WO2020063863A1 - Ezh2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途 - Google Patents

Ezh2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途 Download PDF

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WO2020063863A1
WO2020063863A1 PCT/CN2019/108535 CN2019108535W WO2020063863A1 WO 2020063863 A1 WO2020063863 A1 WO 2020063863A1 CN 2019108535 W CN2019108535 W CN 2019108535W WO 2020063863 A1 WO2020063863 A1 WO 2020063863A1
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cancer
antibody
once
tumor
inhibitor
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French (fr)
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张岚
蒋家骅
廖成
张连山
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Jiangsu Hengrui Medicine Co Ltd
Suzhou Suncadia Biopharmaceuticals Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
Suzhou Suncadia Biopharmaceuticals Co Ltd
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4433—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with oxygen as a ring hetero atom
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol

Definitions

  • the present disclosure relates to the use of an EZH2 inhibitor combined with an immune checkpoint inhibitor in the manufacture of a medicament for treating tumors.
  • Protein programmed death 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 (Agata et al., Supra; Okazaki et al. (2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170: 711-8). The original members of the family, CD28 and ICOS (Hutloff et al. (1999) Nature397: 263-266; Hansen et al.
  • PD-1 was discovered by screening for differential expression in apoptotic cells (Ishida et al. (1992) EMBOJ11: 3887-95).
  • Nivolumab by Ono and Pembrolizumab by Merck have been successfully approved for marketing for the treatment of unresectable or metastatic melanoma, non-small cell lung cancer, advanced renal cell carcinoma, Hodgkin lymphoma, recurrent or metastatic Squamous cell carcinoma.
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (DCs).
  • the expression of PD-L1 on activated cells can be up-regulated.
  • PD-L1 inhibits the immune system by combining with PD-1 and B7-1.
  • Many tumor cells and immune cells in the tumor tissue microenvironment express PD-L1.
  • New research finds high PD-L1 in human tumor tissues such as breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer Protein expression, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients.
  • WO2017084495 discloses a series of PD-L1 antibodies, which can effectively improve the effect of suppressing tumorigenesis and development.
  • the histone methyltransferase encoded by the EZH2 gene is a catalytic component of the polycomb inhibitory complex 2 (PRC2).
  • PRC2 polycomb inhibitory complex 2
  • EZH2 levels are abnormally elevated in cancer tissues, while EZH2 expression levels are highest in advanced or poor prognosis of cancer.
  • overexpression of EZH2 occurs simultaneously with amplification of the EZH2 gene.
  • si / shRNA experiments have found that reducing EZH2 expression in tumor cell lines can inhibit tumor cell proliferation, migration and invasion or angiogenesis, and cause apoptosis.
  • Tazemetostat developed by Eisai is used to treat non-Hodgkin B-cell lymphoma. It is currently in the clinical stage II.
  • -1205 is used for the treatment of B-cell lymphoma, which is currently in the clinical stage I.
  • GSK-2816126 developed by GlaxoSmithKline is used to treat diffuse large B-cell lymphoma and follicular lymphoma, which is currently in the clinical stage I
  • T cell infiltration selectively interacts with high EZH2-PRC2 complex activity in human skin melanoma Related.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • EZH2 inactivation reversed this resistance and inhibited melanoma growth in synergy with anti-CTLA-4 and IL-2 immunotherapy.
  • EZH2 is used as a molecular switch to control melanoma escape during T-cell targeted immunotherapy (Cells Reports, Volume 20, Issue 4, 25 July 2017, Pages 854-867).
  • the present disclosure provides the use of an EZH2 inhibitor combined with an immune checkpoint inhibitor in the manufacture of a medicament for treating tumors.
  • the EZH2 inhibitor may be a polypeptide, including but not limited to a peptide or an antibody.
  • the EZH2 inhibitor may also be a small molecule, and is specifically selected from CPI-0209, CPI-1205, GSK126, valemetostat, tazemetostat, PF-06821497, DS- 3201GSK-2816126, 3-deazaneplanocin A, HKMT-I-005, KM-301 or a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof, preferably a compound represented by formula (I) or a complex thereof or Its pharmaceutically acceptable salts,
  • the immune checkpoint inhibitors described in this disclosure may be selected from the group consisting of a programmed death-1 (PD-1) receptor inhibitor, a programmed death ligand-1 (PD-L1) inhibitor, or a cytotoxic-T-lymph Cell-associated protein-4 (CTLA-4) inhibitor.
  • PD-1 programmed death-1
  • PD-L1 programmed death ligand-1
  • CTLA-4 cytotoxic-T-lymph Cell-associated protein-4
  • the PD-1 receptor inhibitor described in the present disclosure is an antibody or an antigen-binding fragment thereof that specifically binds PD-1 and inhibits PD-1 activity;
  • the PD-L1 inhibitor is An antibody or antigen-binding fragment thereof capable of specifically binding PD-L1 and inhibiting PD-L1 activity;
  • the CTLA-4 inhibitor is an antibody or antigen-binding thereof capable of specifically binding CTLA-4 and inhibiting CTLA-4 activity Fragment.
  • the antibody or antigen-binding fragment thereof that specifically binds PD-1 and inhibits PD-1 activity described in the present disclosure is an anti-PD-1 antibody or an antigen-binding fragment thereof.
  • the antibodies or antigen-binding fragments thereof that can specifically bind PD-L1 and inhibit the activity of PD-L1 described in the present disclosure are anti-PD-L1 antibodies or antigen-binding fragments thereof.
  • the antibody or antigen-binding fragment thereof that specifically binds CTLA-4 and inhibits CTLA-4 activity described in the present disclosure is an anti-CTLA-4 antibody or antigen-binding fragment thereof.
  • the anti-PD-1 antibodies provided in this disclosure may be selected from sintilimab, cemipilimab, pembrolizumab, tisselizumab, nivolumab, JS-001, AK-103, dostarlimab, PD1-PIK, GLS-010, geneolimzumab, BI- 754091, spartalizumab, MGA-012, PF-06801591, XmAb-20717, CS-1003, Sym-021, AGEN-2034, MEDI-5752, MGD-013, AK-105, AK-104, BCD-100, PF- 06753512, HLX-10, AMP-224, LZM-009.
  • the anti-PD-L1 antibodies provided in this disclosure may be selected from the group consisting of avelumab, atezolizumab, durvalumab, CS-1001, M-7824, KL-A167, CX-072, BGB-A333, GNS-1480, CA-170, BMS-936559, preferably avelumab, atezolizumab, durvalumab.
  • the anti-CTLA-4 antibody provided in the present disclosure is selected from the group consisting of ipilimumab, vertremimumab, AGEN-1884, CS-1002, XmAb-20717, REGN-4659, BCD-145, MEDI-5752, AK-104, MK-1308, BMS-986249, BMS-986218, and PF-06753512, preferably ipilimumab and extremelumumab.
  • the light chain variable region of the PD-1 antibody described in the present disclosure 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 PD-1 antibody is a humanized antibody or a fragment thereof.
  • the anti-PD-1 antibody or antigen-binding fragment thereof described in the present disclosure is selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab ') 2 fragments of antibody fragments.
  • Immunoglobulins can be derived from any generally known isotype, including but not limited to IgA, secreted IgA, IgG, and IgM.
  • IgG subclasses are also well known to those skilled in the art and include, but are not limited to, IgG1, IgG2, IgG3, and IgG4.
  • Isotype refers to an Ab species or subclass (eg, IgM or IgG1) encoded by a heavy chain constant region gene.
  • 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 the IgG1 or IgG4 isotype Heavy chain constant region.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a light chain constant region of a light chain constant region of kappa or lambda.
  • the humanized antibody light chain variable region sequence is preferably a sequence as shown in SEQ ID NO: 10 or a variant thereof, and the variant preferably has a 0-10 amino acid change in the light chain variable region, more Preferably it is an amino acid change of A43S; the sequence of the humanized antibody heavy chain variable region is the sequence shown in SEQ ID NO: 9 or a variant thereof, and the variant preferably has 0-10 in the heavy chain variable region
  • the amino acid change is more preferably an amino acid change of G44R.
  • sequences of the heavy and light chains of the aforementioned humanized antibodies are as follows:
  • the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 8 or a variant thereof; the variant preferably has an amino acid change of 0-10 in the variable region of the light chain, more preferably A43S amino acid changes; the humanized antibody heavy chain sequence is the sequence shown in SEQ ID NO: 7 or a variant thereof, the variant preferably has a 0-10 amino acid change in the heavy chain variable region, more An amino acid change of G44R is preferred.
  • the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 8
  • the heavy chain sequence is the sequence shown in SEQ ID NO: 7.
  • sequences of the heavy and light chains of the humanized antibody are as follows:
  • the heavy chain variable region of the PD-L1 antibody or antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11-13, respectively, the PD-L1 antibody or antigen
  • the light chain variable region of the binding fragment comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 14-16, respectively;
  • HCDR1 is selected from:
  • HCDR2 is selected from:
  • HCDR3 is selected from:
  • LCDR1 is selected from:
  • LCDR2 is selected from:
  • LCDR3 is selected from:
  • the PD-L1 antibody or antigen-binding fragment comprises an amino acid sequence: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain variable region CDR sequences, and amino acids Sequence: SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable region CDR sequences.
  • the PD-L1 antibody or antigen-binding fragment may be selected from a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, preferably a humanized antibody.
  • the PD-L1 antibody or antigen-binding fragment comprises and the amino acid sequence SEQ ID NO: 17 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable region sequences, and the amino acid sequence SEQ ID NO: 18 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain variable region sequences.
  • the PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human IgG2 or IgG4 heavy chain constant region More preferably, the IgG4 heavy chain constant region comprises F234A and L235A mutations; the humanized antibody light chain further comprises a constant region of a human-derived kappa, lambda chain, or a variant thereof.
  • the PD-L1 antibody or antigen-binding fragment comprises and the amino acid sequence SEQ ID NO: 19 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain sequence, and the amino acid sequence SEQ ID NO: 21 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain sequences.
  • the heavy chain sequence of the PD-L1 antibody or antigen-binding fragment is SEQ ID NO: 19, and the light chain sequence is SEQ ID NO: 21.
  • EZH2 inhibitors and immune checkpoint inhibitors provided in the present disclosure can adjust the dosage regimen to provide the optimal desired response, for example, maximum therapeutic response and / or minimal adverse effects.
  • the EZH2 inhibitor in the present disclosure is a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof.
  • the EZH2 inhibitor can be used with a uniform dose or a weight-based dose.
  • the EZH2 inhibitor is administered as a uniform dose, specifically selected from 1-1600 mg, 10-800 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 , 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, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg,
  • the dose in order to administer an anti-PD-1 antibody or antigen-binding fragment thereof, may be in the following range: 0.1-10.0 mg / kg, 0.1-5 mg / kg, 1-5 mg / kg, 2- 5mg / kg, for example, the specific dose may 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, for example,
  • the dose may also be in the range of 1-1000 mg, 80-800 mg, 80-700 mg, 80-600 mg, 80-500 mg, 80-400 mg, 80-300 mg in order to administer an anti-PD-1 antibody. , 100-300mg or 200-300mg.
  • the specific dose can be selected from 1.0mg, 1.2mg, 1.4mg, 1.6mg, 1.8mg, 2.0mg, 2.2mg, 2.4mg, 2.6mg, 2.8mg, 3.0mg, 3.2mg, 3.4mg, 3.6mg, 3.8mg, 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 ,
  • An exemplary treatment regimen for the anti-PD-1 antibody or antigen-binding fragment thereof in the present disclosure requires once a week, once every two weeks, once every three weeks, once every four weeks, once a month, every 3-6 Dosing at monthly or longer dosing.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered every 2 weeks.
  • the antibody is administered once every three weeks. The dose may vary during the course of treatment.
  • the dosage regimen of an anti-PD-1 antibody or antigen-binding fragment thereof described in the present disclosure comprises intravenous or subcutaneous or intraperitoneal administration, 0.3-10 mg / kg, 1-5 mg / kg, or 1- 3 mg / kg, or 80-800 mg, the antibody is administered every 14-21 days over a period of up to 6 or 12 weeks until a complete response or confirmation of progressive disease.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is administered at 200 mg every 2 weeks.
  • any combination therapy disclosed in the present disclosure lasts for at least 3 weeks, or at least 1 month, or at least 3 months, or at least 6 months, or 9 months, or at least 1 year, or at least 18 Month, or at least 24 months, or at least 3 years, or at least 5 years.
  • the dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 200-300 mg
  • the frequency of administration is once every two or three weeks
  • the dose of the EZH2 inhibitor is 10-800 mg.
  • the frequency is once a day or twice a day.
  • the dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 200 mg
  • the frequency of administration is once every two or three weeks
  • the dose of EZH2 inhibitor is 10-800 mg
  • the frequency of administration is Once a day or twice a day.
  • the dose of the anti-PD-1 antibody or antigen-binding fragment thereof is 200 mg
  • the frequency of administration is once every 2 weeks or once every 3 weeks
  • the dose of the EZH2 inhibitor is 50 mg, 100 mg, 200 mg, 250 mg, 300mg, 350mg, 400mg, 450mg or 800mg
  • the frequency of administration is once a day or twice a day.
  • the PD-L1 antibody or antigen-binding fragment dose is selected from 50-3000 mg, preferably 180 mg, 225 mg, 600 mg, 750 mg, 1200 mg, 1500 mg, more preferably 600 mg, 750 mg, and the PD-L1 antibody.
  • the frequency of administration of the antigen-binding fragment may be once every week, once every two weeks, once every three weeks or once every four weeks.
  • the anti-PD-L1 antibody or antigen-binding fragment is administered every two weeks at a dose of 600 mg or 750 mg.
  • the dose of the anti-PD-L1 antibody or antigen-binding fragment is selected from 50-3000 mg, and the frequency of administration can be once every two or three weeks; the dose of EZH2 inhibitor is 10-800 mg, and the frequency of administration is Once a day or twice a day.
  • the dose of the anti-PD-L1 antibody or antigen-binding fragment is selected from 180 mg, 225 mg, 600 mg, 750 mg, 1200 mg, and 1500 mg, and the frequency of administration may be once every two weeks and once every three weeks; the dose of the EZH2 inhibitor is 50mg, 100mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg or 800mg, the frequency of administration is once a day or twice a day.
  • the dose of the anti-PD-L1 antibody or antigen-binding fragment is selected from 600 mg, 750 mg, and the frequency of administration is once every two weeks; the EZH2 inhibitor dose is 50 mg, 100 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg Or 800 mg twice daily.
  • the tumor described in the present disclosure is selected from the group consisting of adrenocortical cancer, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, cerebellar astrocytoma, cerebral astrocytoma, basal cell cancer, and skin cancer (non-melanoma).
  • Biliary tract cancer extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, bone and joint cancer, osteosarcoma, malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, ependymal tumor, neural tube Cell tumor, 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 myeloma Cell 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 tumor, gonadal External germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma,
  • the tumor described in the present disclosure refers to the aforementioned tumor that is resistant to the treatment of a single EZH2 inhibitor or an immune checkpoint inhibitor, and the immune checkpoint inhibitor is selected from the PD-1 receptor Inhibitor, PD-L1 inhibitor or CTLA-4 inhibitor.
  • the present disclosure provides the use of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof and an immune checkpoint inhibitor in the preparation of a medicament for treating tumors.
  • the present disclosure provides the use of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof in combination with the above-mentioned anti-PD-1 antibody or an antigen-binding fragment thereof in the manufacture of a medicament for treating tumors.
  • the present disclosure provides the use of a compound represented by formula (I) or a complex thereof or a pharmaceutically acceptable salt thereof and the above-mentioned anti-PD-L1 antibody or an antigen-binding fragment thereof in the manufacture of a medicament for treating tumors.
  • Routes of administration of immune checkpoint inhibitors in the present disclosure include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.
  • the "parenteral administration” refers to an administration mode other than enteral and local administration by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, Intracystic, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, and intracorporeal electricity perforation.
  • the immune checkpoint inhibitor (eg, an anti-PD-1 antibody) is administered by a non-parenteral route, and in certain embodiments, is administered orally.
  • non-parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasally, vaginally, rectally, sublingually or locally.
  • compositions for example, a pharmaceutical composition containing an antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier for the antibody-containing composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, abdominal, spinal or epidermal administration (e.g., by injection or infusion), and the pharmaceutical composition of the present disclosure may include One or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers, and / or adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents.
  • the PD-1 antibody or antigen-binding fragment thereof is administered by injection, for example, subcutaneously or intravenously or intraperitoneally. Before injection, the PD-1 antibody or antigen-binding fragment thereof is required.
  • An injectable form of an anti-PD-1 antibody in a particularly preferred embodiment of the present disclosure is an injection or a lyophilized powder injection, which comprises a PD-1 antibody or an antigen-binding fragment thereof, a buffer, a stabilizer, and optionally a surface Active agent.
  • the buffer may 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, 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 Of these, polysorbate 20 is most preferred.
  • the most preferred injectable form of the PD-1 antibody or antigen-binding fragment thereof comprises the PD-1 antibody or antigen-binding fragment thereof, an acetate buffer, trehalose, and polysorbate 20.
  • the EZH2 inhibitor described in this disclosure may be a hydrochloride, a phosphate, a hydrogen phosphate, a sulfate, a hydrogen sulfate, a sulfite, an acetate, an oxalate, a malonate, a valerate, a valley Carbamate, oleate, palmitate, stearate, laurate, borate, p-toluenesulfonate, mesylate, isethionate, maleate, malate Salt, tartrate, benzoate, paraben, salicylate, vanillate, mandelate, succinate, gluconate, lactobionate or laurylsulfonate.
  • the route of administration of the EZH2 inhibitor described in the present disclosure may be the same as or different from the above immune checkpoint inhibitors, and specifically includes oral, nasal, topical, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other gastrointestinal External application route.
  • the EZH2 inhibitor is administered orally and is formulated in the form of a composition.
  • the carrier of the composition includes, but is not limited to, a filler, a lubricant, a disintegrant, an adhesive, and the like.
  • the present disclosure provides a method for treating a tumor, comprising administering to a patient a therapeutically effective amount of the above-mentioned EZH2 inhibitor and an immune checkpoint inhibitor.
  • the tumor in the method for treating a tumor refers to the above-mentioned tumor that is resistant to the treatment of a single EZH2 inhibitor or an immune checkpoint inhibitor.
  • the method includes administering to a patient a therapeutic effect. Amounts of the above EZH2 inhibitors and immune checkpoint inhibitors.
  • the EZH2 inhibitor and the immune checkpoint inhibitor are administered simultaneously; in one embodiment, the EZH2 inhibitor and the immune checkpoint inhibitor are administered sequentially; in one embodiment, the EZH2 inhibitor The agent is administered before the immune checkpoint inhibitor.
  • the method of treating tumors provided by this disclosure in one embodiment, is administered parenterally with an immune checkpoint inhibitor.
  • the EZH2 inhibitor is administered orally and the immune checkpoint inhibitor is administered intravenously.
  • the PD-L1 antibody or antigen-binding fragment thereof is administered by injection, such as subcutaneously or intravenously.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an EZH2 inhibitor, an immune checkpoint inhibitor, and one or more pharmaceutically acceptable carriers, excipients, and diluents.
  • the pharmaceutical composition can be made into any pharmaceutically acceptable dosage form. For example, they can be formulated as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injections and concentrated solutions for injections), suppositories, inhalants or sprays Agent.
  • frequency of administration means the frequency of the administered dose of a drug disclosed herein at a given time.
  • the frequency of dosing can be indicated as the number of doses per given time, for example, once a week, once every two weeks.
  • flat dose refers to the dose administered to a patient regardless of the patient's weight or body surface area (BSA). For example, a 60 kg person and a 100 kg person will receive the same dose of antibody (eg, 240 mg of anti-PD-1 antibody).
  • BSA body surface area
  • weight-based dose refers to a dose calculated to be administered to a patient based on the weight of the patient. For example: 10.0mg / kg refers to 10.0mg per kg based on the weight of the subject.
  • the “combination” described in the present disclosure is a mode of administration, which means that at least one dose of an immune checkpoint inhibitor and an EZH2 inhibitor is administered within a certain period of time, and both of the drugs show a pharmacological effect.
  • the time period may be within a dosing cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours.
  • the immune checkpoint inhibitor and the EZH2 inhibitor can be administered simultaneously or sequentially. This term includes treatment in which immune checkpoint inhibitors and EZH2 inhibitors are administered by the same route or different routes of administration.
  • 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 Antibodies produced in human germline antibody framework sequences. It can overcome the strong antibody variable antibody response induced by the chimeric antibody because it carries a large amount of mouse protein components.
  • Such framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references.
  • germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc. People, 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the CDR sequence of the PD-1 humanized antibody is selected from the group consisting of SEQ ID NO: 1,2,3,4,5,6.
  • the “antigen-binding fragment” described in the present disclosure refers to a Fab fragment, Fab ′ fragment, F (ab ′) 2 fragment, and Fv fragment sFv fragment that binds to human PD-1;
  • the antibody is selected from one or more CDR regions of SEQ ID NO: 1 to SEQ ID NO: 6.
  • the Fv fragment contains the variable region of the heavy chain and light chain of the antibody, but has no constant region and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called single chain antibodies (single chain antibodies) or single chain Fv (sFv).
  • binding to PD-1 in this disclosure refers to the ability to interact with human PD-1.
  • antigen-binding site in the present disclosure refers to a three-dimensional spatial site on the antigen that is discontinuous and is recognized by the antibody or antigen-binding fragment in the present disclosure.
  • T / C (%) (Ti-T0) / (Ci-C0) * 100%;
  • Ti and Ci are the average tumor volume of the treatment group and the control day, respectively;
  • T0 and C0 are the treatment group and the control group, respectively Day 0 Mean tumor volume.
  • TGI Tumor growth inhibition
  • % TGI (1- (Ti-T0) / (Ci-C0)) * 100%; Ti and Ci are the average tumor volume on the day of the treatment group and control group; T0 and C0 are the treatment group and control group, respectively Day 0 Mean tumor volume.
  • FIG. 1 Effect of different drugs combined on body weight of B16F10 tumor-bearing mice.
  • Example 1 Evaluation of the inhibitory effect of the compound represented by formula (I) (drug A) and anti-PD-1 (drug B) antibody on the growth of huPD-1 humanized BALB / c mice CT-26 colon cancer xenografts
  • mice PD-1HuGEMM mice, 42 females, 4-8 weeks old.
  • the animals used were from Nanjing University-Nanjing Institute of Biomedicine.
  • Mouse-derived colon cancer cells CT-26 WT were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells were digested and passaged with trypsin containing EDTA as usual, passaged twice a week, and placed at 37 ° C, The culture was continued in a 5% CO 2 incubator. Tumor cells in the logarithmic growth phase will be used to establish a model of transplanted tumor in vivo.
  • Drug A prepared according to the method provided in patent application WO2017084494A;
  • Drug B prepared according to the method of patent application WO2017054646A, 200mg, lyophilized powder injection;
  • hIgG negative control, derived from Shanghai Hengrui Pharmaceutical Co., Ltd., diluted with PBS.
  • CT-26 WT cells resuspended in PBS were inoculated subcutaneously in the right flank of PD-1HuGEMM BALB / c mice at a concentration of 5 ⁇ 10 6 cells / 0.1 mL at a volume of 0.1 mL / head. A total of 42 cells were transferred. mouse. When the average tumor volume reached about 100 mm 3 , select mice with moderate tumor volume for each group, 7 mice in each group, and start the administration on the day of the group. The specific dosing schedule is shown in Table 1 below.
  • i.p . intraperitoneal injection
  • p.o . oral gavage
  • BID twice daily
  • Q3D once every three days.
  • Example 2 Efficacy of a combination of a compound represented by formula (I) (drug A) and an anti-PD-1 antibody (drug B) on a subcutaneous transplantation tumor in a mouse melanoma B16F10 model tumor-bearing mouse
  • hIgG negative control, derived from Shanghai Hengrui Pharmaceutical Co., Ltd., diluted with PBS.
  • Drug A is prepared with 0.5% CMC-Na 1% Tween 80 solution
  • Drug B is prepared with 5% glucose solution and diluted with PBS.
  • mice PD-1HuGEMM mice, 6-7 weeks old, tadpoles, purchased from Nanjing Yinhe Biological Medicine Co., Ltd.
  • B16F10 mouse-derived melanoma cells were cultured in RPMI1640 medium containing 10% fetal bovine serum.
  • B16F10 cells resuspended in PBS were inoculated at a concentration of 5 ⁇ 10 5 cells / 0.1 mL in a volume of 0.1 mL / head to PD-1HuGEMM C57BL / 6 mice were subcutaneously in the right posterior flank. After the tumors grew to about 100 mm 3 , the animals were randomly divided into groups (D0) and administered. See Table 3 for the doses and schedules. The tumor volume is measured 2-3 times a week, the weight of the mouse is weighed, and the data is recorded. The combined effect on the subcutaneous transplantation tumor of B16F10 tumor-bearing huPD1 mice is shown in Table 4.
  • i.p . intraperitoneal injection
  • p.o . oral gavage
  • BID twice daily
  • Q3D once every three days.

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Abstract

一种EZH2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途。具体而言，所述的EZH2抑制剂为式（I）所示化合物或其可药用盐，所述的免疫检查点抑制剂选自PD-1抑制剂、PD-L1抑制剂或CTLA-4抑制剂。

Description

EZH2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途 技术领域
本公开中涉及一种EZH2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途。
背景技术
蛋白质程序性死亡1(PD-1)是CD28受体家族的抑制性成员，该家族还包括CD28、CTLA-4、ICOS和BTLA。PD-1在活化的B细胞、T细胞和髓样细胞上表达(Agata等人，同下；Okazaki等人(2002)Curr.Opin.Immunol.14:391779-82；Bennett等人(2003)J Immunol170:711-8)。根据加入单克隆抗体后对提升T细胞增殖的功能性影响发现了家族最初的成员，CD28和ICOS(Hutloff等人(1999)Nature397:263-266；Hansen等人(1980)Immunogenics10:247-260)。通过在凋亡细胞中筛选差异表达发现了PD-1(Ishida等人(1992)EMBOJ11:3887-95)。目前，已有小野公司的Nivolumab及Merck公司的Pembrolizumab成功获批上市，用于治疗不可切除或转移性黑色素瘤、非小细胞肺癌、晚期肾细胞癌、霍奇金淋巴瘤、复发性或转移性鳞状细胞癌等。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell,DC)上，在活化后细胞上的表达能够进行上调。PD-L1通过和PD-1及B7-1的结合抑制免疫系统，很多肿瘤细胞及肿瘤组织微环境的免疫细胞表达PD-L1。新的研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤、非小细胞肺癌、结肠癌、膀胱癌、卵巢癌、胰腺癌及肝癌等人类肿瘤组织中检测到高PD-L1蛋白的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。WO2017084495公开了一系列PD-L1抗体，能够有效地提高抑治肿瘤发生和发展的效果。
EZH2基因编码的组蛋白甲基转移酶是多梳蛋白抑制性复合体2(PRC2)的催化组分。与正常组织相比，EZH2水平在癌组织异常升高，而在癌症晚期或不良预后中，EZH2的表达水平最高。在一些癌症类型中，EZH2表达过剩与EZH2基因的扩增同时发生。大量si/shRNA实验研究发现在肿瘤细胞系中减少EZH2表达，可抑制肿瘤细胞的增殖，迁移和侵袭或血管生成，并导致细胞凋亡。
目前已有进入临床开发阶段的EZH2抑制剂，以下简要列举，卫材开发的Tazemetostat(EPZ-6438)用于治疗非霍奇金B细胞淋巴瘤，目前处于临床Ⅱ期阶段，Constellation公司开发的CPI-1205用于治疗B细胞淋巴瘤，目前处于临床Ⅰ期阶段，葛兰素史克公司开发的GSK-2816126用于治疗弥漫大B细胞淋巴瘤、滤泡性淋巴瘤，目前处于临床Ⅰ期阶段
Figure PCTCN2019108535-appb-000001
PCT申请WO2017084494A中提供了一种EZH2抑制剂，结构如下所示：
Figure PCTCN2019108535-appb-000002
Lu Gan等的综述中提到了EZH2在肿瘤免疫中的新兴作用(Biomarker Research，December 2018,6:10)。
Russell W Jenkins等通过对新出现的临床和临床前数据，确定先天和获得性免疫检查点抑制的新机制(British Journal of Cancer volume 118,pages 9–16(2018))。
DanielZingg等发现诱导组蛋白甲基转移酶EZH2控制几种肿瘤细胞的内在和外在抗性机制，值得注意的是，T细胞浸润选择性地与人皮肤黑色素瘤中的高EZH2-PRC2复合物活性相关。在小鼠抗CTLA-4或IL-2免疫治疗期间，肿瘤内肿瘤坏死因子-α(TNF-α)的产生和T细胞的积累导致黑色素瘤细胞中EZH2表达增加，从而沉默其自身的免疫原性和抗原呈递。EZH2失活逆转了这种抗性并与抗CTLA-4和IL-2免疫疗法协同抑制黑色素瘤的生长。这些抗肿瘤作用依赖于肿瘤内积聚干扰素-γ(IFN-γ)产生PD-1 low CD8 +T细胞和PD-L1下调黑色素瘤细胞。因此，EZH2在T细胞靶向免疫疗法期间用作控制黑色素瘤逃逸的分子开关(Cells Reports,Volume 20,Issue 4,25 July 2017,Pages 854-867)。
发明内容
本公开提供一种EZH2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途。
本公开中EZH2抑制剂可以是多肽，包括但不限于肽或抗体，EZH2抑制剂也可以是小分子，具体选自CPI-0209、CPI-1205、GSK126、valemetostat、tazemetostat、PF-06821497、DS-3201GSK-2816126、3-deazaneplanocin A、HKMT-I-005、KM-301或式(I)所示化合物或其复合物或其可药用盐，优选式(I)所示化合物或其复合物或其可药用盐，
Figure PCTCN2019108535-appb-000003
本公开中所述的免疫检查点抑制剂可选自程序性死亡-1(PD-1)受体抑制剂、程序性死亡配体-1(PD-L1)抑制剂或细胞毒性-T-淋巴细胞相关蛋白-4(CTLA-4)抑制剂。
在一些实施方案中，本公开中所述的PD-1受体抑制剂为可特异性的结合PD-1并抑制PD-1活性的抗体或者其抗原结合片段；所述PD-L1抑制剂为可特异性地结合PD-L1并抑制PD-L1活性的抗体或其抗原结合片段；所述CTLA-4抑制剂为可特异性的结合CTLA-4并抑制CTLA-4活性的抗体或其抗原结合片段。
本公开中所述的可特异性的结合PD-1并抑制PD-1活性的抗体或者其抗原结合片段即为抗PD-1抗体或其抗原结合片段。
本公开中所述的可特异性地结合PD-L1并抑制PD-L1活性的抗体或其抗原结合片段即为抗PD-L1抗体或其抗原结合片段。
本公开中所述的可特异性的结合CTLA-4并抑制CTLA-4活性的抗体或其抗原结合片段即抗CTLA-4抗体其抗原结合片段。
在一些实施方案中，本公开中提供的抗PD-1抗体可选自sintilimab、cemiplimab pembrolizumab、tislelizumab、nivolumab、JS-001、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-105、AK-104、BCD-100、PF-06753512、HLX-10、AMP-224、LZM-009。
在一些实施方案中，本公开中提供的抗PD-L1抗体可选自avelumab、atezolizumab、durvalumab、CS-1001、M-7824、KL-A167、CX-072、、BGB-A333、GNS-1480、CA-170、BMS-936559，优选avelumab、atezolizumab、durvalumab。
在一些实施方案中，本公开中提供的抗CTLA-4抗体选自ipilimumab、tremelimumab、AGEN-1884、CS-1002、XmAb-20717、REGN-4659、BCD-145、MEDI-5752、AK-104、MK-1308、BMS-986249、BMS-986218、PF-06753512，优选ipilimumab、tremelimumab。
在一些实施方案中，本公开所述的PD-1抗体的轻链可变区包含分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3，所述的PD-1抗体的重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
其中，前面所述的各CDR序列如下表所示：
名称 序列 编号
HCDR1 SYMMS SEQID NO：1
HCDR2 TISGGGANTYYPDSVKG SEQID NO：2
HCDR3 QLYYFDY SEQID NO：3
LCDR1 LASQTIGTWLT SEQID NO：4
LCDR2 TATSLAD SEQID NO：5
LCDR3 QQVYSIPWT SEQID NO：6
优选的，所述的PD-1抗体为人源化抗体或其片段。
在可选实施方案中，本公开中所述抗PD-1抗体或其抗原结合片段选自由Fab，Fab’-SH，Fv，scFv，和(Fab’)2片段组成的组的抗体片段。
免疫球蛋白可以来源于任何通常已知的同种型，包括但不限于IgA、分泌型IgA、IgG和IgM。IgG亚类也是本领域技术人员众所周知的，包括但不限于IgG1、IgG2、IgG3和IgG4。“同种型”是指由重链恒定区基因编码的Ab种类或亚类(例如，IgM或IgG1)。在一些可选实施方案中，本公开中所述抗PD-1抗体或其抗原结合片段包含人源IgG1、IgG2、IgG3或IgG4同种型的重链恒定区，优选包含IgG1或IgG4同种型的重链恒定区。
在另一些可选实施方案中，所述抗PD-1抗体或其抗原结合片段包含κ或λ的轻链恒定区的轻链恒定区。
进一步地，优选人源化抗体轻链可变区序列为如SEQ ID NO:10所示的序列或其变体，所述变体优选在轻链可变区有0-10的氨基酸变化，更优选为A43S的氨基酸变化；所述人源化抗体重链可变区序列为如SEQ ID NO:9所示的序列或其变体，所述变体优选在重链可变区有0-10的氨基酸变化，更优选为G44R的氨基酸变化。
前述的人源化抗体重、轻链的序列如下所示：
重链可变区
Figure PCTCN2019108535-appb-000004
轻链可变区
Figure PCTCN2019108535-appb-000005
优选的，所述人源化抗体轻链序列为如SEQ ID NO:8所示的序列或其变体；所述变体优选在轻链可变区有0-10的氨基酸变化，更优选为A43S的氨基酸变化；所述人源化抗体重链序列为如SEQ ID NO:7所示的序列或其变体，所述变体优选 在重链可变区有0-10的氨基酸变化，更优选为G44R的氨基酸变化。
在另一实施方案中，所述人源化抗体的轻链序列为如SEQ ID NO:8所示的序列，重链序列为如SEQ ID NO:7所示的序列。
所述人源化抗体重、轻链的序列如下所示：
重链
Figure PCTCN2019108535-appb-000006
轻链
Figure PCTCN2019108535-appb-000007
在某些实施方式中，所述PD-L1抗体或抗原结合片段的重链可变区包含分别如SEQ ID NO：11-13所示的HCDR1、HCDR2和HCDR3，所述PD-L1抗体或抗原结合片段的轻链可变区包含分别如SEQ ID NO：14-16所示的HCDR1、HCDR2和HCDR3；
具体如下：
HCDR1选自：
SYWMH                     SEQ ID NO：11
HCDR2选自：
RIGPNSGFTSYNEKFKN         SEQ ID NO：12
HCDR3选自：
GGSSYDYFDY                SEQ ID NO：13
LCDR1选自：
RASESVSIHGTHLMH           SEQ ID NO：14
LCDR2选自：
AASNLES                     SEQ ID NO：15
LCDR3选自：
QQSFEDPLT                   SEQ ID NO：16；
在某些实施方式中，所述PD-L1抗体或抗原结合片段包含和氨基酸序列：SEQ ID NO：14，SEQ ID NO：15，SEQ ID NO：16具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链可变区CDR序列，和氨基酸序列：SEQ ID NO：11，SEQ ID NO：12和SEQ ID NO：13具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区CDR序列。
在某些实施方式中，所述PD-L1抗体或抗原结合片段可选自鼠源抗体、嵌合抗体、人源化抗体，人抗体，优选人源化抗体。
在某些实施方式中，所述PD-L1抗体或抗原结合片段包含和氨基酸序列SEQ ID NO：17具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区序列，和氨基酸序列SEQ ID NO：18具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链可变区序列。
在某些实施方式中，所述的PD-L1抗体或抗原结合片段进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，更优选包含引入F234A和L235A突变的IgG4重链恒定区；所述人源化抗体轻链进一步包含人源κ、λ链或其变体的恒定区。
在某些实施方式中，所述PD-L1抗体或抗原结合片段包含和氨基酸序列SEQ ID NO：19具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链序列，和氨基酸序列SEQ ID NO：21具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链序列。
在某些实施方式中，所述的PD-L1抗体或抗原结合片段的重链序列为SEQ ID NO：19，轻链序列为SEQ ID NO：21。
Figure PCTCN2019108535-appb-000008
注：序列中斜体为FR序列；下划线为CDR序列。
重链序列
Figure PCTCN2019108535-appb-000009
重链序列编码基因序列
Figure PCTCN2019108535-appb-000010
Figure PCTCN2019108535-appb-000011
轻链序列
Figure PCTCN2019108535-appb-000012
轻链序列编码基因序列：
Figure PCTCN2019108535-appb-000013
Figure PCTCN2019108535-appb-000014
本公开中提供的EZH2抑制剂与免疫检查点抑制剂的联合可以调节剂量方案以提供最适期望应答，例如，最大治疗应答和/或最小不良作用。
在一些实施方案中，本公开中EZH2抑制剂为式(I)所示化合物或其复合物或其可药用盐。
在一些实施方案中，EZH2抑制剂可以与统一剂量或基于重量的剂量一起使用。在其它实施方案中，所述EZH2抑制剂作为统一剂量施用，具体选自1-1600mg、10-800mg，例如：10mg、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、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，优选50mg、100mg、200mg、250mg、300mg、350mg、400mg、450mg或800mg，给药频率为每天两次、每天一次、每两天一次、每三天一次、每四天一次、每五天一次、每六天一次、每周一次、每二周一次、每三周一次、每四周一次，优选每天两次或每天一次。
在某些实施方案中，为了施用抗PD-1抗体或其抗原结合片段，所述剂量可以在以下范围内：0.1-10.0mg/kg、0.1-5mg/kg、1-5mg/kg、2-5mg/kg，例如，具体剂量可以是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。
在某些实施方案中，为了施用抗PD-1抗体所述剂量也可以在以下范围内1-1000mg、80-800mg、80-700mg、80-600mg、80-500mg、80-400mg、80-300mg、100-300mg或200-300mg。具体剂量可以选自1.0mg、1.2mg、1.4mg、1.6mg、1.8mg、2.0mg、2.2mg、2.4mg、2.6mg、2.8mg、3.0mg、3.2mg、3.4mg、3.6mg、3.8mg、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，优选80mg、100mg、160mg、200mg、240mg、300mg、320mg、400mg、500mg、600mg、700mg或800mg，最优选200mg。
本公开中抗PD-1抗体或其抗原结合片段的一种示例性治疗方案需要以每周一次、每二周一次、每三周一次、每四周一次、1个月一次、每3-6个月或更长一次的给药频率施用。在优选的实施方案中，每2周一次将抗PD-1抗体或其抗原结合片段施用。在优选的实施方案中，每三周一次施用所述抗体。所述剂量可以在治疗过程中变化。
在一些实施方案中，本公开中所述的抗PD-1抗体或其抗原结合片段的剂量方案包含经由静脉或皮下或腹腔给药，0.3-10mg/kg、1-5mg/kg、或1-3mg/kg、或80-800mg，所述抗体在多达6周或12周的周期中每14-21天施用直到完全应答或证实进行性疾病。
在一些实施方案中，每2周以200mg施用抗-PD-1抗体或其抗原结合片段。
在一些实施方案中，本公开公开的任意组合治疗持续至少3周、或至少1个月、或至少3个月、或至少6个月、或9个月、或至少1年、或至少18个月、或至少24个月、或至少3年、或至少5年。
在一些实施方案中，本公开中抗PD-1抗体或其抗原结合片段剂量为200-300mg，给药频率为每二周一次或每三周一次，EZH2抑制剂剂量为10-800mg，给药 频率为每天一次或每天两次。
在一些实施方案中，本公开中抗PD-1抗体或其抗原结合片段剂量为200mg，给药频率为每二周一次或每三周一次，EZH2抑制剂剂量为10-800mg，给药频率为每天一次或每天两次。
在一些实施方案中，本公开中抗PD-1抗体或其抗原结合片段剂量为200mg，给药频率为每2周一次或每3周一次，EZH2抑制剂剂量为50mg、100mg、200mg、250mg、300mg、350mg、400mg、450mg或800mg，给药频率为每天一次或每天两次。
在一些实施方案中，所述的PD-L1抗体或抗原结合片段剂量选自50-3000mg，优选180mg、225mg、600mg、750mg、1200mg、1500mg，更优选600mg、750mg，所述的PD-L1抗体或抗原结合片段的给药频率可以是每一周一次、每二周一次、每三周一次或每四周一次。
在一些实施方案中，抗PD-L1抗体或抗原结合片段的给药频率为每二周给药一次，剂量为600mg或750mg。
在一些实施方案中，抗PD-L1抗体或抗原结合片段剂量选自50-3000mg，给药频率可以是每二周一次、每三周一次；EZH2抑制剂剂量为10-800mg，给药频率为每天一次或每天两次。
在一些实施方案中，抗PD-L1抗体或抗原结合片段剂量选自180mg、225mg、600mg、750mg、1200mg、1500mg，给药频率可以是每二周一次、每三周一次；EZH2抑制剂剂量为50mg、100mg、200mg、250mg、300mg、350mg、400mg、450mg或800mg，给药频率为每天一次或每天两次。
在一些实施方案中抗PD-L1抗体或抗原结合片段剂量选自600mg、750mg，给药频率是每二周一次；EZH2抑制剂剂量为50mg、100mg、200mg、250mg、300mg、350mg、400mg、450mg或800mg，给药频率为每天两次。
本公开中所述肿瘤是选自肾上腺皮质癌、肛门癌、肛门直肠癌、肛管癌、阑尾癌、小脑星形细胞瘤、脑星形细胞瘤、基底细胞癌、皮肤癌(非黑色素瘤)、胆道癌、肝外胆管癌、肝内胆管癌、膀胱癌、骨关节癌、骨肉瘤、恶性纤维组织细胞瘤、脑癌、脑肿瘤、脑干胶质瘤、室管膜瘤、成神经管细胞瘤、视觉通路和下丘脑神经胶质瘤、乳腺癌、支气管腺瘤、神经系统癌、神经系统淋巴瘤、中枢神经系统癌、中枢神经系统淋巴瘤、宫颈癌、慢性淋巴细胞白血病、慢性粒细胞白血病白血病、慢性骨髓增生性疾病、结肠癌、结直肠癌、皮肤T细胞淋巴瘤、淋巴肿瘤、蕈样真菌病、Sezary综合征、子宫内膜癌、食管癌、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、眼癌、眼内黑色素瘤、视网膜母细胞瘤、胆囊癌、胃癌、胃肠道类癌、胃肠道间质瘤(GIST)、生殖细胞肿瘤、卵巢生殖细胞瘤、头颈癌、肝细胞癌、霍奇金淋巴瘤、胰岛细胞瘤、卡波西肉瘤、肾癌、喉癌、急性淋巴细胞白血病、急性髓性白血病、毛细胞白血病、唇和口腔腔癌、肝癌、肺癌、非小 细胞肺癌、小细胞肺癌、非霍奇金淋巴瘤、原发性中枢神经系统淋巴瘤、Waldenstroem巨球蛋白血症、黑色素瘤、间皮瘤、转移性鳞癌、舌癌、多发性内分泌肿瘤综合征、骨髓增生异常综合征、多发性骨髓瘤、鼻咽癌、神经母细胞瘤、口咽癌、卵巢癌、卵巢上皮癌、卵巢低恶性潜能肿瘤、胰腺癌、胰岛细胞胰腺癌、鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体瘤、垂体瘤、浆细胞肿瘤、胸膜肺母细胞瘤、前列腺癌、直肠癌、肾盂和输尿管移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、尤文家族肉瘤、卡波西肉瘤、滑膜肉瘤、子宫癌、子宫肉瘤、小肠癌、软组织肉瘤、鳞状细胞癌、幕上原始神经外胚层肿瘤、睾丸癌、咽喉癌、胸腺瘤、尿道癌、子宫内膜异位症、阴道癌、外阴癌或威尔姆氏肿瘤，优选黑色素瘤或结肠癌。
在可选的实施方案中，本公开中所述的肿瘤是指对单一EZH2抑制剂或免疫检查点抑制剂治疗具有抗性的上述肿瘤，所述免疫检查点抑制剂选自上述PD-1受体抑制剂、PD-L1抑制剂或CTLA-4抑制剂。
本公开提供一种式(I)所示化合物或其复合物或其可药用盐和免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途。
本公开提供一种式(I)所示化合物或其复合物或其可药用盐与上述抗PD-1抗体或其抗原结合片段联合在制备治疗肿瘤的药物中的用途。
本公开提供一种式(I)所示化合物或其复合物或其可药用盐和上述抗PD-L1抗体或其抗原结合片段联合在制备治疗肿瘤的药物中的用途。
本公开中免疫检查点抑制剂的给药途径包括静脉内、肌肉内、皮下、腹膜内、脊柱或其它胃肠外施用途径，例如通过注射或输注。所述“胃肠外施用”是指，通过注射进行的除了肠内和局部施用以外的施用模式，且包括但不限于，静脉内、肌肉内、动脉内、鞘内、淋巴管内、病灶内、囊内、眶内、心内、真皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注、以及体内电穿孔。在某些实施方案中，所述免疫检验点抑制剂(例如抗-PD-1抗体)通过非胃肠外途径施用，在某些实施方案中，口服施用。其它非胃肠外途径包括局部、表皮或粘膜施用途径，例如，鼻内地、阴道地、直肠地、舌下地或局部地。
可以将本公开的免疫检查点抑制剂构成在组合物中，例如，含有抗体和药学上可接受的载体的药物组合物。本文中使用的“药学上可接受的载体”包括生理学上相容的任意的和所有的溶剂、分散介质、包衣剂、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。在一个实施方案中，用于含有抗体的组合物的载体适合静脉、肌肉、皮下、胃肠外、腹腔、脊柱或表皮施用(例如，通过注射或输注)，本公开的药物组合物可以包括一种或多种药学上可接受的盐、抗氧化剂、水性和非水性载体，和/或佐剂，诸如防腐剂、润湿剂、乳化剂和分散剂。
在本公开中优选的实施方案中，所述的PD-1抗体或其抗原结合片段以注射的 方式给药，例如皮下或静脉注射或腹腔，注射前需将PD-1抗体或其抗原结合片段配制成可注射的形式。本公开特别优选的实施方案中抗PD-1抗体的可注射形式是注射液或冻干粉针，其包含PD-1抗体或其抗原结合片段、缓冲剂、稳定剂，任选地还含有表面活性剂。缓冲剂可选自醋酸盐、柠檬酸盐、琥珀酸盐、以及磷酸盐中的一种或几种。稳定剂可选自糖或氨基酸，优选二糖，例如蔗糖、乳糖、海藻糖、麦芽糖。表面活性剂选自聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、聚氧乙烯山梨醇酐脂肪酸酯，优选所述聚氧乙烯山梨醇酐脂肪酸酯为聚山梨酯20、40、60或80，最优选聚山梨酯20。最为优选的PD-1抗体或其抗原结合片段的可注射形式包含PD-1抗体或其抗原结合片段、醋酸盐缓冲剂、海藻糖和聚山梨酯20。
本公开中所述EZH2抑制剂可以是盐酸盐、磷酸盐、磷酸氢盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、丙二酸盐、戊酸盐、谷氨酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、对甲苯磺酸盐、甲磺酸盐、羟乙基磺酸盐、马来酸盐、苹果酸盐、酒石酸盐、苯甲酸盐、双羟萘酸盐、水杨酸盐、香草酸盐、扁桃酸盐、琥珀酸盐、葡萄糖酸盐、乳糖酸盐或月桂基磺酸盐等。
本公开中所述EZH2抑制剂的给药途径可以是与上述免疫检查点抑制剂相同或者不同，具体包括包括口服、鼻腔、局部、静脉内、肌肉内、皮下、腹膜内、脊柱或其它胃肠外施用途径。优选的实施方案中所述EZH2抑制剂口服给药，且以组合物的形式成药，组合物的载体包括但不限于填充剂、润滑剂、崩解剂、粘合剂等。
本公开提供一种治疗肿瘤的方法，包括向患者施用治疗有效量的上述EZH2抑制剂和免疫检查点抑制剂。
在一个优选的方案中，本公开提供的治疗肿瘤的方法中所述肿瘤是指对单一EZH2抑制剂或免疫检查点抑制剂治疗具有抗性的上述肿瘤，所述方法，包括向患者施用治疗有效量的上述EZH2抑制剂和免疫检查点抑制剂。
本公开提供的方法，在一种实施方案中EZH2抑制剂和免疫检查点抑制剂同时施用；在一种实施方案中依次施用EZH2抑制剂和免疫检查点抑制剂；在一种实施方案中EZH2抑制剂在免疫检查点抑制剂之前施用。
本公开提供的治疗肿瘤的方法，在一种实施方案中免疫检查点抑制剂胃肠外施用。
本公开提供的治疗肿瘤的方法，在一种实施方案中口服施用EZH2抑制剂并静脉内施用免疫检查点抑制剂。
在某些实施方式中，所述的PD-L1抗体或其抗原结合片段以注射的方式给药，例如皮下或静脉注射。
本公开还涉及一种包含EZH2抑制剂、免疫检查点抑制剂，以及一种或多种药用载体、赋形剂、稀释剂的药物组合物。所述药物组合物可以制成药学上可接受的任一剂型。例如，可以配制为片剂、胶囊剂、丸剂、颗粒剂、溶液剂、混悬 剂、糖浆剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、栓剂、吸入剂或喷雾剂。
如无相反解释，本公开中术语具有如下含义：
本文中使用的术语“给药频率”表示在给定时间中本文公开的药物的施用剂量的频率。可以将给药频率指示为每个给定时间的剂量的数目，例如，一周一次、二周一次。
术语“统一剂量(flat dose)”的应用是指不考虑患者的重量或体表面积(BSA)施用给患者的剂量。例如，60kg人和100kg人将接受相同剂量的抗体(例如，240mg抗PD-1抗体)。
术语“基于重量的剂量”是指基于患者的重量计算出的、施用给患者的剂量。例如：10.0mg/kg是指基于给药对象的体重，每kg给药10.0mg。
本公开中所述的“联合”是一种给药方式，是指在一定时间期限内给予至少一种剂量的免疫检查点抑制剂和EZH2抑制剂，其中两种药物都显示药理学作用。所述的时间期限可以是一个给药周期内，优选4周内，3周内，2周内，1周内，或24小时以内。可以同时或依次给予免疫检查点抑制剂和EZH2抑制剂。这种期限包括这样的治疗，其中通过相同给药途径或不同给药途径给予给予免疫检查点抑制剂和EZH2抑制剂。
本公开中所述“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。在本公开中一个优选的实施方案中，所述的PD-1人源化抗体的CDR序列选自SEQ ID NO:1，2，3，4，5，6。
本公开中所述“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab‘片段，F(ab’)2片段，以及与人PD-1结合的Fv片段sFv片段；包含本公开中所述抗体的选自SEQ ID NO:1至SEQ ID NO:6中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本公开中的术语“与PD-1结合”，指能与人PD-1相互作用。本公开中的术语“抗原结合位点”指抗原上不连续的，由本公开中抗体或抗原结合片段识别的三维空间位点。
部分缩写词说明
肿瘤体积(TV)
肿瘤体积计算公式为TV＝0.5a*b 2，a，b分别代表肿瘤的长度和宽度。
平均肿瘤体积(MTV)
相对肿瘤增殖率(T/C)
T/C(％)＝(Ti-T0)/(Ci-C0)*100％；Ti和Ci分别为处理组和对照组Day i天的平均肿瘤体积；T0和C0分别为处理组和对照组Day 0天的平均肿瘤体积。
肿瘤生长抑制(TGI)
％TGI＝(1-(Ti-T0)/(Ci-C0))*100％；Ti和Ci分别为处理组和对照组Day i天的平均肿瘤体积；T0和C0分别为处理组和对照组Day 0天的平均肿瘤体积。
附图说明
附图1.不同药物联用在CT-26 WT模型中对肿瘤生长的影响；
附图2.不同药物联用对CT-26 WT荷瘤小鼠体重的影响；
附图3.不同药物联用在B16F10模型中对肿瘤生长的影响；
附图4.不同药物联用对B16F10荷瘤小鼠体重的影响。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制本公开的范围。
实施例1式(I)所示化合物(药物A)及抗PD-1(药物B)抗体对huPD-1人源化BALB/c小鼠CT-26结肠癌移植瘤生长的抑制作用评价
1.实验材料
实验动物：PD-1HuGEMM小鼠，42只，雌性，4-8周龄。使用动物来源于南京大学-南京生物医药研究院。
细胞：将鼠源性结肠癌细胞CT-26 WT培养于含有10％胎牛血清的RPMI1640培养基中，细胞按照常规使用含有EDTA的胰蛋白酶消化传代，每周传代两次，放置于37℃、5％CO 2培养箱中继续培养。对数生长期的肿瘤细胞将用于进行体内移植瘤模型的建立。
药物：
药物A：依据专利申请WO2017084494A中提供方法制备；
药物B：依据专利申请WO2017054646A的方法制备，200mg，冻干粉针；
hIgG：为阴性对照，来源于上海恒瑞医药有限公司，用PBS稀释。
2.实验步骤
将PBS重悬的CT-26 WT细胞以5×10 6个/0.1mL浓度，0.1mL/只体积接种于 PD-1HuGEMM BALB/c小鼠的右侧胁肋部皮下，共转接42只小鼠。当平均肿瘤体积达到约100mm 3时，挑选个体肿瘤体积适中的小鼠入组，每组7只，分组当天开始给药，具体给药方案见下表1，根据体重调整给药量(给药体积＝0.1mL/10g)，受试样品配置见表2，使用游标卡尺每周两次测量肿瘤体积，称小鼠体重，记录数据，检测受试化合物对人源化小鼠CT-26 WT结肠癌细胞移植瘤的生长抑制作用。
表1.转基因小鼠的结肠癌CT-26 WT模型给药途径，给药剂量及分组
Figure PCTCN2019108535-appb-000015
注：i.p.：腹腔注射；p.o.：口服灌胃；BID:每天两次；Q3D:每三天一次。
表2.药物A和药物B联用对CT-26 WT荷瘤huPD1小鼠皮下移植瘤的疗效
Figure PCTCN2019108535-appb-000016
D0:第一次给药时间；P值指与hIgG+溶剂组相比；采用参数分析(parametric statistical analysis)。实验开始时小鼠数目：n＝7。
各组给药后肿瘤体积见附图1，小鼠体重见附图2。
实验结果表明药物A单用(100mg/kg)或药物B(3mg/kg)单用均未表现出显著抑制huPD1小鼠CT-26 WT皮下移植瘤的生长。在给药后第17天时肿瘤抑制率分别为30％和14.3％，p值分别为0.17和0.699。药物A(100mg/kg)与药物B(3mg/kg)联合给药表现出抑瘤作用，给药后第17天的肿瘤抑制率为43.6％(p＝0.002)。各组荷瘤小鼠均没有出现明显的体重下降情况，表明荷瘤小鼠对该剂量下药物联用的耐受性良好。
实施例2.式(I)所示化合物(药物A)及抗PD-1抗体(药物B)二者合用对鼠黑色素瘤B16F10模型荷瘤小鼠皮下移植瘤的疗效
1、实验材料
hIgG：为阴性对照，来源于上海恒瑞医药有限公司，用PBS稀释。
配制方法：药物A用0.5％CMC-Na 1％吐温80溶液配制；药物B用5％葡萄糖溶液配制后用PBS稀释。
实验动物：PD-1HuGEMM小鼠，6-7周龄，♀，购自南京银河生物医药有限公司。
3、实验步骤
B16F10鼠源黑色素瘤细胞培养于含有10％胎牛血清的RPMI1640培养基中，将PBS重悬的B16F10细胞以5×10 5个/0.1mL浓度，0.1mL/只体积接种于PD-1HuGEMM C57BL/6小鼠的右后胁肋部皮下。待肿瘤长至约100mm 3后，将动物随机分组(D0)并给药。给药剂量和给药方案见表3。每周测2-3次测量肿瘤体积，称小鼠体重，记录数据，联用对B16F10荷瘤huPD1小鼠皮下移植瘤的疗效见表4。
表3.转基因小鼠的黑色素瘤B16F10模型药效实验的给药途径，给药剂量及分组
Figure PCTCN2019108535-appb-000017
注：i.p.：腹腔注射；p.o.：口服灌胃；BID:每天两次；Q3D:每三天一次。
表4.联用对B16F10荷瘤huPD1小鼠皮下移植瘤的疗效
Figure PCTCN2019108535-appb-000018
D0:第一次给药时间；P值指与hIgG+溶剂组相比；采用非参数分析(Nonparametric statistical analysis)。实验开始时小鼠数目：n＝5。
各组给药后肿瘤体积见附图3，小鼠体重见附图4。
实验结果表明药物A单用(100和200mg/kg)或药物B(10mg/kg)单用均未表现出显著抑制huPD1小鼠B16F10皮下移植瘤的生长。药物A(100和200mg/kg)分别与药物B(10mg/kg)联合给药表现出抑瘤作用，肿瘤抑制率均大于60％，在给药后第14天时，肿瘤抑制率分别为68％和71％，p值分别为0.056和0.032。各组荷瘤小鼠均没有出现明显的体重下降情况，表明荷瘤小鼠对该剂量下药物联用的耐受性良好。

Claims (24)

  1. 一种EZH2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途。
  2. 根据权利要求1所述的用途，所述的EZH2抑制剂选自CPI-0209、CPI-1205、GSK126、valemetostat、tazemetostat、PF-06821497、DS-3201、GSK-2816126、3-deazaneplanocin A、HKMT-I-005、KM-301或式(I)所示化合物或其复合物或其可药用盐，优选式(I)所示化合物或其复合物或其可药用盐，
    Figure PCTCN2019108535-appb-100001
  3. 根据权利要求2所述的用途，其特征在于所述的免疫检查点抑制剂选自PD-1受体抑制剂、PD-L1抑制剂或CTLA-4抑制剂中的一种。
  4. 根据权利要求3所述的用途，所述PD-1受体抑制剂为抗PD-1抗体或者其抗原结合片段。
  5. 根据权利要求4所述的用途，所述抗PD-1抗体选自sintilimab、cemiplimab、JS-001、nivolumab、tislelizumab、pembrolizumab、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-105、AK-104、BCD-100、PF-06753512、HLX-10、AMP-224、LZM-009。
  6. 根据权利要求4所述的用途，所述抗PD-1抗体的轻链可变区包含分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3，所述的PD-1抗体的重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
  7. 根据权利要求4所述的用途，所述抗PD-1抗体或其抗原结合片段选自人源化抗体或其片段。
  8. 权利要求6或7所述的用途，所述抗PD-1抗体或其抗原结合片段包含人源IgG1、IgG2、IgG3或IgG4同种型的重链恒定区，优选包含IgG1或IgG4同种型 的重链恒定区。
  9. 根据权利要求7所述的用途，所述人源化抗体的轻链可变区序列为如SEQ ID NO:10所示的序列或其变体，所述人源化抗体的重链可变区序列为如SEQ ID NO:9所示的序列或其变体。
  10. 根据权利要求7所述的用途，所述人源化抗体的轻链序列为如SEQ ID NO:8所示的序列或其变体；所述人源化抗体的重链序列为如SEQ ID NO:7所示的序列或其变体。
  11. 根据权利要求7所述的用途，所述的人源化抗体轻链序列为如SEQ ID NO:8所示的序列，重链序列为如SEQ ID NO:7所示的序列。
  12. 根据权利要求3所述的用途，所述PD-L1抑制剂为抗PD-L1抗体或其抗原结合片段，所述抗PD-L1抗体选自avelumab、atezolizumab、durvalumab、CS-1001、M-7824、KL-A167、CX-072、BGB-A333、GNS-1480、CA-170、BMS-936559，优选avelumab、atezolizumab、durvalumab。
  13. 根据权利要求3所述的用途，所述PD-L1抑制剂为抗PD-L1抗体或其抗原结合片段，所述抗PD-L1抗体或抗原结合片段的重链可变区包含分别如SEQ ID NO：11-13所示的HCDR1、HCDR2和HCDR3，所述抗PD-L1抗体或抗原结合片段的轻链可变区包含分别如SEQ ID NO：14-16所示的HCDR1、HCDR2和HCDR3。
  14. 根据权利要求13所述的用途，所述抗PD-L1抗体或其抗原结合片段包含SEQ ID NO：17所示的重链可变区或其变体，和SEQ ID NO：18所示的轻链可变区或其变体。
  15. 根据权利要求13或14所述的用途，于所述的PD-L1抗体或抗原结合片段进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，更优选包含引入F234A和L235A突变的IgG4重链恒定区；所述人源化抗体轻链进一步包含人源κ、λ链或其变体的恒定区。
  16. 根据权利要求15所述的用途，所述的PD-L1抗体或抗原结合片段包含如序列为SEQ ID NO：19所示重链，和如序列为SEQ ID NO：21所示轻链。
  17. 根据权利要求13所述的用途，所述的PD-L1抗体或抗原结合片段选自鼠源抗体、嵌合抗体、人源化抗体，人抗体，优选人源化抗体。
  18. 根据权利要求3所述的用途，所述CTLA-4抑制剂为抗CTLA-4抗体或其抗原结合片段，所述抗CTLA-4抗体选自ipilimumab、tremelimumab、AGEN-1884、CS-1002、XmAb-20717、REGN-4659、BCD-145、MEDI-5752、AK-104、MK-1308、BMS-986249、BMS-986218、PF-06753512，优选ipilimumab、tremelimumab。
  19. 根据权利要求1-18任一项所述的用途，所述肿瘤选自肾上腺皮质癌、肛门癌、肛门直肠癌、肛管癌、阑尾癌、小脑星形细胞瘤、脑星形细胞瘤、基底细胞癌、皮肤癌(非黑色素瘤)、胆道癌、肝外胆管癌、肝内胆管癌、膀胱癌、骨关节癌、骨肉瘤、恶性纤维组织细胞瘤、脑癌、脑肿瘤、脑干胶质瘤、室管膜瘤、成神经管细胞瘤、视觉通路和下丘脑神经胶质瘤、乳腺癌、支气管腺瘤、神经系统癌、神经系统淋巴瘤、中枢神经系统癌、中枢神经系统淋巴瘤、宫颈癌、慢性淋巴细胞白血病、慢性粒细胞白血病白血病、慢性骨髓增生性疾病、结肠癌、结直肠癌、皮肤T细胞淋巴瘤、淋巴肿瘤、蕈样真菌病、Sezary综合征、子宫内膜癌、食管癌、颅外生殖细胞肿瘤、性腺外生殖细胞肿瘤、眼癌、眼内黑色素瘤、视网膜母细胞瘤、胆囊癌、胃癌、胃肠道类癌、胃肠道间质瘤(GIST)、生殖细胞肿瘤、卵巢生殖细胞瘤、头颈癌、肝细胞癌、霍奇金淋巴瘤、胰岛细胞瘤、卡波西肉瘤、肾癌、喉癌、急性淋巴细胞白血病、急性髓性白血病、毛细胞白血病、唇和口腔腔癌、肝癌、肺癌、非小细胞肺癌、小细胞肺癌、非霍奇金淋巴瘤、原发性中枢神经系统淋巴瘤、Waldenstroem巨球蛋白血症、黑色素瘤、间皮瘤、转移性鳞癌、舌癌、多发性内分泌肿瘤综合征、骨髓增生异常综合征、多发性骨髓瘤、鼻咽癌、神经母细胞瘤、口咽癌、卵巢癌、卵巢上皮癌、卵巢低恶性潜能肿瘤、胰腺癌、胰岛细胞胰腺癌、鼻窦和鼻腔癌、甲状旁腺癌、阴茎癌、咽癌、嗜铬细胞瘤、松果体瘤、垂体瘤、浆细胞肿瘤、胸膜肺母细胞瘤、前列腺癌、直肠癌、肾盂和输尿管移行细胞癌、视网膜母细胞瘤、横纹肌肉瘤、唾液腺癌、尤文家族肉瘤、卡波西肉瘤、滑膜肉瘤、子宫癌、子宫肉瘤、小肠癌、软组织肉瘤、鳞状细胞癌、幕上原始神经外胚层肿瘤、睾丸癌、咽喉癌、胸腺瘤、尿道癌、子宫内膜异位症、阴道癌、外阴癌或威尔姆氏肿瘤，优选黑色素瘤或结肠癌。
  20. 根据权利要求19所述的用途，所述的肿瘤对单一EZH2抑制剂或免疫检查点抑制剂治疗具有抗性，所述免疫检查点抑制剂选自PD-1受体抑制剂、PD-L1抑制剂或CTLA-4抑制剂。
  21. 根据权利要求2-20任一项所述的用途，其特征在于所述EZH2抑制剂为 式(I)所示化合物。
  22. 根据权利要求2-21任一项所述的用途，其特征在于所述EZH2抑制剂为的剂量选自1-1600mg，给药频率为每天两次、每天一次、每两天一次、每三天一次、每四天一次、每五天一次、每六天一次、每周一次、每二周一次、每三周一次、每四周一次，优选每天一次或每天两次。
  23. 根据权利要求22所述的用途，其特征在于所述抗PD-1抗体或其抗原结合片段剂量选自0.1-10.0mg/kg或1-1000mg，给药频率为每周一次、每二周一次、每三周一次、每四周一次、1个月一次、每3-6个月一次，优选每二周一次或每三周一次。
  24. 根据权利要求3、12-17、19-22任一项所述的PD-L1抗体或其抗原结合片段剂量选自50-3000mg，优选600mg、750mg，给药频率为每周一次、每二周一次、每三周一次、每四周一次，优选每二周一次或每三周一次。
PCT/CN2019/108535 2018-09-29 2019-09-27 Ezh2抑制剂与免疫检查点抑制剂联合在制备治疗肿瘤的药物中的用途 Ceased WO2020063863A1 (zh)

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