B的ELISA检测
实验材料
供试品见表9。
表9：
名称 盐酸多柔比星脂质体注射液 IN10018
代号 Doxil IN10018
供应商 常州金远药业制造有限公司 WO2010058032中的方法合成即可
浓度 10ml：20mg  
性状 红色粘稠液体 白色粉末
溶媒 专用溶剂&PBS DPBS
储存条件 4℃ 常温
本次使用量 10mg 190mg
实验试剂信息见表10。
表10：
名称 品牌 货号 批号
Mouse IFN-γELISA Kit 碧云天 P1507 051922220705
Mouse Granzyme B ELISA Kit 博士德 EK1115 66117109707
实验方法与步骤
BALB/c小鼠(来源上海灵畅生物科技有限公司)，构建CT26肿瘤细胞 模型。当肿瘤体积达到40～60mm 3左右时，根据肿瘤体积进行随机分组并给药，分组信息见表11。
表11：实验动物分组及给药方案
Figure PCTCN2022137545-appb-000032
注：
1.N：每组小鼠数目
2.给药体积：根据小鼠体重10mL/kg，如果体重下降超过15％，动物停止给药；待体重恢复至降低10％，再恢复给药
小鼠血清采集及制备
Doxil第二次给药后的24小时，4组动物进行眼眶采血，将血液置于不含抗凝剂的收集管内，室温下静置自然凝集30～60分钟，待血液凝固，1000rpm 4℃离心10分钟。将上层血清转移至新的收集管内，放于负80℃冰箱保存。
小鼠血清中IFN-γ的检测
将样品按照100μl/孔加入相应孔中(样本事先用样本稀释液进行5倍稀释)，用封板膜(透明)封住反应孔，室温孵育120分钟；洗板5次，且最后一次置于厚吸水纸上拍干；加入生物素化抗体100μl/孔，用封板膜(透明)封住反应孔，室温孵育60分钟；洗板5次，且最后一次置于厚吸水纸上拍干；加入辣根过氧化物酶标记Streptavidin 100μl/孔，用封板膜(白色)封住反应孔，室温避光孵育20分钟。室温偏低时(低于25℃)，需要适当延长孵育时间；洗板5次，且最后一次置于厚吸水纸上拍干；加入显色剂TMB溶液100μl/孔，用封板膜(白色)封住反应孔，室温避光孵育15-20分钟，室温偏低时需要适当延长孵育时间，此时可以孵育至标准品出现非常显著的颜色变化，若样品浓度足够高也会出现显著的颜色变化；加入终止液50μl/孔，混匀后立 即测量A450值。
小鼠血清中Granzyme B检测
将样品按照100μl/孔加入相应孔中(样本事先用样本稀释液进行5倍稀释)，用封板膜(透明)封住反应孔，37℃孵育90分钟，不洗；加入生物素化抗体100μl/孔，用封板膜(透明)封住反应孔，37℃孵育60分钟；洗板3次，且最后一次置于厚吸水纸上拍干；加入Avidin-Biotin-Peroxidase Complex(ABC)100μl/孔，用封板膜(白色)封住反应孔，37℃避光孵育30分钟；洗板5次，且最后一次置于厚吸水纸上拍干；加入显色剂TMB溶液90μl/孔，用封板膜(白色)封住反应孔，室温避光孵育15-20分钟。室温偏低时需要适当延长孵育时间，此时可以孵育至标准品出现非常显著的颜色变化，若样品浓度足够高也会出现显著的颜色变化；加入终止液100μl/孔，混匀后立即测量A450值。
统计分析
统计分析运用Prism Graphpad软件进行分析。多组间比较，采用Two-way ANOVA，Kruskal-Wallis test法检验进行分析。P<0.05认为有显著性差异。
实验结果
小鼠血清中IFN-γ的检测结果
各组IFN-γ蛋白浓度为空白对照363.10±4.06pg/ml，IN10018 367.24±7.34pg/ml，Doxil 359.13±5.71pg/ml，Doxil+IN10018 387.00±4.04pg/ml，综合蛋白浓度与Doxil+IN10018进行比较，Doxil+IN10018两药联用组比Doxil单药组有更高的蛋白表达，进行统计学分析，P值为p<0.001，详见图17。
小鼠血清中Granzyme B检测结果
各组Granzyme B蛋白浓度为空白对照34.06±47.49pg/ml，IN10018  50.48±53.08pg/ml，Doxil 17.24±30.80pg/ml，Doxil+IN10018 72.21±35.40pg/ml，综合蛋白浓度与Doxil+IN10018进行比较，Doxil+IN10018两药联用组比Doxil单药组有更高的蛋白表达，进行统计学分析，P值为p<0.05，详见图18。
实验结论
在小鼠血清IFN-γ蛋白浓度检测实验中，各组与Doxil+IN10018组相比。Doxil+IN10018两药联用组比Doxil单药组有更高的蛋白表达，进行统计学分析，P值为p<0.001。
在小鼠血清Granzyme B蛋白浓度检测实验中，各组与Doxil+IN10018组相比。Doxil+IN10018两药联用组比Doxil单药组有更高的蛋白表达，进行统计学分析，P值为p<0.05。
实施例9：待测化合物阿霉素和AMP945对小鼠乳腺癌4T1细胞体外免疫原性细胞死亡靶点诱导的研究
实验材料：
1)本实验所用药物
阿霉素由MCE提供,Lot No.:97451
AMP945由MCE提供,Lot No.:143253
2)本实验所用抗体
重组Alexa
Figure PCTCN2022137545-appb-000033
647荧光Anti-Calreticulin抗体(Abcam，Cat No.:ab196159,Lot No.:CR33676773)。Annexin V-调亡检测试剂盒(Beyotime,Cat No.:C1062L,Lot No.:122221220706)。
实验方法：
4T1细胞(来源南京科佰生物科技有限公司，货号：CBP60352)用RPMI1640(上海元培,Cat No.:L210KJ,Lot No.:F210916)+10％FBS(Gibco,Cat No.: 10099-141c,Lot No.:2158737cp)培养，培养条件为37℃、5％CO 2。一周两到三次用胰蛋白酶进行常规消化传代处理。当细胞处于指数生长期，贴壁汇合至80％-90％时，收取细胞并进行铺板。4T1细胞用胰蛋白酶进行消化后收取细胞并进行计数，根据计数结果，用RPMI1640+10％FBS对细胞进行稀释，稀释浓度为5万个细胞每毫升，然后进行12孔细胞培养板铺板，每孔铺2ml细胞悬液即10万个细胞。铺板完成后，将细胞放于37℃、5％CO 2培养箱中培养。细胞铺展24小时后，设立六组，第一组为对照组，添加培养基，第二组为AMP945，浓度为3μM，第三组为AMP945，浓度为6μM，第四组为阿霉素(阿霉素，MCE)，浓度为0.5uM，第五组为AMP945(3μM)与阿霉素(0.5μM)联用，第六组为AMP945(6μM)与阿霉素(0.5μM)联用。将药物混合，并在37℃下在5％CO 2培养箱中培养48小时。
实验结果
药物作用48h后，收集细胞进行流动分析，并使用流动缓冲液(PBS+2％FBS)和0.5μl AF647清洗细胞两次，每个孔中加入抗钙网蛋白抗体(abcam)，混合。在4℃且避光的条件下孵育，孵育20min后加入流动缓冲液，使用膜联蛋白染色试剂盒(Beyotime)，加入195μl Annexin-V-FITC结合物，与细胞进行吹打混合后，加入5μl Annexin-V-FITC抗体，轻轻混合，最后加入10μl PI染料混合，室温避光孵育15min，将样品送至流式细胞仪上进行信号测定。
流式细胞仪分析结果显示两药合用组的CRT阳性率和Annexin-V阳性率显著优于单药组及对照组，见图19a和19b。
实施例10：待测化合物阿霉素和AMP945对小鼠结直肠癌CT26细胞体外免疫原性细胞死亡靶点诱导的研究
实验材料：
1)本实验所用药物
阿霉素由MCE提供,Lot No.:97451
AMP945由MCE提供,Lot No.:143253
2)本实验所用抗体
重组Alexa
Figure PCTCN2022137545-appb-000034
647荧光Anti-Calreticulin抗体(Abcam，Cat No.:ab196159,Lot No.:CR33676773)。Annexin V-调亡检测试剂盒(Beyotime,Cat No.:C1062L,Lot No.:122221220706)。
实验方法：
CT26细胞(来源南京科佰生物科技有限公司，货号：CBP60043)用RPMI 1640(上海元培,Cat No.:L210KJ,Lot No.:F210916)+10％FBS(Gibco,Cat No.:10099-141c,Lot No.:2158737cp)培养，培养条件为37℃、5％CO 2。一周两到三次用胰蛋白酶进行常规消化传代处理。当细胞处于指数生长期，贴壁汇合至80％-90％时，收取细胞并进行铺板。CT26细胞用胰蛋白酶进行消化后收取细胞并进行计数，根据计数结果，用RPMI1640+10％FBS对细胞进行稀释，稀释浓度为5万个细胞每毫升，然后进行12孔细胞培养板铺板，每孔铺2ml细胞悬液即10万个细胞。铺板完成后，将细胞放于37℃、5％CO 2培养箱中培养。细胞铺展24小时后，设立六组，第一组为对照组，添加培养基，第二组为AMP945，浓度为0.75μM，第三组为AMP945，浓度为1.5μM，第四组为阿霉素(阿霉素，MCE)，浓度为0.5uM，第五组为AMP945(0.75μM)与阿霉素(0.5μM)联用，第六组为AMP945(1.5μM)与阿霉素(0.5μM)联用。将药物混合，并在37℃下在5％CO 2培养箱中培养48小时。
实验结果
药物作用48h后，然后收集细胞进行流动分析，并使用流动缓冲液(PBS +2％FBS)和0.5μl AF647清洗细胞两次，每个孔中加入抗钙网蛋白抗体(abcam)，混合。在4℃且避光的条件下孵育，孵育20min后加入流动缓冲液，使用膜联蛋白染色试剂盒(Beyotime)，加入195μl Annexin-V-FITC结合物，与细胞进行吹打混合后，加入5μl Annexin-V-FITC抗体，轻轻混合，最后加入10μl PI染料混合，室温避光孵育15min，将样品送至流式细胞仪上进行信号测定。
流式细胞仪分析结果显示两药合用组的CRT阳性率和Annexin-V阳性率显著优于单药组及对照组，见图20a和20b。
实施例11：IN10018联合PLD和抗PD-1治疗卵巢癌的临床研究
IN10018联合聚乙二醇脂质体多柔比星(PLD)和抗PD-1抗体治疗铂耐药复发卵巢癌目前正在一项在中国开展的Ib/II期临床研究(IN10018-010)中进行评估。所述IN10018使用的是其酒石酸盐，所述PLD使用的是市售的盐酸多柔比星脂质体注射液(通用名)，所述抗PD-1抗体研究中采用的是市售的特瑞普利单抗。研究受试者将接受IN10018 100mg每天一次(QD)口服给药联合PLD 40mg/m 2每4周一次(Q4W)以及特瑞普利单抗3mg/kg每2周一次(Q2W)静脉输注给药。一个治疗周期为28天。
截至2022年11月30日，研究共入组41例各种实体瘤患者接受3药联合(IN10018+PLD+抗PD-1)治疗，其中24例为铂耐药复发卵巢癌患者，这41例患者均接受了至少一次药物治疗，纳入安全性分析。入组的24例铂耐药复发卵巢癌患者中有13例患者有首次肿瘤影像学评估数据，纳入疗效分析。
入组的这24例铂耐药复发卵巢癌患者的基线特征总结见表12。
表12：正在进行研究中26例铂耐药复发卵巢癌患者的基线特征总结
Figure PCTCN2022137545-appb-000035
疗效
研究基于肿瘤影像学检查评估肿瘤负荷随时间的变化，以评价3药联合(IN10018+PLD+抗PD-1)治疗在铂耐药复发卵巢癌受试者中的抗肿瘤疗效。研究首选通过对比增强计算机断层扫描(CT)进行影像学检查。对于腹部和盆腔，当碘化对比剂CT有使用禁忌时，可使用对比增强核磁共振成像(MRI)检查。MRI是脑部成像的首选检查，如有需要，还可以采用其他成像技术，如PET/CT和骨扫描。
筛选期的影像学检查必须在研究治疗分配前28天内进行。研究期间受试者将接受每8周(56±7天)一次肿瘤影像学检查，直至研究者评估受试者发现影像学确定的疾病进展、开始了新的抗肿瘤治疗、退出研究或死亡(以先发生者为准)。
研究将基于实体瘤疗效评价标准第1.1版(RECIST 1.1)来进行肿瘤的疗效评估。疗效指标包括客观缓解率(ORR)、疾病控制率(DCR)、缓解持续时间(DOR)、无进展生存期(PFS)和总生存期(OS)。
三药联合(IN10018+PLD+抗PD-1)治疗在铂耐药复发卵巢癌研究中共有13例疗效可评估患者，其中绝大部分只有首次肿瘤影像学评估结果，因而只呈现了首次肿瘤评估的疗效数据。在13例疗效可评估患者肿，首次肿瘤评估时观察到7例部分缓解(PR)，ORR为53.8％％(7/13)，DCR为100％(13/13)，83.5％(11/13)的患者有肿瘤靶病灶缩小。
既往2药联合(IN10018+PLD)治疗研究中共有52例疗效可评估患者，其中观察到24例PR和1例完全缓解(CR)，最佳整体疗效的ORR为48.1％，然而首次肿瘤评估时的2药联合治疗铂耐药复发卵巢癌的ORR为28.8％。与两药联合相比，三药联合(IN10018+PLD+抗PD-1)治疗显著提高了首次肿评时的疗效，更多疗效有待数据进一步成熟。
具体数据详见表13、表14及图21。
表13：两药和三药联合抗肿瘤疗效总结
Figure PCTCN2022137545-appb-000036
表14：三药联合首次肿评疗效列表
Figure PCTCN2022137545-appb-000037
Figure PCTCN2022137545-appb-000038
靶病灶总径相对基线最佳变化＝(治疗后最小靶病灶总径–基线靶病灶总径)/基线靶病灶总径x 100％。
安全性
研究在包括卵巢癌在内的实体瘤中评估了三药联合(IN10018+PLD+抗PD-1)治疗的安全性。共入组了41例实体瘤患者，其中铂耐药复发卵巢癌患者24例。
安全性分析表明三药联合(IN10018+PLD+抗PD-1)治疗的安全性与二药联合(IN10018+PLD)治疗总体相当，安全性特征与各自药物历史数据相当，未观察到联用导致不良事件有显著增加。两药和三药联合的安全性总结如下表15所示。
表15：两药和三药联合安全性总结
Figure PCTCN2022137545-appb-000039
通过引用将本发明中所提及的所有参考文献均完整合并入本文，就如同每一篇文献均单独列出一样。应理解，在阅读了本发明的公开内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落入本申请所附权利要求书所限定的范围内。
本申请要求于2021年12月10日递交的中国专利申请202111508073.8、于2022年9月22日递交的中国专利申请202211160418.X和于2022年12月2日递交的中国专利申请202211543036.5的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。

Claims (24)

  1. FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂在制备用于在对象中治疗肿瘤的药物中的用途，其中所述FAK抑制剂、所述蒽环类化疗药和所述免疫检查点抑制剂被同时或依次施用于所述对象。
  2. FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂的药物组合产品，其用于在对象中治疗肿瘤，其中所述FAK抑制剂、所述蒽环类化疗药和所述免疫检查点抑制剂被同时或依次施用于所述对象。
  3. 一种治疗肿瘤的方法，该方法包括向有需要的对象同时或依次施用治疗有效量的FAK抑制剂、蒽环类化疗药和免疫检查点抑制剂。
  4. 如权利要求1-3任一项所述的用途、药物组合产品或者方法，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
    Figure PCTCN2022137545-appb-100001
  5. 如权利要求1-4任一项所述的用途、药物组合产品或者方法，其中所述蒽环类化疗药为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
  6. 如权利要求1-5任一项所述的用途、药物组合产品或者方法，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
  7. 如权利要求1-6任一项所述的用途、药物组合产品或者方法，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类化疗药为阿 霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
  8. 如权利要求1-7任一项所述的用途、药物组合产品或者方法，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结肠癌，直肠癌，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结肠癌。
  9. 一种试剂盒或药学上可接受的组合物，其包括：
    (a)FAK抑制剂；
    (b)蒽环类化疗药；和
    (c)免疫检查点抑制剂。
  10. 如权利要求9所述的试剂盒或组合物，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
    Figure PCTCN2022137545-appb-100002
  11. 如权利要求9或10所述的试剂盒或组合物，其中所述蒽环类化疗药为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
  12. 如权利要求9-11任一项所述的试剂盒或组合物，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
  13. 如权利要求9-12任一项所述的试剂盒或组合物，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述蒽环类化疗药为阿霉素，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
  14. 如权利要求9-13任一项所述的试剂盒或组合物，其用作药物。
  15. FAK抑制剂，其在治疗肿瘤中用于增强蒽环类化疗药诱导的免疫原性细胞死亡。
  16. 如权利要求15所述的用途、FAK抑制剂或方法，其中所述蒽环类化疗药为阿霉素、表阿霉素、柔红霉素，优选为阿霉素。
  17. 如权利要求15-16任一项所述的用途、FAK抑制剂或方法，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
    Figure PCTCN2022137545-appb-100003
  18. FAK抑制剂、阿霉素和免疫检查点抑制剂在制备用于在对象中治疗肿瘤的药物中的用途，其中所述FAK抑制剂、所述阿霉素和所述免疫检查点抑制剂被同时或依次施用于所述对象。
  19. FAK抑制剂、阿霉素和免疫检查点抑制剂的药物组合产品，其用于在对象中治疗肿瘤，其中所述FAK抑制剂、所述阿霉素和所述免疫检查点抑制剂被同时或依次施用于所述对象。
  20. 一种治疗肿瘤的方法，该方法包括向有需要的对象同时或依次施用治疗有效量的FAK抑制剂、阿霉素和免疫检查点抑制剂。
  21. 如权利要求18-20任一项所述的用途、药物组合产品或者方法，其中所述FAK抑制剂为IN10018、Defactinib、GSK2256098、PF-00562271、VS-4718、APG-2449、AMP945、AMP886或其药学上可接受的盐，优选为IN10018、Defactinib、AMP945或其药学上可接受的盐，进一步优选为IN10018或其药学上可接受的盐，尤其是IN10018酒石酸盐，所述IN10018结构如下：
    Figure PCTCN2022137545-appb-100004
  22. 如权利要求18-21任一项所述的用途、药物组合产品或者方法，其中所述免疫检查点抑制剂为抗PD-1/PD-L1抗体、PD-1/PD-L1小分子抑制剂或者TIGIT抑制剂。
  23. 如权利要求18-22任一项所述的用途、药物组合产品或者方法，其中所述FAK抑制剂为IN10018或其药学上可接受的盐，所述免疫检查点抑制剂为抗PD-1/PD-L1抗体或PD-1/PD-L1小分子抑制剂，尤其是特瑞普利单抗。
  24. 如权利要求18-23任一项所述的用途、药物组合产品或者方法，其中所述肿瘤为霍奇金淋巴瘤，非霍奇金淋巴瘤，非小细胞肺癌，小细胞肺癌，肝细胞癌，胆管癌，骨髓增生异常综合征，急性淋巴细胞白血病，急性髓系白血病，慢性髓系白血病，甲状腺癌，神经胶质瘤，结肠癌，直肠癌，结直肠癌，卵巢癌，膀胱癌，前列腺癌，乳腺癌，脂肪肉瘤，纤维肉瘤，横纹肌 肉瘤，平滑肌肉瘤，血管肉瘤，神经母细胞瘤，肾细胞癌，头颈部癌，胃癌，食管癌，胃食管结合部癌，胸腺癌，胰腺癌，子宫内膜癌，宫颈癌，黑色素瘤，葡萄球膜黑色素瘤，皮肤癌，生殖细胞癌，鼻咽癌，口咽癌，或喉癌；进一步的所述肿瘤为急性髓系白血病，黑色素瘤，甲状腺癌，结肠癌，食道癌，肝细胞癌，卵巢癌，纤维肉瘤，胃癌，非小细胞肺癌或胆管癌；更进一步的为卵巢癌或结肠癌。
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