WO2020119758A1 - Il-15蛋白复合物联合pd-l1抗体用于治疗肿瘤疾病的用途 - Google Patents

Il-15蛋白复合物联合pd-l1抗体用于治疗肿瘤疾病的用途 Download PDF

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WO2020119758A1
WO2020119758A1 PCT/CN2019/124833 CN2019124833W WO2020119758A1 WO 2020119758 A1 WO2020119758 A1 WO 2020119758A1 CN 2019124833 W CN2019124833 W CN 2019124833W WO 2020119758 A1 WO2020119758 A1 WO 2020119758A1
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antibody
seq
fusion protein
use according
antigen
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PCT/CN2019/124833
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English (en)
French (fr)
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孙星
马晨
杨昌永
廖成
张连山
邹建军
施薇
杜园媛
周玉洁
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江苏恒瑞医药股份有限公司
苏州盛迪亚生物医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 苏州盛迪亚生物医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CA3122333A priority Critical patent/CA3122333A1/en
Priority to AU2019399720A priority patent/AU2019399720A1/en
Priority to JP2021532250A priority patent/JP2022512139A/ja
Priority to US17/311,642 priority patent/US20220111008A1/en
Priority to MX2021006589A priority patent/MX2021006589A/es
Priority to EP19897174.9A priority patent/EP3896089A4/en
Priority to KR1020217020567A priority patent/KR20210102917A/ko
Priority to BR112021010999-4A priority patent/BR112021010999A2/pt
Priority to CN201980072011.1A priority patent/CN112969718A/zh
Publication of WO2020119758A1 publication Critical patent/WO2020119758A1/zh

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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure belongs to the field of medicine, and relates to the use of IL-15 protein complex combined with PD-L1 antibody in the preparation of drugs for preventing or treating tumor diseases.
  • Tumor immunotherapy has been a hot topic in the field of cancer therapy in recent years, and T cell tumor immunotherapy is at its core position. Tumor immunotherapy is to make full use of and mobilize the killer T cells in tumor subjects to kill tumors. It may be the most effective and safe way to treat tumors. Tumor escape is a huge obstacle faced by tumor immunotherapy. Tumor cells use their own inhibitory effect on the immune system to promote the crazy growth of tumors. There is a very complicated relationship between the tumor's immune escape mechanism and the body's immune response to the tumor. In the early stage of tumor immunotherapy, tumor-specific killer T cells have their biological activity, but they lose their killing function as the tumor grows later. Therefore, the current stage of tumor immunotherapy is to maximize the subject's own immune system response to the tumor. It must not only activate the original immune system response in the body, but also maintain the duration and intensity of the immune system response. Is the key to immunotherapy for tumors.
  • Programmed death receptor 1 (PD-1) antibody can specifically recognize and bind to PD-1 on the surface of lymphocytes, blocking the PD-1/PD-L1 signaling pathway, thereby activating the immune killing effect of T cells on tumors, and mobilizing the body
  • the immune system clears tumor cells in the body.
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages, and dendritic cells (DC), and the expression on activated cells can be up-regulated after activation.
  • PD-L1 suppresses the immune system by combining with PD-1 and B7-1, and many tumor cells and immune cells in the tumor tissue microenvironment express PD-L1.
  • New research finds high PD-L1 detected in human tumor tissues such as breast cancer, lung cancer, gastric cancer, intestinal cancer, renal cancer, melanoma, non-small cell lung cancer, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer
  • the expression of protein, and the expression level of PD-L1 are closely related to the clinical and prognosis of patients. Because PD-L1 acts as a second signaling pathway to inhibit T cell proliferation, blocking PD-L1/PD-1 binding has become a very promising emerging target in the field of tumor immunotherapy.
  • the combination of PD-1, PD-L1 antibody and other immune checkpoint inhibitors with other drugs is also a hot research area.
  • a series of anti-PD-L1 antibodies have been disclosed.
  • WO2017084495 discloses a series of PD-L1 antibodies, which can effectively improve the effect of inhibiting tumorigenesis and development.
  • Interleukin 15 is a cytokine of about 12-14kD discovered by Grabstein et al in 1994, which can play a role in the body's normal immune response, such as promoting T cells, B cells, natural killing (NK) Cell proliferation.
  • IL-15 needs to exert its biological activity by binding to its receptor.
  • the IL-15 receptor consists of three receptor subunits: IL-15 receptor ⁇ (IL-15R ⁇ ), IL-2 receptor ⁇ (IL-2R ⁇ ) and ⁇ c.
  • IL-15R ⁇ contains a Sushi domain that can bind to IL-15 and is necessary for the IL-15 to perform its biological functions. In recent years, it has been found that IL-15 and its receptor IL-15R ⁇ form a complex, which can significantly enhance the biological activity of IL-15.
  • IL-15-hIgG4Fc homodimer involved in the CN100334112C patent
  • the protein is used for the treatment of antimicrobial infections;
  • CN103370339B discloses an IL-15N72D:IL-15R ⁇ Su/Fc fusion protein complex whose IL-15 polypeptide contains a N72D mutation, which exhibits a reduced effect on IL compared to the original IL-15 -15 ⁇ C receptor binding activity;
  • WO2016095642 discloses an IL-15 protein complex composed of IL-15 polypeptide and IL-15R ⁇ /Fc, which introduces a disulfide bond between IL-15 and IL-15R ⁇ , which can improve Molecular stability and biological activity can also simplify the preparation process.
  • the present disclosure provides the use of an IL-15 protein complex combined with PD-L1 antibody in the preparation of a medicament for preventing or treating tumor diseases, and shows a good tumor suppressing effect.
  • the present disclosure provides the use of IL-15 or its protein complex in combination with PD-L1 antibody or antigen-binding fragment in the preparation of a medicament for preventing or treating tumor diseases.
  • any one of the PD-L1 antibodies or antigen-binding fragments is selected from the following CDR region sequences or mutant sequences thereof: antibody heavy chain variable region HCDR region sequence: SEQ ID NO: 1-3 ; And antibody light chain variable region LCDR region sequence: SEQ ID NO: 4-6;
  • HCDR1 is selected from:
  • HCDR2 is selected from:
  • HCDR3 is selected from:
  • LCDR1 is selected from:
  • LCDR2 is selected from:
  • LCDR3 is selected from:
  • X 1 is selected from H or G, preferably G;
  • X 2 is selected from G or F, preferably F.
  • the PD-L1 antibody or antigen-binding fragment comprises and amino acid sequence: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain variable region CDR sequences, and amino acids Sequence: SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, or 99% sequence identity of heavy chain variable region CDR sequences.
  • the PD-L1 antibody or antigen-binding fragment may be selected from murine antibodies, chimeric antibodies, humanized antibodies, human antibodies, and preferably humanized antibodies.
  • the PD-L1 antibody or antigen-binding fragment comprises and the amino acid sequence SEQ ID NO: 7 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of the heavy chain variable region sequence, and the amino acid sequence SEQ ID NO: 8 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of the light chain variable region sequence.
  • the PD-L1 antibody or antigen-binding fragment further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human heavy chain constant region of IgG2 or IgG4 It is more preferable to include an IgG4 heavy chain constant region that introduces F234A and L235A mutations; the humanized antibody light chain further includes a constant region of a human ⁇ , ⁇ chain, or a variant thereof.
  • the PD-L1 antibody or antigen-binding fragment comprises and the amino acid sequence SEQ ID NO: 9 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity of the heavy chain variable region sequence, and the amino acid sequence SEQ ID NO: 11 has at least 85%, 86%, Light chain sequences with 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the heavy chain sequence of the PD-L1 antibody or antigen-binding fragment is SEQ ID NO: 9, and the light chain sequence is SEQ ID NO: 11.
  • the light chain sequence encodes the gene sequence:
  • the IL-15 protein complex consists of a soluble fusion protein (I) and a soluble fusion protein ( ⁇ ); wherein the soluble fusion protein (I) comprises an IL-15 polypeptide or a functional fragment thereof; soluble Fusion protein ( ⁇ ) contains IL-15R ⁇ polypeptide or its functional fragments; soluble fusion protein (I) or soluble fusion protein ( ⁇ ) has one or more amino acid sites mutated to Cys, and the corresponding soluble fusion protein ( ⁇ ) or soluble fusion protein (I), the mutated Cys at the amino acid position forms a disulfide bond.
  • the soluble fusion protein (I) comprises an IL-15 polypeptide or a functional fragment thereof
  • soluble Fusion protein ( ⁇ ) contains IL-15R ⁇ polypeptide or its functional fragments
  • soluble fusion protein (I) or soluble fusion protein ( ⁇ ) has one or more amino acid sites mutated to Cys, and the corresponding soluble fusion protein ( ⁇ ) or soluble fusion protein (I),
  • the soluble fusion protein ( ⁇ ) further comprises an Fc fragment or a mutant thereof; preferably, the soluble fusion protein ( ⁇ ) is composed of an IL-15R ⁇ polypeptide or a functional fragment linked to the N-terminus of the Fc fragment ; More preferably, the Fc fragment is SEQ ID NO: 13.
  • sequence of the soluble fusion protein (I) is SEQ ID NO: 14.
  • amino acid Cys mutation site of the IL-15 protein complex occurs on L45, Q48, V49, L52, E53, C88, or E89 on the IL-15 polypeptide or functional fragment thereof, It preferably occurs on L52, E53 or E89, more preferably L52.
  • amino acid Cys mutation site of the IL-15 protein complex occurs on K34, L42, A37, G38, or S40 on the IL-15R ⁇ polypeptide or a functional fragment thereof, preferably occurs on A37, For G38 or S40, S40 is more preferred.
  • sequence of the soluble fusion protein ( ⁇ ) is selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.
  • the IL-15 protein complex is selected from the following combinations of soluble fusion protein (I) and soluble fusion protein ( ⁇ ):
  • the IL-15 protein complex is selected from IL-15 (L52C) (SEQ ID NO: 14) soluble fusion protein (I) and IL-15R ⁇ -Sushi+ (S40C)-Fc (SEQ ID NO: 17) Combination of soluble fusion protein ( ⁇ ).
  • the tumor is selected from malignant tumors and benign tumors.
  • the malignant tumor is selected from melanoma, skin cancer, renal cell carcinoma, liver cancer, gastric cancer, breast cancer, colorectal cancer, glioblastoma, ovarian cancer, prostate cancer, blood system cancer, urothelial/bladder cancer, Lung cancer, esophageal cancer, head and neck cancer.
  • the hematological cancers described in this disclosure include but are not limited to acute and chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, bone marrow tissue proliferative diseases, multiple myeloma, and Hodgkin's disease , Non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular central cell lymphoma, chronic myeloid leukemia.
  • the tumor is selected from advanced tumors, relapsed refractory tumors, failed and/or relapsed tumors treated with chemotherapy drugs, failed and/or relapsed tumors after radiation therapy, failed and/or targeted drug therapies Relapsed tumor, failed immunotherapy and/or relapsed tumor.
  • the tumor is selected from advanced/metastatic malignancy.
  • the dose of IL-15 or its protein complex is selected from 1-100ug/kg, preferably from 1ug/kg, 2ug/kg, 3ug/kg, 4ug/kg, 5ug/kg, 6ug /kg, 7ug/kg, 8ug/kg, 9ug/kg, 10ug/kg, 11ug/kg, 12ug/kg, 13ug/kg, 14ug/kg, 15ug/kg, 16ug/kg, 17ug/kg, 18ug/kg , 19ug/kg, 20ug/kg, 21ug/kg, 22ug/kg, 23ug/kg, 24ug/kg, 25ug/kg, preferably 1 ⁇ g/kg, 3 ⁇ g/kg, 6 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg, 20 ⁇ g/kg.
  • the dose of the PD-L1 antibody or antigen-binding fragment is selected from 50-3000mg, preferably 490-2000mg, more preferably 490mg, 500mg, 550mg, 600mg, 750mg, 1200mg, 1280mg, 1500mg, most preferably 600mg, 750mg.
  • the combined administration route of the present disclosure is selected from oral administration, parenteral administration, and transdermal administration.
  • the parenteral administration includes but is not limited to intravenous injection, subcutaneous injection, and intramuscular injection.
  • the present disclosure further relates to the use of IL-15 or its protein complex in combination with PD-L1 antibody or antigen-binding fragment in the preparation of a medicament for preventing or treating tumor diseases, wherein the frequency of administration of IL-15 or its protein complex may be one week Once, once every two weeks, once every three weeks.
  • the frequency of administration of the PD-L1 antibody or antigen-binding fragment may be once a week, once every two weeks, once every three weeks, or once every four weeks.
  • the frequency of administration of IL-15 or its protein complex is once a week, and the dose is 1 ug/kg, 3 ⁇ g/kg, 6 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg or 20 ⁇ g/ kg; PD-L1 antibody or antigen-binding fragment is administered every two weeks at a dose of 600 mg or 750 mg.
  • IL-15 or its protein complex prior to administration of IL-15 or its protein complex in combination with PD-L1 antibody or antigen-binding fragment, IL-15 or its protein complex is administered as a single drug loading.
  • the introduction cycle may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, preferably 2 weeks or 4 weeks.
  • the dosage may be 1 ug/kg, 3 ⁇ g/kg, 6 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg or 20 ⁇ g/kg each time, and the frequency of administration is once a week.
  • the present disclosure further relates to the use of IL-15 or its protein complex in combination with PD-L1 antibody or antigen-binding fragment in the preparation of a medicament for preventing or treating tumor diseases, wherein IL-15 or its Protein complex alone for 2 weeks or 4 weeks, the dose can be 1ug/kg, 3 ⁇ g/kg, 6 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg or 20 ⁇ g/kg each time, the frequency of administration is once a week; Then, IL-15 or its protein complex and PD-L1 antibody or antigen-binding fragment are co-administered.
  • the frequency of administration of IL-15 or its protein complex may be once a week, and the dose may be 1ug/kg, 3 ⁇ g each time /kg, 6 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg or 20 ⁇ g/kg, the frequency of PD-L1 antibody or antigen-binding fragment administration is once every 2 weeks or once every 3 weeks, the dose is 600mg or 750mg.
  • the administration period may be 1 day, 3 days, 1 week, 2 weeks, 3 weeks (21 days), 3-4 weeks (21-28 days), 4 weeks (28 days), 5 Week, 6 weeks.
  • the dosing cycle is 4 weeks, where the first 2 weeks are the introduction cycle, and IL-15 or its protein complex is administered on days 1, 8, 15, 22 of each dosing cycle; in each PD-L1 antibody or antigen-binding fragment was administered on the 15th day of the dosing cycle.
  • the dosing cycle is 4 weeks, and IL-15 or its protein complex is administered on days 1, 8, 15, 22 of each dosing cycle; on the 1st, 15th of each dosing cycle PD-L1 antibody or antigen-binding fragment is administered daily.
  • the dosing cycle is 5 weeks, and IL-15 or its protein complex is administered on days 1, 8, 15, 22 of each dosing cycle; on the 1st, 15th of each dosing cycle PD-L1 antibody or antigen-binding fragment is administered daily.
  • the dosing cycle is 6 weeks, and IL-15 or its protein complex is administered on days 1, 8, 15, 22 of each dosing cycle; on the 1st, 15th of each dosing cycle PD-L1 antibody or antigen-binding fragment is administered daily.
  • the dosing cycle is 6 weeks, and IL-15 or its protein complex is administered on days 1, 8, 15, 22, and 29 of each dosing cycle; on the 1st of each dosing cycle 15 days to give PD-L1 antibody or antigen-binding fragment.
  • the PD-L1 antibody or antigen-binding fragment thereof is administered by injection, for example, subcutaneously or intravenously.
  • the IL-15 or its protein complex is administered by injection, such as subcutaneously or intravenously, preferably subcutaneously.
  • the dosage of the drug and the frequency of administration may be adjusted appropriately.
  • the combination optionally further includes other components, including but not limited to other anti-tumor drugs.
  • the present disclosure also provides a method of treating tumor diseases, which includes administering IL-15 or a protein complex thereof and a PD-L1 antibody or antigen-binding fragment to a patient.
  • the patient fails or does not tolerate standard treatment, or there is no standard treatment after the disease relapses/progresses.
  • the present disclosure also provides a method of treating tumor diseases, including administering PD-L1 antibodies or antigen-binding fragments to patients.
  • the PD-L1 antibody or antigen-binding fragment may be administered alone or in combination with other drugs (eg, chemotherapy drugs).
  • the PD-L1 antibody or antigen-binding fragment is the aforementioned PD-L1 antibody or antigen-binding fragment.
  • the method includes administering a fixed dose of PD-L1 antibody or antigen-binding fragment to the patient.
  • the dose administered is not related to the patient's weight.
  • the fixed-dose PD-L1 antibody or antigen-binding fragment dose is selected from 490-2000 mg, preferably 490 mg, 500 mg, 550 mg, 600 mg, 750 mg, 1200 mg, 1280 mg, 1500 mg, more preferably 600 mg, 750 mg.
  • the frequency of PD-L1 antibody or antigen-binding fragment administration is every 2 weeks or every 3 weeks.
  • the fixed-dose PD-L1 antibody or antigen-binding fragment dose is 490-2000 mg, and the administration frequency is once every 2 weeks or every 3 weeks; the preferred dose is 490 mg, 500 mg, 550mg, 600mg, 750mg, 1200mg, 1280mg, 1500mg, the frequency of administration is once every 2 weeks, or the dose is 490mg, 500mg, 550mg, 600mg, 750mg, 1200mg, 1280mg, 1500mg, once every 3 weeks; more The preferred dosage is 600 mg or 750 mg, and the frequency of administration is once every 2 weeks or every 3 weeks.
  • the administration brings the target patient population to the following average pharmacokinetic (pk) distribution:
  • the pharmacokinetic (pk) distribution of the PD-L1 antibody or antigen-binding fragment it has an average C max of about 269.75 ⁇ g/mL ( ⁇ 20%), and/or about 0.54 hours ( ⁇ 20%), such as 0.08
  • the median time to maximum concentration (T max ) to about 2 hours and/or has an average plasma concentration of about 1985.61 ⁇ g.day/mL ( ⁇ 20%) from the time of administration to about 21 hours after administration -Area under the time curve (AUC 0-21 ), and/or an average plasma concentration-area under the time curve of about 3568.48 ⁇ g.day/mL ( ⁇ 20%) from the time of administration to the infinite time after administration (AUC inf ).
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising IL-15 or its protein complex, PD-L1 antibody or antigen-binding fragment, and one or more pharmaceutically acceptable carriers, excipients, and diluents.
  • the pharmaceutical composition can be made into any pharmaceutically acceptable dosage form. For example, it can be formulated as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injection and concentrated solutions for injection), suppositories, inhalants or sprays Agent.
  • composition containing IL-15 or its protein complex, PD-L1 antibody or antigen-binding fragment described in this disclosure can be administered alone or in combination with one or more therapeutic agents.
  • the present disclosure also provides a pharmaceutical packaging box in which the IL-15 or protein complex thereof and the PD-L1 antibody or antigen-binding fragment described in the present disclosure are packaged.
  • the present disclosure co-administers IL-15 or its protein complex and PD-L1 antibody or antigen-binding fragment, thereby enhancing anti-tumor activity and improving the therapeutic effect of tumor diseases.
  • the "antibody” described in this disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain and ⁇ chain, respectively. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig can have a ⁇ chain or a ⁇ chain.
  • the antibody light chain described in the present disclosure may further comprise a light chain constant region, the light chain constant region comprising a human- or murine-derived kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present disclosure may further include a heavy chain constant region, which comprises human or murine IgG1, IgG2, IgG3, IgG4, or a variant thereof.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences.
  • the three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment described in the present disclosure conform to the known Kabat numbering rules.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the transplantation of mouse CDR sequences into the framework of human antibody variable regions, that is, different types of human germ lines Antibodies produced in antibody framework sequences. It can overcome the strong antibody variable antibody reaction induced by the chimeric antibody due to carrying 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.
  • germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • the humanized antibodies of the present disclosure also include humanized antibodies after further affinity affinity maturation of CDRs by phage display.
  • the "antigen-binding fragment” described in this disclosure refers to a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab') 2 fragment, and an Fv fragment ScFv fragment that binds to human PD-L1; it includes the present disclosure
  • One or more CDR regions of the antibody are selected from SEQ ID NO: 1 to SEQ ID NO: 12.
  • the Fv fragment contains the antibody heavy chain variable region and light chain variable region, but has no constant region, and has the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding.
  • Different linkers can also be used to connect the variable regions of two antibodies into a single polypeptide chain, called single chain antibody (single chain antibody) or single chain Fv (sFv).
  • single chain antibody single chain antibody
  • single chain Fv single chain Fv
  • the term "associated with PD-L1" of the present disclosure refers to the ability to interact with human PD-L1.
  • antigen binding site of the present disclosure refers to a discontinuous three-dimensional site on the antigen recognized by the antibody or antigen-binding fragment of the present disclosure.
  • the "protein complex” or “complex protein” described in the present disclosure refers to a protein formed by combining two different monomeric proteins.
  • the “monomer protein” ie, soluble fusion protein (I), soluble fusion protein ( ⁇ ) constituting the protein complex in the present disclosure may be a fusion protein or a non-fusion protein.
  • the "fusion protein” described in the present disclosure refers to a protein product obtained by expressing gene recombination by connecting coding regions of two or more genes by gene recombination method, chemical method or other appropriate method, under the control of the same regulatory sequence.
  • the soluble fusion protein (I) is a monomeric protein obtained by fusion or non-fusion expression of IL-15 or a variant thereof with a biologically active polypeptide such as an Fc fragment;
  • the soluble fusion protein ( ⁇ ) is IL- 15R ⁇ or its variants are monomeric proteins obtained by fusion or non-fusion expression with biologically active polypeptides such as Fc fragments.
  • the coding regions of two or more genes may be fused at one or several positions by sequences encoding peptide linkers. Peptide linkers can also be used to construct fusion proteins of the present disclosure.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, subjects Subjects, cells, tissues, organs or biological fluids.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes the contact of reagents with cells and the contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering and “treatment” also mean in vitro and ex vivo treatment of, for example, cells by an agent, diagnosis, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, preventive or preventative measures, research and diagnostic applications.
  • Treatment means administration of a therapeutic agent for internal or external use to a patient, such as a composition containing any of the binding compounds of the present disclosure, the patient has one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to relieve one or more disease symptoms in the treated patient or group to induce the regression of such symptoms or inhibit the development of such symptoms to the extent of any clinical measurement.
  • the amount of therapeutic agent effective to relieve the symptoms of any specific disease can vary based on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce a desired therapeutic effect in the patient.
  • embodiments of the present disclosure may be ineffective in relieving the symptoms of each target disease, according to any statistical test methods known in the art such as Student's test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the target disease symptoms in a statistically significant number of patients.
  • the "combination" described in this disclosure is a mode of administration, which means that at least one dose of IL-15 or its protein complex and at least one dose of PD-L1 antibody or antigen-binding fragment are administered within a certain period of time, Two of these drugs show pharmacological effects.
  • the time period may be within one administration cycle, preferably within 4 weeks, within 3 weeks, within 2 weeks, within 1 week, or within 24 hours, more preferably within 12 hours.
  • IL-15 or its protein complex and PD-L1 antibody or antigen-binding fragment can be administered simultaneously or sequentially. This period includes treatments in which IL-15 or its protein complex and PD-L1 antibody or antigen-binding fragment are administered by the same route of administration or different routes of administration.
  • the combined administration method of the present disclosure is selected from simultaneous administration, independently formulated and co-administered, or independently formulated and sequentially administered.
  • 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 may vary depending on factors such as the condition to be treated, the patient's general health, the route and dosage of the method of administration, and the severity of side effects.
  • the effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Figure 1 Effect of IL-15 fusion protein and PD-L1 antibody administered alone or in combination on the tumor volume of mouse colon cancer cell MC38/H11 subcutaneously transplanted tumor model in Example 1.
  • Figure 2 The effect of IL-15 fusion protein and PD-L1 antibody in Example 1 alone or in combination on the tumor volume of mouse colon cancer cell MC38/H11 subcutaneously transplanted tumor model;
  • Example 1 Therapeutic effect of IL-15 protein complex and anti-PD-L1 antibody co-administration and separate administration on transplanted tumors of mouse colon cancer cells MC38/H11 mice
  • IL-15 fusion protein The sequence is described as No. 3, which is composed of soluble fusion protein (I) (SEQ ID NO: 14) and soluble fusion protein ( ⁇ ) (SEQ ID NO: 17).
  • Preparation method Take 1.0 mL of water for injection into IL-15 fusion protein lyophilized powder (1.0 mg), fully dissolve it into 1.0 mg/mL solution, take 1.5 mL 1.0 mg/mL IL-15 fusion protein solution and add 13.5 mL of physiological saline was inverted and mixed to obtain 20 mL of 0.075 mg/mL solution. Take 0.25 mL of 1.0 mg/mL IL-15 fusion protein solution and add 9.75 mL of physiological saline, mix to obtain 10 mL of 0.025 mg/mL solution.
  • PD-L1 antibody its heavy chain sequence is SEQ ID NO: 9, light chain sequence is SEQ ID NO: 11.
  • Preparation method Take 0.8mL of 50mg/mL PD-L1 antibody solution, add 19.2mL of 5% glucose solution, and mix to obtain 20mL of 2mg/mL solution.
  • hIgG1 Use physiological saline to prepare a 0.075mg/mL solution.
  • hIgG4-Fc Use 5% glucose solution to prepare 2mg/mL solution.
  • mice 8-10 weeks old, female, were purchased from Zhejiang Viton Lihua Experimental Animal Technology Co., Ltd. Laboratory animal certificate number: 1806280013.
  • Feeding environment SPF level; temperature: control temperature 20 ⁇ 26°C; relative humidity: control relative humidity 40% ⁇ 70%; light: automatic light, alternating light and dark every 12h.
  • mice 1 ⁇ 10 6 mouse colon cancer cells MC38/H11 (the mouse colon cancer cell line MC38 was knocked out of mouse endogenous PD-L1 using CRISPR/Cas9 technology, and then transferred to human PD-L1 gene, and then Make MC38 cells stably express human-derived PD-L1, H-11 is a monoclonal cell line selected after construction.) Injected into the left armpit of mice.
  • mice are divided into 7 groups: negative control group (Control), PD-L1 antibody 20mg/kg group, IL-15 fusion protein 0.75mg/kg (iv) Group, IL-15 fusion protein 0.75 mg/kg (sc) group, PD-L1 antibody + IL-15 fusion protein 20 + 0.25 mg/kg (iv) group, PD-L1 antibody + IL-15 fusion protein 20 + 0.75 9 mg/kg (iv) group and PD-L1 antibody + IL-15 fusion protein 20+0.75 mg/kg (sc) group.
  • Each group was administered the test substance at the corresponding concentration according to the administration volume of 10 mL/kg.
  • TV (tumor volume) 1/2 ⁇ a ⁇ b 2 , where a and b represent the length and width of the tumor, respectively;
  • RTV (relative tumor volume) V t /V 0 , where V 0 is the tumor volume measured at the time of group administration (ie d 0 ), and V t is the tumor volume at each measurement;
  • T/C(%) T RTV /C RTV ⁇ 100%, where T RTV is the RTV of the treatment group and C RTV is the RTV of the control group;
  • TGI (Tumor Growth Inhibition) (%) (1-T RTV /C RTV ) ⁇ 100%, where T RTV is the RTV of the treatment group and C RTV is the RTV of the control group.
  • mice show that IL-15 fusion protein 0.25mg/kg (iv), 0.75mg/kg (iv and sc) combined with PD-L1 antibody can significantly improve PD-L1 antibody inhibition of mouse colon cancer cells MC38/H11 small
  • IL-15 fusion protein 0.25mg/kg (iv), 0.75mg/kg (iv and sc) combined with PD-L1 antibody can significantly improve PD-L1 antibody inhibition of mouse colon cancer cells MC38/H11 small
  • Example 2 Phase I clinical study of tolerance, safety, pharmacokinetics and pharmacodynamics of IL-15 fusion protein combined with PD-L1 antibody in patients with advanced malignant tumors
  • IL-15 fusion protein The sequence is described as No. 3, which is composed of soluble fusion protein (I) (SEQ ID NO: 14) and soluble fusion protein ( ⁇ ) (SEQ ID NO: 17). Specification: 1mg/support.
  • the heavy chain sequence is SEQ ID NO: 9
  • the light chain sequence is SEQ ID NO: 11. Specification: 600mg/support.
  • Subjects who passed the screening were given IL-15 fusion protein and PD-L1 antibody.
  • IL-15 fusion protein is administered intravenously once a week for 1 ⁇ g/kg, 3 ⁇ g/kg, 6 ⁇ g/kg, 10 ⁇ g/kg or subcutaneously for 3 ⁇ g/kg, 6 ⁇ g/kg, 10 ⁇ g/kg, 15 ⁇ g/kg, 20 ⁇ g/kg , PD-L1 antibody was given 600 mg intravenously every two weeks.
  • the IL-15 fusion protein is administered for 2 weeks or 4 weeks after single drug loading, and then the IL-15 fusion protein and PD-L1 antibody are co-administered.
  • Example 3 Phase I clinical study of tolerability, safety, pharmacokinetics and pharmacodynamics of PD-L1 antibody in patients with advanced solid tumors
  • the heavy chain sequence is SEQ ID NO: 9
  • the light chain sequence is SEQ ID NO: 11. Specification: 600mg/support.
  • Qualified subjects are given PD-L1 antibody, every three weeks is a dosing cycle, the first day of each cycle is administered, within 30 minutes at 1mg/kg, 3mg/kg, 10mg/kg or 20mg /kg dose of PD-L1 antibody intravenously.
  • the treatment effects and side effects of each dose group are shown in the following table.
  • the treatment effects and side effects of PD-L1 antibody have no obvious correlation with the dose increase.
  • the minimum blood drug concentration of PD-L1 antibody Comparing the blood drug concentration of PD-L1 antibody with the lowest drug effect concentration, except for the Q3W, 490mg program, the minimum blood drug concentration of other programs is higher than the minimum effective concentration of PD-L1 antibody, that is, these modes of administration can Meet the needs of clinical treatment. Therefore, the PD-L1 antibody can be administered in a fixed dose.

Abstract

提供一种IL-15蛋白复合物联合PD-L1抗体用于治疗肿瘤疾病的用途。

Description

IL-15蛋白复合物联合PD-L1抗体用于治疗肿瘤疾病的用途 技术领域
本公开属于医药领域，涉及IL-15蛋白复合物联合PD-L1抗体在制备预防或治疗肿瘤疾病的药物中的用途。
背景技术
肿瘤免疫治疗是近年来肿瘤治疗领域的热点话题，其中T细胞的肿瘤免疫治疗又处于其核心位置。肿瘤免疫治疗是充分利用、调动肿瘤受试者体内的杀伤性T细胞，对肿瘤进行杀伤作用，它可能是最有效的也是最安全的治疗肿瘤的途径。肿瘤逃逸是肿瘤免疫治疗面临的一个巨大障碍，肿瘤细胞利用其自身对免疫系统的抑制作用促进了肿瘤的疯狂生长。肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。肿瘤免疫治疗早期，肿瘤特异性的杀伤性T细胞是有其生物活性的，但随着肿瘤生长后期失去了杀伤的功能。所以现阶段肿瘤免疫治疗是为了最大限度的提高受试者自身对肿瘤的免疫系统反应，它不但要在体内激活原有的免疫系统反应，更要维持免疫系统反应的持续时间和反应强度，才是免疫治疗肿瘤的关键。
程序性死亡受体1(PD-1)抗体可以特异性识别并结合淋巴细胞表面PD-1，阻断PD-1/PD-L1信号通路，进而激活T细胞对肿瘤的免疫杀伤作用，调动机体免疫系统而清除体内肿瘤细胞。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell,DC)上，在活化后细胞上的表达能够进行上调。PD-L1通过和PD-1及B7-1的结合抑制免疫系统，很多肿瘤细胞及肿瘤组织微环境的免疫细胞表达PD-L1。新的研究发现乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤、非小细胞肺癌、结肠癌、膀胱癌、卵巢癌、胰腺癌及肝癌等人类肿瘤组织中检测到高PD-L1蛋白的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。由于PD-L1起到第二信号通路抑制T细胞增殖的作用，所以阻断PD-L1/PD-1之间结合成为了肿瘤免疫治疗领域一个非常有潜力的新兴靶点。而PD-1、PD-L1抗体等免疫检查点抑制剂与其他药物联合治疗目前也是热门的研究领域。目前已公开了一系列抗PD-L1抗体，其中，WO2017084495公开了一系列PD-L1抗体，能够有效地提高抑治肿瘤发生和发展的效果。
白细胞介素15(IL-15)是Grabstein等人于1994年发现的一种约为12-14kD的细胞因子，可在机体正常的免疫应答中发挥作用，如促进T细胞、B细胞、自然杀伤(NK) 细胞的增殖。IL-15需通过与其受体结合发挥生物学活性。IL-15受体由三个受体亚基组成：IL-15受体α(IL-15Rα)、IL-2受体β(IL-2Rβ)和γc。IL-15Rα内含一个Sushi结构域，能与IL-15结合，并且是使结合后的IL-15发挥生物学功能所必需的。近年发现，IL-15与其受体IL-15Rα形成复合物后，可显著增强IL-15的生物学活性。
由于IL-15在肿瘤免疫治疗领域的良好预期，国内外有不少公司或研究机构在从事IL-15免疫治疗方面的相关研究，比如CN100334112C专利所涉及的IL-15-hIgG4Fc同源二聚体蛋白用于抗微生物感染的治疗；CN103370339B公开了一种IL-15N72D:IL-15RαSu/Fc融合蛋白复合物，其IL-15多肽包含N72D突变，该突变体较原始IL-15显示减少的对IL-15βγC受体结合活性；WO2016095642公开了一种IL-15蛋白复合物，由IL-15多肽和IL-15Rα/Fc组成，其在IL-15和IL-15Rα之间引入二硫键，能够提高分子稳定性及生物活性，同时也能够简化制备工艺。
目前已开展了一些免疫检查点抑制剂与IL-15联合治疗的研究。John M Wrangle等(The Lancet Oncology,Volume 19,Issue 5,May 2018,Pages 694-704)评估了IL-15复合物ALT-803联合抗PD-1抗体nivolumab治疗晚期非鳞癌非小细胞肺癌的安全性。
本公开提供了一种IL-15蛋白复合物联合PD-L1抗体在制备预防或治疗肿瘤疾病的药物中的用途，并显示了良好的抑瘤效果。
发明内容
本公开提供了IL-15或其蛋白复合物联合PD-L1抗体或抗原结合片段在制备预防或治疗肿瘤疾病的药物中的用途。
在某些实施方式中，所述的PD-L1抗体或抗原结合片段任意1个选自以下的CDR区序列或其突变序列：抗体重链可变区HCDR区序列：SEQ ID NO：1-3；和抗体轻链可变区LCDR区序列：SEQ ID NO：4-6；
具体如下：
HCDR1选自：
Figure PCTCN2019124833-appb-000001
HCDR2选自：
Figure PCTCN2019124833-appb-000002
HCDR3选自：
Figure PCTCN2019124833-appb-000003
LCDR1选自：
Figure PCTCN2019124833-appb-000004
LCDR2选自：
Figure PCTCN2019124833-appb-000005
LCDR3选自：
Figure PCTCN2019124833-appb-000006
其中X 1选自H或G，优选G；X 2选自G或F，优选F。
在某些实施方式中，所述PD-L1抗体或抗原结合片段包含和氨基酸序列：SEQ ID NO：4，SEQ ID NO：5，SEQ ID NO：6具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链可变区CDR序列，和氨基酸序列：SEQ ID NO：1，SEQ ID NO：2和SEQ ID NO：3具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区CDR序列。
在某些实施方式中，所述PD-L1抗体或抗原结合片段可选自鼠源抗体、嵌合抗体、人源化抗体，人抗体，优选人源化抗体。
在某些实施方式中，所述PD-L1抗体或抗原结合片段包含和氨基酸序列SEQ ID NO：7具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区序列，和氨基酸序列SEQ ID NO：8具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链可变区序列。
在某些实施方式中，所述的PD-L1抗体或抗原结合片段进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，更优选包含引入F234A和L235A突变的IgG4重链恒定区；所述人源化抗体轻链进一步包含人源κ、λ链或其变体的恒定区。
在某些实施方式中，所述PD-L1抗体或抗原结合片段包含和氨基酸序列SEQ ID NO：9具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的重链可变区序列，和氨基酸序列SEQ ID NO：11具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，或99％序列一致性的轻链序列。
在某些实施方式中，所述的PD-L1抗体或抗原结合片段的重链序列为SEQ ID NO： 9，轻链序列为SEQ ID NO：11。
Figure PCTCN2019124833-appb-000007
注：序列中斜体为FR序列；下划线为CDR序列。
重链序列
Figure PCTCN2019124833-appb-000008
重链序列编码基因序列
Figure PCTCN2019124833-appb-000009
Figure PCTCN2019124833-appb-000010
轻链序列
Figure PCTCN2019124833-appb-000011
轻链序列编码基因序列：
Figure PCTCN2019124833-appb-000012
Figure PCTCN2019124833-appb-000013
在某些实施方式中，所述IL-15蛋白复合物由可溶性融合蛋白(I)和可溶性融合蛋白(Π)组成；其中可溶性融合蛋白(I)包含IL-15多肽或其功能性片段；可溶性融合蛋白(Π)包含IL-15Rα多肽或其功能性片段；可溶性融合蛋白(I)或可溶性融合蛋白(Π)上具有一个或多个氨基酸位点突变成Cys，与对应的可溶性融合蛋白(Π)或可溶性融合蛋白(I)氨基酸位点上突变成的Cys配对形成二硫键。
在某些实施方式中，其中可溶性融合蛋白(Π)还包含Fc片段或其突变体；优选地，可溶性融合蛋白(Π)由IL-15Rα多肽或其功能性片段连接在Fc片段的N端组成；更优选地，所述的Fc片段为SEQ ID NO：13。
Figure PCTCN2019124833-appb-000014
在某些实施方式中，其中所述的可溶性融合蛋白(I)的序列为SEQ ID NO：14。
Figure PCTCN2019124833-appb-000015
在某些实施方式中，其中所述IL-15蛋白复合物的氨基酸Cys突变位点发生在IL-15多肽或其功能性片段上的L45、Q48、V49、L52、E53、C88或E89上，优选发生在L52、E53或E89上，更优选L52。
在某些实施方式中，其中所述IL-15蛋白复合物氨基酸Cys突变位点发生在IL-15Rα多肽或其功能性片段上的K34、L42、A37、G38或S40上，优选发生在A37、G38或S40上，更优选S40。
在某些实施方式中，其中可溶性融合蛋白(Π)的序列选自SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18。
Figure PCTCN2019124833-appb-000016
Figure PCTCN2019124833-appb-000017
在某些实施方式中，所述IL-15蛋白复合物选自如下可溶性融合蛋白(I)和可溶性融合蛋白(Π)的组合：
Figure PCTCN2019124833-appb-000018
Figure PCTCN2019124833-appb-000019
在某些实施方式中，所述IL-15蛋白复合物选自IL-15(L52C)(SEQ ID NO：14)可溶性融合蛋白(I)和IL-15Rα-Sushi+(S40C)-Fc(SEQ ID NO:17)可溶性融合蛋白(Π)的组合。
在某些实施方式中，所述肿瘤选自恶性肿瘤、良性肿瘤。所述恶性肿瘤选自黑色素瘤、皮肤癌、肾细胞癌、肝癌、胃癌、乳腺癌、结直肠癌、胶质母细胞瘤、卵巢癌、前列腺癌、血液系统癌症、尿路上皮/膀胱癌、肺癌、食道癌、头颈癌。
本公开所述的血液系统癌症包括但不限于急性及慢性骨髓性白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、骨髓组织增殖性疾病、多发性骨髓瘤、何杰金氏(Hodgkin)疾病、非何杰金氏淋巴瘤、B细胞淋巴瘤、T细胞淋巴瘤、滤泡中心细胞淋巴瘤、慢性粒细胞白血病。
在某些实施方式中，所述肿瘤选自晚期肿瘤、复发难治性肿瘤、经化疗药物治疗失败和/或复发肿瘤、经放疗失败和/或复发肿瘤、经靶向药物治疗失败和/或复发肿瘤、经免疫治疗失败和/或复发肿瘤。
在某些实施方式中，所述肿瘤选自晚期/转移性恶性肿瘤。
在某些实施方式中，所述的IL-15或其蛋白复合物剂量选自1-100ug/kg，优选自1ug/kg、2ug/kg、3ug/kg、4ug/kg、5ug/kg、6ug/kg、7ug/kg、8ug/kg、9ug/kg、10ug/kg、11ug/kg、12ug/kg、13ug/kg、14ug/kg、15ug/kg、16ug/kg、17ug/kg、18ug/kg、19ug/kg、20ug/kg、21ug/kg、22ug/kg、23ug/kg、24ug/kg、25ug/kg，优选1μg/kg、3μg/kg、6μg/kg、10μg/kg、15μg/kg、20μg/kg。
在某些实施方式中，所述的PD-L1抗体或抗原结合片段剂量选自50-3000mg，优选490-2000mg，更优选490mg、500mg、550mg、600mg、750mg、1200mg、1280mg、1500mg，最优选600mg、750mg。
本公开所述联合的给药途径选自经口给药、胃肠外给药、经皮给药，所述胃肠外给药包括但不限于静脉注射、皮下注射、肌肉注射。
本公开进一步涉及IL-15或其蛋白复合物联合PD-L1抗体或抗原结合片段在制备预防或治疗肿瘤疾病的药物中的用途，其中IL-15或其蛋白复合物的给药频次可以是一周一次、二周一次、三周一次。PD-L1抗体或抗原结合片段的给药频次可以是一周一次、二周一次、三周一次、四周一次。
在某些实施方式中，IL-15或其蛋白复合物的给药频次为每周给药一次，剂量为1ug/kg、3μg/kg、6μg/kg、10μg/kg、15μg/kg或20μg/kg；PD-L1抗体或抗原结合片段的给药频次为每2周给药一次，剂量为600mg或750mg。
在某些实施方式中，在IL-15或其蛋白复合物联合PD-L1抗体或抗原结合片段给药之前，先给予IL-15或其蛋白复合物单药导入(loading)。导入周期可以是1周、2周、3周、4周、5周、6周，优选2周或4周。剂量可以是每次1ug/kg、3μg/kg、6μg/kg、10μg/kg、15μg/kg或20μg/kg，给药频次为每周给药一次。
在某些实施方式中，本公开进一步涉及IL-15或其蛋白复合物联合PD-L1抗体或抗原结合片段在制备预防或治疗肿瘤疾病的药物中的用途，其中，先给予IL-15或其蛋白复合物单药2周或4周，剂量可以是每次1ug/kg、3μg/kg、6μg/kg、10μg/kg、15μg/kg或20μg/kg，给药频次为每周给药一次；再将IL-15或其蛋白复合物与PD-L1抗体或抗原结合片段联合给药，IL-15或其蛋白复合物的给药频次可以是一周一次，剂量可以是每次1ug/kg、3μg/kg、6μg/kg、10μg/kg、15μg/kg或20μg/kg，PD-L1抗体或抗原结合片段的给药频次为每2周给药一次或每3周给药一次，剂量为600mg或750mg。
在本公开中，所述给药周期可为1天、3天、1周、2周、3周(21天)、3-4周(21-28天)、4周(28天)、5周、6周。
在某些实施方式中，给药周期为4周，其中前2周为导入周期，在每个给药周期的1、8、15、22天给予IL-15或其蛋白复合物；在每个给药周期的第15天给予PD-L1抗体或抗原结合片段。
在某些实施方式中，给药周期为4周，在每个给药周期的1、8、15、22天给予IL-15或其蛋白复合物；在每个给药周期的第1、15天给予PD-L1抗体或抗原结合片段。
在某些实施方式中，给药周期为5周，在每个给药周期的1、8、15、22天给予IL-15或其蛋白复合物；在每个给药周期的第1、15天给予PD-L1抗体或抗原结合片段。
在某些实施方式中，给药周期为6周，在每个给药周期的1、8、15、22天给予IL-15或其蛋白复合物；在每个给药周期的第1、15天给予PD-L1抗体或抗原结合片段。
在某些实施方式中，给药周期为6周，在每个给药周期的1、8、15、22、29天给予IL-15或其蛋白复合物；在每个给药周期的第1、15天给予PD-L1抗体或抗原结合片段。
在某些实施方式中，所述的PD-L1抗体或其抗原结合片段以注射的方式给药，例如皮下或静脉注射。
在某些实施方式中，所述的IL-15或其蛋白复合物以注射的方式给药，例如皮下或静脉注射，优选皮下给药。
在某些实施方式中，在施用IL-15或其蛋白复合物、PD-L1抗体或抗原结合片段时由于毒副作用等原因导致患者不能耐受的，可适当调整药物的剂量以及给药频次。
本公开所述的方案中，所述的联合任选的还包含其他组分，所述其他组分包括但不限于其他抗肿瘤药等。
本公开还提供了一种治疗肿瘤疾病的方法，包括向患者施用IL-15或其蛋白复合物以及PD-L1抗体或抗原结合片段。
在某些实施方式中，所述患者经标准治疗失败或不耐受，或疾病复发/进展后再无标准治疗。
本公开还提供了一种治疗肿瘤疾病的方法，包括向患者施用PD-L1抗体或抗原结合片段。
所述PD-L1抗体或抗原结合片段可单独给予或与其他药物(例如化疗药物)联合给予。
其中，PD-L1抗体或抗原结合片段为前述的PD-L1抗体或抗原结合片段。
在某些实施方式中，所述方法包括向患者施用固定剂量的PD-L1抗体或抗原结合片段。
所述给药的剂量与患者的体重不相关。
所述的固定剂量的PD-L1抗体或抗原结合片段的剂量选自490-2000mg，优选490mg、500mg、550mg、600mg、750mg、1200mg、1280mg、1500mg，更优选600mg、750mg。
PD-L1抗体或抗原结合片段的给药频次为每2周或每3周给药一次。
在某些实施方式中，所述的固定剂量的PD-L1抗体或抗原结合片段的剂量为490-2000mg，给药频次为每2周或每3周给药一次；优选剂量为490mg、500mg、550mg、600mg、750mg、1200mg、1280mg、1500mg，给药频次为每2周给药一次，或剂量为490mg、500mg、550mg、600mg、750mg、1200mg、1280mg、1500mg，每3周给药一次；更优选剂量为600mg或750mg，给药频次为每2周或每3周给药一次。
在某些实施方式中，所述给药使得目标患者群体达到以下药物代谢动力学(pk)分布平均值：
对于PD-L1抗体或抗原结合片段的药物代谢动力学(pk)分布，其具有约269.75μg/mL(±20％)的平均C max，和/或约0.54小时(±20％)，例如0.08至约2小时的到达最大 浓度的中位时间(T max)，和/或具有从给药零时至在给后约21小时的约1985.61μg.day/mL(±20％)的平均血浆浓度-时间曲线下面积(AUC 0-21)，和/或具有从给药零时至在给药后无穷大时间的约3568.48μg.day/mL(±20％)的平均血浆浓度-时间曲线下面积(AUC inf)。
本公开还涉及一种包含IL-15或其蛋白复合物、PD-L1抗体或抗原结合片段，以及一种或多种药用载体、赋形剂、稀释剂的药物组合物。所述药物组合物可以制成药学上可接受的任一剂型。例如，可以配制为片剂、胶囊剂、丸剂、颗粒剂、溶液剂、混悬剂、糖浆剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、栓剂、吸入剂或喷雾剂。
本公开所述的含IL-15或其蛋白复合物、PD-L1抗体或抗原结合片段的药物组合物，可以单独给药，或者与一种或多种治疗剂联合使用。
本公开还提供了一种药物包装盒，其中包装有本公开所述的IL-15或其蛋白复合物以及PD-L1抗体或抗原结合片段。
本公开将IL-15或其蛋白复合物和PD-L1抗体或抗原结合片段联合给药，从而增强了抗肿瘤活性，以及改善了肿瘤疾病的治疗效果。
发明详述
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
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。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
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。
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。
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。
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。
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。
本公开中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab‘片段，F(ab’)2片段，以及与人PD-L1结合的Fv片段ScFv片段；其包含本公开所述抗体的选自SEQ ID NO:1至SEQ ID NO:12中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本公开的术语“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。
本公开所述的“蛋白复合物”或“复合蛋白”是指两个不同的单体蛋白质结合而成的蛋白。本公开中组成蛋白复合物的“单体蛋白质”(即可溶性融合蛋白(I)、可溶性融合蛋白(Π))可以是融合蛋白或非融合蛋白。
本公开所述的“融合蛋白”是指，通过用基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物。在本公开的一些实施方案中，可溶性融合蛋白(I)为IL-15或其变体与生物活性多肽如Fc片段融合或非融合表达得到的单体蛋白质；可溶性融合蛋白(Π)为IL-15Rα或其变体与生物活性多肽如Fc片段融合或非融合表达得到的单体蛋白质。本公开的融合蛋白中，两个或多个基因的编码区之间可由编码肽接头的序列于一个或数个位置融合。肽接头也可用于构建本公开的融合蛋白。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
本公开所述的“联合”是一种给药方式，是指一定时间期限内给予至少一种剂量的IL-15或其蛋白复合物和至少一种剂量的PD-L1抗体或抗原结合片段，其中两种药物都显示药理学作用。所述的时间期限可以是一个给药周期内，优选4周内，3周内，2周内，1周内，或24小时以内，更优选12小时以内。可以同时或依次给予IL-15或其蛋白复合物以及PD-L1抗体或抗原结合片段。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IL-15或其蛋白复合物以及PD-L1抗体或抗原结合片段。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。
“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。
附图说明
图1.实施例1中IL-15融合蛋白和PD-L1抗体单独或联合给药对小鼠结肠癌细胞MC38/H11皮下移植瘤模型瘤体积的影响；
图2.实施例1中IL-15融合蛋白和PD-L1抗体单独或联合给药对小鼠结肠癌细胞MC38/H11皮下移植瘤模型相对瘤体积的影响；
图3.实施例3中PD-L1抗体的C1D15受体占有率；
图4.实施例3中PD-L1抗体的C1D21受体占有率；
图5.实施例3中PD-L1抗体的观测值和模拟值与最低有效浓度的比较。
具体实施方式
实施例1：IL-15蛋白复合物与抗PD-L1抗体联合给药与各自单独给药对小鼠结肠癌细胞MC38/H11小鼠移植瘤的治疗作用研究
1.受试药物
IL-15融合蛋白：序列如编号3所述，其由可溶性融合蛋白(I)(SEQ ID NO：14)和可溶性融合蛋白(Π)(SEQ ID NO:17)组成。配制方法：取1.0mL注射用水注入IL-15融合蛋白冻干粉(1.0mg)中，充分溶解后配制为1.0mg/mL溶液，取1.5mL 1.0mg/mL的IL-15融合蛋白溶液加入13.5mL的生理盐水颠倒混匀得到20mL的0.075mg/mL溶液。取0.25mL 1.0mg/mL的IL-15融合蛋白溶液加入9.75mL的生理盐水，混匀得到10mL的0.025mg/mL溶液。
PD-L1抗体：其重链序列为SEQ ID NO：9，轻链序列为SEQ ID NO：11。配制方法：取0.8mL 50mg/mL的PD-L1抗体溶液加入19.2mL的5％葡萄糖溶液，混匀得到20mL的2mg/mL溶液。
hIgG1：用生理盐水配置成0.075mg/mL溶液。
hIgG4-Fc：用5％葡萄糖溶液配置成2mg/mL溶液。
2.实验动物
C57BL/6小鼠，8～10周龄，雌性，购自浙江维通利华实验动物技术有限公司。实验动物合格证号：1806280013。
饲养环境：SPF级；温度：控制温度20～26℃；相对湿度：控制相对湿度40％～70％；光照：自动光照，每12h明暗交替。
3.实验步骤
将1×10 6个小鼠结肠癌细胞MC38/H11(对小鼠结肠癌细胞系MC38采用CRISPR/Cas9技术敲除小鼠内源性PD-L1后再转入人源PD-L1基因，继而使得MC38细胞稳定表达人源PD-L1，H-11为构建好之后挑选的单克隆细胞系。)注射入小鼠左侧腋下。待肿瘤生长至平均体积50-100mm 3后，将63只小鼠分为7组：阴性对照组(Control)、PD-L1抗体20mg/kg组、IL-15融合蛋白0.75mg/kg(i.v.)组、IL-15融合蛋白0.75mg/kg(s.c.)组、PD-L1抗体+IL-15融合蛋白20+0.25mg/kg(i.v.)组、PD-L1抗体+IL-15融合蛋白20+0.75mg/kg(i.v.)组和PD-L1抗体+IL-15融合蛋白20+0.75mg/kg(s.c.)组，每组9只。各组按10mL/kg的给药容量给予相应浓度的受试物。每周称重和测量肿瘤体积2次，给药周期25天，于第26天称量体重测量肿瘤体积后计算相对肿瘤体积(RTV)、相对肿瘤增值率(T/C)，做统计学检测。计算公式如下：
(1)TV(tumor volume)＝1/2×a×b 2，其中a、b分别表示肿瘤的长和宽；
(2)RTV(relative tumor volume)＝V t/V 0，其中V 0为分组给药时(即d 0)所测得的肿瘤体积，V t为每一次测量时的肿瘤体积；
(3)T/C(％)＝T RTV/C RTV×100％，其中T RTV为治疗组的RTV，C RTV为对照组的RTV；
(4)TGI(Tumor Growth Inhibition)(％)＝(1-T RTV/C RTV)×100％，其中T RTV为治疗组的RTV，C RTV为对照组的RTV。
4.实验结果
Figure PCTCN2019124833-appb-000020
Figure PCTCN2019124833-appb-000021
与对照组相比， *：P<0.05， **：P<0.01， ***：P<0.001(One-way ANOVA)；与PD-L1抗体20mg/kg组相比， ##：P<0.01， ###：P<0.001(One-way ANOVA)；与IL-15融合蛋白0.75mg/kg(i.v.)组相比PD-L1抗体+IL-15融合蛋白20+0.75mg/kg(i.v.)组无统计学差异(T-test)；与IL-15融合蛋白0.75mg/kg(s.c.)组相比， □：P<0.05(T-test)；与PD-L1抗体+IL-15融合蛋白20+0.75mg/kg(i.v.)组相比， △：P<0.05(T-test)。
实验结果显示，IL-15融合蛋白0.25mg/kg(i.v.)、0.75mg/kg(i.v.和s.c.)与PD-L1抗体联用可以显著提高PD-L1抗体抑制小鼠结肠癌细胞MC38/H11小鼠皮下移植瘤生长的作用。
实施例2：IL-15融合蛋白联合PD-L1抗体在晚期恶性肿瘤患者中耐受性、安全性、药代动力学和药效学的I期临床研究
1、试验药物
IL-15融合蛋白：序列如编号3所述，其由可溶性融合蛋白(I)(SEQ ID NO：14)和可溶性融合蛋白(Π)(SEQ ID NO:17)组成。规格：1mg/支。
PD-L1抗体，其重链序列为SEQ ID NO：9，轻链序列为SEQ ID NO：11。规格：600mg/支。
2、入组受试者
(1)年龄18～70岁，男女皆可；
(2)经组织病理学或细胞学确诊的晚期/转移性恶性肿瘤患者；
(3)标准治疗失败或不耐受，疾病复发/进展后再无标准治疗的患者。
3、给药方法
经筛选合格的受试者给予IL-15融合蛋白和PD-L1抗体。
IL-15融合蛋白每周一次静脉滴注给予1μg/kg、3μg/kg、6μg/kg、10μg/kg或者皮下注射给予3μg/kg、6μg/kg、10μg/kg、15μg/kg、20μg/kg，PD-L1抗体每两周一次静脉滴注给予600mg。给药时，先给予2周或4周IL-15融合蛋白单药导入(loading)后，再进行IL-15融合蛋白和PD-L1抗体的联合给药。
实施例3：PD-L1抗体在晚期实体瘤患者中耐受性、安全性、药代动力学和药效学的I期临床研究
1、试验药物
PD-L1抗体，其重链序列为SEQ ID NO：9，轻链序列为SEQ ID NO：11。规格：600mg/支。
2、入组受试者
(1)年龄18岁以上；
(2)患有组织学或细胞学证实的转移性或局部晚期实体肿瘤；
(3)标准治疗失败或没有已知的有效抗肿瘤治疗方法。
3、给药方法
经筛选合格的受试者给予PD-L1抗体，每三周为一个给药周期，每个周期的第1天给药，在30分钟内以1mg/kg、3mg/kg、10mg/kg或20mg/kg的剂量静脉内输注PD-L1抗体。
4、实验结果
在任何剂量水平均未观察到剂量限制性毒性(DLT)。疾病控制率为40.0％，客观应答率为8.6％。其药代动力学数据见下表。
Figure PCTCN2019124833-appb-000022
受试患者的体重情况：
参数 数值
人数 37
平均体重(kg) 74.30
中位数体重(kg) 73.78
Std.(kg) 16.95
CV(％) 22.82
各剂量组的治疗效果和副作用情况如下表，PD-L1抗体的治疗效果、副作用与剂量增长并无明显相关性。
剂量 人数 SD PR PD 未评估
3mg/kg 12 5(41％) 1(8.3％) 6(50％) 0
10mg/kg 12 6(50％) 0 4(33.3％) 2(16.7％)
20mg/kg 12 5(41％) 0 2(16.7％) 5(41％)
Figure PCTCN2019124833-appb-000023
收集病人给药后的全血样本，采用流式细胞仪技术检测给予PD-L1抗体后的外周血T细胞表面PD-L1受体占有率(RO)情况(图3)，确认PD-L1抗体的最小有效浓度为约70μg/mL。用二房式模型对病人血药浓度进行拟合，得到一系列采用固定剂量给药的给药方式和剂量下的谷浓度，如下表所示。
Figure PCTCN2019124833-appb-000024
将PD-L1抗体血药浓度和最低的药效浓度进行比较，除了Q3W，490mg方案外，其他方案的最小血药浓度均高于PD-L1抗体的最小有效浓度，即这些给药方式均能满足临床治疗需求。因此，PD-L1抗体可采用固定剂量的方式给药。

Claims (18)

  1. IL-15或其蛋白复合物联合PD-L1抗体或抗原结合片段在制备预防或治疗肿瘤疾病的药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于，所述的PD-L1抗体或抗原结合片段任意1个选自以下的CDR区序列或其突变序列：抗体重链可变区HCDR区序列：SEQ ID NO：1-3；和抗体轻链可变区LCDR区序列：SEQ ID NO：4-6；具体如下：
    HCDR1选自：
    SYWMH                      SEQ ID NO：1
    HCDR2选自：
    RI X 1PNSG X 2TSYNEKFKN      SEQ ID NO：2
    HCDR3选自：
    GGSSYDYFDY                 SEQ ID NO：3
    LCDR1选自：
    RASESVSIHGTHLMH            SEQ ID NO：4
    LCDR2选自：
    AASNLES                    SEQ ID NO：5
    LCDR3选自：
    QQSFEDPLT                  SEQ ID NO：6；
    其中X 1选自H或G，优选G；X 2选自G或F，优选F。
  3. 根据权利要求2所述的用途，其特征在于，所述的PD-L1抗体或抗原结合片段的重链可变区序列为SEQ ID NO：7，轻链可变区序列为SEQ ID NO：8。
  4. 根据权利要求3所述的用途，其特征在于，所述的PD-L1抗体或抗原结合片段进一步包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，更优选包含引入F234A和L235A突变的IgG4重链恒定区；所述人源化抗体轻链进一步包含人源κ、λ链或其变体的恒定区。
  5. 根据权利要求4所述的用途，其特征在于，所述的PD-L1抗体或抗原结合片段的重链序列为SEQ ID NO：9，轻链序列为SEQ ID NO：11。
  6. 根据权利要求1所述的用途，其特征在于，所述的PD-L1抗体或抗原结合片段选自鼠源抗体、嵌合抗体、人源化抗体，人抗体，优选人源化抗体。
  7. 根据权利要求1所述的用途，其中所述IL-15蛋白复合物由可溶性融合蛋白(I)和可溶性融合蛋白(Π)组成；其中可溶性融合蛋白(I)包含IL-15多肽或其功能性片段；可溶性融合蛋白(Π)包含IL-15Rα多肽或其功能性片段；可溶性融合蛋白(I)或可溶性融合蛋白(Π)上具有一个或多个氨基酸位点突变成Cys，与对应的可溶性融合蛋白(Π)或可溶性融合蛋白(I)氨基酸位点上突变成的Cys配对形成二硫键。
  8. 根据权利要求7所述的用途，其中可溶性融合蛋白(Π)还包含Fc片段或其突变体；优选地，可溶性融合蛋白(Π)由IL-15Rα多肽或其功能性片段连接在Fc片段的N端组成；更优选地，所述的Fc片段为SEQ ID NO：13。
  9. 根据权利要求7所述的用途，其中所述的可溶性融合蛋白(I)的序列为SEQ ID NO：14。
  10. 根据权利要求7至9任一项所述的用途，其中可溶性融合蛋白(Π)的序列选自SEQ ID NO：15、SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18。
  11. 根据权利要求7所述的用途，所述IL-15蛋白复合物选自如下可溶性融合蛋白(I)和可溶性融合蛋白(Π)的组合：
    Figure PCTCN2019124833-appb-100001
  12. 根据权利要求8所述的用途，所述IL-15蛋白复合物选自IL-15(L52C) (SEQ ID NO：14)可溶性融合蛋白(I)和IL-15Rα-Sushi+(S40C)-Fc(SEQ ID NO:17)可溶性融合蛋白(Π)的组合。
  13. 根据权利要求1-12任意一项所述的用途，其特征在于，所述肿瘤疾病选自恶性肿瘤、良性肿瘤，优选黑色素瘤、皮肤癌、肾细胞癌、肝癌、胃癌、乳腺癌、结直肠癌、胶质母细胞瘤、卵巢癌、前列腺癌、血液系统癌症、尿路上皮/膀胱癌、肺癌、食道癌、头颈癌。
  14. 根据权利要求1-13任意一项所述的用途，其特征在于，所述肿瘤选自晚期肿瘤、复发难治性肿瘤、经化疗药物治疗失败和/或复发肿瘤、经放疗失败和/或复发肿瘤、经靶向药物治疗失败和/或复发肿瘤、经免疫治疗失败和/或复发肿瘤。
  15. 根据权利要求1-14任意一项所述的用途，其特征在于，所述肿瘤选自晚期或转移性恶性肿瘤。
  16. 根据权利要求1-15任意一项所述的用途，其特征在于，所述的IL-15或其蛋白复合物剂量选自1-100ug/kg，优选1μg/kg、3μg/kg、6μg/kg、10μg/kg、15μg/kg、20μg/kg。
  17. 根据权利要求1-16任意一项所述的用途，其特征在于，所述PD-L1抗体或抗原结合片段剂量选自50-3000mg，优选600mg、750mg。
  18. 药物组合物，包含权利要求1所述的IL-15或其蛋白复合物、PD-L1抗体或抗原结合片段，以及一种或多种可药用的赋形剂、稀释剂或载体。
PCT/CN2019/124833 2018-12-13 2019-12-12 Il-15蛋白复合物联合pd-l1抗体用于治疗肿瘤疾病的用途 WO2020119758A1 (zh)

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AU2019399720A AU2019399720A1 (en) 2018-12-13 2019-12-12 Use of IL-15 protein complex joint PD-L1 antibody for treating tumor diseases
JP2021532250A JP2022512139A (ja) 2018-12-13 2019-12-12 腫瘍性疾患の治療のためのｐｄ－ｌ１抗体と組み合わせたｉｌ－１５タンパク質複合体の使用
US17/311,642 US20220111008A1 (en) 2018-12-13 2019-12-12 Use of il-15 protein complex joint pd-l1 antibody for treating tumor diseases
MX2021006589A MX2021006589A (es) 2018-12-13 2019-12-12 Uso de un complejo de proteinas il-15 junto con un anticuerpo contra pd-l1 para el tratamiento de enfermedades tumorales.
EP19897174.9A EP3896089A4 (en) 2018-12-13 2019-12-12 USE OF IL-15 PROTEIN COMPLEXES IN CONJUNCTION WITH PD-L1 ANTIBODIES FOR THE TREATMENT OF TUMOR DISEASES
KR1020217020567A KR20210102917A (ko) 2018-12-13 2019-12-12 종양 질환을 치료하기 위한 il-15 단백질 복합체 연합 pd-l1 항체의 용도
BR112021010999-4A BR112021010999A2 (pt) 2018-12-13 2019-12-12 Uso de anticorpo pd-l1 de articulação do complexo de proteína il-15 para tratamento de doenças tumorais
CN201980072011.1A CN112969718A (zh) 2018-12-13 2019-12-12 Il-15蛋白复合物联合pd-l1抗体用于治疗肿瘤疾病的用途

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100334112C (zh) 2004-10-15 2007-08-29 上海海欣生物技术有限公司 人白细胞介素15与Fc融合蛋白
CN103370339A (zh) 2010-09-21 2013-10-23 阿尔托生物科学有限公司 多聚体il-15可溶性融合分子与其制造与使用方法
WO2016095642A1 (zh) 2014-12-19 2016-06-23 江苏恒瑞医药股份有限公司 白细胞介素15蛋白复合物及其用途
WO2017084495A1 (zh) 2015-11-17 2017-05-26 江苏恒瑞医药股份有限公司 Pd-l1抗体、其抗原结合片段及其医药用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9925247B2 (en) * 2014-06-30 2018-03-27 Altor Bioscience Corporation IL-15-based molecules and methods of use thereof
RU2019122408A (ru) * 2017-01-20 2021-02-20 Новартис Аг Средство комбинированной терапии для лечения рака

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100334112C (zh) 2004-10-15 2007-08-29 上海海欣生物技术有限公司 人白细胞介素15与Fc融合蛋白
CN103370339A (zh) 2010-09-21 2013-10-23 阿尔托生物科学有限公司 多聚体il-15可溶性融合分子与其制造与使用方法
WO2016095642A1 (zh) 2014-12-19 2016-06-23 江苏恒瑞医药股份有限公司 白细胞介素15蛋白复合物及其用途
CN106459219A (zh) * 2014-12-19 2017-02-22 江苏恒瑞医药股份有限公司 白细胞介素15蛋白复合物及其用途
WO2017084495A1 (zh) 2015-11-17 2017-05-26 江苏恒瑞医药股份有限公司 Pd-l1抗体、其抗原结合片段及其医药用途

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CAROLINE JOCHEMS, ET AL: "The multi-functionality of N-809, a novel fusion protein encompassing anti-PD-L1 and the IL-15 superagonist fusion complex", ONCOIMMUNOLOGY, vol. 8, no. 2, 1 February 2019 (2019-02-01), pages e1532764, XP055719145, DOI: 10.1080/2162402X.2018.1532764 *
J. BIOL. CHEM, vol. 243, 1968, pages 3558
JOHN M WRANGLE ET AL., THE LANCET ONCOLOGY, vol. 19, May 2018 (2018-05-01), pages 694 - 704
KABAT, EA ET AL.: "Sequences of Proteins of Immunological Interest", 1991
See also references of EP3896089A4

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