TW202126302A - Use of ezh2 inhibitor in combination with immune checkpoint inhibitor for preparation of medicament for treating tumor diseases - Google Patents

Abstract

The disclosure relates to use of EZH2 inhibitor and immune checkpoint inhibitor in combination with tyrosine kinase inhibitor for preparation of medicament for treating tumor diseases. Specifically, the said EZH2 inhibitor is the compound of formula (I) or its pharmaceutical salt, the said immune checkpoint inhibitor is anti-PD-1 antibody or its antigen binding fragment, the said tyrosine kinase inhibitor is the compound of formula (II) or its pharmaceutical salt.

Description

Use of combination of EZH2 inhibitor, immune checkpoint inhibitor and tyrosine kinase inhibitor in the preparation of tumor drugs

The present disclosure relates to the use of the combination of EZH2 inhibitors, immune checkpoint inhibitors, and multi-target tyrosine kinase inhibition in the preparation of drugs for treating tumors.

Protein programmed death 1 (PD-1) is an inhibitory member of the CD28, receptor family, which also includes CD28, CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells. The first members of the family, CD28 and ICOS, were discovered based on the functional effect of the addition of monoclonal antibodies on the promotion of T cell proliferation. PD-1 was discovered by screening for differential expression in apoptotic cells. Currently, Ono’s Nivolumab and Merck’s Pembrolizumab have been successfully approved for the market for the treatment of unresectable or metastatic melanoma, non-small cell lung cancer, advanced renal cell carcinoma, Hodgkin’s lymphoma, recurrent or metastatic disease Squamous cell carcinoma and so on. PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (dendritic cells, DC), and the expression on cells can be up-regulated after activation. PD-L1 inhibits the immune system by binding to PD-1 and B7-1 In general, many tumor cells and immune cells in the tumor tissue microenvironment express PD-L1. New research found high PD-L1 was detected in human tumor tissues such as breast cancer, lung cancer, stomach cancer, bowel cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer. The expression of protein and the expression level of PD-L1 are closely related to the clinical and prognosis of patients.

The histone methyltransferase encoded by EZH2 gene is the catalytic component of Polycomb inhibitory complex 2 (PRC2). Compared with normal tissues, the level of EZH2 is abnormally increased in cancer tissues, and the expression level of EZH2 is the highest in advanced cancer or poor prognosis. In some cancer types, overexpression of EZH2 occurs simultaneously with amplification of the EZH2 gene. A large number of si/shRNA experimental studies have found that reducing EZH2 expression in tumor cell lines can inhibit tumor cell proliferation, migration and invasion or angiogenesis, and lead to cell apoptosis.

At present, EZH2 inhibitors have entered the clinical development stage. The following is a brief list. Tazemetostat (EPZ-6438) developed by Eisai is used for the treatment of non-Hodgkin B-cell lymphoma. It is currently in phase II clinical phase. CPI developed by Constellation -1205 is used to treat B-cell lymphoma and is currently in clinical phase I. GSK-2816126 developed by GlaxoSmithKline is used to treat diffuse large B-cell lymphoma and follicular lymphoma and is currently in clinical phase I.

WO2017084494A provides an EZH2 inhibitor, the structure is as follows:

The review by Lu Gan et al. mentioned the emerging role of EZH2 in tumor immunity (Biomarker Research, December 2018, 6:10).

The anti-tumor mechanism of tyrosine kinase inhibitors (TKIs) may be achieved through the following ways: inhibiting tumor cell damage and repair, blocking cell division in the G1 phase, inducing and maintaining cell apoptosis, and anti-angiogenesis. Overexpression of EGFR often indicates poor prognosis, rapid metastasis, resistance to chemotherapy drugs, hormone resistance, and shorter survival time. The FDA has approved a variety of multi-target TKIs, such as sorafenib, vandetanib, and sunitinib (Sutent, SU-11248). WO2007085188 discloses a compound similar to Sunitinib, as shown in the following formula (II), which may be better applied to the treatment of the above-mentioned tumors. The chemical name of the compound is 5-(2-diethylamino-ethyl)-2-(5-fluoro-2-oxo-1,2-dihydro-indole-3-ylidene-methyl)- 3-Methyl-1,5,6,7-tetrahydro-pyrrole [3,2-c]pyridin-4-one, which is known to inhibit tumor proliferation and angiogenesis, and can selectively inhibit vascular endothelial growth factor The kinase activity of (VEGF) receptors can be used clinically for the treatment of renal cancer, gastrointestinal stromal tumors, colorectal cancer and pancreatic neuroendocrine tumors.

WO2018068691 discloses the use of a combination of an anti-PD-1 antibody and a VEGFR inhibitor in the preparation of a medicine for the treatment of cancer.

WO2019097369A discloses a method for administering an EZH2 inhibitor and a platinum-based anti-tumor agent to a patient in combination to treat cancer. Further, other anti-cancer agents can be administered, and the other anti-cancer agents can be selected from anti-PD-1 antibodies and anti-PD-L1 Antibodies and anti-CTLA-4 antibodies, etc.

WO2018223030A Example 1 discloses tissue testing for patients with epithelioid sarcoma and renal medullary carcinoma and found that PD-L1 will be up-regulated after EZH2 inhibition. The data indicate that these tumors are triggered due to the presence of T cell-mediated immune responses in the tumors, but the immune response It may be inhibited by the increased expression of PD-L1 in tumors, indicating that this tumor can be treated by a combination of EZH2 inhibition and PD-1/PD-L1 inhibition. In Example 2, patients with soft tissue sarcoma were given a combination therapy of tazemetostat and atezolizumab, but no specific drug efficacy information was disclosed.

WO2017210395A claims a method for treating cancer in a subject in need, comprising administering to the subject a therapeutically effective amount of an EZH2 inhibitor and a therapeutically effective amount of an immune checkpoint inhibitor, the immune checkpoint inhibitor can The anti-PD-L1 antibody is selected. The examples disclose the combination of tazemetostat and atezolizumab for patients with locally advanced or metastatic urothelial cancer, patients with diffuse large B-cell lymphoma, and The treatment plan for non-Hodgkin's lymphoma, but no specific information on the efficacy is given.

The present disclosure provides a use of an EZH2 inhibitor, an immune checkpoint inhibitor, and a multi-target tyrosine kinase inhibitor in the preparation of a drug for treating tumors.

In certain embodiments, the EZH2 inhibitor in the present disclosure may be selected from CPI-0209, CPI-1205, GSK126, valemetostat, tazemetostat, PF-06821497, DS-3201 At least one of GSK-2816126, 3-deazaneplanocin A, HKMT-I-005, and KM-301.

In certain embodiments, the EZH2 inhibitor described in the present disclosure is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

The pharmaceutically acceptable salt of the compound represented by formula (I) in the present disclosure can be hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalate, malonate, pentane Acid salt, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, isethionate, maleate , Malate, tartrate, benzoate, pamoate, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or lauryl sulfonate, etc. .

In certain embodiments, the immune checkpoint inhibitor described in the present disclosure may be an anti-PD-1 antibody or an antigen-binding fragment thereof.

In certain embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof described in the present disclosure can be selected from sintilimab, cemiprimab, JS-001, nivolumab Anti-(nivolumab), tislelizumab, pebrolizumab, AK-103, dostarlimab, PD1-PIK, GLS-010, genolimzumab ), BI-754091, spartalizumab, MGA-012, PF-06801591, XmAb-20717, CS-1003, Sym-021, AGEN-2034, MEDI-5752, MGD-013, AK- At least one of 105, AK-104, BCD-100, PF-06753512, HLX-10, AMP-224, LZM-009.

In certain embodiments, the light chain variable region of the PD-1 antibody described in the present disclosure comprises the variable regions shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. LCDR1, LCDR2 and LCDR3, the heavy chain variable region of the PD-1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

Among them, the aforementioned CDR sequences are shown in Table 1 below:

Preferably, the PD-1 antibody is a humanized antibody or a fragment thereof.

In an alternative embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof in the present disclosure is selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') 2 fragments.

Immunoglobulins can be derived from any commonly known isotype, including but not limited to IgA, secreted IgA, IgG, and IgM. The IgG subclass is also well known to those skilled in the art, including but not limited to IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (for example, IgM or IgG1) encoded by the heavy chain constant region gene. In some alternative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof described in the present disclosure comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype, preferably comprising IgG1 or IgG4 isotype. Constant region of the heavy chain.

In other alternative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain constant region of kappa or lambda.

Further, the preferred humanized antibody light chain variable region sequence is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the variant preferably has 0-10 amino acid changes in the light chain variable region , More preferably the amino acid change of A43S; the humanized antibody heavy chain variable region sequence is the sequence shown in SEQ ID NO: 9 or a variant thereof, and the variant preferably has a heavy chain variable region The amino acid change of 0-10 is more preferably the amino acid change of G44R.

The sequences of the heavy and light chains of the aforementioned humanized antibodies are as follows:

Heavy chain variable region

SEQID NO: 9

Light chain variable region

SEQID NO: 10

Preferably, the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 8 or a variant thereof; this variant preferably has an amino acid change of 0-10 in the light chain variable region, and more Preferably, the amino acid change of A43S; the humanized antibody heavy chain sequence is the sequence shown in SEQ ID NO: 7 or a variant thereof, and the variant preferably has 0-10 amines in the variable region of the heavy chain The base acid change is more preferably the amino acid change of G44R.

In some embodiments, the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8, and the heavy chain sequence is the sequence shown in SEQ ID NO: 7.

The sequences of the heavy and light chains of the humanized antibody are as follows:

Heavy chain

SEQID NO: 7

Light chain

SEQID NO: 8

In certain embodiments, the multi-target tyrosine kinase inhibition may be selected from the group consisting of Sunitinib, Pazopanib, Lenvatinib, Regorafenib , Cabozantinib, Dovitinib, Sorafenib, Vandetanib, Crizotinib, Anlotinib, General At least one of Ponatinib, Imatinib, ENMD-2076, Afatinib, Dasatinib, and nintedanib.

In some embodiments, the multi-target tyrosine kinase inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

In the present disclosure, the pharmaceutically acceptable salt of the compound represented by formula (II) may be hydrochloride, phosphate, hydrogen phosphate, sulfate, hydrogen sulfate, sulfite, acetate, oxalate, and malonate. , Valerate, glutamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, methanesulfonate, isethionate, maleate Acid salt, malate, tartrate, benzoate, pamoate, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or lauryl sulfonic acid Salt, etc. In an alternative embodiment, the pharmaceutically acceptable salt of the compound represented by formula (II) is malate.

In some embodiments, the dosage of EZH2 is selected from 1 to 1600 mg, for example: 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg , 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290mg, 300mg, 310mg, 320mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg, 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg, 510mg, 520mg, 530mg, 540mg, 550mg, 560mg, 570mg, 580mg, 590mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, 1000mg, 1025mg, 1050mg, 1075mg, 1100mg, 1125mg, 1150mg, 1175mg, 1200mg, 1225mg, 1250mg, 1275mg, 1300mg, 1325mg, 1350mg, 1375mg, 1400mg, 1425mg, 1450mg, 1475mg, 1500mg, 1525mg, 1550mg, 1575mg, 1600mg, the frequency of administration is 2 times a day, once a day, once every 2 days, Once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks, preferably twice a day or once a day.

In some embodiments, the dosage of EZH2 is selected from 1-800mg, and the frequency of administration is 2 times a day, once a day, once every 2 days, once every 3 days, once every 4 days, every 5 days Once, once every 6 days, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks, preferably twice a day or once a day.

In some embodiments, the dosage of EZH2 is selected from 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, and the frequency of administration is 2 times a day, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, every 6 days Once, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks, preferably twice a day or once a day.

In some embodiments, the dosage of EZH2 is selected from 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, and the frequency of administration is It is 2 times a day or 1 time a day.

In some embodiments, the dosage of EZH2 is selected from 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, and the frequency of administration is 2 times a day, once a day, once every 2 days, every 3 days Once, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks or once every 4 weeks.

In some embodiments, the dosage of EZH2 is selected from 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, and the frequency of administration is twice a day or once a day.

In some embodiments, the administration dose of the immune checkpoint inhibitor may be in the following range: 0.1-10.0 mg/kg, 0.1-5 mg/kg, 1-5 mg/kg, 2-5 mg/kg, for example, the dose It can be 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 0.6mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/ kg, 1.2mg/kg, 1.4mg/kg, 1.6mg/kg, 1.8mg/kg, 2.0mg/kg, 2.2mg/kg, 2.4mg/kg, 2.6mg/kg, 2.8mg/kg, 3.0mg/ kg, 3.2mg/kg, 3.4mg/kg, 3.6mg/kg, 3.8mg/kg, 4.0mg/kg, 4.2mg/kg, 4.4mg/kg, 4.6mg/kg, 4.8mg/kg, 5.0mg/ kg, 5.2mg/kg, 5.4mg/kg, 5.6mg/kg, 5.8mg/kg, 6.0mg/kg, 6.2mg/kg, 6.4mg/kg, 6.6mg/kg, 6.8mg/kg, 7.0mg/ kg, 7.2mg/kg, 7.4mg/kg, 7.6mg/kg, 7.8mg/kg, 8.0mg/kg, 8.2mg/kg, 8.4mg/kg, 8.6mg/kg, 8.8mg/kg, 9.0mg/kg, 9.2mg/kg, 9.4mg/kg, 9.6mg/kg, 9.8mg/kg, 10.0mg/kg, the frequency of administration is once a week, every 2 It is administered at a frequency of once a week, once every 3 weeks, once every 4 weeks, once a month, once every 3-6 months or longer.

In some embodiments, the dose range of the immune checkpoint inhibitor is selected from 1-1000 mg, 80-800 mg, 80-700 mg, 80-600 mg, 80-500 mg, 80-400 mg, 80-300 mg, 100-300 mg, or 200 mg. -300mg. For example, the dosage can be 1.0 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2.0 mg, 2.2 mg, 2.4 mg, 2.6 mg, 2.8 mg, 3.0 mg, 3.2 mg, 3.4 mg, 3.6 mg, 3.8 mg, 4.0mg, 4.2mg, 4.4mg, 4.6mg, 4.8mg, 5.0mg, 5.2mg, 5.4mg, 5.6mg, 5.8mg, 6.0mg, 6.2mg, 6.4mg, 6.6mg, 6.8mg, 7.0mg, 7.2mg , 7.4mg, 7.6mg, 7.8mg, 8.0mg, 8.2mg, 8.4mg, 8.6mg, 8.8mg, 9.0mg, 9.2mg, 9.4mg, 9.6mg, 9.8mg, 10.0mg, 15mg, 20mg, 25mg, 30mg , 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg , 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 275mg, 280mg , 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 345mg, 350mg, 355mg, 360mg, 365mg, 370mg, 375mg, 380mg, 385mg, 390mg, 395mg, 400mg, 405mg , 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg, 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg, 505mg, 510mg, 515mg, 520mg, 525mg, 530mg, 535mg, 540mg, 545mg, 550mg, 555mg, 560mg, 565mg, 570mg, 575mg, 580mg, 585mg, 590mg, 595mg, 600mg, the frequency of administration is once a week, once every 2 weeks, once every 3 weeks, It is administered every 4 weeks, once a month, every 3-6 months or longer.

In an alternative embodiment, the dose of the immune checkpoint inhibitor is 80 mg, 100 mg, 160 mg, 200 mg, 240 mg, 300 mg, 320 mg, 400 mg, 500 mg, 600 mg, 700 mg or 800 mg, and the frequency of administration is once a week, every 2 weeks It is administered once, once every 3 weeks, once every 4 weeks, once a month, once every 3-6 months or longer.

In an alternative embodiment, the amount of immune checkpoint inhibitor is 200 mg, and the frequency of administration is once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, every 3-6 It is administered frequently every month or more.

In an alternative embodiment, the frequency of administration of the immune checkpoint inhibitor is once every 2 weeks or once every 3 weeks.

In an alternative embodiment, the dose of the multi-target tyrosine kinase inhibitor is selected from 0.1-1000 mg, and the frequency of administration may be once a day, twice a day, or 3 times a day, preferably once a day.

In an optional embodiment, the dose of the multi-target tyrosine kinase inhibitor is selected from 0.1-100 mg, specifically 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9mg, 1.0mg, 2mg, 3mg, 4mg, 5mg, 6mg, 7mg, 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, 25mg, 26mg, 27mg, 28mg, 29mg, 30mg, 31mg, 32mg, 33mg, 34mg, 35mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74mg, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 94mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg, the frequency of administration may be once a day, twice a day, 3 times a day, preferably once a day.

In an alternative embodiment, the multi-target tyrosine kinase inhibitor is administered by body weight, and the dose is selected from 0.1-10.0 mg/kg.

In the above regimen of administration according to body weight, the dose of the multi-target tyrosine kinase inhibitor can be 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7mg/kg, 0.8mg/kg, 0.9mg/kg, 1.0mg/kg, 1.2mg/kg, 1.4mg/kg, 1.6mg/kg, 1.8mg/kg, 2.0mg/kg, 2.2 mg/kg, 2.4mg/kg, 2.6mg/kg, 2.8mg/kg, 3.0mg/kg, 3.2mg/kg, 3.4mg/kg, 3.6mg/kg, 3.8mg/kg, 4.0mg/kg, 4.2 mg/kg, 4.4mg/kg, 4.6mg/kg, 4.8mg/kg, 5.0mg/kg, 5.2mg/kg, 5.4mg/kg, 5.6mg/kg, 5.8mg/kg, 6.0mg/kg, 6.2 mg/kg, 6.4mg/kg, 6.6mg/kg, 6.8mg/kg, 7.0mg/kg, 7.2mg/kg, 7.4mg/kg, 7.6mg/kg, 7.8mg/kg, 8.0mg/kg, 8.2 mg/kg, 8.4mg/kg, 8.6mg/kg, 8.8mg/kg, 9.0mg/kg, 9.2mg/kg, 9.4mg/kg, 9.6mg/kg, 9.8mg/kg, 10.0mg/kg, give The frequency of medication may be once a day, twice a day, or 3 times a day, preferably once a day.

In an alternative embodiment, the dose of the multi-target tyrosine kinase inhibitor is selected from 1-25 mg, specifically 15 mg, 20 mg, 25 mg, and the frequency of administration may be once a day, twice a day, or 3 times a day. Times, preferably once a day.

In an alternative embodiment, the multi-target tyrosine kinase inhibitor is the compound shown in (II) malate.

The tumor described in the present disclosure is selected from the group consisting of adrenal cortical cancer, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, cerebellar astrocytoma, brain astrocytoma, basal cell carcinoma, skin cancer (non-melanoma ), biliary tract cancer, extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma, bladder cancer, bone and joint cancer, osteosarcoma, malignant fibrous histiocytoma, brain cancer, brain tumor, brainstem glioma, ependymoma, adult Neuroblastoma, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic Myelocytic leukemia, leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer, skin T-cell lymphoma, lymphoma, mycosis fungoides, Sezary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, Extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, head and neck cancer, Hepatocellular carcinoma, Hodgkin’s lymphoma, islet cell tumor, Kaposi’s sarcoma, kidney cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non- Small cell lung cancer, small cell lung cancer, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstroem macroglobulinemia, melanoma, mesothelioma, metastatic squamous cell carcinoma, tongue cancer, multiple endocrine tumors Syndrome, myelodysplastic syndrome, multiple myeloma, nasopharyngeal carcinoma, neuroblastoma, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential Tumor, pancreatic cancer, islet cell pancreatic cancer, sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal tumor, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, Prostate cancer, rectal cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Juventus sarcoma, Kaposi sarcoma, synovial sarcoma, uterine cancer, uterine sarcoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma Stomatocell carcinoma, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma, urethral cancer, endometriosis, vaginal cancer, vulvar cancer or Wilm’s tumor, preferably colorectal cancer or colon cancer.

The immune checkpoint inhibitor of the present disclosure can be constituted in a composition, for example, a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents and absorption delaying agents that are physiologically compatible. In one embodiment, the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraperitoneal, spinal or epidermal administration (for example, by injection or infusion), and the pharmaceutical composition of the present disclosure may include a Or a variety of pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents.

In a preferred embodiment of the present disclosure, the PD-1 antibody or its antigen-binding fragment is administered by injection, such as subcutaneous or intravenous injection or intraperitoneal injection. The PD-1 antibody or its antigen-binding fragment must be prepared before injection. Made in an injectable form. In a particularly preferred embodiment of the present disclosure, the injectable form of the anti-PD-1 antibody is an injection or a lyophilized powder injection, which contains the PD-1 antibody or its antigen-binding fragment, a buffer, a stabilizer, and optionally a surface Active agent. The buffer can be selected from one or more of acetate, citrate, succinate, and phosphate. The stabilizer may be selected from sugars or amino acids, preferably disaccharides, such as sucrose, lactose, trehalose, and maltose. The surfactant is selected from polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, Preferably, the polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60 or 80, most preferably polysorbate 20. The most preferred injectable form of PD-1 antibody or antigen-binding fragment thereof includes PD-1 antibody or antigen-binding fragment thereof, acetate buffer, trehalose and polysorbate 20.

The present disclosure provides a method for treating tumors, which includes administering to a patient a therapeutically effective amount of the above-mentioned EZH2 inhibitor, immune checkpoint inhibitor, and multi-target tyrosine kinase inhibitor.

Another aspect of the present disclosure provides an EZH2 inhibitor for the treatment of tumors, which is used in combination with the immune checkpoint inhibitor and the multi-target tyrosine kinase inhibitor.

Another aspect of the present disclosure provides an immune checkpoint inhibitor for the treatment of tumors, which is used in combination with an EZH2 inhibitor and a multi-target tyrosine kinase inhibitor.

Another aspect of the present disclosure provides a multi-target tyrosine kinase inhibitor, which is used in combination with an EZH2 inhibitor and an immune checkpoint inhibitor.

In an alternative embodiment, in the method for treating tumors provided in the present disclosure, the patient is a human.

The tyrosine kinase inhibitor described in the present disclosure refers to a drug that can block tyrosine kinase, and the multi-target tyrosine kinase inhibitor means that it can inhibit at least two receptor tyrosine kinases at the same time. Drugs, receptor tyrosine kinases include but are not limited to PDGF-R, VEGFR, flt3, EGFR, HER2, FGFR, etc.

The “combination” described in the present disclosure is a mode of administration, which refers to the administration of at least one dose of immune checkpoint inhibitor, EZH2 inhibitor and multi-target tyrosine kinase inhibitor within a certain period of time, of which three drugs Both show pharmacological effects. The time period can be within a dosing cycle, for example, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours. Immune checkpoint inhibitors, EZH2 inhibitors and multi-target tyrosine kinase inhibitors can be administered simultaneously or in no particular order. This period includes treatments in which the same route of administration or different The route of administration is to give immune checkpoint inhibitors, EZH2 inhibitors and multi-target tyrosine kinase inhibitors.

The "humanized antibody" in the present disclosure, also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types Of human germline antibody framework sequences. It can overcome the strong variable antibody response induced by the chimeric antibody due to the large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc. Human, 1991 Sequences of Proteins of Immunologioal Interest, found in 5th edition. In a preferred embodiment of the present disclosure, the CDR sequence of the PD-1 humanized antibody is selected from SEQ ID NO: 1, 2, 3, 4, 5, 6.

The "antigen-binding fragment" in the present disclosure refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments that bind to human PD-1, sFv fragments that have antigen-binding activity; The antibody is selected from one or more CDR regions in SEQ ID NO:1 to SEQ ID NO:6. The Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all the antigen binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into a polypeptide chain, which is called single chain antibody or single chain Fv (sFv). The term "and PD-1" in this disclosure Binding" refers to the ability to interact with human PD-1. The term "antigen-binding site" in the present disclosure refers to a three-dimensional site that is discontinuous on the antigen and recognized by the antibody or antigen-binding fragment in the present disclosure.

Figure 1 is a graph showing changes in body weight of mice in each group;

Figure 2 is a graph of tumor volume change in CT-26 mouse allograft model.

The following examples are used to further describe the present disclosure, but these examples do not limit the scope of the present disclosure.

Example 1. Verification of the efficacy of the test drug in the colorectal cancer cell line CT-26 female huPD-1 mouse allograft model

1. Experimental materials

1) Experimental animals

Mice, huPD-1 BALB/c, Jiangsu Jicui Yaokang Biotechnology Co., Ltd., 6-8 weeks old, 18-20g, 48 mice plus 40% redundancy, a total of 68 mice.

2) Experimental cells

CT-26, from the Chinese Academy of Sciences, colorectal cancer cells, F3 generation; the required cell volume is 8.2X10E7.

3) Experimental drugs

EZH2 inhibitor: compound represented by formula (I); multi-target tyrosine kinase inhibitor: compound represented by formula (II) malate; anti-PD-1 antibody: disclosed in WO2017054646A The heavy and light chain sequences of the PD-1 antibody are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

2. Experimental methods and procedures

1) CT-26 cells were resuscitated and cultured in vitro to obtain 1X10E8 cells; 68 6-8 week old female huPD-1 were adaptively reared for 1 week, weighed, and each mouse was inoculated subcutaneously at the bilateral scapula for 5* 10E5 cells, each seeding volume is 0.1mL. The tumor volume and body weight were measured once a week ^{, and mice with tumor volumes between 60-120 mm 3} on both sides were selected and randomly divided into 6 groups according to the tumor volume and body weight on the right side, with 8 mice in each group. The administration was started immediately after grouping. The start date of administration is regarded as day 0, and the administration and grouping information is shown in Table 2. After the start of the administration, the mouse body weight and tumor volume were measured 3 times a week.

2) The experimental drug configuration is shown in Table 3.

3) Experimental observation, data collection and statistical analysis

After inoculation, observe the morbidity and death of animals every working day. Daily observations include tumor growth and the effects of drugs on laboratory animals, such as changes in activity, changes in food and water, weight loss, changes in hair, eye appearance, death, and other clinical symptoms. After inoculation to before grouping, the weight of the experimental animals was measured once a week. After grouping, measure the weight of experimental animals 3 times a week or change the frequency of measuring the weight of mice according to customer requirements. After inoculation to before grouping, when the tumor is visible, the tumor volume of the experimental animal is measured once a week. After the inoculation and grouping, the tumor volume of the experimental animal is measured once a week.

Tumor volume is measured by a two-way measurement method. First, the length and short diameter of the tumor are measured with a vernier caliper, and then the tumor volume is calculated ^{using the formula TV=0.5×a×b 2.} Where a is the long diameter of the tumor and b is the short diameter of the tumor.

At the end of the experiment, the following analysis methods will be used for data analysis: %△T/C=(mean(T)-mean(T0))/(mean(C)-mean(C0))* 100%; T-dosing Group tumor volume, T0-administration group initial tumor volume, C-control group tumor volume, C0-control group initial tumor volume. Mean%△Inhibition(TGI%)=((mean(C)-mean(C0))-(mean(T)-mean(T0)))/(mean(C)-mean(C0))* 100%; T-administration group tumor volume, T0-administration group initial tumor volume, C-control group tumor volume, C0-control group initial tumor volume.

3. Experimental results

1) Mouse body weight

The average weight of each group's body weight at different time points is shown in Table 4.

See Table 5 for the percentage of weight change in each group.

The body weight change curve of each group of mice is shown in Figure 1.

2) Tumor volume data

In this experiment, there was no intratumoral administration in groups 1, 2, 3, and 4, so the left and right tumors were treated in the same way, and the data were combined for analysis. The volume of the tumor on the left side of the 34 and 35# mice in the fourth group was analyzed by Prism as an abnormal value, which was excluded from the statistics. The tumor volume of each treatment group at each time point is shown in Table 6.

3) Tumor growth inhibition

See Table 7 for tumor growth inhibition in each group.

4) The results of pairwise comparison between each single-drug group of tumors and the corresponding combination-drug group are shown in Table 8.

5) Tumor growth curve

The tumor growth curves on the left and right sides of each group are shown in Figure 2. In the fourth group, the left tumor volume of 34 and 35# mice was analyzed as an abnormal value by Prism abnormal value, which was excluded from the chart.

discuss:

The average tumor volume of EZH2 inhibitor (group 2) on the 14th day after po administration was 1871.8±263.53mm ³ , which was not significant compared with the control group (group 1, the tumor volume on the 14th day was 2020.2±177.82mm ³⁾ Difference (TGI%=8%, p=0.6439).

The anti-PD-1 antibody and multi-target tyrosine kinase inhibitor combined administration group (group 3) had an average tumor volume of 918.4±206.16mm ^{3 on} the 14th day after administration, which was significantly different from the control group (In group 1, the tumor volume on day 14 was 2020.2±177.82mm ³ , TGI%=57%, p=0.0003).

The EZH2 inhibitor, anti-PD-1 antibody and multi-target tyrosine kinase inhibitor combination group (group 4) had an average tumor volume of 425.5±110.32mm ^{3 on} the 14th day after administration, which was significantly higher than that of the control group. Significant difference (group 1, tumor volume on day 14 is 2020.2±177.82mm ³ , TGI%=82%, p<0.0001). Compared with the EZH2 inhibitor alone group (group 2, p<0.0001) and the anti-PD-1 antibody & multi-target tyrosine kinase inhibitor group (group 3), the three-drug combination has anti-tumor effect More significant (p<0.0001 and p=0.0486).

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Claims (17)

The use of an EZH2 inhibitor, an immune checkpoint inhibitor, and a multi-target tyrosine kinase inhibitor in the preparation of a drug for treating tumors. The use according to claim 1, wherein the EZH2 inhibitor is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

The use according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof. The use according to claim 3, wherein the light chain variable region of the anti-PD-1 antibody comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively The heavy chain variable region of the PD-1 antibody comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively. The use according to claim 3, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is selected from humanized antibodies or fragments thereof. The use according to claim 4 or 5, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype, preferably comprising IgG1 or IgG4 isotype Constant region of the heavy chain. The use according to claim 5, wherein the light chain variable region sequence of the humanized antibody is the sequence shown in SEQ ID NO: 10 or a variant thereof, and the heavy chain variable region sequence of the humanized antibody It is the sequence shown in SEQ ID NO: 9 or a variant thereof. The use according to claim 5, wherein the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8 or a variant thereof; the heavy chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: The sequence shown in 7 or its variants. The use according to claim 5, wherein the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8 and the heavy chain sequence is the sequence shown in SEQ ID NO: 7. The use according to claim 1, wherein the multi-target tyrosine kinase inhibitor is a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

The use according to claim 10, wherein the multi-target tyrosine kinase inhibitor is the compound shown in (II) malate. The use according to any one of claims 2 to 11, wherein the EZH2 inhibitor is a compound represented by formula (I). The use according to any one of claims 1 to 12, wherein the tumor is selected from the group consisting of adrenocortical cancer, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, cerebellar astrocytoma, brain astrocytoma , Basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma, bladder cancer, bone and joint cancer, osteosarcoma, malignant fibrous histiocytoma, brain cancer, brain tumor, Brainstem glioma, ependymoma, medulloblastoma, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, nervous system cancer, nervous system lymphoma, central nervous system cancer, central nervous system lymphoma Tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disease, colon cancer, colorectal cancer, skin T-cell lymphoma, lymphoma, mycosis fungoides, Sezary syndrome, endometrial Cancer, food Tube cancer, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST), germ cell tumor , Ovarian germ cell tumor, head and neck cancer, hepatocellular carcinoma, Hodgkin’s lymphoma, islet cell tumor, Kaposi’s sarcoma, kidney cancer, laryngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, hairy cell leukemia, lip and Oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstroem macroglobulinemia, melanoma, mesothelioma, metastatic squamous cells Cancer, tongue cancer, multiple endocrine tumor syndrome, myelodysplastic syndrome, multiple myeloma, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian low-malignant potential tumors, pancreas Cancer, islet cell pancreatic cancer, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal tumor, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, prostate cancer, Rectal cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Juventus sarcoma, Kaposi's sarcoma, synovial sarcoma, uterine cancer, uterine sarcoma, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma , Supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma, urethral cancer, endometriosis, vaginal cancer, vulvar cancer or Wilm’s tumor, preferably colorectal cancer, colon cancer. The use according to any one of claims 1 to 13, wherein the dose of the EZH2 inhibitor is selected from 1 to 1600 mg, and the frequency of administration is 2 times a day, once a day, once every 2 days, and once every 3 days , Once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, preferably 2 times a day or once a day. The use according to claim 14, wherein the dose of the immune checkpoint inhibitor is selected from 0.1-10.0mg/kg or 1-1000mg, and the frequency of administration is once a week, once every 2 weeks, Once every 3 weeks, once every 4 weeks, once a month, once every 3-6 months, preferably once every 2 weeks or once every 3 weeks. The use according to claim 15, wherein the dose of the multi-target tyrosine kinase inhibitor is 0.1-1000 mg or 0.1-10 mg/kg, and the frequency of administration is once a day, twice a day, or 3 times a day, which is more Good 1 time a day. The use according to claim 16, wherein the dose of the multi-target tyrosine kinase inhibitor is selected from 15mg, 20mg, 25mg, and the frequency of administration is once a day, twice a day or 3 times a day, Preferably once a day.

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