WO2021063331A1 - USE OF COMBINATION OF EZH2 INHIBITOR AND TGF-β RECEPTOR-CONTAINING FUSION PROTEIN IN PREPARATION OF DRUGS FOR TREATING TUMORS - Google Patents

USE OF COMBINATION OF EZH2 INHIBITOR AND TGF-β RECEPTOR-CONTAINING FUSION PROTEIN IN PREPARATION OF DRUGS FOR TREATING TUMORS Download PDF

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WO2021063331A1
WO2021063331A1 PCT/CN2020/118609 CN2020118609W WO2021063331A1 WO 2021063331 A1 WO2021063331 A1 WO 2021063331A1 CN 2020118609 W CN2020118609 W CN 2020118609W WO 2021063331 A1 WO2021063331 A1 WO 2021063331A1
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once
tgf
cancer
weeks
antibody
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French (fr)
Chinese (zh)
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张岚
廖成
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江苏恒瑞医药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • 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
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present disclosure belongs to the field of pharmacy, and specifically relates to the use of an EZH2 inhibitor combined with a fusion protein containing a TGF- ⁇ receptor in preparing a medicine for treating tumors.
  • PD-1 Programmed death receptor 1
  • PD-1 is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid cells, and it has two ligands, namely programmed death ligand-1 (PD-L1) and PD-L2. PD-L1 interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of immune response.
  • the expression of PD-L1 protein can be detected in many human tumor tissues.
  • the microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells.
  • the expressed PD-L1 is conducive to the occurrence and growth of tumors, and induces anti-tumor Apoptosis of T cells.
  • PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, and restore the activity of T cells, thereby enhancing the immune response. Therefore, with PD-1/PD- The immunomodulation with L1 as the target is of great significance to tumor suppression.
  • Transforming growth factor- ⁇ (transforming growth factor- ⁇ , TGF- ⁇ ) belongs to the TGF- ⁇ superfamily that regulates cell growth and differentiation. TGF- ⁇ transmits signals through a heterotetrameric receptor complex, which is composed of two type I and two type II transmembrane serine/threonine kinase receptors.
  • TGF- ⁇ signaling pathway can reduce tumor metastasis.
  • the truncated Smad2/3 negative mutant was used to inhibit the TGF- ⁇ signaling pathway of breast tumor cell lines, and it was found that the metastatic ability of tumor cells was inhibited.
  • the microsatellite instability study of colon cancer found that the inactive mutation of TGF- ⁇ RII reduced metastasis and increased the survival rate of patients after surgery.
  • inhibitors that inhibit the TGF- ⁇ signaling pathway alone have little effect in clinical treatment, which may be related to the abnormally high expression of TGF- ⁇ mainly in tumor cells, while pure TGF- ⁇ signaling pathway inhibitors are very effective. The difficulty of focusing on tumors leads to low efficacy or bioavailability of signal pathway inhibitors.
  • inhibiting the PD-1/PD-L1 pathway on the basis of TGF- ⁇ that targets and neutralizes the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of suppressing the occurrence and development of tumors.
  • the PD-L1/TGF- ⁇ double antibody M-7824 developed by Merck has now entered clinical phase II.
  • this product has also been developed in combination with eribulin mesylate to treat metastases Triple-negative breast cancer (NCT03579472); combined with gemcitabine for the treatment of previously treated advanced pancreatic adenocarcinoma (NCT03451773); combined with topotecan or temozolomide for the treatment of recurrent small cell lung cancer (NCT0-3554473) and other indications .
  • 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.
  • Example 2 patients with soft tissue sarcoma were given the combination therapy of tazemetostat and atezolizumab, but no specific pharmacodynamic 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
  • the inhibitor can be an anti-PD-L1 antibody.
  • the example discloses a treatment plan for the combination of tazemetostat and atezolizumab for patients with locally advanced or metastatic urothelial cancer, patients with diffuse large B-cell lymphoma, and non-Hodgkin’s lymphoma. However, no specific efficacy information was given.
  • WO2017084494A provides an EZH2 inhibitor, the structure is as follows:
  • WO2018205985A discloses a new PD-L1/TGF- ⁇ fusion protein, and there is no relevant information about its drug combination.
  • the present disclosure relates to the use of a combination of an EZH2 inhibitor and a fusion protein containing a TGF- ⁇ receptor in the preparation of drugs for treating tumors or cancers.
  • the EZH2 inhibitor is selected from CPI-0209, CPI-1205, GSK126, valemetostat, tazemetostat, PF-06821497, DS-3201GSK-2816126, HKMT-I-005, 3-deazaneplanocin A, KM-301 or the compound represented by formula (I) or its pharmaceutically acceptable salt, preferably the compound represented by formula (I) or its pharmaceutically acceptable salt,
  • the fusion protein of the TGF- ⁇ receptor comprises a PD-L1 antibody or an antigen-binding fragment thereof and a TGF- ⁇ receptor, wherein the TGF- ⁇ receptor part is the extracellular domain of TGF- ⁇ RII N-terminal truncated form.
  • the TGF- ⁇ receptor fusion protein is represented by the general formula (II):
  • TGF- ⁇ RII ECD is a truncated form of the extracellular region of TGF- ⁇ RII;
  • Ab is the PD-L1 antibody or its antigen-binding fragment
  • L is the connection sequence.
  • connection sequence is (G4S)xG, where x is 3-6, preferably 4.
  • the PD-L1 antibody or antigen-binding fragment thereof comprises:
  • HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively LCDR1, LCDR2 and LCDR3,
  • HCDR1 SYWMH SEQ ID NO: 1
  • HCDR2 RIX1PNSGX2TSYNEKFKN SEQ ID NO: 2
  • HCDR3 GGSSYDYFDY SEQ ID NO: 3
  • LCDR1 RASESVSIHGTHLMH SEQ ID NO: 4
  • the PD-L1 antibody or antigen-binding fragment thereof is a chimeric antibody or a functional fragment thereof, a humanized antibody or a functional fragment thereof, or a human antibody or a functional fragment thereof.
  • sequences of the heavy and light chains of the humanized PD-L1 antibody are as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence is FR sequence in italics; the underline is the CDR sequence, and the double-underlined position is the position obtained after affinity maturation screening.
  • the heavy chain amino acid sequence of the PD-L1 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 9 or has at least 85%, 86% with the sequence shown in SEQ ID NO: 9, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity
  • the PD- The light chain amino acid sequence of the L1 antibody or its antigen-binding fragment is shown in SEQ ID NO: 10 or has at least 85%, 86%, 87%, 88%, 89%, 90% with the sequence shown in SEQ ID NO: 10 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, where
  • PD-L1 antibody heavy chain sequence Ig G4 (AA) (S228P)
  • Non-limiting example sequences of the TGF- ⁇ RII extracellular domain and its truncated form in the present disclosure are as follows:
  • TGF- ⁇ RII extracellular domain sequence ECD(1-136)
  • TGF- ⁇ RII extracellular domain sequence has a 19 amino acid truncation or deletion at the N-terminus: ECD(20-136)
  • the TGF- ⁇ RII extracellular domain sequence has a 21 amino acid truncation or deletion at the N-terminus: ECD(22-136)
  • TGF- ⁇ RII extracellular domain sequence has a truncation or deletion of 14 amino acids at the N-terminus: ECD(15-136)
  • Ab is the PD-L1 antibody described in the present disclosure.
  • ECD (n-136) is the full length or truncated form of the extracellular region of TGF- ⁇ RII, and n is the amino acid after truncation of the extracellular region of TGF- ⁇ RII. Starting digits.
  • the PD-L1 antibody or antigen-binding fragment thereof is selected from avelumab, atezolizumab, durvalumab, CS-1001, M-7824, KL-A167, CX-072, BGB-A333, GNS-1480 , CA-170, BMS-936559, preferably avelumab, atezolizumab, durvalumab.
  • the administration dose of EZH2 is selected from 1-1600 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, 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, 10 mg, 15 mg
  • the dosage of EZH2 is selected from 1-800mg, and the frequency of administration is twice a day, once a day, once every two days, once every three days, once every four days, and every five days. Once a day, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.
  • the administration dose of EZH2 is selected from 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, administration
  • the frequency is twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or Once every four weeks, preferably twice a day or once a day.
  • the dosage of EZH2 is selected from 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, and the frequency of administration is twice a day, once a day, once every two days, every three times. Once a day, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.
  • the dosage of EZH2 is 350 mg twice a day.
  • the dosage of the TGF- ⁇ receptor-containing fusion protein is selected from 0.1-500 mg/kg, and the dosage frequency is once a week, once every two weeks, once every three weeks or every four weeks.
  • the specific dosage of the fusion protein containing TGF- ⁇ receptor can be selected from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg.
  • the dosage of the TGF- ⁇ receptor-containing fusion protein is selected from 0.1-200 mg/kg, and the frequency of administration is once a week, once every two weeks, once every three weeks or every four weeks once.
  • the dosage of the fusion protein containing TGF- ⁇ receptor is selected from 0.1-100 mg/kg, and the frequency of administration is once a week, once every two weeks, once every three weeks or every four weeks once.
  • the dosage of the fusion protein containing TGF- ⁇ receptor is selected from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg. kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/ kg, 33mg/kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 31m
  • the dosage of the fusion protein containing TGF- ⁇ receptor is selected from 1 mg/kg, 3 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg. kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, the frequency of administration is once every two weeks, once every three weeks or once every four weeks.
  • the administration dose of the fusion protein containing TGF- ⁇ receptor is selected from the group consisting of 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, and administration
  • the frequency is once every two weeks, once every three weeks or once every four weeks.
  • the dosage of the TGF- ⁇ receptor-containing fusion protein is 30 mg/kg, once every 3 weeks.
  • the dosage of the fusion protein containing TGF- ⁇ receptor is 1-4000 mg, specifically 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, 2000mg, 2050mg, 2100mg, 2150mg, 2200mg, 2250mg, 2300mg, 2350mg, 2400mg, 2450mg, 2500mg, 2550mg, 2600mg
  • the dosage of the fusion protein containing TGF- ⁇ receptor is 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg, 3000 mg, 3300 mg, 3600 mg, and the dosage frequency is every Once every two weeks, once every three weeks or once every four weeks.
  • the dosage of the TGF- ⁇ receptor-containing fusion protein is 600 mg, 1200 mg, 1800 mg, 2400 mg, 3000 mg, and the administration frequency is once every two weeks or once every three weeks.
  • the TGF- ⁇ receptor-containing fusion protein is administered by injection, such as subcutaneous or intravenous injection, and the TGF- ⁇ receptor-containing fusion protein needs to be formulated as an injectable before injection.
  • the injectable form of the fusion protein containing the TGF- ⁇ receptor can be an injection or a lyophilized powder injection, which contains the fusion protein containing the TGF- ⁇ receptor, 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, maltose.
  • the surfactant is selected from polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like.
  • the present disclosure provides a method for treating tumor or cancer, which comprises administering to a patient an effective amount of the above-mentioned EZH2 inhibitor and an effective amount of the above-mentioned TGF- ⁇ receptor-containing fusion protein.
  • Another aspect of the present disclosure provides an EZH2 inhibitor for the treatment of tumors or cancers, which is used in combination with a fusion protein containing a TGF- ⁇ receptor.
  • TGF- ⁇ receptor-containing fusion protein for treating tumors or cancers, and the TGF- ⁇ receptor-containing fusion protein is used in combination with an EZH2 inhibitor.
  • the patient in the method for treating tumor or cancer provided by the present disclosure, is a human.
  • the tumor or cancer in the present disclosure is selected from: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterus Endometrial cancer, uterine cancer, bladder, neuroendocrine tumor, head and neck cancer, liver cancer, cholangiocarcinoma, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell Shape cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • the “combination” in the present disclosure refers to the administration of at least one dose of EZH2 inhibitor and at least one dose of fusion protein containing TGF- ⁇ receptor within a certain period of time, of which two substances Both show pharmacological effects.
  • the time limit can be within one administration cycle, optionally within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 24 hours, or within 2 hours.
  • the EZH2 inhibitor and the fusion protein containing the TGF- ⁇ receptor can be administered simultaneously or sequentially. This period includes treatments in which the EZH2 inhibitor and the TGF- ⁇ receptor-containing fusion protein are administered through the same route of administration or different routes of administration.
  • the mode of administration of the combination described in the present disclosure is selected from simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the antibody light chain described in the present disclosure may further include a light chain constant region, and the light chain constant region includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain described in the present disclosure may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known Kabat numbering rules (LCDR1-3, HCDE2-3), or comply with the numbering rules of Kabat and Chothia (HCDR1).
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • the "antibody or its antigen binding” or “functional fragment” as used in the present disclosure refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and ScFv fragments that bind to antibodies.
  • the Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but has no constant region and is the smallest antibody fragment with all antigen binding sites.
  • an Fv antibody also contains 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).
  • binding to PD-L1 refers to the ability to interact with human PD-L1.
  • antigen-binding site refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • murine antibody in the present disclosure refers to a monoclonal antibody to human PD-L1 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with PD-L1 antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed.
  • the gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the PCSK-9 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the PCSK-9 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising human IgG2 or IgG4 heavy chain constant region, or using amino acid mutations without ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody 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 germlines The antibody produced in the antibody framework sequence. It can overcome the strong variable antibody response induced by the chimeric antibody because it carries a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the 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 www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.
  • identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the sequence identity in the present disclosure may be at least 85%, 90% or 95%, preferably at least 95%.
  • Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%.
  • Sequence comparison and determination of the percent identity between two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • TGF- ⁇ receptor II or “TGF ⁇ RII” or “transforming growth factor ⁇ receptor II” refers to binding ligands (including but not limited to TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3) and thereby triggering intracellular signal transduction Pathway of cell surface receptors.
  • PD-L1 refers to programmed death ligand 1, also known as CD274 and B7H1.
  • PD-L1 is a 290 amino acid protein with extracellular IgV-like and IgC-like domains (amino acids 19-239 of full length PD-L1), a transmembrane domain, and an intracellular domain of about 30 amino acids.
  • PD-L1 is constitutively expressed on many cells such as antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells), as well as hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and immune pardon sites) .
  • antigen-presenting cells e.g., dendritic cells, macrophages, and B cells
  • hematopoietic and non-hematopoietic cells e.g., vascular endothelial cells, pancreatic islets, and
  • PD-L1 is also expressed on a variety of tumors and virus-infected cells, and is a component of an immunosuppressive milieu (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of the two T cell co-inhibitors PD-1 and B7-1.
  • the fusion protein described in the present disclosure is a protein product in which two genes are co-expressed through DNA recombination.
  • the methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15.
  • mice can be immunized with human PD-L1 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region.
  • the human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT ImmunoGeneTics
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • FIG. 1 Schematic diagram of mouse tumor inoculation position
  • FIG. 1 Mouse body weight changes in the BALB/C-hPDL1 humanized mouse subcutaneous CT26-hPDL1 colon cancer model.
  • Example 1 The efficacy of the test drug in the humanized mouse BALB/c-hPDL1 subcutaneous CT26-hPDL1 colon cancer tumor model at the immune checkpoint was evaluated.
  • mice mice, BALB/C-hPDL1, 7.57-8.14 weeks (weeks old when tumor cells were inoculated), female, 90 animals, from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • TGF- ⁇ receptor fusion protein fusion protein 9, 50 mg/ml
  • EZH2 inhibitor compound 1 represented by formula (I).
  • mice are randomly divided into 4 groups according to the tumor volume, with 7 mice in each group.
  • the grouping day was defined as D0 day, and the administration was started on D0 day.
  • Dosing volume Adjust the administration volume to 10 ⁇ l/g according to body weight. In the combined administration, the oral small molecules are given first, and then the antibody injections are given after all animals are given the small molecules.
  • tumor volume (mm 3 ) 0.5 (tumor long diameter ⁇ tumor short diameter 2 ).
  • tumor volume change TGItv
  • average weight change TGItw
  • TGItv relative tumor inhibition rate
  • T0 Initial tumor volume in the administration group
  • C Tuor volume in the control group
  • C0 Initial tumor volume in the control group
  • Tumor weight change (TGItw) calculation formula: TGItw (1-meanTW treat/meanTW vehicle)x100%; where meanTWtreat: mean value of tumor weight at the end-point treatment of mice in the administration group, meanTW vehicle: end-point treatment of mice in the vehicle group Average tumor weight
  • the experimental results of tumor volume, mouse body weight, tumor weight of each group of animals are expressed as mean ⁇ standard error (Mean ⁇ SEM).
  • the independent sample T test method was used to compare whether there were significant differences between the different treatment groups and the control group. All data are analyzed with SPSS 18.0. P ⁇ 0.05 indicates a significant difference.
  • the drawing software is Graphpad prism 6.0.
  • TGI data shows that the combination group (group 4) has significant differences compared with the control group and the single-drug group (groups 2 and 3).
  • TGF- ⁇ receptor fusion The combined administration of protein (3mg/kg) and EZH2 inhibitor (100mg/kg) has a significant anti-tumor effect.
  • Example 2 A clinical trial of PL1/TGF- ⁇ fusion protein used in multiple tumors
  • Test drug EZH2 inhibitor: compound 1 of formula (I), 350 mg orally 2 times/day; PD-L1/TGF ⁇ fusion protein: fusion protein 9,30 mg/kg, intravenous infusion 1 time/3 weeks; 21 days for 1 cycle.
  • EGFR/ALK/ROS-1 negative advanced non-small cell lung cancer
  • gastrointestinal tumors esophageal cancer, gastric cancer, intestinal cancer
  • biliary pancreatic tumors bile duct cancer, pancreatic cancer
  • Breast cancer HER2/ER/PR negative
  • gynecological tumors ovarian cancer, cervical cancer
  • Solid tumors have received at least first-line treatment and failed (except for cholangiocarcinoma and pancreatic cancer), and lymphoma has received at least two-line treatment and failed.
  • Anti-PD-1/PD-L1 antibodies in previous treatments are not limited. At least 18 to 75 years old, regardless of gender. It is expected that 50 patients will eventually be included.

Abstract

The present disclosure relates to use of a combination of a EZH2 inhibitor and a TGF-β receptor-containing fusion protein in preparation of drugs for treating tumors. Specifically, a EZH2 inhibitor involved in the present disclosure is selected from a compound represented by formula (I) and a pharmaceutically acceptable salt thereof. Compared with a single drug, a combination of a EZH2 inhibitor and a TGF-β receptor-containing fusion protein further enhances the antitumor effect.

Description

EZH2抑制剂与含有TGF-β受体的融合蛋白联合在制备***的药物中的用途Use of combination of EZH2 inhibitor and fusion protein containing TGF-β receptor in preparing medicine for treating tumor
本申请要求申请日为2019/9/30的中国专利申请2019109407105和申请日为2020/2/10的中国专利申请2020100841430的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of the Chinese patent application 2019109407105 with the filing date of 2019/9/30 and the Chinese patent application 2020100841430 with the filing date of 2020/2/10. This application quotes the full text of the aforementioned Chinese patent application.
技术领域Technical field
本公开属于制药领域，具体的涉及一种EZH2抑制剂与含有TGF-β受体的融合蛋白联合在制备***的药物中的用途。The present disclosure belongs to the field of pharmacy, and specifically relates to the use of an EZH2 inhibitor combined with a fusion protein containing a TGF-β receptor in preparing a medicine for treating tumors.
背景技术Background technique
程序性死亡受体1(programmed death 1,PD-1)为CD28超家族成员。PD-1表达于活化的T细胞，B细胞及髓系细胞，其有两个配体，即程序性死亡配体-1(programmed death ligand 1,PD-L1)和PD-L2。PD-L1与T细胞上的受体PD-1相互作用，在免疫应答的负调控方面发挥着重要作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达，肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答，因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。Programmed death receptor 1 (PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid cells, and it has two ligands, namely programmed death ligand-1 (PD-L1) and PD-L2. PD-L1 interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of immune response. The expression of PD-L1 protein can be detected in many human tumor tissues. The microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells. The expressed PD-L1 is conducive to the occurrence and growth of tumors, and induces anti-tumor Apoptosis of T cells. PD-1/PD-L1 pathway inhibitors can block the combination of PD-1 and PD-L1, block negative regulatory signals, and restore the activity of T cells, thereby enhancing the immune response. Therefore, with PD-1/PD- The immunomodulation with L1 as the target is of great significance to tumor suppression.
转化生长因子-β(transforming growth factor-β,TGF-β)属于调节细胞生长和分化的TGF-β超家族。TGF-β通过异源四聚体受体复合物传递信号，这个受体复合物是由两个I型和两个II型的跨膜丝氨酸/苏氨酸激酶受体组成。Transforming growth factor-β (transforming growth factor-β, TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation. TGF-β transmits signals through a heterotetrameric receptor complex, which is composed of two type I and two type II transmembrane serine/threonine kinase receptors.
研究发现阻断TGF-β信号传导通路能够减少肿瘤的转移。运用截短的Smad2/3显负性突变体抑制乳腺肿瘤细胞系的TGF-β信号通路，结果发现肿 瘤细胞的转移能力被抑制。结肠癌的微卫星不稳定性研究发现，TGF-βRII无活性的突变，使转移减少，增加了患者术后的存活率。但总体而言，抑制TGF-β信号通路的抑制剂单用在临床治疗中效果微弱，可能跟TGF-β主要在肿瘤细胞内异常性高表达，而单纯的TGF-β信号通路的抑制剂很难集中靶向于肿瘤导致药效不高或信号通路抑制剂生物利用度有关。Studies have found that blocking the TGF-β signaling pathway can reduce tumor metastasis. The truncated Smad2/3 negative mutant was used to inhibit the TGF-β signaling pathway of breast tumor cell lines, and it was found that the metastatic ability of tumor cells was inhibited. The microsatellite instability study of colon cancer found that the inactive mutation of TGF-βRII reduced metastasis and increased the survival rate of patients after surgery. However, in general, inhibitors that inhibit the TGF-β signaling pathway alone have little effect in clinical treatment, which may be related to the abnormally high expression of TGF-β mainly in tumor cells, while pure TGF-β signaling pathway inhibitors are very effective. The difficulty of focusing on tumors leads to low efficacy or bioavailability of signal pathway inhibitors.
因此，在靶向中和肿瘤微环境的TGF-β基础上抑制PD-1/PD-L1通路，可以使T细胞恢复活性，增强免疫应答，更有效地提高抑治肿瘤发生和发展的效果。Merck公司开发的PD-L1/TGF-β双抗M-7824目前已经进入临床II期，除单药用于***外，该产品还开发了与艾日布林甲磺酸盐联用治疗转移性三阴性乳腺癌(NCT03579472)；与吉西他滨联用治疗在先接受过治疗的晚期胰腺腺癌(NCT03451773)；与拓扑替康或者替莫唑胺联用治疗复发性小细胞肺癌(NCT0-3554473)等适应症。Therefore, inhibiting the PD-1/PD-L1 pathway on the basis of TGF-β that targets and neutralizes the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of suppressing the occurrence and development of tumors. The PD-L1/TGF-β double antibody M-7824 developed by Merck has now entered clinical phase II. In addition to single-drug treatment for tumors, this product has also been developed in combination with eribulin mesylate to treat metastases Triple-negative breast cancer (NCT03579472); combined with gemcitabine for the treatment of previously treated advanced pancreatic adenocarcinoma (NCT03451773); combined with topotecan or temozolomide for the treatment of recurrent small cell lung cancer (NCT0-3554473) and other indications .
WO2018223030A实施例1公开针对上皮样肉瘤和肾髓样癌患者的组织检测发现EZH2抑制后PD-L1会发生上调，数据表明这些肿瘤被引发由于肿瘤中存在T细胞介导的免疫应答，但是免疫应答可能受到肿瘤中PD-L1表达增加的抑制，这表明这种肿瘤可通过EZH2抑制和PD-1/PD-L1抑制的组合来治疗。实施例2中针对软组织肉瘤的患者给与tazemetostat和atezolizumab的组合治疗，但未公开具体药效信息。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 the combination therapy of tazemetostat and atezolizumab, but no specific pharmacodynamic information was disclosed.
WO2017210395A要求保护一种在有需要的受试者中治疗癌症的方法，包括向所述受试者施用治疗有效量的EZH2抑制剂和治疗有效量的免疫检查点抑制剂，所述的免疫检查点抑制剂可选抗PD-L1抗体，实施例公开了tazemetostat和atezolizumab联用针对局部晚期或转移性尿路上皮癌患者、弥漫性大B细胞淋巴瘤患者及非霍奇金淋巴瘤的治疗方案，但是未给出具体的药效信息。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 The inhibitor can be an anti-PD-L1 antibody. The example discloses a treatment plan for the combination of tazemetostat and atezolizumab for patients with locally advanced or metastatic urothelial cancer, patients with diffuse large B-cell lymphoma, and non-Hodgkin’s lymphoma. However, no specific efficacy information was given.
WO2017084494A中提供了一种EZH2抑制剂，结构如下所示：WO2017084494A provides an EZH2 inhibitor, the structure is as follows:
Figure PCTCN2020118609-appb-000001
Figure PCTCN2020118609-appb-000001
WO2018205985A公开了一种新的PD-L1/TGF-β融合蛋白，目前尚未见其药物联用的相关信息。WO2018205985A discloses a new PD-L1/TGF-β fusion protein, and there is no relevant information about its drug combination.
发明内容Summary of the invention
本公开涉及一种EZH2抑制剂与含有TGF-β受体的融合蛋白联合在制备***或癌症的药物中的用途。The present disclosure relates to the use of a combination of an EZH2 inhibitor and a fusion protein containing a TGF-β receptor in the preparation of drugs for treating tumors or cancers.
在某些实施方式中，所述的EZH2抑制剂选自CPI-0209、CPI-1205、GSK126、valemetostat、tazemetostat、PF-06821497、DS-3201GSK-2816126、HKMT-I-005、3-deazaneplanocin A、KM-301或式(I)所示化合物或其可药用盐，优选式(I)所示化合物或其可药用盐，In some embodiments, the EZH2 inhibitor is selected from CPI-0209, CPI-1205, GSK126, valemetostat, tazemetostat, PF-06821497, DS-3201GSK-2816126, HKMT-I-005, 3-deazaneplanocin A, KM-301 or the compound represented by formula (I) or its pharmaceutically acceptable salt, preferably the compound represented by formula (I) or its pharmaceutically acceptable salt,
Figure PCTCN2020118609-appb-000002
Figure PCTCN2020118609-appb-000002
在某些实施方式中，所述的TGF-β受体的融合蛋白包含PD-L1抗体或其抗原结合片段和TGF-β受体，其中TGF-β受体部分为TGF-βRII胞外区的N端截短形式。In some embodiments, the fusion protein of the TGF-β receptor comprises a PD-L1 antibody or an antigen-binding fragment thereof and a TGF-β receptor, wherein the TGF-β receptor part is the extracellular domain of TGF-βRII N-terminal truncated form.
在某些实施方式中，所述TGF-β受体融合蛋白如通式(II)所示：In some embodiments, the TGF-β receptor fusion protein is represented by the general formula (II):
Ab-L-TGF-βRII ECD       (II)Ab-L-TGF-βRII ECD (II)
其中TGF-βRII ECD为TGF-βRII胞外区的截短形式；Among them, TGF-βRII ECD is a truncated form of the extracellular region of TGF-βRII;
Ab为PD-L1抗体或其抗原结合片段；Ab is the PD-L1 antibody or its antigen-binding fragment;
L为连接序列。L is the connection sequence.
在某些实施方式中，所述的连接序列为(G4S)xG，其中x为3-6，优选为4。In some embodiments, the connection sequence is (G4S)xG, where x is 3-6, preferably 4.
在某些实施方式中，所述的PD-L1抗体或其抗原结合片段包含：In some embodiments, the PD-L1 antibody or antigen-binding fragment thereof comprises:
分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3，HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively LCDR1, LCDR2 and LCDR3,
其中，前面所述的各CDR序列如下所示：Among them, the aforementioned CDR sequences are as follows:
HCDR1：SYWMH                     SEQ ID NO：1HCDR1: SYWMH SEQ ID NO: 1
HCDR2：RIX1PNSGX2TSYNEKFKN       SEQ ID NO：2HCDR2: RIX1PNSGX2TSYNEKFKN SEQ ID NO: 2
HCDR3：GGSSYDYFDY                SEQ ID NO：3HCDR3: GGSSYDYFDY SEQ ID NO: 3
LCDR1：RASESVSIHGTHLMH           SEQ ID NO：4LCDR1: RASESVSIHGTHLMH SEQ ID NO: 4
LCDR2：AASNLES                   SEQ ID NO：5LCDR2: AASNLES SEQ ID NO: 5
LCDR3：QQSFEDPLT                 SEQ ID NO：6LCDR3: QQSFEDPLT SEQ ID NO: 6
其中X1为G，X2为F。Where X1 is G and X2 is F.
在某些实施方式中，所述PD-L1抗体或其抗原结合片段谓嵌合抗体或其功能片段、人源化抗体或其功能片段或人抗体或其功能片段。In some embodiments, the PD-L1 antibody or antigen-binding fragment thereof is a chimeric antibody or a functional fragment thereof, a humanized antibody or a functional fragment thereof, or a human antibody or a functional fragment thereof.
在某些实施方式中，所述人源化PD-L1抗体重、轻链的序列如下所示：In some embodiments, the sequences of the heavy and light chains of the humanized PD-L1 antibody are as follows:
PD-L1抗体重链可变区：PD-L1 antibody heavy chain variable region:
Figure PCTCN2020118609-appb-000003
Figure PCTCN2020118609-appb-000003
PD-L1抗体轻链可变区：PD-L1 antibody light chain variable region:
Figure PCTCN2020118609-appb-000004
Figure PCTCN2020118609-appb-000004
                           
Figure PCTCN2020118609-appb-000005
Figure PCTCN2020118609-appb-000005
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列；下划线为CDR序列，其中双下划线的位点为亲和力成熟筛选后得到的位点。Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the sequence is FR sequence in italics; the underline is the CDR sequence, and the double-underlined position is the position obtained after affinity maturation screening.
在某些实施方案中，所述PD-L1抗体或其抗原结合片段的重链氨基酸序列如SEQ ID NO：9所示或与SEQ ID NO：9所示的序列具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性，所述PD-L1抗体或其抗原结合片段的轻链氨基酸序列如SEQ ID NO：10所示或与SEQ ID NO：10所示的序列具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同一性，其中In some embodiments, the heavy chain amino acid sequence of the PD-L1 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 9 or has at least 85%, 86% with the sequence shown in SEQ ID NO: 9, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, the PD- The light chain amino acid sequence of the L1 antibody or its antigen-binding fragment is shown in SEQ ID NO: 10 or has at least 85%, 86%, 87%, 88%, 89%, 90% with the sequence shown in SEQ ID NO: 10 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, where
PD-L1抗体重链序列：Ig G4(AA)(S228P)PD-L1 antibody heavy chain sequence: Ig G4 (AA) (S228P)
Figure PCTCN2020118609-appb-000006
Figure PCTCN2020118609-appb-000006
PD-L1抗体轻链序列：PD-L1 antibody light chain sequence:
Figure PCTCN2020118609-appb-000007
Figure PCTCN2020118609-appb-000007
Figure PCTCN2020118609-appb-000008
Figure PCTCN2020118609-appb-000008
本公开中TGF-βRII胞外结构域及其截短形式的非限制性实施例序列如下：Non-limiting example sequences of the TGF-βRII extracellular domain and its truncated form in the present disclosure are as follows:
TGF-βRII胞外结构域序列：ECD(1-136)TGF-βRII extracellular domain sequence: ECD(1-136)
Figure PCTCN2020118609-appb-000009
Figure PCTCN2020118609-appb-000009
TGF-βRII胞外结构域序列在N端有19个氨基酸的截短或缺失：ECD(20-136)TGF-βRII extracellular domain sequence has a 19 amino acid truncation or deletion at the N-terminus: ECD(20-136)
Figure PCTCN2020118609-appb-000010
Figure PCTCN2020118609-appb-000010
TGF-βRII胞外结构域序列在N端有21个氨基酸的截短或缺失：ECD(22-136)The TGF-βRII extracellular domain sequence has a 21 amino acid truncation or deletion at the N-terminus: ECD(22-136)
Figure PCTCN2020118609-appb-000011
Figure PCTCN2020118609-appb-000011
TGF-βRII胞外结构域序列在N端有14个氨基酸的截短或缺失：ECD(15-136)TGF-βRII extracellular domain sequence has a truncation or deletion of 14 amino acids at the N-terminus: ECD(15-136)
Figure PCTCN2020118609-appb-000012
Figure PCTCN2020118609-appb-000012
利用同源重组技术将PD-L1抗体的重链C末端氨基酸通过(G 4S) xG连接不同长度TGF-βRII胞外区，与轻链一起，通过293表达***进行常规表达，得到如表1所示的融合蛋白： Using homologous recombination technology, the C-terminal amino acids of the heavy chain of the PD-L1 antibody were connected to the extracellular region of TGF-βRII of different lengths through (G 4 S) x G, together with the light chain, were routinely expressed through the 293 expression system to obtain the table Fusion protein shown in 1:
表1.PD-L1抗体/TGF-βRII胞外区融合蛋白Table 1. PD-L1 antibody/TGF-βRII extracellular domain fusion protein
Figure PCTCN2020118609-appb-000013
Figure PCTCN2020118609-appb-000013
注：Ab为本公开所述PD-L1抗体，序列描述中ECD(n-136)为TGF-βRII胞外区的全长或截短形式，n为TGF-βRII胞外区截短后的氨基酸起始位数。Note: Ab is the PD-L1 antibody described in the present disclosure. In the sequence description, ECD (n-136) is the full length or truncated form of the extracellular region of TGF-βRII, and n is the amino acid after truncation of the extracellular region of TGF-βRII. Starting digits.
在某些实施方式中，所述PD-L1抗体或其抗原结合片段选自avelumab、 atezolizumab、durvalumab、CS-1001、M-7824、KL-A167、CX-072、、BGB-A333、GNS-1480、CA-170、BMS-936559，优选avelumab、atezolizumab、durvalumab。In certain embodiments, the PD-L1 antibody or antigen-binding fragment thereof is selected from avelumab, atezolizumab, durvalumab, CS-1001, M-7824, KL-A167, CX-072, BGB-A333, GNS-1480 , CA-170, BMS-936559, preferably avelumab, atezolizumab, durvalumab.
在某些实施方式中，所述EZH2的给药剂量选自1-1600mg，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，给药频率为一日两次，一日一次，每两日一次，每三日一次，每四日一次，每五日一次，每六日一次，每周一次，每两周一次，每三周一次或每四周一次，优选一日两次或一日一次。In some embodiments, the administration dose of EZH2 is selected from 1-1600 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, 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, the frequency of administration is twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, weekly Once, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.
在某些实施方式中，所述EZH2的给药剂量选自1-800mg，给药频率为一日两次，一日一次，每两日一次，每三日一次，每四日一次，每五日一次，每六日一次，每周一次，每两周一次，每三周一次或每四周一次，优选一日两次或一日一次。In some embodiments, the dosage of EZH2 is selected from 1-800mg, and the frequency of administration is twice a day, once a day, once every two days, once every three days, once every four days, and every five days. Once a day, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.
在某些实施方式中，所述EZH2的给药剂量选自50mg、100mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg、500mg、550mg、600mg、 650mg、700mg、750mg、800mg，给药频率为一日两次，一日一次，每两日一次，每三日一次，每四日一次，每五日一次，每六日一次，每周一次，每两周一次，每三周一次或每四周一次，优选一日两次或一日一次。In some embodiments, the administration dose of EZH2 is selected from 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, administration The frequency is twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or Once every four weeks, preferably twice a day or once a day.
在某些实施方式中，所述EZH2的给药剂量选自150mg、200mg、250mg、300mg、350mg、400mg、450mg，给药频率为一日两次，一日一次，每两日一次，每三日一次，每四日一次，每五日一次，每六日一次，每周一次，每两周一次，每三周一次或每四周一次，优选一日两次或一日一次。In some embodiments, the dosage of EZH2 is selected from 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, and the frequency of administration is twice a day, once a day, once every two days, every three times. Once a day, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably twice a day or once a day.
在某些实施方式中，所述EZH2的给药剂量为350mg，每日两次。In some embodiments, the dosage of EZH2 is 350 mg twice a day.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量选自0.1-500mg/kg，给药频率为每周一次，每两周一次，每三周一次或每四周一次，具体的含有TGF-β受体的融合蛋白的给药剂量可以选自1mg/kg、2mg/kg、3mg/kg、4mg/kg、5mg/kg、6mg/kg、7mg/kg、8mg/kg、9mg/kg、10mg/kg、11mg/kg、12mg/kg、13mg/kg、14mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg、20mg/kg、21mg/kg、22mg/kg、23mg/kg、24mg/kg、25mg/kg、26mg/kg、27mg/kg、28mg/kg、29mg/kg、30mg/kg、31mg/kg、32mg/kg、33mg/kg、34mg/kg、35mg/kg、36mg/kg、37mg/kg、38mg/kg、39mg/kg、40mg/kg、41mg/kg、42mg/kg、43mg/kg、44mg/kg、45mg/kg、46mg/kg、47mg/kg、48mg/kg、49mg/kg、50mg/kg、51mg/kg、52mg/kg、53mg/kg、54mg/kg、55mg/kg、56mg/kg、57mg/kg、58mg/kg、59mg/kg、60mg/kg、61mg/kg、62mg/kg、63mg/kg、64mg/kg、65mg/kg、66mg/kg、67mg/kg、68mg/kg、69mg/kg、70mg/kg、71mg/kg、72mg/kg、73mg/kg、74mg/kg、75mg/kg、76mg/kg、77mg/kg、78mg/kg、79mg/kg、80mg/kg、81mg/kg、82mg/kg、83mg/kg、84mg/kg、85mg/kg、86mg/kg、87mg/kg、88mg/kg、89mg/kg、90mg/kg、91mg/kg、92mg/kg、93mg/kg、94mg/kg、95mg/kg、96mg/kg、97mg/kg、98mg/kg、99mg/kg、100mg/kg、105mg/kg、110mg/kg、115mg/kg、120mg/kg、125mg/kg、130mg/kg、135mg/kg、140mg/kg、145mg/kg、150mg/kg、155mg/kg、160mg/kg、165mg/kg、170mg/kg、175mg/kg、180mg/kg、185mg/kg、190mg/kg、195mg/kg、 200mg/kg、205mg/kg、210mg/kg、215mg/kg、220mg/kg、225mg/kg、230mg/kg、235mg/kg、240mg/kg、245mg/kg、250mg/kg、260mg/kg、270mg/kg、280mg/kg、290mg/kg、300mg/kg、310mg/kg、320mg/kg、330mg/kg、340mg/kg、350mg/kg、360mg/kg、370mg/kg、380mg/kg、390mg/kg、400mg/kg、410mg/kg、420mg/kg、430mg/kg、440mg/kg、450mg/kg、460mg/kg、470mg/kg、480mg/kg、490mg/kg、500mg/kg。In some embodiments, the dosage of the TGF-β receptor-containing fusion protein is selected from 0.1-500 mg/kg, and the dosage frequency is once a week, once every two weeks, once every three weeks or every four weeks. Once, the specific dosage of the fusion protein containing TGF-β receptor can be selected from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg. kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/kg, 33mg/ kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42mg/kg, 43mg/kg, 44mg/kg, 45mg/kg, 46mg/kg, 47mg/kg, 48mg/kg, 49mg/kg, 50mg/kg, 51mg/kg, 52mg/kg, 53mg/kg, 54mg/kg, 55mg/kg, 56mg/kg, 57mg/kg, 58mg/ kg, 59mg/kg, 60mg/kg, 61mg/kg, 62mg/kg, 63mg/kg, 64mg/kg, 65mg/kg, 66mg/kg, 67mg/kg, 68mg/kg, 69mg/kg, 70mg/kg, 71mg/kg, 72mg/kg, 73mg/kg, 74mg/kg, 75mg/kg, 76mg/kg, 77mg/kg, 78mg/kg, 79mg/kg, 80mg/kg, 81mg/kg, 82mg/kg, 83mg/ kg, 84mg/kg, 85mg/kg, 86mg/kg, 87mg/kg, 88mg/kg, 89mg/kg, 90mg/kg, 91mg/kg, 92mg/kg, 93mg/kg, 94mg/kg, 95mg/kg, 96mg/kg, 97mg/kg, 98mg/kg, 99mg/kg, 100mg/kg, 105mg/kg, 110mg/kg, 115mg/kg, 120mg/kg, 125mg/kg, 130mg/kg, 135mg/kg, 140mg/ kg, 145mg/kg, 150mg/kg, 155mg/kg, 160mg/kg, 1 65mg/kg, 170mg/kg, 175mg/kg, 180mg/kg, 185mg/kg, 190mg/kg, 195mg/kg, 200mg/kg, 205mg/kg, 210mg/kg, 215mg/kg, 220mg/kg, 225mg/ kg, 230mg/kg, 235mg/kg, 240mg/kg, 245mg/kg, 250mg/kg, 260mg/kg, 270mg/kg, 280mg/kg, 290mg/kg, 300mg/kg, 310mg/kg, 320mg/kg, 330mg/kg, 340mg/kg, 350mg/kg, 360mg/kg, 370mg/kg, 380mg/kg, 390mg/kg, 400mg/kg, 410mg/kg, 420mg/kg, 430mg/kg, 440mg/kg, 450mg/ kg, 460mg/kg, 470mg/kg, 480mg/kg, 490mg/kg, 500mg/kg.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量选自0.1-200mg/kg，给药频率为每周一次，每两周一次，每三周一次或每四周一次。In some embodiments, the dosage of the TGF-β receptor-containing fusion protein is selected from 0.1-200 mg/kg, and the frequency of administration is once a week, once every two weeks, once every three weeks or every four weeks once.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量选自0.1-100mg/kg，给药频率为每周一次，每两周一次，每三周一次或每四周一次。In some embodiments, the dosage of the fusion protein containing TGF-β receptor is selected from 0.1-100 mg/kg, and the frequency of administration is once a week, once every two weeks, once every three weeks or every four weeks once.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量选自1mg/kg、2mg/kg、3mg/kg、4mg/kg、5mg/kg、6mg/kg、7mg/kg、8mg/kg、9mg/kg、10mg/kg、11mg/kg、12mg/kg、13mg/kg、14mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg、20mg/kg、21mg/kg、22mg/kg、23mg/kg、24mg/kg、25mg/kg、26mg/kg、27mg/kg、28mg/kg、29mg/kg、30mg/kg、31mg/kg、32mg/kg、33mg/kg、34mg/kg、35mg/kg、36mg/kg、37mg/kg、38mg/kg、39mg/kg、40mg/kg、41mg/kg、42mg/kg、43mg/kg、44mg/kg、45mg/kg、46mg/kg、47mg/kg、48mg/kg、49mg/kg、50mg/kg、51mg/kg、52mg/kg、53mg/kg、54mg/kg、55mg/kg、56mg/kg、57mg/kg、58mg/kg、59mg/kg、60mg/kg，给药频率为每两周一次，每三周一次或每四周一次。In some embodiments, the dosage of the fusion protein containing TGF-β receptor is selected from 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg. kg, 8mg/kg, 9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/ kg, 33mg/kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42mg/kg, 43mg/kg, 44mg/kg, 45mg/kg, 46mg/kg, 47mg/kg, 48mg/kg, 49mg/kg, 50mg/kg, 51mg/kg, 52mg/kg, 53mg/kg, 54mg/kg, 55mg/kg, 56mg/kg, 57mg/ kg, 58mg/kg, 59mg/kg, 60mg/kg, the frequency of administration is once every two weeks, once every three weeks or once every four weeks.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量选自1mg/kg、3mg/kg、10mg/kg、15mg/kg、20mg/kg、25mg/kg、30mg/kg、35mg/kg、40mg/kg、45mg/kg、50mg/kg、55mg/kg、60mg/kg，给药频率为每两周一次，每三周一次或每四周一次。In some embodiments, the dosage of the fusion protein containing TGF-β receptor is selected from 1 mg/kg, 3 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg. kg, 35mg/kg, 40mg/kg, 45mg/kg, 50mg/kg, 55mg/kg, 60mg/kg, the frequency of administration is once every two weeks, once every three weeks or once every four weeks.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量选自10mg/kg、20mg/kg、30mg/kg、40mg/kg、50mg/kg、60mg/kg，给药频率为每两周一次，每三周一次或每四周一次。In some embodiments, the administration dose of the fusion protein containing TGF-β receptor is selected from the group consisting of 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, and administration The frequency is once every two weeks, once every three weeks or once every four weeks.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的给药剂量为30mg/kg，每3周一次。In some embodiments, the dosage of the TGF-β receptor-containing fusion protein is 30 mg/kg, once every 3 weeks.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的剂量为1-4000mg，具体可以为50mg、100mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg、500mg、550mg、600mg、650mg、700mg、750mg、800mg、850mg、900mg、950mg、1000mg、1050mg、1100mg、1150mg、1200mg、1250mg、1300mg、1350mg、1400mg、1450mg、1500mg、1550mg、1600mg、1650mg、1700mg、1750mg、1800mg、1850mg、1900mg、1950mg、2000mg、2050mg、2100mg、2150mg、2200mg、2250mg、2300mg、2350mg、2400mg、2450mg、2500mg、2550mg、2600mg、2650mg、2700mg、2750mg、2800mg、2850mg、2900mg、2950mg、3000mg、3050mg、3100mg、3150mg、3200mg、3250mg、3300mg、3350mg、3400mg、3450mg、3500mg、3550mg、3600mg、3650mg、3700mg、3750mg、3800mg、3850mg、3900mg、3950mg、4000mg，给药频率为每周一次，每两周一次，每三周一次或每四周一次。In some embodiments, the dosage of the fusion protein containing TGF-β receptor is 1-4000 mg, specifically 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, 2000mg, 2050mg, 2100mg, 2150mg, 2200mg, 2250mg, 2300mg, 2350mg, 2400mg, 2450mg, 2500mg, 2550mg, 2600mg, 2650mg, 2700mg, 2750mg, 2800mg, 2850mg, 2900mg, 2950mg, 3000mg, 3050mg, 3100mg, 3150mg, 3200mg, 3250mg, 3300mg, 3350mg, 3400mg, 3450mg, 3500mg, 3550mg, 3600mg, 3650mg, 3700mg, 3750mg, 3800mg, 3850mg, 3900mg, 3950mg, 4000mg, dosing frequency is once a week, once every two weeks , Once every three weeks or once every four weeks.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的剂量为300mg、600mg、900mg、1200mg、1500mg、1800mg、2100mg、2400mg、2700mg、3000mg、3300mg、3600mg，给药频率为每两周一次，每三周一次或每四周一次。In some embodiments, the dosage of the fusion protein containing TGF-β receptor is 300 mg, 600 mg, 900 mg, 1200 mg, 1500 mg, 1800 mg, 2100 mg, 2400 mg, 2700 mg, 3000 mg, 3300 mg, 3600 mg, and the dosage frequency is every Once every two weeks, once every three weeks or once every four weeks.
在某些实施方式中，所述含有TGF-β受体的融合蛋白的剂量为600mg、1200mg、1800mg、2400mg、3000mg，给药频率为每两周一次或每三周一次。In some embodiments, the dosage of the TGF-β receptor-containing fusion protein is 600 mg, 1200 mg, 1800 mg, 2400 mg, 3000 mg, and the administration frequency is once every two weeks or once every three weeks.
可选的实施方案中，所述的含有TGF-β受体的融合蛋白以注射的方式给药，例如皮下或静脉注射，注射前需将含有TGF-β受体的融合蛋白配制成可注射的形式，含有TGF-β受体的融合蛋白的可注射形式可以是是注射液或冻 干粉针，其包含含有TGF-β受体的融合蛋白、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等。In an alternative embodiment, the TGF-β receptor-containing fusion protein is administered by injection, such as subcutaneous or intravenous injection, and the TGF-β receptor-containing fusion protein needs to be formulated as an injectable before injection. The injectable form of the fusion protein containing the TGF-β receptor can be an injection or a lyophilized powder injection, which contains the fusion protein containing the TGF-β receptor, 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, maltose. The surfactant is selected from polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester and the like.
本公开提供一种***或癌症的方法，包括向患者施用有效量上述EZH2抑制剂和有效量上述含有TGF-β受体的融合蛋白。The present disclosure provides a method for treating tumor or cancer, which comprises administering to a patient an effective amount of the above-mentioned EZH2 inhibitor and an effective amount of the above-mentioned TGF-β receptor-containing fusion protein.
本公开另一方面提供一种用于***或癌症的EZH2抑制剂，所述EZH2抑制剂与含有TGF-β受体的融合蛋白联合使用。Another aspect of the present disclosure provides an EZH2 inhibitor for the treatment of tumors or cancers, which is used in combination with a fusion protein containing a TGF-β receptor.
本公开另一方面提供一种用于***或癌症的含有TGF-β受体的融合蛋白，所述含有TGF-β受体的融合蛋白与EZH2抑制剂联合使用。Another aspect of the present disclosure provides a TGF-β receptor-containing fusion protein for treating tumors or cancers, and the TGF-β receptor-containing fusion protein is used in combination with an EZH2 inhibitor.
可选的实施方案中，本公开提供的***或癌症的方法，所述患者为人类。In an alternative embodiment, in the method for treating tumor or cancer provided by the present disclosure, the patient is a human.
本公开中所述肿瘤或癌症选自：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、食管癌、***癌、肾癌、***、骨髓瘤、淋巴瘤、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱、神经内分泌瘤、头部颈部癌、肝癌、胆管癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合征。The tumor or cancer in the present disclosure is selected from: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, uterus Endometrial cancer, uterine cancer, bladder, neuroendocrine tumor, head and neck cancer, liver cancer, cholangiocarcinoma, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell Shape cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
本公开中所述“联合”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EZH2抑制剂和至少一种剂量的含有TGF-β受体的融合蛋白，其中两种物质都显示药理学作用。所述的时间期限可以是一个给药周期内，可选4周内，3周内，2周内，1周内，24小时以内，2小时以内。可以同时或依次给予EZH2抑制剂和含有TGF-β受体的融合蛋白。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EZH2抑制剂和含有TGF-β受体的融合蛋白。本公开所述联合的给药方式选自同时给药、独立地配制 并共给药或独立地配制并相继给药。The “combination” in the present disclosure refers to the administration of at least one dose of EZH2 inhibitor and at least one dose of fusion protein containing TGF-β receptor within a certain period of time, of which two substances Both show pharmacological effects. The time limit can be within one administration cycle, optionally within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, within 24 hours, or within 2 hours. The EZH2 inhibitor and the fusion protein containing the TGF-β receptor can be administered simultaneously or sequentially. This period includes treatments in which the EZH2 inhibitor and the TGF-β receptor-containing fusion protein are administered through the same route of administration or different routes of administration. The mode of administration of the combination described in the present disclosure is selected from simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration.
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。The "antibody" described in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
在本公开中，本公开所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。In the present disclosure, the antibody light chain described in the present disclosure may further include a light chain constant region, and the light chain constant region includes human or murine κ, λ chains or variants thereof.
在本公开中，本公开所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。In the present disclosure, the antibody heavy chain described in the present disclosure may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDE2-3)，或者符合kabat和chothia的编号规则(HCDR1)。The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions. The sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known Kabat numbering rules (LCDR1-3, HCDE2-3), or comply with the numbering rules of Kabat and Chothia (HCDR1).
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。The antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
本公开中所述的“抗体或其抗原结合”或“功能片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与抗体结合的Fv片段ScFv片段。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)。本公开的术语“与PD-L1结合”，指能与人PD-L1相互作用。本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原 结合片段识别的三维空间位点。The "antibody or its antigen binding" or "functional fragment" as used in the present disclosure refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and ScFv fragments that bind to antibodies. The Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but has no constant region and is the smallest antibody fragment with all antigen binding sites. Generally, an Fv antibody also contains 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 "binding to PD-L1" in the present disclosure refers to the ability to interact with human PD-L1. The term "antigen-binding site" in the present disclosure refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人PD-L1的单克隆抗体。制备时用PD-L1抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。The term "murine antibody" in the present disclosure refers to a monoclonal antibody to human PD-L1 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with PD-L1 antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的PCSK-9嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的PCSK-9嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源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，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。The term "chimeric antibody" is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma secreting murine-specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed. The gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the present disclosure, the antibody light chain of the PCSK-9 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the PCSK-9 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof. The constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising human IgG2 or IgG4 heavy chain constant region, or using amino acid mutations without ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
术语“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，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包 括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。 The term "humanized antibody", 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 germlines The antibody produced in the antibody framework sequence. It can overcome the strong 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 public DNA databases or published references that include germline antibody gene sequences. For example, the 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 www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc. Human, 1991 Sequences of Proteins of Immunological Interest, found in 5th edition. In order to avoid the decrease of immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.
本公开中所述“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。本公开中的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。In the present disclosure, "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. The sequence identity in the present disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparison and determination of the percent identity between two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
术语“TGF-β受体II”或“TGFβRII”或“转化生长因子β受体II”是指结合配体(包括但不限于TGFβ1、TGFβ2和TGFβ3)、并且由此引发细胞内的信号转导途径的细胞表面受体。The term "TGF-β receptor II" or "TGFβRII" or "transforming growth factor β receptor II" refers to binding ligands (including but not limited to TGFβ1, TGFβ2 and TGFβ3) and thereby triggering intracellular signal transduction Pathway of cell surface receptors.
术语“PD-L1”是指程序性死亡配体1，也称为CD274和B7H1。PD-L1是具有胞外IgV样和IgC样结构域(全长PD-L1的氨基酸19-239)、跨膜结构域和约30个氨基酸的胞内结构域的290个氨基酸的蛋白质。PD-L1在许多细胞例如抗原呈递细胞(例如，树突细胞、巨噬细胞和B细胞)上以及造血细胞和非造血细胞(例如，血管内皮细胞、胰岛、和免疫赦免部位)上组成型表达。PD-L1也在多种肿瘤和病毒感染的细胞上表达，并且是免疫抑制环境(immunosuppressive milieu)的组成(Ribas 2012，NEJM 366：2517-2519)。PD-L1与两种T细胞共抑制剂PD-1和B7-1之一结合。The term "PD-L1" refers to programmed death ligand 1, also known as CD274 and B7H1. PD-L1 is a 290 amino acid protein with extracellular IgV-like and IgC-like domains (amino acids 19-239 of full length PD-L1), a transmembrane domain, and an intracellular domain of about 30 amino acids. PD-L1 is constitutively expressed on many cells such as antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells), as well as hematopoietic and non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islets, and immune pardon sites) . PD-L1 is also expressed on a variety of tumors and virus-infected cells, and is a component of an immunosuppressive milieu (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of the two T cell co-inhibitors PD-1 and B7-1.
本公开中所述的融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，小鼠可以用人PD-L1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免 疫球蛋白杂志，2001ISBN012441351上获得。The fusion protein described in the present disclosure is a protein product in which two genes are co-expressed through DNA recombination. The methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human PD-L1 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to the non-human CDR region. The human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。"Effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
附图说明Description of the drawings
图1.小鼠肿瘤接种位置示意图；Figure 1. Schematic diagram of mouse tumor inoculation position;
图2.在BALB/C-hPDL1人源化小鼠皮下CT26-hPDL1结肠癌模型中的肿瘤体积变化，*,v.s.对照，#v.s.TGF-β受体融合蛋白单药；Figure 2. Tumor volume changes in the BALB/C-hPDL1 humanized mouse subcutaneous CT26-hPDL1 colon cancer model, *, v.s. control, #v.s. TGF-β receptor fusion protein single agent;
图3.在BALB/C-hPDL1人源化小鼠皮下CT26-hPDL1结肠癌模型中的小鼠体重变化。Figure 3. Mouse body weight changes in the BALB/C-hPDL1 humanized mouse subcutaneous CT26-hPDL1 colon cancer model.
具体实施方式Detailed ways
实施例1、评价了测试药物在免疫检测点人源化小鼠BALB/c-hPDL1皮下CT26-hPDL1结肠癌肿瘤模型中的药效。Example 1. The efficacy of the test drug in the humanized mouse BALB/c-hPDL1 subcutaneous CT26-hPDL1 colon cancer tumor model at the immune checkpoint was evaluated.
1、实验材料1. Experimental materials
实验动物：小鼠，BALB/C-hPDL1，7.57-8.14周(接种肿瘤细胞时周龄)，雌性，90只，来源于江苏集萃药康生物科技有限公司。Experimental animals: mice, BALB/C-hPDL1, 7.57-8.14 weeks (weeks old when tumor cells were inoculated), female, 90 animals, from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
实验细胞：来源于江苏集萃药康生物科技有限公司。Experimental cells: from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
实验药物：TGF-β受体融合蛋白：融合蛋白9，50mg/ml；EZH2抑制剂：式(I)所示化合物l。Experimental drug: TGF-β receptor fusion protein: fusion protein 9, 50 mg/ml; EZH2 inhibitor: compound 1 represented by formula (I).
2、实验方法2. Experimental method
1)人源化CT26-hPDL1细胞(该细胞表达人的hPDL1并敲除小鼠的mPDL1)复苏并记录复苏代次(Pn+4代)。细胞在体外培养时添加200ng/ml  G418进行抗性维持，培养基为1640+10％FBS+1％P/S+200ng/ml G418。于当天收集对数生长期的CT26-hPDL1细胞，去除培养液并用PBS洗两次后接种(荷瘤前、荷瘤后CT26细胞存活率分别为：97.6％及83.3％)，接种量：5×10 5/100ul/只，接种位置：右侧背部大腿上方位置(图1所示1号位)。 1) Humanized CT26-hPDL1 cells (the cells express human hPDL1 and knock out mPDL1 in mice) were resuscitated and the resuscitation generations (Pn+4 generation) were recorded. When cells are cultured in vitro, 200ng/ml G418 is added to maintain resistance, and the medium is 1640+10% FBS+1% P/S+200ng/ml G418. CT26-hPDL1 cells in the logarithmic growth phase were collected on the same day, the culture medium was removed and washed twice with PBS, and then inoculated (the survival rate of CT26 cells before and after tumor was 97.6% and 83.3%, respectively). Inoculation amount: 5× 10 5 /100ul/only, inoculation position: the upper thigh of the right back (position 1 shown in Figure 1).
2)实验方案设计如表3所示，当平均肿瘤体积达到88.16mm 3时，小鼠根据肿瘤体积随机分成4组，每组7只。分组当天定义为D0天，并于D0天开始给药。 2) The experimental design is shown in Table 3. When the average tumor volume reaches 88.16 mm 3 , the mice are randomly divided into 4 groups according to the tumor volume, with 7 mice in each group. The grouping day was defined as D0 day, and the administration was started on D0 day.
表2.实验方案设计Table 2. Experimental design
Figure PCTCN2020118609-appb-000014
Figure PCTCN2020118609-appb-000014
备注：1.N:动物只数；G:Group，i.p为腹腔注射，p.o为灌胃，BID每天两次，Q3D每三天1次2.给药体积：
Figure PCTCN2020118609-appb-000015
按体重调整给药体积10μl/g。联合给药时，先给与口服的小分子，待全部动物给完小分子之后，再给与抗体注射。 Remarks: 1.N: number of animals; G: Group, ip means intraperitoneal injection, po means gavage, BID twice a day, Q3D once every three days 2. Dosing volume:
Figure PCTCN2020118609-appb-000015
Adjust the administration volume to 10μl/g according to body weight. In the combined administration, the oral small molecules are given first, and then the antibody injections are given after all animals are given the small molecules.
3)药物配置3) Drug configuration
表3.实验药物配置Table 3. Experimental drug configuration
Figure PCTCN2020118609-appb-000016
Figure PCTCN2020118609-appb-000016
4)实验观察和数据采集4) Experimental observation and data collection
细胞接种后，每周常规监测肿瘤对动物正常行为的影响。具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。开始给药后，每周称量体重三次。每周测量瘤体积三次，瘤体积计算方式为：肿瘤体积(mm 3)＝0.5(肿瘤长径×肿瘤短径 2)。 After cell inoculation, the impact of tumors on the normal behavior of animals was routinely monitored every week. The specific contents include the mobility of the experimental animal, food and water consumption, weight gain or loss, eyes, coat and other abnormal conditions. After starting the administration, the body weight was weighed three times a week. The tumor volume was measured three times a week, and the tumor volume calculation method was: tumor volume (mm 3 )=0.5 (tumor long diameter×tumor short diameter 2 ).
实验结束时，分析下列指标：肿瘤体积变化(TGItv)、平均体重变化、肿瘤重量变化(TGItw)At the end of the experiment, analyze the following indicators: tumor volume change (TGItv), average weight change, tumor weight change (TGItw)
TGItv(相对肿瘤抑制率)计算公式：Mean％ΔInhibition＝((mean(C)-mean(C0))-(mean(T)-mean(T0)))/(mean(C)-mean(C0))*100％；其中T–给药组肿瘤体积，TGItv (relative tumor inhibition rate) calculation formula: Mean%ΔInhibition=((mean(C)-mean(C0))-(mean(T)-mean(T0)))/(mean(C)-mean(C0) )*100%; where T-administration group tumor volume,
T0–给药组初始肿瘤体积，C–对照组肿瘤体积，C0–对照组初始肿瘤体积T0—Initial tumor volume in the administration group, C—Tumor volume in the control group, C0—Initial tumor volume in the control group
肿瘤重量变化(TGItw)计算公式：TGItw＝(1-meanTW treat/meanTW vehicle)x100％；其中meanTWtreat:给药组小鼠终点处理时肿瘤重量的平均值，meanTW vehicle:Vehicle组小鼠终点处理时肿瘤重量的平均值Tumor weight change (TGItw) calculation formula: TGItw=(1-meanTW treat/meanTW vehicle)x100%; where meanTWtreat: mean value of tumor weight at the end-point treatment of mice in the administration group, meanTW vehicle: end-point treatment of mice in the vehicle group Average tumor weight
各组动物的肿瘤体积、小鼠体重、肿瘤重量等实验结果以平均值±标准误差(Mean±SEM)表示。采用独立样本T检验方法比较不同治疗组与对照组相比有无显著性差异。所有的数据均用SPSS 18.0进行分析。P<0.05为具有显著性差异。作图软件为Graphpad prism 6.0。The experimental results of tumor volume, mouse body weight, tumor weight of each group of animals are expressed as mean±standard error (Mean±SEM). The independent sample T test method was used to compare whether there were significant differences between the different treatment groups and the control group. All data are analyzed with SPSS 18.0. P<0.05 indicates a significant difference. The drawing software is Graphpad prism 6.0.
3、实验结果3. Experimental results
1)肿瘤体积及瘤重变化1) Changes in tumor volume and tumor weight
不同组别小鼠的肿瘤体积变化如图2所示。The tumor volume changes of mice in different groups are shown in Figure 2.
2)肿瘤抑制率(TGI)及P值分析2) Analysis of tumor inhibition rate (TGI) and P value
表4.不同组别肿瘤抑制率变化(TGI)及P值Table 4. Changes in tumor inhibition rate (TGI) and P value in different groups
Figure PCTCN2020118609-appb-000017
Figure PCTCN2020118609-appb-000017
3)体重变化3) Weight change
不同组别小鼠的体重变化如图3。The body weight changes of mice in different groups are shown in Figure 3.
结论：TGI数据显示联用组(第4组)相比于对照组、单药组(第2组及第3组)均有显著性差异，在当前的测试***下，TGF-β受体融合蛋白(3mg/kg)和EZH2抑制剂(100mg/kg)联合给药有显著的抗肿瘤效果。Conclusion: TGI data shows that the combination group (group 4) has significant differences compared with the control group and the single-drug group (groups 2 and 3). Under the current test system, TGF-β receptor fusion The combined administration of protein (3mg/kg) and EZH2 inhibitor (100mg/kg) has a significant anti-tumor effect.
实施例2：一项PL1/TGF-β融合蛋白联合用于多种肿瘤的临床试验Example 2: A clinical trial of PL1/TGF-β fusion protein used in multiple tumors
试验药物：EZH2抑制剂：式(I)所示化合物l，350mg口服2次/日；PD-L1/TGFβ融合蛋白：融合蛋白9,30mg/kg，静滴1次/3周；21天为1个周期。Test drug: EZH2 inhibitor: compound 1 of formula (I), 350 mg orally 2 times/day; PD-L1/TGFβ fusion protein: fusion protein 9,30 mg/kg, intravenous infusion 1 time/3 weeks; 21 days for 1 cycle.
入组以下患者：有明确病理学依据的晚期非小细胞肺癌(EGFR/ALK/ROS-1阴性)、消化道肿瘤(食管癌、胃癌、肠癌)、胆道胰腺肿瘤(胆管癌、胰腺癌)、乳腺癌(HER2/ER/PR阴性)、妇科肿瘤(卵巢癌、***)及淋巴瘤。实体肿瘤至少接受过一线治疗且失败(胆管癌、胰腺癌除外)，淋巴瘤至少接受过两线治疗且失败。既往治疗中抗PD-1/PD-L1抗体不限。年满18～75周岁，性别不限。预计最终纳入50例患者。The following patients were included: advanced non-small cell lung cancer (EGFR/ALK/ROS-1 negative) with clear pathological evidence, gastrointestinal tumors (esophageal cancer, gastric cancer, intestinal cancer), biliary pancreatic tumors (bile duct cancer, pancreatic cancer) , Breast cancer (HER2/ER/PR negative), gynecological tumors (ovarian cancer, cervical cancer) and lymphoma. Solid tumors have received at least first-line treatment and failed (except for cholangiocarcinoma and pancreatic cancer), and lymphoma has received at least two-line treatment and failed. Anti-PD-1/PD-L1 antibodies in previous treatments are not limited. At least 18 to 75 years old, regardless of gender. It is expected that 50 patients will eventually be included.

Claims (18)

  1. 一种EZH2抑制剂与含有TGF-β受体的融合蛋白联合在制备***或癌症的药物中的用途。The use of an EZH2 inhibitor and a fusion protein containing a TGF-β receptor in the preparation of a medicine for treating tumors or cancers.
  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)所示化合物或其可药用盐，优选式(I)所示化合物或其可药用盐，The use according to claim 1, wherein the EZH2 inhibitor is selected from CPI-0209, CPI-1205, GSK126, valemetostat, tazemetostat, PF-06821497, DS-3201 GSK-2816126, 3-deazaneplanocin A, HKMT-I -005, KM-301 or a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, preferably a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2020118609-appb-100001
    Figure PCTCN2020118609-appb-100001
  3. 根据权利要求2所述的用途，所述的TGF-β受体的融合蛋白包含PD-L1抗体或其抗原结合片段和TGF-β受体，其中TGF-β受体部分为TGF-βRII胞外区的N端截短形式。The use according to claim 2, wherein the fusion protein of the TGF-β receptor comprises a PD-L1 antibody or an antigen-binding fragment thereof and a TGF-β receptor, wherein the part of the TGF-β receptor is TGF-βRII extracellular N-terminal truncated form of the region.
  4. 根据权利要求3所述的用途，所述TGF-β受体融合蛋白如通式(II)所示：The use according to claim 3, the TGF-β receptor fusion protein is represented by the general formula (II):
    Ab-L-TGF-βRII ECD   (II)Ab-L-TGF-βRII ECD (II)
    其中TGF-βRII ECD为TGF-βRII胞外区的截短形式；Among them, TGF-βRII ECD is a truncated form of the extracellular region of TGF-βRII;
    Ab为PD-L1抗体或其抗原结合片段；Ab is the PD-L1 antibody or its antigen-binding fragment;
    L为连接序列。L is the connection sequence.
  5. 根据权利要求4所述的用途，其中所述的连接序列为(G4S)xG，其中x为3-6，优选为4。The use according to claim 4, wherein the linking sequence is (G4S)xG, wherein x is 3-6, preferably 4.
  6. 根据权利要求4所述的用途，所述的TGF-βRII胞外区的序列如SEQ ID NO：11、12、13和14所示；优选包含SEQ ID NO：12所示的序列。The use according to claim 4, the sequence of the extracellular region of TGF-βRII is shown in SEQ ID NO: 11, 12, 13 and 14, and preferably comprises the sequence shown in SEQ ID NO: 12.
  7. 根据权利要求4所述的用途，所述的PD-L1抗体或其抗原结合片段 包含：The use according to claim 4, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises:
    分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively LCDR1, LCDR2 and LCDR3.
  8. 根据权利要求4-7任一项所述的用途，所述PD-L1抗体或其抗原结合片段谓嵌合抗体或其功能片段、人源化抗体或其功能片段或人抗体或其功能片段。The use according to any one of claims 4-7, the PD-L1 antibody or antigen-binding fragment thereof is a chimeric antibody or a functional fragment thereof, a humanized antibody or a functional fragment thereof, or a human antibody or a functional fragment thereof.
  9. 根据权利要求8所述的用途，所述PD-L1抗体或其抗原结合片段包含包含如SEQ ID NO:7所示的重链可变区，和如SEQ ID NO:8所示的轻链可变区。The use according to claim 8, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 7, and a light chain variable region as shown in SEQ ID NO: 8 Variable area.
  10. 根据权利要求1-9任一项所述的用途，所述PD-L1抗体或其抗原结合片段的重链氨基酸序列如SEQ ID NO：9所示或与SEQ ID NO：9所示的氨基酸序列具有至少95％的同一性，所述PD-L1抗体或其抗原结合片段的轻链氨基酸序列如SEQ ID NO：10所示或与SEQ ID NO：10所示氨基酸序列具有至少95％的同一性。The use according to any one of claims 1-9, the heavy chain amino acid sequence of the PD-L1 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 9 or is the same as the amino acid sequence shown in SEQ ID NO: 9 Have at least 95% identity, and the light chain amino acid sequence of the PD-L1 antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 10 or has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 10 .
  11. 根据权利要求4所述的用途，所述PD-L1抗体或其抗原结合片段选自avelumab、atezolizumab、durvalumab、CS-1001、M-7824、KL-A167、CX-072、、BGB-A333、GNS-1480、CA-170、BMS-936559，优选avelumab、atezolizumab、durvalumab。The use according to claim 4, wherein the PD-L1 antibody or antigen-binding fragment thereof is 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.
  12. 根据权利要求1-11任一项所述的用途，所述EZH2抑制剂的给药剂量选自1-1600mg，给药频率为一日两次，一日一次，每两日一次，每三日一次，每四日一次，每五日一次，每六日一次，每周一次，每两周一次，每三周一次或每四周一次，优选一日一次或一日两次。The use according to any one of claims 1-11, the dosage of the EZH2 inhibitor is selected from 1 to 1600 mg, and the frequency of administration is twice a day, once a day, once every two days, every three days Once, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks or once every four weeks, preferably once a day or twice a day.
  13. 根据权利要求12所述的用途，所述EZH2的给药剂量选自150mg、200mg、250mg、300mg、350mg、400mg、450mg，给药频率为一日一次或一日两次。The use according to claim 12, wherein the dosage of EZH2 is selected from 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, and the frequency of administration is once a day or twice a day.
  14. 根据权利要求1-13任一项所述的用途，所述含有TGF-β受体的融合蛋白的给药剂量选自0.1-500mg/kg，给药频率为每周一次，每两周一次，每三周一次或每四周一次。The use according to any one of claims 1-13, wherein the dosage of the TGF-β receptor-containing fusion protein is selected from 0.1-500mg/kg, and the frequency of administration is once a week, once every two weeks, Once every three weeks or once every four weeks.
  15. 根据权利要求14所述的用途，所述含有TGF-β受体的融合蛋白的给药剂量选自10mg/kg、20mg/kg、30mg/kg、40mg/kg、50mg/kg、60mg/kg，给药频率为每两周一次，每三周一次或每四周一次。The use according to claim 14, wherein the dosage of the TGF-β receptor-containing fusion protein is selected from the group consisting of 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, The frequency of administration is once every two weeks, once every three weeks or once every four weeks.
  16. 根据权利要求1-13任一项所述的用途，所述含有TGF-β受体的融合蛋白的给药剂量选自1-4000mg，给药频率为每周一次，每两周一次，每三周一次或每四周一次。The use according to any one of claims 1-13, wherein the dosage of the TGF-β receptor-containing fusion protein is selected from 1-4000 mg, and the dosage frequency is once a week, once every two weeks, and every three Once a week or once every four weeks.
  17. 根据权利要求16所述的用途，所述含有TGF-β受体的融合蛋白的给药剂量选自300mg、600mg、900mg、1200mg、1500mg、1800mg、2100mg、2400mg、2700mg、3000mg、3300mg、3600mg，给药频率为每两周一次，每三周一次或每四周一次。The use according to claim 16, wherein the dosage of the TGF-β receptor-containing fusion protein is selected from the group consisting of 300mg, 600mg, 900mg, 1200mg, 1500mg, 1800mg, 2100mg, 2400mg, 2700mg, 3000mg, 3300mg, 3600mg, The frequency of administration is once every two weeks, once every three weeks or once every four weeks.
  18. 根据权利要求1-17任一项所述用途，所述肿瘤或癌症选自：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、食管癌、***癌、肾癌、***、骨髓瘤、淋巴瘤、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱、神经内分泌瘤、头部颈部癌、肝癌、胆管癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合征。The use according to any one of claims 1-17, wherein the tumor or cancer is selected from: colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, kidney cancer, cervical cancer, myeloma , Lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder, neuroendocrine tumor, head and neck cancer, liver cancer, cholangiocarcinoma, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, Melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome .
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023030299A1 (en) * 2021-08-30 2023-03-09 江苏恒瑞医药股份有限公司 Use of ezh2 inhibitor in preparation of drug for treating t-cell lymphoma

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017084494A1 (en) * 2015-11-19 2017-05-26 江苏恒瑞医药股份有限公司 Benzofuran derivative, preparation method thereof and use thereof in medicine
WO2017210395A1 (en) * 2016-06-01 2017-12-07 Epizyme, Inc. Use of ezh2 inhibitors for treating cancer
WO2018144798A1 (en) * 2017-02-02 2018-08-09 Epizyme, Inc. Cancer treatment modalities
WO2018205985A1 (en) * 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 FUSION PROTEIN CONTAINING TGF-β RECEPTOR AND MEDICINAL USES THEREOF
WO2018223030A1 (en) * 2017-06-02 2018-12-06 Epizyme, Inc. Use of ezh2 inhibitors for treating cancer
WO2019097369A1 (en) * 2017-11-14 2019-05-23 Pfizer Inc. Ezh2 inhibitor combination therapies

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017084494A1 (en) * 2015-11-19 2017-05-26 江苏恒瑞医药股份有限公司 Benzofuran derivative, preparation method thereof and use thereof in medicine
WO2017210395A1 (en) * 2016-06-01 2017-12-07 Epizyme, Inc. Use of ezh2 inhibitors for treating cancer
WO2018144798A1 (en) * 2017-02-02 2018-08-09 Epizyme, Inc. Cancer treatment modalities
WO2018205985A1 (en) * 2017-05-12 2018-11-15 江苏恒瑞医药股份有限公司 FUSION PROTEIN CONTAINING TGF-β RECEPTOR AND MEDICINAL USES THEREOF
WO2018223030A1 (en) * 2017-06-02 2018-12-06 Epizyme, Inc. Use of ezh2 inhibitors for treating cancer
WO2019097369A1 (en) * 2017-11-14 2019-05-23 Pfizer Inc. Ezh2 inhibitor combination therapies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MURAI FUMIHIKO, KOINUMA DAIZO, SHINOZAKI-USHIKU AYA, FUKAYAMA MASASHI, MIYAOZONO KOHEI, EHATA SHOGO: "EZH2 promotes progression of small cell lung cancer by suppressing the TGF-β-Smad-ASCL1 pathway", CELL DISCOVERY, vol. 1, no. 1, 1 December 2015 (2015-12-01), XP055796791, DOI: 10.1038/celldisc.2015.26 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023030299A1 (en) * 2021-08-30 2023-03-09 江苏恒瑞医药股份有限公司 Use of ezh2 inhibitor in preparation of drug for treating t-cell lymphoma

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