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WO2022184148A1 - Il-21-抗白蛋白单域抗体融合蛋白药物组合物及其用途 - Google Patents

Il-21-抗白蛋白单域抗体融合蛋白药物组合物及其用途 Download PDF

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WO2022184148A1
WO2022184148A1 PCT/CN2022/079127 CN2022079127W WO2022184148A1 WO 2022184148 A1 WO2022184148 A1 WO 2022184148A1 CN 2022079127 W CN2022079127 W CN 2022079127W WO 2022184148 A1 WO2022184148 A1 WO 2022184148A1
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fusion protein
domain antibody
single domain
albumin
pharmaceutical composition
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PCT/CN2022/079127
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English (en)
French (fr)
Inventor
刘洪川
刘辉
张静
孟琴
刘沛想
李秀鹏
赵文亭
冯辉
姚盛
Original Assignee
上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Publication of WO2022184148A1 publication Critical patent/WO2022184148A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
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    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/2818—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 CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of therapeutic pharmaceutical compositions.
  • the present invention relates to the field of pharmaceutical preparations, the pharmaceutical composition contains IL-21-anti-albumin single domain antibody fusion protein, and the IL-21 fusion protein in the treatment of tumors and enhancing the anti-tumor activity of immune checkpoint inhibitors. use.
  • Cytokine therapy is an effective strategy to stimulate the immune system to induce an immune response against disease such as cancer or infection.
  • cytokines administered to patients typically have short half-lives.
  • interleukin-21 can stimulate various immune cells (such as T cells, B cells, and NK cells) and enhance their antitumor activity.
  • the half-life of recombinant IL-21 has been reported to be only about one to three hours after intravenous administration (see Schmidt H, Clin. Cancer Res. 2010 Nov 1; Vol 16 No 21: pp. 5312-5319) .
  • TILs tumor-infiltrating T lymphocytes
  • CTLA-4 activation-induced inhibitory receptors
  • PD-1 PD-1 ligand 1
  • B7-H1 or CD274 immunosuppressive ligands
  • PD-1/PD-L1 pathway blockade has been shown to be an effective way to induce durable antitumor responses in various cancer indications.
  • Monoclonal antibodies (mAbs) that block the PD/PD-L1 pathway can enhance tumor-specific T cell activation and effector function, reduce tumor burden, and improve survival.
  • the FDA has approved two anti-PD1 monoclonal antibodies (nivolumab) and three anti-PD-L1 monoclonal antibodies (atezolizumab, avelumab, and durvalumab) for the treatment of human tumors.
  • IL-21 a member of the gamma chain cytokine family, can stimulate CD8 + T cell expansion and increase cytotoxicity, enhance T cell-dependent B cell proliferation and antibody production, promote NK cell differentiation and activation, and reduce Treg cells.
  • Recombinant human IL-21 has demonstrated antitumor activity as monotherapy or in combination with targeted therapy or monoclonal antibodies in nonclinical and clinical studies.
  • rIL-21 recombinant IL-21
  • RCC renal cell carcinoma
  • non-Hodgkin's lymphoma recombinant IL-21
  • the pharmaceutical composition disclosed in the present invention is a highly stable pharmaceutical composition containing IL-21-anti-albumin single domain antibody fusion protein (IL-21 fusion protein).
  • IL-21 fusion protein IL-21 fusion protein
  • the present invention found that the combination containing trehalose has an unexpected characteristic, namely high stability.
  • the half-life and the exposure level in the blood of the IL-21-anti-albumin single domain antibody fusion protein pharmaceutical composition of the present invention are significantly better than those of recombinant interleukin 21, and the single drug and its combination with PD-1 single drug in the mouse subcutaneous tumor model Anti-combination showed superior anti-tumor activity.
  • JS-EC21 nanobody targeting human serum albumin (HSA) was fused to the C-terminus of human IL-21 to form a fusion referred to herein as JS-EC21
  • HSA human serum albumin
  • JS-EC21 significantly enhanced the antitumor effect of PD-1 mAb or TIGIT mAb and provided long-term protective antitumor immunity.
  • the combination of JS-EC21 and TIGIT mAb induced a significant enrichment of the KEGG signaling pathway associated with tumor immune responses and increased the expression levels of genes associated with CD8 + T cell and NK cytotoxicity.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (1) a buffer; and (2) an IL-21-anti-albumin single domain antibody fusion protein.
  • the IL-21-anti-albumin single domain antibody fusion protein described above comprises: (a) the cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds to albumin, the IL-21 described above Including the amino acid sequence shown in SEQ ID NO: 1, the above-mentioned single domain antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively.
  • the single domain antibody (sdAb) described above comprises the amino acid sequence set forth in SEQ ID NO:5.
  • the single domain antibody described above is fused to the C-terminus of the cytokine, and the cytokine described above and the single domain antibody are directly linked.
  • the concentration of the IL-21-anti-albumin single domain antibody fusion protein in the above pharmaceutical composition is about 0.1-100 mg/mL, preferably about 0.2-20 mg/mL, preferably about 0.2-10 mg/mL, Preferably about 0.5-5 mg/mL, preferably about 1-5 mg/mL, more preferably about 0.5-1 mg/mL; more preferably, the concentration of the above-mentioned IL-21-anti-albumin single domain antibody fusion protein is about 0.5 mg /mL, 0.8mg/mL, 1mg/mL, 1.2mg/mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL or 10 mg/mL, preferably about 1 mg/mL.
  • the above-mentioned buffer is selected from one or more of acetate buffer, citrate buffer and histidine buffer; preferably, the above-mentioned buffer is selected from acetic acid-sodium acetate buffer, citric acid- One or more of sodium citrate buffer, histidine-acetate buffer and histidine-hydrochloride buffer; preferably, the above-mentioned buffer is acetic acid-sodium acetate buffer and histidine-acetate buffer more preferably, the above-mentioned buffer is acetic acid-sodium acetate buffer.
  • the above-mentioned buffer is histidine buffer, preferably, the above-mentioned histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer, preferably histidine -HCl buffer.
  • the histidine-hydrochloride buffers described above are made from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride.
  • the histidine buffer is made from 1-30 mM L-histidine and 1-30 mM L-histidine monohydrochloride.
  • the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4.
  • the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1:1. In some protocols, the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine formulation is a histidine buffer at a pH of about 5.0 made from about 1.7 mM L-histidine and about 18.3 mM L-histidine monohydrochloride. In some embodiments, the histidine formulation is a histidine buffer at a pH of about 5.5 made from about 4.5 mM L-histidine and about 15.5 mM L-histidine monohydrochloride.
  • the histidine formulation is a histidine buffer at a pH of about 5.5 made from about 7.5 mM L-histidine and about 22.5 mM L-histidine monohydrochloride. In some embodiments, the histidine formulation is a histidine buffer at a pH of about 6.0 made from about 15 mM histidine and about 15 mM histidine hydrochloride.
  • the above-mentioned histidine buffer is a histidine-acetate buffer, preferably, the molar ratio of the two is about 1:1 to 1.5:1, and preferably, the pH of such a buffer is 5.5 ⁇ 0.3, preferably about 5.5, preferably such buffers contain 15-20 mM histidine and 12-15 mM acetic acid.
  • the above buffer is acetate buffer, preferably, the above acetate buffer is acetic acid-sodium acetate buffer or acetic acid-potassium acetate buffer, preferably acetic acid-sodium acetate buffer.
  • the acetate buffer is made up of 1-30 mM acetic acid and 1-30 mM sodium acetate. In some protocols, the acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 2:3. In some protocols, the acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:2.1.
  • the acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:5.7. In some embodiments, the acetate buffer is: acetate buffer at a pH of about 4.8 made from about 8 mM acetic acid and about 12 mM sodium acetate. In some embodiments, the acetate buffer is an acetate buffer at a pH of about 5.0 made from about 6.5 mM acetic acid and about 13.5 mM sodium acetate. In some embodiments, the acetate buffer is an acetate buffer at a pH of about 5.5 made from about 3 mM acetic acid and about 17 mM sodium acetate.
  • the above-mentioned buffer is a citrate buffer, preferably, the above-mentioned citrate buffer is a citric acid-sodium citrate buffer.
  • the concentration of the above-mentioned buffer is about 1-200 mM, preferably about 5-200 mM, preferably about 10-50 mM, preferably about 10-30 mM; preferably about 10-20 mM, the above-mentioned buffer concentration is not limiting Typical examples are about 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM or 180 mM or a range formed by any two values within these ranges as endpoints, preferably 10 mM, 15 mM, 20 mM or 30 mM.
  • the pH of the above buffer is about 4.0-6.5, preferably about 4.0-6.0, preferably about 4.5-6.0, preferably about 4.5-5.5, preferably about 4.5-5.1, preferably about 4.5-5.0 , preferably about 4.7-5.0
  • non-limiting examples of pH of the above buffers are about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 6.0, preferably about 4.7 or 4.8.
  • the above-mentioned pharmaceutical composition also includes a stabilizer, and the above-mentioned stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose, and trehalose, preferably, the above-mentioned stabilizer is Trehalose.
  • the concentration of the aforementioned stabilizer is about 10 mM to 400 mM, preferably 20 mM to 300 mM, more preferably 30 mM to 200 mM.
  • the above-mentioned stabilizer is mannitol at a concentration of about 100-300 mM; or the above-mentioned stabilizer is sucrose at a concentration of about 100-300 mM; or the above-mentioned stabilizer is trehalose at a concentration of about 100-300 mM; or the above-mentioned stabilizer is About 30-200 mM sodium chloride in combination with about 30-200 mM mannitol; or the above stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM sucrose; preferably, the above stabilizer is about 100-300 mM trehalose; more preferably, the above stabilizer is about 200-280 mM trehalose.
  • the aforementioned stabilizer is mannitol.
  • the aforementioned stabilizer is mannitol at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the aforementioned mannitol concentration is about 200 mM , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 240mM.
  • the aforementioned stabilizer is sucrose.
  • the aforementioned stabilizer is sucrose at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-280 mM, non-limiting examples of the aforementioned sucrose concentrations are about 200 mM, 210 mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 235mM.
  • the aforementioned stabilizer is trehalose.
  • the above-mentioned stabilizer is trehalose at a concentration of about 100-300 mM, preferably about 150-300 mM, preferably about 200-280 mM, and a non-limiting example of the above-mentioned trehalose concentration is about 180 mM , 200mM, 210mM, 220mM, 230mM, 235mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 235mM.
  • the above stabilizer is a combination of sodium chloride and mannitol. In some aspects, the above stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM mannitol, preferably about 40-150 mM sodium chloride in combination with about 40-180 mM mannitol, preferably about 40-180 mM mannitol.
  • a combination of 40-100 mM sodium chloride and about 40-150 mM mannitol, non-limiting examples of the above stabilizers are about 50 mM sodium chloride and about 120 mM mannitol, or about 50 mM sodium chloride In combination with about 140 mM mannitol.
  • the above stabilizer is a combination of sodium chloride and sucrose.
  • the aforementioned stabilizer is about 30-200 mM sodium chloride in combination with about 30-200 mM sucrose, preferably about 40-150 mM sodium chloride in combination with about 40-180 mM sucrose, preferably about 40- A combination of 100 mM sodium chloride and about 40-150 mM sucrose, non-limiting examples of the above stabilizers are about 50 mM sodium chloride and about 140 mM sucrose.
  • the above pharmaceutical composition further comprises a surfactant selected from the group consisting of polysorbate 80, polysorbate 20 or poloxamer 188.
  • the above-mentioned surfactant is selected from polysorbate 80.
  • the above-mentioned surfactant is selected from polysorbate 20.
  • the above-mentioned surfactant concentration is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.02%-0.08%, preferably about 0.01%-0.05%, on a w/v basis ;
  • the concentration of the above-mentioned surfactant is about 0.02%, 0.04% or 0.08%, preferably 0.02%.
  • the above-mentioned IL-21 fusion protein comprises: (a) the cytokine IL-21, and (b) a single domain antibody (sdAb) that specifically binds to albumin, wherein the above-mentioned IL-21 comprises as SEQ ID NO: The amino acid sequence shown in 1, and the sdAb comprises the amino acid sequence shown in SEQ ID NO:5.
  • the IL-21-anti-albumin single domain antibody fusion protein described above comprises or consists of the amino acid sequence set forth in SEQ ID NO:6.
  • the above-mentioned pharmaceutical composition comprises the components shown in any one of the following (1)-(6), wherein the IL-21-anti-albumin single domain antibody fusion protein is according to any embodiment of the present invention Said:
  • the above pharmaceutical compositions are liquid or lyophilized formulations.
  • the pharmaceutical compositions described above are liquid formulations.
  • the above-described liquid or lyophilized formulations are stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.
  • the aqueous or lyophilized formulations described above are stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicine for treating or preventing cancer; preferably, the above-mentioned cancer includes mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, Leukemia, head and neck cancer, liver cancer, esophageal cancer, stomach cancer and colorectal cancer.
  • the above-mentioned cancer includes mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, Leukemia, head and neck cancer, liver cancer, esophageal cancer, stomach cancer and colorectal cancer.
  • the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for treating or preventing inflammatory diseases.
  • the present invention provides IL-21 fusion protein (also known as IL-21-anti-albumin single domain antibody fusion protein), or the above pharmaceutical composition, or IL-21 fusion protein and immune checkpoint modulator
  • IL-21 fusion protein also known as IL-21-anti-albumin single domain antibody fusion protein
  • immune checkpoint modulator The combination of or the combination of the above-mentioned pharmaceutical composition and the immune checkpoint modulator is used in the preparation of a medicine for preventing or treating tumors, or the IL-21-anti-albumin single-domain antibody fusion protein or the above-mentioned pharmaceutical composition is enhanced in preparation Use of immune checkpoint modulators for antitumor activity in medicine.
  • the present invention provides a method of preventing or treating tumors, comprising administering to an individual in need thereof an effective amount of an IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or an IL-21 fusion protein and immune checkpoint modulation A combination of agents or a combination of the above pharmaceutical composition and an immune checkpoint modulator.
  • the present invention provides an IL-21 fusion protein, or the aforementioned pharmaceutical composition, or a combination of an IL-21 fusion protein and an immune checkpoint modulator or a combination of the aforementioned pharmaceutical composition and an immune checkpoint modulator, which is used for the treatment or prevention of tumors.
  • the tumor is an IL-21-associated tumor, or an immune checkpoint-associated tumor.
  • the tumor is selected from the group consisting of mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, kidney cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-Hodgkin lymphoma, esophageal cancer, gastric cancer and colorectal cancer.
  • the tumor is colorectal cancer.
  • the IL-21-anti-albumin single-domain antibody fusion proteins disclosed herein comprise: (a) the cytokine IL-21, and (b) a single-domain antibody that specifically binds albumin ( sdAb), IL-21 includes the amino acid sequence shown in SEQ ID NO: 1, and the single domain antibody (sdAb) includes the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively HCDR1, HCDR2 and HCDR3.
  • the single domain antibodies (sdAbs) disclosed herein comprise the amino acid sequence set forth in SEQ ID NO:5.
  • the single domain antibodies disclosed herein are fused to the C-terminus of the cytokine, and the cytokine and single domain antibody are directly linked.
  • the IL-21 fusion proteins disclosed herein comprise the amino acid sequence shown in SEQ ID NO:6.
  • the IL-21 fusion proteins disclosed herein are used in combination with immune checkpoint modulators
  • the immune checkpoint modulator disclosed herein is selected from PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27 and B7H4 immune checkpoint inhibitors.
  • the immune checkpoint modulator disclosed herein is an anti-PD-1 antibody.
  • the anti-PD-1 antibodies disclosed herein comprise:
  • the anti-PD-1 antibody disclosed in the present invention is selected from one or more of nivolumab, pembrolizumab, toripalimab, Sintilimab, Camrelizumab, Tislelizumab, and Cemiplimab; preferably toripalimab.
  • the IL-21 fusion protein disclosed herein and the anti-PD-1 antibody used in combination have one or more properties selected from the group consisting of:
  • the immune checkpoint modulator disclosed herein is an anti-TIGIT antibody.
  • the anti-TIGIT antibodies disclosed herein are selected from the group consisting of: Tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, and BMS-986207.
  • the use of the IL-21 fusion protein disclosed herein in combination with the anti-TIGIT antibody has one or more properties selected from the group consisting of:
  • the present invention provides a pharmaceutical combination comprising:
  • the present invention provides a kit comprising:
  • kits further comprise instructions for the method of use of the pharmaceutical compositions therein.
  • Figure 1 Inhibitory effect of fusion protein JS-EC21 of the present invention on MC38 tumor.
  • Figure 2 Pharmacokinetic profiles of low, medium and high doses of JS-EC21 in cynomolgus monkeys.
  • Figure 3 Inhibitory effect of fusion protein JS-EC21 of the present invention combined with PD-1 monoclonal antibody on MC38 tumor.
  • Figure 4 Changes of tumor volume in mice after the fusion protein JS-EC21 of the present invention and toripalimab were administered alone or in combination.
  • Figure 5 Changes in mouse tumor weight after the fusion protein JS-EC21 of the present invention and toripalimab were administered alone or in combination.
  • Figure 6 Changes of immune cells in mouse tumors after the fusion protein JS-EC21 of the present invention and toripalimab were administered alone or in combination.
  • composition refers to a mixture comprising one or more of the antibodies described herein in admixture with other components such as physiologically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • liquid formulation refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation.
  • the liquid formulations of the present invention are stable on storage, and their stability is independent of lyophilization (or other state-changing methods, such as spray drying).
  • aqueous liquid formulation refers to a liquid formulation using water as a solvent.
  • aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
  • excipient refers to an agent that can be added to a formulation to provide desired properties (eg, consistency, increased stability) and/or to adjust osmotic pressure.
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
  • buffer pH of about 4.0-6.5 refers to an agent which, through the action of its acid/base conjugate component, renders a solution containing the agent resistant to pH changes.
  • the buffer used in the formulations of the present invention may have a pH in the range of about 4.0 to about 6.5, or a pH in the range of about 4.5 to about 6.5, or a pH in the range of about 4.5 to about 5.5.
  • buffers that control pH within this range include acetate (eg, sodium acetate), succinate (eg, sodium succinate), gluconic acid, histidine, histidine hydrochloride , methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.
  • acetate eg, sodium acetate
  • succinate eg, sodium succinate
  • gluconic acid histidine
  • histidine hydrochloride e.g, methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.
  • Hetidine buffer is a buffer containing histidine ions.
  • histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like, such as histidine-containing Histidine buffer solution of acid and histidine hydrochloride; histidine buffer solution of the present invention also includes histidine buffer solution containing histidine and acetate salt (such as sodium salt or potassium salt) or glacial acetic acid .
  • a “citrate buffer” is a buffer that includes citrate ions.
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • a preferred citrate buffer is a citric acid-sodium citrate buffer.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate buffer.
  • stabilizer means a pharmaceutically acceptable excipient which protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application.
  • Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites, as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine, or the like.
  • Salts such as arginine hydrochloride), glycine, alanine (alpha-alanine, beta-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline acid, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanine opine, octopine, glycine strombine) and N- of trimethylamine oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), alpha-casein, globulin, alpha-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAaseA.
  • TMAO trimethylamine oxide
  • human serum albumin hsa
  • bovine serum albumin bsa
  • alpha-casein globulin
  • alpha-lactalbumin LDH
  • lysozyme my
  • Some stabilizers such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure.
  • the stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars.
  • the preferred salt is sodium chloride
  • the preferred sugars are sucrose and trehalose
  • the preferred polyols are sorbitol and mannitol.
  • Preferred amino acids are arginine or a salt thereof (eg arginine hydrochloride), glycine, proline.
  • Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose , sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, more preferably arginine hydrochloride, sodium chloride-sucrose, arginine hydrochloride-mannitol, arginine hydrochloride- Sucrose, more preferably sucrose or trehalose.
  • surfactant generally includes agents that protect proteins, such as antibodies, from air/solution interface induced stress, solution/surface induced stress to reduce aggregation of the antibody or to minimize the formation of particulate matter in the formulation.
  • exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (eg, polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polysorbate Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Poloxamer, Pluronic), sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (eg, polysorbate 20 and polysorbate 80), poly
  • isotonic means that the formulation has substantially the same osmotic pressure as human blood.
  • Isotonic formulations generally have an osmolarity of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.
  • stable formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage.
  • a pharmaceutical formulation may be stable even if the contained antibody fails to retain 100% of its chemical structure or biological function after a certain period of storage.
  • an antibody that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after storage for a certain period of time may also be considered “" stable”.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, edited by Vincent Lee, Marcel Dekker, Inc. ., New York, N.Y., Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10:29-90 (both incorporated by reference).
  • the stability of a formulation can be measured by determining the percentage (among other methods) of native antibody remaining in a formulation after storage at a given temperature for a given period of time.
  • the percentage of native antibody can be measured by size exclusion chromatography (eg, size exclusion high performance liquid chromatography [SEC-HPLC]), with "native" meaning unaggregated and undegraded.
  • protein stability is determined as the percentage of monomeric protein in solution with a low percentage of degraded (eg, fragmented) and/or aggregated protein.
  • the formulations are stable for storage at room temperature, about 25-30°C, or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or Longer, up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.
  • Stability can be measured by determining (among other methods) the percentage of antibody (“acidic form") that migrates in the more acidic fraction of this antibody main fraction ("predominantly charged form”) during ion exchange, where stability is related to The percentage of antibody in the acidic form is inversely proportional.
  • the percentage of "acidified” antibody can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]).
  • an acceptable degree of stability means that upon storage of the formulation at a certain temperature and for a certain period of time, the detectable acidic form of the antibody therein does not exceed at most about 49%, 45%, 40%, 35% %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.
  • the certain period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.
  • the temperature at which the pharmaceutical formulation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C , about 2-8°C, about 5°C, about 25°C, or about 40°C.
  • the antibody shows substantially no evidence of, for example, aggregation, precipitation and/or denaturation upon visual inspection of color and/or clarity or as measured by UV light scattering or by size exclusion chromatography, then the antibody described above is not present in the pharmaceutical composition. "maintain its physical stability”. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where a visible precipitate is formed.
  • the stability of the formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurements correlate with the turbidity of the formulation.
  • the turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution, and is typically measured by nephelometric methods and measured in nephelometric turbidity units (NTU).
  • the level of turbidity as a function of, for example, the concentration of one or more components in solution is also referred to as the "opaque” or "opaque appearance" of the formulation.
  • Turbidity levels can be calculated with reference to a standard curve generated using suspensions of known turbidity.
  • Reference standards for determining the turbidity level of pharmaceutical compositions can be based on the European Pharmacopoeia standards (European Pharmacopoeia, 4th edition, "Directorate for the Quality of Medicine”). of the Council of Europe) (EDQM), France).
  • a clear solution is defined as a solution with a turbidity lower than or equal to that of a reference suspension according to the European Pharmacopoeia standard of about 3.
  • Nephelometric turbidity measurements can detect Rayleigh scattering in the absence of associative or non-ideal effects, which typically vary linearly with concentration. Other methods for assessing physical stability are known in the art.
  • Such an antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter.
  • Chemical stability can be assessed by, for example, detecting or quantifying chemically altered forms of the antibody.
  • Chemical changes can include size changes (eg, clipping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS).
  • Other types of chemical changes include charge changes (eg occur as a result of deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.
  • Such an antibody "retains its biological activity" in a pharmaceutical composition if the antibody in the pharmaceutical composition is biologically active for its intended purpose. For example, if the formulation is stored at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (eg, 1 to 12 months), the IL-21 fusion protein contained in the formulation binds IL-17A with the same affinity as before the aforementioned storage.
  • a formulation of the invention is considered stable if the binding affinity of the antibody is at least 90%, 95% or more. Binding affinity can also be determined using techniques such as ELISA or plasmon resonance.
  • a “therapeutically effective amount” or “effective amount” of an antibody in the pharmacological sense refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody can effectively treat.
  • a "therapeutically effective amount” or “therapeutically effective dose” or “effective amount” of a drug or therapeutic agent is one that, when used alone or in combination with another therapeutic agent, protects a subject from disease onset or promotes disease Any amount of the drug that resolves as evidenced by a reduction in the severity of symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability caused by the suffering of the disease.
  • the ability of a drug or therapeutic agent to promote disease regression can be assessed using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by in vitro The activity of the agent is determined in an assay.
  • a therapeutically effective amount includes a "prophylactically effective amount", that is, when administered alone or, for example, in combination with other therapeutic agents (eg, antineoplastic agents) to a subject at risk of developing a disease (eg, developing cancer) or having a recurrence of the disease (eg, having a cancer). Any amount of a drug that inhibits the development or recurrence of a disease (eg, cancer) in a subject with cancer recurrence.
  • therapeutic agents eg, antineoplastic agents
  • subject is intended to include mammalian organisms.
  • subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • administering refers to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art.
  • Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion.
  • Parenter administration refers to modes of administration other than enteral or topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular , intra-frame, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion and in vivo electroporation.
  • antibody is used in its broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies, and antigens thereof Binding fragments so long as they exhibit the desired antigen-binding activity.
  • antibody portion refers to a full-length antibody or antigen-binding fragment thereof.
  • full-length antibody or "intact antibody molecule” as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25 kDa in full length) are interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and spaced by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus.
  • Amino acids are generally assigned to each domain according to the following definitions: Sequences of Proteins of Immunological Interest, Kabat et al; National Institutes of Health, Bethesda, Md.; 5th edition; NIH Publication No. 91-3242 (1991) : Kabat (1978) Adv. Prot. Chem.
  • variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • CDR refers to the complementarity determining regions within antibody variable sequences. There are three CDRs in each variable region of the heavy and light chains, which are designated HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently by different systems.
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or delineated according to the conventions of Kabat, Chothia or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chain are inserted between flanking stretches called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are classified based on the amino acid sequence of their heavy chain constant region.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • an "antigen-binding fragment” includes a fragment of an antibody or derivative thereof, typically including at least a fragment of the antigen-binding or variable region (eg, one or more CDRs) of the parent antibody, which retains at least some of the parent antibody binding specificity.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as sc-Fv; nanobodies formed from antibody fragments and multispecific antibodies.
  • the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not significantly alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of an antibody).
  • the CDRs of the antibodies of the invention can be bounded by one skilled in the art according to any scheme in the art (eg, different assignment systems or combinations).
  • Framework regions or "FRs” as used herein refer to immunoglobulin variable regions that do not include CDR regions.
  • Chimeric antibody refers to antibodies and fragments thereof in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in an antibody derived from a particular species (eg, human) or belonging to a particular antibody class or subclass , while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species (eg, mouse) or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity.
  • Human antibody refers to an antibody comprising only human immunoglobulin sequences. If the human antibody is produced in a mouse, a mouse cell, or a mouse cell-derived hybridoma, it may contain a murine carbohydrate chain. Similarly, “mouse antibody” or “rat antibody” refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively.
  • Humanized antibody refers to a form of antibody that contains sequences from non-human (eg, murine) antibodies as well as from human antibodies. Such antibodies contain minimal sequence derived from the unilateral side of the non-human immunoglobulin. Typically, a humanized antibody will comprise substantially all of at least one and usually both variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all The FR regions are those of human immunoglobulins. The humanized antibody optionally also includes at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.
  • Fc immunoglobulin constant region
  • Fv is the smallest antibody fragment that contains a complete antigen recognition site and an antigen binding site.
  • the fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. Folding of these two domains creates six hypervariable loops (3 loops each for the heavy and light chains) that contribute amino acid residues to antigen binding and confer antigen-binding specificity to the antibody.
  • six hypervariable loops (3 loops each for the heavy and light chains) that contribute amino acid residues to antigen binding and confer antigen-binding specificity to the antibody.
  • a single variable domain or half an Fv comprising only three CDRs specific for an antigen
  • HCAb heavy chain antibody
  • HCAb refers to a functional antibody that includes a heavy chain but lacks the light chain typically found in 4-chain antibodies.
  • Camelids such as camels, llamas or alpacas are known to produce HCAbs.
  • single domain antibody refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the sdAbs are capable of binding to antigen alone without pairing with the corresponding CDR-containing polypeptide.
  • the single domain antibody is engineered from a camelid HCAb, and its heavy chain variable domain is referred to herein as "VHH" (variable domain of the heavy chain of a heavy chain antibody).
  • VHH variable domain of the heavy chain of a heavy chain antibody.
  • Camelid sdAbs are among the smallest known antigen-binding antibody fragments (see, eg, Hammers-Casterman et al., Nature, vol. 363: pp. 446-448, 1993; Greenberg et al., Nature, vol.
  • Basic VHHs have the following structure from N-terminal to C-terminal: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 1 3.
  • the numbering of residues in immunoglobulin heavy chains herein refers to the EU index numbering of Kabat et al. "Kabat's EU index” refers to the residue numbering of human IgGl EU antibodies.
  • Therapeutic anti-PD-1 monoclonal antibody refers to an antibody that specifically binds to a specific mature form of PD-1 expressed on the surface of certain mammalian cells. Mature PD-1 has no prosecretory leader sequence, or leader peptide.
  • the terms “PD-1” and “mature PD-1” are used interchangeably herein and are to be understood to mean the same molecule unless expressly defined otherwise, or clear from the context.
  • a therapeutic anti-human PD-1 antibody or anti-hPD-1 antibody refers to a monoclonal antibody that specifically binds to mature human PD-1.
  • isolated antibody or antigen-binding fragment thereof refers to a purified state and in which case the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials such as cellular debris or growth culture medium).
  • cytokine should be understood to refer to any protein or peptide, analog or functional fragment thereof, which is capable of stimulating or inducing in mammals the targeting of a preselected cell type (eg, cancer cells or virus-infected cells) ) of the cytocidal immune response. Accordingly, it is contemplated that a variety of cytokines may be incorporated into this application. Useful cytokines include, for example, tumor necrosis factor (TNF), interleukin (IL), lymphokine (L), colony stimulating factor (CSF), interferon (IFN), including those capable of stimulating or inducing such killing. Species variants, truncated analogs thereof for immune responses.
  • TNF tumor necrosis factor
  • IL interleukin
  • L lymphokine
  • CSF colony stimulating factor
  • IFN interferon
  • Useful tumor necrosis factors include, for example, TNF ⁇ .
  • Useful lymphokines include, for example, LT.
  • Useful colony stimulating factors include, for example, GM-CSF and M-CSF.
  • Useful interleukins include, for example, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15, IL-18, IL-21, IL22, and IL-33.
  • Useful interferons include, for example, IFN-alpha, IFN-beta and IFN-gamma.
  • cytokine should also be understood to include any variant of a wild-type cytokine (such as IL-21, IL-7, IL-15, etc.) that includes modification and retains at least a substantial portion of any desired function thereof (such as at least about 50%).
  • a wild-type cytokine such as IL-21, IL-7, IL-15, etc.
  • Immuno checkpoints refer to a series of molecules that are expressed on immune cells and can regulate the degree of immune activation. They play an important role in preventing autoimmunity (abnormal immune function, attacking normal cells). Common immune checkpoints include, but are not limited to, PD-1, PD-L1, PD-L2, CTLA-4, 4-1BB, CD47, TIGIT, GITR, CD112R, BTLA, TIM3, LAG3, CD27, and B7H4.
  • the immune checkpoint modulators described herein refer to agents, such as inhibitors, that can specifically bind to these immune checkpoints and enhance, attenuate or block their biological activity.
  • Such agents include, inter alia, antibodies or antigen-binding fragments thereof that specifically bind to these immune checkpoints.
  • the immune checkpoint-related tumors described herein refer to tumors whose biological activities can be attenuated or blocked by binding to immune checkpoints, thereby achieving tumor prevention and treatment.
  • the immune checkpoint-related tumors described herein are PD-1 or PD-L1 or TIGIT-related tumors.
  • AE adverse effect
  • a medical treatment may have one or more associated AEs, and each AE may be of the same or a different level of severity.
  • Tumor burden refers to the total amount of tumor material distributed throughout the body. Tumor burden refers to the total number of cancer cells or the total size of a tumor throughout the body. Tumor burden can be determined by a variety of methods known in the art, such as the use of calipers after the tumor is removed from the subject, or imaging techniques (eg, ultrasound, bone scan, computed tomography) when in vivo (CT) or Magnetic Resonance Imaging (MRI) scans) to measure its size.
  • imaging techniques eg, ultrasound, bone scan, computed tomography
  • CT in vivo
  • MRI Magnetic Resonance Imaging
  • tumor size refers to the total size of the tumor, which can be measured as the length and width of the tumor. Tumor size can be determined by a variety of methods known in the art, such as using calipers after the tumor is removed from the subject, or using imaging techniques such as bone scans, ultrasound, CT or MRI scans when in vivo. size.
  • subject include any organism, preferably an animal, more preferably a mammal (eg, rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.
  • subject and patient are used interchangeably herein.
  • PFS (also called "time to tumor progression”) refers to the length of time during and after treatment that the cancer does not grow, and includes the amount of time a patient experiences CR or PR as well as the amount of time a patient experiences SD.
  • DFS refers to the length of time a patient remains disease free during and after treatment.
  • OS refers to an increase in life expectancy compared to an initial or untreated individual or patient.
  • Treatment regimens for the combinations of the invention that are effective in treating cancer patients can vary depending on factors such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anticancer response in the subject. Although embodiments of the present invention may not achieve an effective positive therapeutic effect in every subject, they should be effective and achieve a positive therapeutic effect in a statistically significant number of subjects.
  • administration mode and “dosing regimen” are used interchangeably and refer to the dosage and timing of use of each therapeutic agent in the combination of the present invention.
  • cancer refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division, growth division and growth leads to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream.
  • IL-21-associated tumors refers to tumors that benefit from the administration of IL-21 or IL-21 fusion protein in the treatment or prevention of tumors.
  • tumors or cancers suitable for treatment or prevention with the methods, medicaments and kits of the invention refer in particular to tumors or cancers that benefit from IL-21, or immune checkpoint-related tumors, especially PD-1 or PD-L1 or TIGIT-related tumors, including but not limited to mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, kidney cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-Hodgkin lymphoma, esophageal cancer, Stomach and colorectal cancer.
  • the tumor/cancer disclosed herein refers to colorectal cancer.
  • the IL-21-anti-albumin single domain antibody fusion protein (IL-21 fusion protein) disclosed in the present invention includes any one of the IL-21 fusion proteins described in Publication No. WO2019246004, the entire contents of which are hereby incorporated by reference way to be incorporated into this article.
  • the amino acid sequence of the antibody used in the methods and compositions of the invention includes the amino acid sequence from the IL-21 fusion protein P798 described in WO2019246004.
  • the non-limiting, exemplary antibody used in the examples herein is selected from the IL-21-anti-albumin single domain antibody fusion protein P798 (JS-EC21) described in WO2019246004, the amino acid sequence of which is shown below:
  • HCDR1 GSTWSINT (SEQ ID NO: 2)
  • HCDR2 ISSGGST (SEQ ID NO: 3)
  • HCDR3 YAQSTWYPPS (SEQ ID NO: 4)
  • PD-1 antibody refers to binding to the PD-1 receptor, blocking the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T, B, NK cells) and preferably Any chemical compound or biomolecule that also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed on immune cells.
  • the PD-1 antibody blocks the binding of human PD-L1 to human PD-1, and preferably blocks both human PD-L1 and PD-L2 from binding to human PD1 binding.
  • the human PD-1 amino acid sequence can be found at NCBI locus number: NP_005009.
  • Human PD-L1 and PD-L2 amino acid sequences can be found at NCBI locus numbers: NP_054862 and NP_079515, respectively.
  • anti-PD-1 antibody when referring to an "anti-PD-1 antibody”, unless otherwise specified or described, the term includes antigen-binding fragments thereof.
  • anti-PD-1 antibody or its antigen-binding fragment suitable for any use, therapy, medicine and kit of the present invention binds PD-1 with high specificity and high affinity, and blocks PD-L1/2 and PD-1 , and inhibit PD-1 signal transduction, thereby achieving immunosuppressive effect.
  • anti-PD-1 antibodies include full-length antibodies themselves, as well as those that bind to the PD-1 receptor and exhibit similarities to the full Ab in terms of inhibiting ligand binding and upregulating the immune system.
  • the anti-PD-1 antibody is an anti-PD-1 antibody that cross-competes with toripalimab for binding to human PD-1.
  • the anti-PD-1 antibody is a murine, chimeric, humanized or human Ab or antigen-binding fragment thereof.
  • the Ab is a humanized Ab.
  • the constant region is selected from the group consisting of human IgGl, IgG2, IgG3 and IgG4 constant regions; preferably, an anti-PD-1 antibody suitable for use in any of the uses, therapies, medicaments and kits described herein
  • a heavy chain constant region comprising a human IgGl or IgG4 isotype, more preferably a human IgG4 constant region.
  • the sequence of the IgG4 heavy chain constant region of the anti-PD-1 antibody comprises a S228P mutation that replaces a serine residue in the hinge region with a proline residue normally present at the corresponding position in an IgG1 isotype antibody .
  • the anti-PD-1 antibodies disclosed in the present invention include any one of the anti-PD-1 antibodies or antigen-binding fragments thereof described in Publication No. WO2014206107, the entire disclosure of which is incorporated herein by reference .
  • the anti-PD-1 antibodies useful in any of the uses, therapies, medicaments and kits disclosed herein are selected from the humanized antibodies 38, 39, 41 and 48 described in WO2014206107.
  • the anti-PD-1 antibody useful in any of the uses, therapies, medicaments and kits disclosed herein is Toripalimab (an antibody with WHO Drug Information (Vol. 32) , Issue 2, pp. 372-373 (2016)).
  • the non-limiting, exemplary antibody used in the examples herein is selected from the humanized antibody Toripalimab described in WO2014206107, whose amino acid CDR sequence, variable region sequence and full-length amino acid sequence are as follows (KABAT):
  • LCDR2 KVSNRFS (SEQ ID NO: 8)
  • LCDR3 FQGSHVPLT (SEQ ID NO: 9)
  • HCDR1 DYEMH (SEQ ID NO: 10)
  • HCDR2 VIESETGGTAYNQKFKG (SEQ ID NO: 11)
  • HCDR3 EGITTVATTYYWYFDV (SEQ ID NO: 12)
  • Toripalimab heavy chain (SEQ ID NO: 16)
  • the anti-PD-1 antibody is a monoclonal antibody or an antigen-binding fragment thereof, and its light chain CDRs are SEQ ID NOs: 7, 8 and the amino acids shown in 9, the heavy chain CDRs are the amino acids shown in SEQ ID NOs: 10, 11 and 12.
  • the anti-PD-1 antibody comprises: (a) a light chain variable region comprising SEQ ID NO: 13, and (b) ) monoclonal antibody comprising the heavy chain variable region of SEQ ID NO: 14.
  • the anti-PD-1 antibody is a light chain comprising: (a) a light chain comprising SEQ ID NO: 15, and (b) a light chain comprising Monoclonal antibody to the heavy chain of SEQ ID NO: 16.
  • Anti-PD-1 antibodies useful in any of the uses, therapies, medicaments, and kits disclosed herein also include FDA-approved Nivolumab and Pembrolizumab.
  • anti-PD-1 antibodies useful in any of the uses, therapies, medicaments, and kits disclosed herein also include anti-PD-1 antibodies that specifically bind to PD-L1 to block the binding of PD-L1 to PD-1 PD-L1 monoclonal antibodies such as nivolumab, pembrolizumab, toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Cemiplimab.
  • TIGIT short for T cell Ig and ITIM domain proteins, refers to proteins derived from any vertebrate (including mammals such as primates (e.g., humans) and rodents ( For example, any native TIGIT of mouse and rat) unless otherwise specified.
  • the term encompasses "full-length” unprocessed TIGIT as well as any form of TIGIT or any fragment thereof produced by intracellular processing.
  • the term also includes native TIGIT Variants of TIGIT that exist, for example, splice variants or allelic variants.
  • TIGIT refers to full-length TIGIT from human and cynomolgus monkeys or a fragment thereof (such as a mature fragment thereof lacking a signal peptide).
  • human TIGIT refers to the mature TIGIT (amino acid residues 1-21 being the leader peptide) identical to the sequence of amino acid residues 22-244 of Genbank Accession No. NP_776160.2. In one embodiment, human TIGIT refers to Genbank accession number NP_776160.2 TIGIT ectodomain sequence identical to amino acid residues 22-141. In one embodiment, cynomolgus monkey (Wacaca fascicuiaris) TIGIT refers to the sequence of amino acid residues 22-245 of Genbank accession number XP_005548158.1 Consistent mature TIGIT.
  • anti-TIGIT antibody refers to a TIGIT protein or fragment thereof capable of binding with sufficient affinity such that the antibody can be used as a diagnostic agent targeting TIGIT and/or therapeutic agents.
  • anti-TIGIT antibody when referring to an "anti-TIGIT antibody”, unless otherwise specified or described, the term includes antigen-binding fragments thereof.
  • the anti-TIGIT antibodies or antigen-binding fragments thereof disclosed herein include any one of the anti-TIGIT antibodies or antigen-binding fragments thereof described in Application No. PCT/CN2020/101883 (WO2021/008523), all of which are described herein. The entire disclosure is incorporated herein by reference.
  • the CDR sequences of anti-TIGIT antibodies used in the methods, uses and compositions of the invention include CDR sequences from the antibodies hu20 or hu3 described in PCT/CN2020/101883.
  • the light and heavy chain variable regions of anti-TIGIT antibodies used in the methods, uses and compositions of the invention comprise those from the antibodies hu20 or hu3 described in PCT/CN2020/101883 Light chain variable region and heavy chain variable region.
  • the anti-TIGIT antibody used in the methods, uses and compositions of the invention is the antibody hu20 or hu3 described in PCT/CN2020/101883.
  • the anti-TIGIT antibodies disclosed herein are selected from: Tiragolumab, Etigilimab, Vibostolimab, Domvanalimab, EOS-884448, or BMS-986207.
  • the pharmaceutical composition disclosed in the present invention is a highly stable pharmaceutical composition containing IL-21-anti-albumin single domain antibody fusion protein.
  • the present invention finds that the combination of trehalose can obviously improve the stability of the pharmaceutical composition.
  • the pharmaceutical composition disclosed in the present invention is more sensitive to pH, and when the pH range is controlled to 4.5-5.1, the pharmaceutical composition disclosed in the present invention has higher stability.
  • the present invention provides a pharmaceutical composition, comprising: (1) buffer; (2) IL-21-anti-albumin single domain antibody fusion protein.
  • the IL-21 fusion protein in the pharmaceutical composition of the present invention comprises: (a) the cytokine IL-21, and (b) a single-domain antibody (sdAb) that specifically binds to albumin, and the above-mentioned IL-21 comprises, for example, SEQ ID NO: 1
  • the amino acid sequence shown, the above-mentioned single domain antibody comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively.
  • the fusion protein in the pharmaceutical composition of the present invention comprises the cytokine IL-21 whose amino acid sequence is shown in SEQ ID NO: 1, and the sdAb whose amino acid sequence is shown in SEQ ID NO: 5; more preferably, the present invention
  • the fusion protein in the pharmaceutical composition comprises or consists of the amino acid sequence shown in SEQ ID NO:6.
  • the concentration of the IL-21-anti-albumin single domain antibody fusion protein is about 0.1-100 mg/mL, preferably about 0.2-20 mg/mL, preferably about 0.2-10 mg/mL, preferably about 0.5-5mg/mL, more preferably 1-5mg/mL; more preferably, the above-mentioned IL-21-anti-albumin single domain antibody fusion protein concentration is about 0.5mg/mL, 0.8mg/mL, 1mg/mL, 1.2mg /mL, 1.5mg/mL, 1.8mg/mL, 2mg/mL, 2.5mg/mL, 3mg/mL, 3.5mg/mL, 4mg/mL, 4.5mg/mL, 5mg/mL, 6mg/mL, 7mg/mL mL, 8 mg/mL or 10 mg/mL, preferably about 1 mg/mL.
  • the buffer in the pharmaceutical composition of the present invention can be selected from acetate buffer, citrate buffer and histidine buffer to provide the pharmaceutical composition of the present invention with 4.0 to 6.5, preferably 4.5 to 5.5, more preferably 4.8 ⁇ 0.3 , more preferably a pH of about 4.8.
  • the pH of the buffer used in the pharmaceutical composition of the present invention may be 4.0-6.5, preferably 4.5-5.5, more preferably 4.8 ⁇ 0.3, more preferably about 4.8.
  • a particularly preferred buffer in the pharmaceutical composition of the present invention is an acetate buffer, preferably, the above-mentioned acetate buffer is an acetic acid-sodium acetate buffer or an acetic acid-potassium acetate buffer, preferably an acetic acid-sodium acetate buffer.
  • the acetate buffer is made up of 1-30 mM acetic acid and 1-30 mM sodium acetate. In some protocols, the acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 2:3. In some protocols, the acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:2.1.
  • the acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:5.7. In some embodiments, the acetate buffer is: acetate buffer at a pH of about 4.8 made from about 8 mM acetic acid and about 12 mM sodium acetate. In some embodiments, the acetate buffer is an acetate buffer at a pH of about 5.0 made from about 6.5 mM acetic acid and about 13.5 mM sodium acetate. In some embodiments, the acetate buffer is an acetate buffer at a pH of about 5.5 made from about 3 mM acetic acid and about 17 mM sodium acetate.
  • the pharmaceutical composition of the present invention may contain: an acetic acid-sodium acetate buffer with a pH of 4.5-5.5 (preferably 4.5-5.0) in a concentration of 10-30 mM in the pharmaceutical composition; and 0.5-5 mg/mL of
  • the IL-21-anti-albumin single domain antibody fusion protein of any of the preceding embodiments is preferably JS-EC21 disclosed herein.
  • the pharmaceutical compositions of the present invention also contain stabilizers.
  • the above-mentioned stabilizer is selected from one or more of sodium chloride, mannitol, sorbitol, sucrose and trehalose, preferably, the above-mentioned stabilizer is trehalose.
  • the concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10 mM to 400 mM, preferably 20 mM to 300 mM, more preferably 30 mM to 200 mM.
  • the above-mentioned stabilizer is mannitol at a concentration of about 100-300 mM, preferably 200-300 mM; or the above-mentioned stabilizer is sucrose at a concentration of about 100-300 mM, preferably 200-300 mM; or the above-mentioned stabilizer is at a concentration of about 100- 300mM, preferably 200-300mM trehalose; or the above stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the above stabilizer is about 30-200mM sodium chloride and about 30 -200mM sucrose in combination.
  • the above-mentioned stabilizer is about 100-300 mM trehalose or 100-300 mM sucrose; more preferably, the above-mentioned stabilizer is about 200-280 mM trehalose.
  • the pharmaceutical composition of the present invention contains: acetic acid-sodium acetate buffer at pH 4.5-5.5 (preferably 4.5-5.0) at a concentration of 10-30 mM in the pharmaceutical composition; 0.5- 5mg/mL of the IL-21-anti-albumin single domain antibody fusion protein of any of the preceding embodiments, preferably JS-EC21 disclosed herein; and 20mM-300mM of a stabilizer, preferably, the stabilizer includes sodium chloride One or more of , mannitol, sucrose and trehalose, preferably 100-300 mM trehalose or 100-300 mM sucrose, more preferably 200-280 mM trehalose.
  • the pharmaceutical compositions of the present invention further include a surfactant.
  • Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188.
  • the most preferred surfactant is polysorbate 80.
  • the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.001%-0.1%, preferably about 0.02%-0.08%, preferably about 0.02%-0.04%, on a w/v basis.
  • the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.04% or 0.08%, preferably 0.02%.
  • the pharmaceutical composition of the present invention contains: acetic acid-sodium acetate buffer at pH 4.5-5.5 (preferably 4.5-5.0) at a concentration of 10-30 mM in the pharmaceutical composition; 0.5- 5mg/mL of the IL-21-anti-albumin single domain antibody fusion protein of any of the preceding embodiments, preferably JS-EC21 disclosed herein; and 20mM-300mM of a stabilizer, preferably, the stabilizer includes sodium chloride , one or more of mannitol, sucrose and trehalose, preferably 100-300 mM trehalose or 100-300 mM sucrose, more preferably 200-280 mM trehalose; and 0.02% w/v - 0.04% polysorbate 80.
  • the pharmaceutical composition of the present invention can be a liquid preparation or a lyophilized preparation.
  • the present invention also provides a pharmaceutical combination comprising the IL-21 fusion protein described herein and an immune checkpoint modulator.
  • the immune checkpoint modulator disclosed in the present invention can be as described in any of the foregoing embodiments; preferably, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-TIGIT antibody.
  • the above-mentioned anti-IL-21 fusion protein and immune checkpoint modulator can be provided in the form of a mixture (that is, in the form of a pharmaceutical composition), or each can be provided in the form of independent preparations.
  • each formulation contains, in addition to the active ingredient, a pharmaceutically acceptable carrier.
  • the pharmaceutical composition When provided in the form of a pharmaceutical composition, the pharmaceutical composition usually contains, in addition to the active ingredient, a pharmaceutically acceptable carrier.
  • the pharmaceutical combinations of the present invention may also contain one or more additional therapeutic agents.
  • Additional therapeutic agents can be, for example, chemotherapeutic agents, biotherapeutic agents, immunogenic agents (eg, attenuated cancer cells, tumor antigens, antigen-presenting cells such as tumor-derived antigens or nucleic acid-pulsed dendritic cells) , immunostimulatory cytokines (eg, IL-2, IFN-tumor, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines such as, but not limited to, GM-CSF).
  • immunogenic agents eg, attenuated cancer cells, tumor antigens, antigen-presenting cells such as tumor-derived antigens or nucleic acid-pulsed dendritic cells
  • immunostimulatory cytokines eg, IL-2, IFN-tumor, GM-CSF
  • cells transfected with genes encoding immunostimulatory cytokines such as, but not limited to,
  • the present invention provides IL-21 fusion protein, or the above pharmaceutical composition, or the combination of IL-21 fusion protein and immune checkpoint modulator or the combination of the above pharmaceutical composition and immune checkpoint modulator in preparation for prevention or treatment Use in cancer medicine.
  • the present invention also provides use of the above-mentioned pharmaceutical composition or IL-21 fusion protein in a medicament for enhancing or improving the antitumor activity of an immune checkpoint modulator.
  • the present invention provides methods for preventing or treating tumors, comprising administering to an individual in need thereof an effective amount of an IL-21 fusion protein, or the above pharmaceutical composition, or a combination of an IL-21 fusion protein and an immune checkpoint modulator or the above A combination of a pharmaceutical composition and an immune checkpoint modulator.
  • the effective amount includes a prophylactically effective amount and a therapeutically effective amount.
  • the present invention provides IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or the combination of IL-21 fusion protein and immune checkpoint modulator, or the combination of the above-mentioned pharmaceutical composition and immune checkpoint modulator, which is used for treating or preventing tumors .
  • the tumors disclosed herein may be as described in any of the preceding embodiments.
  • the tumors described herein are tumors that benefit from IL-21 treatment, or are PD-1 or TIGIT-related tumors, preferably selected from mesothelioma, lung cancer, breast cancer, ovarian cancer, melanoma, kidney cancer, Pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, non-Hodgkin lymphoma, esophageal cancer, gastric cancer and colorectal cancer; preferably, the tumor is metastatic melanoma, renal cell carcinoma, colorectal cancer and non-Hodgkin's Hodgkin lymphoma, more preferably colorectal cancer.
  • the immune checkpoint modulator disclosed in the present invention can be as described in any of the foregoing embodiments; preferably, the immune checkpoint modulator is an anti-PD-1 antibody or an anti-TIGIT antibody.
  • the two-tailed unpaired Student's t test is used for difference analysis between the two groups of the present invention, * means P ⁇ 0.05; ** means P ⁇ 0.01, *** means P ⁇ 0.001, ns means no Significant difference; P ⁇ 0.05 means significant increase or significant increase.
  • the IL-21 fusion protein disclosed in the present invention and the anti-PD-1 antibody used in combination have one or more selected from the following: Various properties:
  • the application provides the use of a combination of an IL-21 fusion protein disclosed herein and an anti-PD-1 antibody in the manufacture of a reagent for one or more of the following uses:
  • the agents are used in the treatment or prevention of tumors disclosed herein.
  • the IL-21 fusion protein of the present invention in combination with an anti-TIGIT antibody has one or more properties selected from the group consisting of:
  • the application provides the use of a combination of an IL-21 fusion protein disclosed herein and an anti-TIGIT antibody as described above in the manufacture of a reagent for one or more of the following uses:
  • the agents are used in the treatment or prevention of tumors disclosed herein.
  • a preferred IL-21 fusion protein for use in the tumors disclosed herein may be as described in any of the embodiments herein, preferably comprising: (a) the cytokine IL-21, and (b) a single domain that specifically binds albumin Antibody (sdAb), cytokine IL-21 comprises the amino acid sequence as shown in SEQ ID NO: 1, and single domain antibody (sdAb) comprises the amino acid sequence as SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 4; more preferably comprise the amino acid sequence shown in SEQ ID NO:1, and the amino acid sequence shown in SEQ ID NO:5; more preferably comprise the amino acid sequence shown in SEQ ID NO:6 amino acid sequence shown.
  • sdAb single domain antibody
  • Preferred anti-PD-1 antibodies for the tumors disclosed herein may be as described in any of the embodiments herein, more preferably comprising the light chain CDRs of amino acids set forth in SEQ ID NOs: 7, 8 and 9, and the heavy chain
  • a cloned antibody more preferably a monoclonal antibody comprising the light chain shown in SEQ ID NO: 15 and the heavy chain shown in SEQ ID NO: 16, more preferably the humanized antibodies 38, 39, 41 and WO2014206107 described in 48. Toripalimab is most preferred.
  • the present invention provides a method for preventing or treating tumors, the method comprising administering to a tumor patient a therapeutically effective amount of IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or IL-21 fusion protein and anti-PD-
  • a therapeutically effective amount of IL-21 fusion protein, or the above-mentioned pharmaceutical composition, or IL-21 fusion protein and anti-PD- The combination of 1 antibody or the combination of the above-mentioned pharmaceutical composition and anti-PD-1 antibody;
  • the tumor patient is a colorectal cancer patient;
  • the IL-21 fusion protein comprises the amino acid sequence shown in SEQ ID NO:6 ;
  • the anti-PD-1 antibody is toripalimab.
  • the present invention provides an IL-21 fusion protein, or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an anti-PD-1 antibody or a combination of the above pharmaceutical composition and an anti-PD-1 antibody
  • an IL-21 fusion protein or a pharmaceutical composition as described above, or a combination of an IL-21 fusion protein and an anti-PD-1 antibody or a combination of the above pharmaceutical composition and an anti-PD-1 antibody
  • the tumor patient is a colorectal cancer patient
  • the IL-21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 6;
  • the PD-1 antibody was toripalimab.
  • the present invention provides a kit comprising:
  • the present disclosure discloses a kit, which further comprises instructions for directing the method of use of the pharmaceutical composition.
  • the present invention also provides the pharmaceutical composition disclosed in any embodiment of the present invention for treating or preventing cancer, the use of the pharmaceutical composition disclosed in any embodiment of the present invention in the preparation of a medicament for treating or preventing cancer, and the need for administration A method of treating or preventing cancer in an individual or patient of a therapeutically effective amount of a pharmaceutical composition disclosed in any of the embodiments of the present invention.
  • diseases suitable for the treatment and prevention of the pharmaceutical composition of the present invention include but are not limited to mesothelioma, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, lymphoma, leukemia, head and neck cancer, liver cancer, esophageal cancer, gastric cancer and colorectal cancer.
  • the present invention adopts the following abbreviations:
  • his-tag stands for histidine tag
  • HRP stands for horseradish peroxidase
  • TMB stands for 3,3',5,5'-tetramethylbenzidine
  • HBSS Hank's Balanced Salt Solution, that is, Hank's Balanced Salt Solution
  • HEPES stands for N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, Good's buffer reagent.
  • BID represents one dose, 2 times a day
  • CDR stands for complementarity determining region
  • FR stands for framework region
  • IgG stands for Immunoglobulin G
  • the scanning temperature is 20°C-95°C, and the heating rate is 1°C/min.
  • the instrument run ends processing and analyzing the data.
  • the appearance is detected by visual inspection. Make sure that the light intensity of the clarity detector is kept between 1000lx-1500lx. Hold the sample at eye level and gently shake or invert to avoid creating air bubbles. Visual inspection was performed in front of a black background and a white background, respectively. Look for color, opalescence, and visible foreign matter.
  • Protein concentration was detected using a UV spectrophotometer.
  • the percent extinction coefficient (E1%) was set at 1.527 (g/ml) -1 cm -1 .
  • BIO MATE 3S instrument wash the cuvette with ultrapure water, add 150 ⁇ L of ultrapure water, click to measure, and use ultrapure water for blank correction. Each sample was measured in 2 replicates and each solution was measured in 3 replicates, and the average was taken. The protein concentration of the corresponding sample was calculated by the extinction coefficient and OD value.
  • Chromatographic conditions Chromatographic parameters Detector wavelength 280nm Autosampler temperature 25 ⁇ 3°C column temperature 5 ⁇ 3°C flow rate 0.50ml/min Injection volume 25 ⁇ l operating mode isocratic mode operation hours 30.00min
  • NR-CE-SDS non-reducing capillary electrophoresis
  • the NR-CE-SDS purity was checked using a capillary electrophoresis apparatus (Maurice Instruments) equipped with a CE-SDS cartridge. Take 50 ⁇ l of diluent (1 ⁇ sample buffer Maurice CE-SDS), add 5.0 ⁇ l of 0.8M iodoacetamide and mix well, then add 50 ⁇ l of sample (1 mg/ml), and vortex to mix; at the same time, take 50 ⁇ l of diluent, add Mix 5.0 ⁇ l of 0.8M iodoacetamide evenly, incubate at 70°C for 5 min, and calculate by the area normalization method, the percentage of the main peak to the sum of all corrected peak areas is the main peak purity.
  • a capillary electrophoresis apparatus Maurice Instruments
  • the purity of R-CE-SDS was detected using a capillary electrophoresis apparatus (Maurice Instruments) equipped with a CE-SDS cartridge. Take 50 ⁇ l of diluent (1 ⁇ sample buffer Maurice CE-SDS), add 5.0 ⁇ l of 2-mercaptoethanol and mix evenly, then add 50 ⁇ l of sample (1 mg/ml), and vortex to mix; at the same time, take 50 ⁇ l of diluent, add 2 - Mix 5.0 ⁇ l of mercaptoethanol evenly, incubate at 70°C for 15 minutes, and calculate by the area normalization method. The percentage of the main peak in the sum of all corrected peak areas is the purity of the main peak.
  • iCIEF was detected using a capillary electrophoresis apparatus (Maurice Instruments) equipped with an iCIEF cartridge. After the samples were diluted with iCIEF mixture, they were detected by capillary electrophoresis (Maurice). Different isoelectric point components are focused at different positions to achieve the effect of focusing and separation.
  • the iCIEF mixture is prepared as follows:
  • the recombinant human IL-21R protein His tag (Acro company, product number ILR-H5226) was diluted to 4.0 ⁇ g/ml for coating, and incubated in a constant temperature incubator at 37 ⁇ 2°C for 90 min. Plates were washed and blocked with 2% skim milk. Add different concentrations of test samples (starting at 4 ⁇ g/mL, 3-fold serial dilution), incubate for 1 hour and wash the plate. It was then incubated with HRP-conjugated rabbit anti-camel VHH antibody (GenScript, Cat. No. A01861-200) diluted 1:5000 for 1 hour, and then incubated with HRP substrate TMB (Sigma, Cat. No. T2885) for 30 minutes for color development, and the detection was to be tested. The binding signal of the sample. The EC50 was fitted (GraphPad Prism) using a logarithmic (agonist) slope curve fit against the response variable.
  • test sample analysis buffer HBSS+10nM HEPES
  • test sample analysis buffer HBSS+10nM HEPES
  • the Pfeiffer cell density was adjusted to 4.0 ⁇ 10 6 cells/ml, 25 ⁇ l/well was added to the 96-well round bottom plate, and 5 ⁇ l/well of the pre-diluted sample to be tested was added to the 96-well round bottom plate, placed at 37.0°C, Incubate for 30 min in a 5.0% carbon dioxide incubator.
  • the protein concentration closely affects the stability of the antibody, and each fusion protein with unique physicochemical properties has the most suitable concentration.
  • the purpose of this example is to screen an optimal protein concentration so that the IL-21 fusion protein disclosed in the present invention has the best stability and is suitable for clinical application.
  • pH 4.5 and pH 5.0 histidine buffer were used to investigate the stability of IL-21-fusion protein at different concentrations.
  • concentration of 4.0mg/ml IL-21 fusion protein (number JS-EC21) was dialyzed and exchanged to the corresponding buffer solution, and the solution was changed three times, and the time of each solution change was greater than 4 hours.
  • Tween 80 was added and filtered. Fill it into 2R vials with a specification of 0.5ml/bottle, and conduct stability staking and testing.
  • the IL-21-fusion proteins of 1 mg/ml, 3 mg/ml and 5 mg/ml were screened respectively, as shown in Table 1.
  • Evaluation indicators include: 1. Size exclusion chromatography (SEC-HPLC) to determine the percentage of protein monomers and aggregated forms; 2. CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method, in reducing and non-reducing Under the conditions, the purity of the protein was detected; 3. ELISA was used to detect the antibody binding activity and cell activity. The effects of different concentrations on the stability of IL-21-fusion protein were investigated, and the results are shown in Table 2-Table 6.
  • acetate buffer, histidine buffer and citrate buffer were screened with pH ranging from 4.7 to 6.5.
  • the IL-21-fusion protein (number JS-EC21) dialysis bag was replaced with the corresponding buffer. After the liquid exchange, the sample was in the corresponding prescription, and the final protein concentration was about 1 mg/ml. Aseptically filled into a 2R vial with the specification of 2.5ml/bottle for stability testing (as shown in Table 6).
  • Evaluation indicators include: 1. DSC (capillary micro-differential scanning fluorescence method) to determine the Tm value (melting temperature) of the protein; 2. Visual appearance; 3. UV spectrophotometry to determine the protein content; 4. Size exclusion chromatography (SEC) -HPLC) to determine the percentage of protein monomers, aggregates and fragments; 5. iCIEF (imaging capillary isoelectric focusing) method to detect the percentage of the main charge, acidic charge or basic charge of the protein; 6. CE-SDS (dodecyl sulfate) Sodium capillary electrophoresis) method, under reducing and non-reducing conditions, to detect the purity of protein; 7. ELISA method to detect antibody binding activity and cell activity.
  • DSC capillary micro-differential scanning fluorescence method
  • Tm value melting temperature
  • UV spectrophotometry to determine the protein content
  • Size exclusion chromatography (SEC) -HPLC) to determine the percentage of protein monomers, aggregates and fragments
  • FS2-7 and FS2-8 groups found white spot precipitation at T0; after 8 weeks under accelerated conditions or long-term conditions, only FS2-1 and FS2-3 groups showed no abnormal appearance. .
  • sucrose, trehalose or mannitol were selected as stabilizers for comparative testing.
  • the IL-21-fusion protein was exchanged with Millipore Pellicon3 0.11m 2 membrane for UF/DF and diluted to make the sample in the corresponding recipe, and the protein concentration was about 1 mg/ml.
  • the specific recipe information is shown in Table 14. The samples were filled into 2R vials and 6R vials with specifications of 2.0ml/bottle and 3.0ml/bottle, respectively, for stability stakeout and testing.
  • Evaluation indicators include: 1. Visual appearance; 2. UV spectrophotometry to detect protein content; 3. Size exclusion chromatography (SEC-HPLC) to determine the percentage of protein monomers, aggregates and fragments; 4. Cation exchange chromatography (CEX - HPLC) to measure the main charge, acidic charge or basic charge of the antibody; 5. CE-SDS (sodium dodecyl sulfate capillary electrophoresis) method, under reducing and non-reducing conditions, to detect the purity of the protein; 6. ELISA method Antibody binding activity was detected.
  • SEC-HPLC Size exclusion chromatography
  • CEX - HPLC Cation exchange chromatography
  • the binding activity of all samples showed no significant change after being placed under high temperature, accelerated or long-term conditions for 4 weeks, as well as 5 freeze-thaw cycles, 3 days of shaking or 10 days of light.
  • the IL-21 fusion protein of the present invention showed good stability under the conditions of acetate-sodium acetate buffer (pH 4.5-5.0), trehalose or sucrose and polysorbate 80 as stabilizers.
  • the difference in the pharmacokinetic properties of the IL-21-anti-albumin single domain antibody fusion protein JS-EC21 of the present invention and recombinant human IL-21 was evaluated by intraperitoneal administration in mice.
  • the IL-21-anti-albumin single domain antibody fusion protein JS-EC21 in this example was prepared according to the prescription FS3-3.
  • mice Twelve Balb/c female mice were selected and randomly divided into 2 groups with 6 animals in each group, and were injected intraperitoneally with 0.15 mg/kg JS-EC21 group and 0.15 mg/kg recombinant human IL-21 respectively.
  • JS-EC21 group 0.15 mg/kg recombinant human IL-21 respectively.
  • the blood of mice was collected in EDTA-K2 anticoagulation tubes, centrifuged at 3500rpm for 10 minutes, and the plasma was collected and stored in a -80°C refrigerator until Measurement.
  • the half-life of IL-21-fusion protein JS-EC21 was 15.5 h, Cmax was 185.2 ⁇ g/L, and AUC (0-t) was 3368.1 ⁇ g/L*h; the half-life of recombinant human IL-21 is 2.7h, the Cmax is 3.1 ⁇ g/L, and the AUC (0-t) is 11.5 ⁇ g/L*h.
  • the IL-21-fusion protein JS-EC21 of the present invention has greater drug exposure and longer half-life, which is significantly better than that of recombinant human IL-21.
  • Table 20 The mouse intraperitoneal single administration PK analysis table of fusion protein JS-EC21 of the present invention and recombinant human IL-21
  • Example 5 Inhibitory effect of IL-21 fusion protein alone on the growth of mouse MC38 subcutaneous transplanted tumor
  • JS-EC21 of the present invention in the mouse MC38 subcutaneous transplantation model was evaluated and compared with recombinant human IL-21 (rhIL-21).
  • rhIL-21 recombinant human IL-21
  • the IL-21-anti-albumin single domain antibody fusion protein JS-EC21 in this example was prepared according to the prescription FS3-3.
  • mice 6-8 week old female C57BL/6WT mice (Zhaoyan (Suzhou) New Drug Research Center Co., Ltd.) were subcutaneously inoculated with 1 ⁇ 10 6 (cells/0.1 mL) MC38 cells on the right back.
  • Solvent control group physiological saline
  • Recombinant human IL-21 0.625mg/kg.
  • the route of administration in all groups was intraperitoneal injection, twice a week, 6 times in a row, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • the mice were euthanized, and the tumor inhibition rate TGI was calculated.
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%, where Ti: the mean tumor volume of the treatment group on the ith day of administration, T0: the treatment group on the 0th day of administration The mean tumor volume of the day; Vi: the mean tumor volume of the solvent control group on the ith day of administration, V0: the mean tumor volume of the solvent control group on the 0th day of administration.
  • the mean tumor volume in the saline group was 2446 mm 3 .
  • the mean tumor volume of the JS-EC21 (0.42 mg/kg) group was 1482 mm 3 , and the TGI was 41.0%;
  • the mean tumor volume of the JS-EC21 (1.25 mg/kg) group was 599 mm 3 , and the TGI was 78.4%;
  • the mean tumor volume of the JS-EC21 (3.75 mg/kg) group was 390 mm 3 and the TGI was 87.3%.
  • the mean tumor volume of the recombinant human IL-21 (0.625 mg/kg) group was 1698 mm 3 and the TGI was 31.8%.
  • JS-EC21 Compared with the normal saline group, administration of 1.25mg/kg and 3.75mg/kg JS-EC21 significantly inhibited the tumor growth in mice, and showed a good dose effect. 1.25mg/kg of JS-EC21 was significantly better than the equimolar mass of recombinant human IL-21 (0.625mg/kg).
  • a total of 18 cynomolgus monkeys (6 in each group, half male and female, monkey age 3.8-4.3 years old) were randomly assigned to fusion protein JS-EC21 low-dose, middle-dose and high-dose groups.
  • animals were intravenously injected with 0.05 mg/kg in the low-dose group, 0.15 mg/kg in the middle-dose group, and 0.5 mg/kg in the high-dose group.
  • t 1/2 terminal elimination half-life
  • T max time to maximum concentration
  • C max peak drug concentration
  • AUC last area under the drug concentration-time curve from administration time to the last sample collection
  • V d table Observed volume of distribution
  • CL clearance rate
  • MRT mean residence time.
  • Example 7 Inhibitory effect of IL-21 fusion protein combined with PD-1 monoclonal antibody on the growth of mouse MC38 subcutaneous transplanted tumor
  • JS-EC21 combined with PD-1 monoclonal antibody in the mouse MC38 subcutaneous transplantation model.
  • Solvent control group anti-KLH hIgG4, 0.3 mg/kg;
  • PD-1 mAb 0.3mg/kg
  • Anti-KLH hIgG4+JS-EC21 0.3 mg/kg+1 mg/kg.
  • the IL-21-anti-albumin single domain antibody fusion protein JS-EC21 is formulated according to the prescription FS3-3;
  • the PD-1 monoclonal antibody is Toripalimab, which has WHO Drug Information (No. 32) Vol. 2, pp. 372-373 (2016)), a humanized IgG4 mAb comprising the light and heavy chain amino acid sequences set forth in the sequences SEQ ID NOs: 15 and 16.
  • the route of administration in all groups was intraperitoneal injection, twice a week, 6 times in a row, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • the mice were euthanized, and the tumor inhibition rate TGI was calculated.
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%, where Ti: the mean tumor volume of the treatment group on the ith day of administration, T0: the treatment group on the 0th day of administration The mean tumor volume of ; Vi: the mean tumor volume of the solvent control group on the ith day of administration, V0: the mean tumor volume of the solvent control group on the 0th day of administration.
  • the mean tumor volume in the anti-KLH hIgG4 group was 2008 mm 3 on day 21 after administration.
  • the mean tumor volume in the PD-1 mAb group was 1229 mm 3 and the TGI was 41.3%.
  • the mean tumor volume of the JS-EC21 group was 1216 mm 3 and the TGI was 42.0%.
  • the mean tumor volume of the PD-1 mAb combined with JS-EC21 group was 673 mm 3 and the TGI was 70.8%.
  • the experimental results show that the combination of the IL-21-fusion protein JS-EC21 of the present invention and PD-1 has a significant tumor inhibitory effect, and the efficacy is better than that of PD-1 antibody (p ⁇ 0.001) or JS-EC21 monotherapy ( p ⁇ 0.05), with a certain synergistic effect.
  • Example 8 Inhibitory effect and mechanism of IL-21 fusion protein combined with Toripalimab on the growth of hPD-1 humanized mouse MC38 subcutaneous xenograft tumor
  • mice 6-8-week-old female hPD-1 humanized mice (Biocyto Jiangsu Gene Biotechnology Co., Ltd.) were subcutaneously inoculated with 1 ⁇ 10 6 (cells/0.1 mL) MC38 cells on the right back.
  • the tumor size was about 91 mm 3
  • mice were randomly divided into 5 groups of 7 mice (see Table 22), and dosing was started (see Table 23).
  • Toripalimab was manufactured by Shanghai Junshi Biotechnology.
  • the route of administration in all groups was intraperitoneal injection, twice a week, 6 times in a row, and the experiment ended 3 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the body weight and tumor volume of the mice were recorded.
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%, where Ti: the mean tumor volume of the treatment group on the ith day of administration, T0: the treatment group on the 0th day of administration The mean tumor volume of ; Vi: the mean tumor volume of the solvent control group on the ith day of administration, V0: the mean tumor volume of the solvent control group on the 0th day of administration.
  • group 1 in Table 22 is the solvent control group, and groups 2, 3 and 4 are treatment groups.
  • mice tumor On the 21st day after administration, the mouse tumor was immediately transferred to a 50 mL centrifuge tube placed on ice, and pre-cooled PBS solution was added until the tumor disappeared.
  • Preparation of single cells Take an equal amount (about 0.5 g) of tumor tissue, cut the tumor into small pieces of 2-4 mm, and prepare the tumor tissue into single cell suspension by Tumor Dissociation Kit (mouse: 130-096-730). TIL cells were then enriched using CD45(TIL) MicroBeads (murine: 130-110-618).
  • the mean tumor volume in the hIgG4 isotype control group was 2008 mm 3 on day 21 after administration.
  • the mean tumor volume in the toripalimab group was 1229 mm3 and the TGI was 41.3%.
  • the mean tumor volume of the JS-EC21 group was 1216 mm 3 and the TGI was 42.0%.
  • the mean tumor volume of the toripalimab combined with JS-EC21 group was 673 mm 3 , which was significantly lower than that of the toripalimab group (p ⁇ 0.001) and the JS-EC21 group (p ⁇ 0.05), and the TGI was 70.8%.
  • the combined administration group was significantly higher than toripril Monoclonal antibody administration group (p ⁇ 0.01) or JS-EC21 administration group (p ⁇ 0.01); for the ratio of CD8 + T cells in CD3 + T cells, as shown in Figure 6(B), the combined administration group was significantly higher than toripalimab administration group (p ⁇ 0.05) or JS-EC21 administration group (p ⁇ 0.01); for the proportion of CD8 + T cells in a proliferating state (Ki 67 + ), as shown in Figure 6 ( As shown in C), the combined administration group was significantly higher than the toripalimab administration group (p ⁇ 0.05) or the JS-EC21 administration group (p ⁇ 0.05); The ratio, as shown in Figure 6(D), was significantly higher in the combined administration group than in the toripalimab administration group (p ⁇ 0.05) or the JS-EC21 administration group (p ⁇ 0.05);

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Abstract

提供IL-21-抗白蛋白单域抗体融合蛋白药物组合物及其用途。该药物组合物含有IL-21-抗白蛋白单域抗体融合蛋白、缓冲液，还可以含有至少一种稳定剂，任选的还可以含有表面活性剂。所述IL-21-抗白蛋白单域抗体融合蛋白具有高稳定性，其单药及其与PD-1单抗联用均表现出优越的抗肿瘤活性。

Description

IL-21-抗白蛋白单域抗体融合蛋白药物组合物及其用途 技术领域
本发明涉及治疗性药物组合物领域。尤其是，本发明涉及医药制剂领域，该药物组合物含有IL-21-抗白蛋白单域抗体融合蛋白，以及IL-21融合蛋白在治疗肿瘤和增强免疫检查点抑制剂的抗肿瘤活性中的用途。
背景技术
细胞因子疗法是刺激免疫系统以诱导针对疾病(例如癌症或感染)的免疫应答的有效策略。然而，施用给患者的细胞因子通常半衰期较短。例如，白细胞介素-21可刺激各种免疫细胞(诸如T细胞、B细胞和NK细胞)并增强其抗肿瘤活性。据报道，重组IL-21经静脉给药后半衰期仅为约一到三小时(参见Schmidt H，Clin.Cancer Res.2010年11月1日；第16卷第21期：第5312-5319页)。
免疫逃逸是癌症的特征之一。Ahmadzadeh,M.等，Blood,114:1537-44中公开了肿瘤特异性T淋巴细胞常存在于肿瘤微环境、引流淋巴结和外周血中，但由于肿瘤微环境中存在的免疫抑制机制网络，其通常无法控制肿瘤的进展。CD8 +肿瘤浸润T淋巴细胞(TIL)通常表达活化诱导的抑制受体，包括CTLA-4和PD-1，而肿瘤细胞经常表达免疫抑制配体，包括PD-1配体1(PD-L1，也叫B7-H1或CD274)，该配体抑制T细胞激活和效应功能。在抑制机制中，PD-1及其配体已成为肿瘤细胞利用其抑制肿瘤微环境中激活的T细胞的重要途径。
近十年来，PD-1/PD-L1通路阻断已被证明是在各种癌症适应症中诱导持久抗肿瘤应答的有效途径。阻断PD/PD-L1通路的单克隆抗体(mAbs)可以增强肿瘤特异性T细胞的活化和效应功能，减轻肿瘤负担，提高生存率。2014年至2017年之间，FDA已经批准了2款抗-PD1单克隆抗体(nivolumab)和3款抗-PD-L1单克隆抗体(atezolizumab、avelumab和durvalumab)用于治疗人类肿瘤。
尽管在接受免疫检查点抑制剂治疗的10-30％的癌症患者中观察到惊人的效果，但很大一部分患者最初对抗PD-1单抗治疗耐药，而且超过半数的最初有响应的患者最终会复发肿瘤。因此，通过免疫疗法的协同组合以扩大受益者群体并提高疗效 受到了业界的广泛关注。IL-21是γ链细胞因子家族的成员，可以刺激CD8 +T细胞扩增和增加细胞毒性，增强T细胞依赖型B细胞增殖和抗体产生，促进NK细胞分化和激活，并减少Treg细胞。重组人IL-21已在非临床和临床研究中证明作为单一疗法或与靶向疗法或单克隆抗体联合使用可产生抗肿瘤活性。例如，已使用重组IL-21(rIL-21)对转移性黑色素瘤、肾细胞癌(RCC)、结肠直肠癌和非霍奇金淋巴瘤进行了I期和II期临床试验。但最近的研究表明，与其它细胞因子一样，IL-21的抗肿瘤效果在临床试验中受到半衰期短的限制。
因此，需要开发能有效治疗疾病的新细胞因子治疗剂，以及探讨其与免疫检查点抑制剂联用的效果。
发明内容
本发明公开的药物组合物是一种含有IL-21-抗白蛋白单域抗体融合蛋白(IL-21融合蛋白)的高稳定性药物组合物。特别地，本发明发现含有海藻糖的组合具有出人意料的特征，即具有高稳定性。
本发明的IL-21-抗白蛋白单域抗体融合蛋白药物组合物的半衰期和血液中的暴露水平显著优于重组白介素21，在小鼠皮下移植瘤模型中单药及其与PD-1单抗联用均表现出优越的抗肿瘤活性。
本发明发现，与rhIL-21(重组人IL-21)相比，靶向人血清白蛋白(HSA)的纳米抗体融合到人IL-21的C末端而形成的本文称为JS-EC21的融合蛋白在食蟹猴中半衰期显著延长，暴露量显著增加(t 1/2和AUC分别比rhIL-21高近10倍和50倍)。此外，JS-EC21显著增强了PD-1单抗或TIGIT单抗的抗肿瘤作用，并提供了长期保护性抗肿瘤免疫。JS-EC21与TIGIT单抗联合使用可诱导与肿瘤免疫应答相关的KEGG信号通路的显著富集，并增加与CD8 +T细胞和NK细胞毒性相关基因的表达水平。
一方面，本发明提供了一种药物组合物，包含：(1)缓冲液；和(2)IL-21-抗白蛋白单域抗体融合蛋白。
在一些方案中，上述IL-21-抗白蛋白单域抗体融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，上述IL-21包括如SEQ ID NO:1所示的氨基酸序列，上述单域抗体包含氨基酸序列分别如SEQ ID NO:2、SEQ ID  NO:3和SEQ ID NO:4所示的HCDR1、HCDR2和HCDR3。
在一些方案中，上述单域抗体(sdAb)包含如SEQ ID NO:5所示的氨基酸序列。
在一些方案中，上述单域抗体融合至细胞因子的C末端，且上述细胞因子和单域抗体直接连接。
在一些方案中，上述药物组合物中IL-21-抗白蛋白单域抗体融合蛋白的浓度约为0.1-100mg/mL，优选约为0.2-20mg/mL，优选约为0.2-10mg/mL，优选约为0.5-5mg/mL，优选约为1-5mg/mL，更优选约为0.5-1mg/mL；更优选地，上述IL-21-抗白蛋白单域抗体融合蛋白浓度约为0.5mg/mL，0.8mg/mL，1mg/mL，1.2mg/mL，1.5mg/mL，1.8mg/mL，2mg/mL，2.5mg/mL，3mg/mL，3.5mg/mL，4mg/mL，4.5mg/mL，5mg/mL，6mg/mL，7mg/mL，8mg/mL或10mg/mL，优选约为1mg/mL。
在一些方案中，上述缓冲液选自醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液中的一种或多种；优选地，上述缓冲液选自醋酸-醋酸钠缓冲液、柠檬酸-柠檬酸钠缓冲液、组氨酸-醋酸缓冲液和组氨酸-盐酸盐缓冲液中一种或多种；优选地，上述缓冲液为醋酸-醋酸钠缓冲液和组氨酸-醋酸缓冲液；更优选地，上述缓冲液为醋酸-醋酸钠缓冲液。
在一些方案中，上述缓冲液为组氨酸缓冲液，优选地，上述组氨酸缓冲液选自组氨酸-盐酸盐缓冲液或组氨酸-醋酸盐缓冲液，优选组氨酸-盐酸盐缓冲液。在一些方案中，上述组氨酸-盐酸盐缓冲液由组氨酸和组氨酸盐酸盐制成，优选L-组氨酸和L-组氨酸单盐酸盐。在一些方案中，组氨酸缓冲液由1-30mM的L-组氨酸和1-30mM的L-组氨酸单盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:1到1:4的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:1组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为1:3的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸制剂为：由约1.7mM的L-组氨酸和约18.3mM的L-组氨酸单盐酸盐制成的pH约为5.0的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由约4.5mM的L-组氨酸和约15.5mM的L-组氨酸单盐酸盐制成的pH约为5.5的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由约7.5mM的L-组氨酸和约22.5mM的L-组氨酸单盐酸盐制成的pH约为5.5的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由约15mM的组氨酸和约15mM的组氨酸盐 酸盐制成的pH约为6.0的组氨酸缓冲液。
在一些方案中，上述组氨酸缓冲液为组氨酸-醋酸缓冲液，优选地，两者的摩尔比约为1:1到1.5:1，优选地，此类缓冲液的pH为5.5±0.3，优选约为5.5，优选地，这类缓冲液含有15-20mM的组氨酸和12-15mM的醋酸。
在一些方案中，上述缓冲液为醋酸缓冲液，优选地，上述醋酸缓冲液为醋酸-醋酸钠缓冲液或醋酸-醋酸钾缓冲液，优选醋酸-醋酸钠缓冲液。在一些方案中，醋酸缓冲液由1-30mM的醋酸和1-30mM的醋酸钠制成。在一些方案中，醋酸缓冲液由摩尔比为约2:3的醋酸和醋酸钠制成。在一些方案中，醋酸缓冲液由摩尔比为约1:2.1的醋酸和醋酸钠制成。在一些方案中，醋酸缓冲液由摩尔比为约1:5.7的醋酸和醋酸钠制成。在一些方案中，醋酸缓冲液为：由约8mM的醋酸和约12mM的醋酸钠制成的pH约为4.8的醋酸缓冲液。在一些方案中，醋酸缓冲液为：由约6.5mM的醋酸和约13.5mM的醋酸钠制成的pH约为5.0的醋酸缓冲液。在一些方案中，醋酸缓冲液为：由约3mM的醋酸和约17mM的醋酸钠制成的pH约为5.5的醋酸缓冲液。
在一些方案中，上述缓冲液为柠檬酸缓冲液，优选地，上述柠檬酸缓冲液为柠檬酸-柠檬酸钠缓冲液。
在一些方案中，上述缓冲液的浓度约为1-200mM，优选约为5-200mM，优选约为10-50mM，优选约为10-30mM；优选约为10-20mM，上述缓冲液浓度非限制性实施例约为5mM，10mM，15mM，20mM，25mM，30mM、40mM、45mM、50mM、60mM、70mM、80mM、90mM、100mM、105mM、110mM、115mM、120mM、130mM、140mM、150mM、160mM、170mM或180mM或这些范围内任意两个数值作为端点形成的范围，优选为10mM、15mM、20mM或30mM。
在一些方案中，上述缓冲液的pH约为4.0-6.5，优选约为4.0-6.0，优选约为4.5-6.0，优选约为4.5-5.5，优选约为4.5-5.1，优选约为4.5-5.0，优选约为4.7-5.0，上述缓冲液的pH非限制性实施例约为4.0，4.1，4.2，4.3，4.4，4.5，4.6，4.7，4.8，4.9，5.0，5.1，5.2，5.3，5.4，5.5，6.0，优选约为4.7或4.8。
在一些方案中，上述的药物组合物还包括稳定剂，上述稳定剂选自氯化钠、甘露醇、山梨醇、蔗糖、和海藻糖中的一种或多种，优选地，上述稳定剂为海藻糖。
在一些方案中，上述稳定剂的浓度为约10mM-400mM，优选20mM-300mM， 更优选30mM-200mM。
在一些方案中，上述稳定剂为浓度约100-300mM的甘露醇；或上述稳定剂为浓度约100-300mM的蔗糖；或上述稳定剂为浓度约100-300mM的海藻糖；或上述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合；或上述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖的组合；优选地，上述稳定剂为约100-300mM的海藻糖；更优选地，上述稳定剂为约200-280mM的海藻糖。
在一些方案中，上述稳定剂为甘露醇。在一些方案中，上述稳定剂为浓度约100-300mM的甘露醇，上述甘露醇的浓度优选约为150-300mM，优选约为200-280mM，上述甘露醇浓度的非限制性实施例为约200mM，210mM，220mM，230mM，240mM，250mM，260mM，270mM，280mM，优选为240mM。
在一些方案中，上述稳定剂为蔗糖。在一些方案中，上述稳定剂为浓度约100-300mM的蔗糖，上述蔗糖的浓度优选约为150-300mM，优选约为200-280mM，上述蔗糖浓度的非限制性实施例为约200mM，210mM，220mM，230mM，235mM，240mM，250mM，260mM，270mM，280mM，优选为235mM。
在一些方案中，上述稳定剂为海藻糖。在一些方案中，上述稳定剂为浓度约100-300mM的海藻糖，上述海藻糖的浓度优选约为150-300mM，优选约为200-280mM，上述海藻糖浓度的非限制性实施例为约180mM，200mM，210mM，220mM，230mM，235mM，240mM，250mM，260mM，270mM，280mM，优选为235mM。
在一些方案中，上述稳定剂为氯化钠与甘露醇的组合。在一些方案中，上述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合，优选约40-150mM的氯化钠与约40-180mM的甘露醇的组合，优选约40-100mM的氯化钠与约40-150mM的甘露醇的组合，上述稳定剂的非限制性实施例约为50mM的氯化钠与约120mM的甘露醇的组合，或约50mM的氯化钠与约140mM的甘露醇的组合。
在一些方案中，上述稳定剂为氯化钠与蔗糖的组合。在一些方案中，上述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖的组合，优选约40-150mM的氯化钠与约40-180mM的蔗糖的组合，优选约40-100mM的氯化钠与约40-150mM的蔗糖的组合，上述稳定剂的非限制性实施例约为50mM的氯化钠与约140mM的蔗糖的组合。
在一些方案中，上述药物组合物还包括表面活性剂，表面活性剂选自聚山梨醇 酯80、聚山梨醇酯20或泊洛沙姆188。
在一些方案中，上述表面活性剂选自聚山梨醇酯80。
在一些方案中，上述表面活性剂选自聚山梨醇酯20。
在一些方案中，以w/v计算，上述表面活性剂浓度约为0.001％-0.1％，优选约为0.01％-0.1％，优选约为0.02％-0.08％，优选约为0.01％-0.05％；作为非限制性实施例，上述表面活性剂的浓度约为0.02％，0.04％或0.08％，优选0.02％。
在一些方案中，上述IL-21融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，其中上述IL-21包括如SEQ ID NO:1所示的氨基酸序列，且sdAb包含如SEQ ID NO:5所示的氨基酸序列。
在一些方案中，上述IL-21-抗白蛋白单域抗体融合蛋白包含如SEQ ID NO:6所示的氨基酸序列或由其组成。
在一些方案中，上述药物组合物，其包含如下(1)-(6)任一项所示的组分，其中，IL-21-抗白蛋白单域抗体融合蛋白如本发明任一实施方案所述：
(1)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM醋酸缓冲液，pH约为4.5-5.5(优选4.5-5.0)；(c)约100-300mM的海藻糖；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
(2)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM醋酸缓冲液，pH约为4.5-5.5(优选4.5-5.0)；(c)约100-300mM的蔗糖；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
(3)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM醋酸缓冲液，pH约为4.5-5.5(优选4.5-5.0)；(c)约100-300mM的甘露醇；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
(4)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM组氨酸缓冲液，pH约为4.5-5.5(优选4.5-5.0)；(c)约30-200mM的氯化钠和约30-200mM的甘露醇；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
(5)(a)约1mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约20mM醋酸缓冲液，pH约为4.8；(c)约235mM的海藻糖；(d)以及约0.2％的聚山梨醇酯80；或
(6)(a)约1mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约20mM醋 酸缓冲液，pH约为4.8；(c)约235mM的蔗糖；(d)以及约0.2％的聚山梨醇酯80。
在一些方案中，上述药物组合物为液体制剂或冻干制剂。
在一些方案中，上述药物组合物为液体制剂。
在一些方案中，上述液体制剂或冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。
在一些方案中，上述水溶液或冻干制剂于40℃稳定至少7天，至少14天或至少28天。
在又一个方面，本发明还提供了上述药物组合物在制备治疗或预防癌症的药物中的用途；优选地，上述癌症包括间皮瘤、肺癌、乳腺癌、卵巢癌、胰腺癌、淋巴瘤、白血病、头颈癌、肝癌、食管癌、胃癌和结肠直肠癌。
在又一个方面，本发明还提供了上述药物组合物在制备治疗或预防炎性疾病的药物中的用途。
在又一个方面，本发明提供了IL-21融合蛋白(又称为IL-21-抗白蛋白单域抗体融合蛋白)、或上述药物组合物、或IL-21融合蛋白和免疫检查点调节剂的组合或上述药物组合物和免疫检查点调节剂的组合在制备用于预防或治疗肿瘤的药物中的用途，或IL-21-抗白蛋白单域抗体融合蛋白或上述药物组合物在制备增强免疫检查点调节剂的抗肿瘤活性的药物中的用途。
在又一个方面，本发明提供了预防或治疗肿瘤的方法，其包含向有需要的个体施用有效量的IL-21融合蛋白，或上述药物组合物，或IL-21融合蛋白和免疫检查点调节剂的组合或上述药物组合物和免疫检查点调节剂的组合。
在又一个方面，本发明提供IL-21融合蛋白，或上述药物组合物，或IL-21融合蛋白与免疫检查点调节剂的组合或上述药物组合物和免疫检查点调节剂的组合，其用于治疗或预防肿瘤。
在一些方案中，肿瘤为与IL-21相关的肿瘤，或为免疫检查点相关的肿瘤。
在一些方案中，肿瘤选自间皮瘤、肺癌、乳腺癌、卵巢癌、黑色素瘤、肾癌、胰腺癌、淋巴瘤、白血病、头颈癌、肝癌、非霍奇金淋巴瘤、食管癌、胃癌和结肠直肠癌。
在一些方案中，肿瘤为结肠直肠癌。
在一个或多个实施方案中，本发明公开的IL-21-抗白蛋白单域抗体融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，IL-21包括如SEQ ID NO:1所示的氨基酸序列，单域抗体(sdAb)包含氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的HCDR1、HCDR2和HCDR3。
在一个或多个实施方案中，本发明公开的单域抗体(sdAb)包含如SEQ ID NO:5所示的氨基酸序列。
在一个或多个实施方案中，本发明公开的单域抗体融合至细胞因子的C末端，且细胞因子和单域抗体直接相连。
在一个或多个实施方案中，本发明公开的IL-21融合蛋白包含如SEQ ID NO:6所示的氨基酸序列。
在一个或多个实施方案中，本发明公开的IL-21融合蛋白与免疫检查点调节剂组合使用
在一个或多个实施方案中，本发明公开的免疫检查点调节剂选自PD-1、PD-L1、PD-L2、CTLA-4、4-1BB、CD47、TIGIT、GITR、CD112R、BTLA、TIM3、LAG3、CD27和B7H4免疫检查点抑制剂。
在一个或多个实施方案中，本发明公开的免疫检查点调节剂是抗PD-1抗体。
在一个或多个实施方案中，本发明公开的抗PD-1抗体包含：
(1)氨基酸序列如SEQ ID NO：7、8和9所示的轻链互补决定区，和氨基酸序列如SEQ ID NO：10、11和12所示的重链互补决定区；或
(2)氨基酸序列如SEQ ID NO:13所示的轻链可变区，和氨基酸序列如SEQ ID NO:14所示的重链可变区；或
(3)氨基酸序列如SEQ ID NO:15所示的轻链，和氨基酸序列如SEQ ID NO:16所示的重链。
在一个或多个实施方案中，本发明公开的抗PD-1抗体选自nivolumab、pembrolizumab、toripalimab、Sintilimab、Camrelizumab、Tislelizumab、Cemiplimab中的一种或一种以上；优选为toripalimab。
在一些方案中，相比IL-21融合蛋白或抗PD-1抗体单独使用，本发明公开的IL-21融合蛋白与抗PD-1抗体联合使用具有选自以下的一种或多种性质：
(1)免疫细胞中CD3 +T细胞的比例显著提高；
(2)T细胞中CD8 +T细胞的比例显著提高；
(3)增殖状态(Ki 67 +)的CD8 +T细胞的比例显著提高；和
(4)免疫细胞中NK细胞的比例显著提高。
在一个或多个实施方案中，本发明公开的免疫检查点调节剂是抗TIGIT抗体。
在一个或多个实施方案中，本发明公开的抗TIGIT抗体选自：Tiragolumab、Etigilimab、Vibostolimab、Domvanalimab、EOS-884448和BMS-986207。
在一些方案中，相比IL-21融合蛋白或抗TIGIT抗体单独使用，本发明公开的IL-21融合蛋白与抗TIGIT抗体联合使用具有选自以下的一种或多种性质：
(1)显著增加干扰素γ的表达水平；
(2)显著增加颗粒酶A的表达水平；
(3)显著增加颗粒酶B的表达水平；
(4)显著增加穿孔素1的表达水平；
(5)富集更多具有差异基因表达的KEGG信号通路；和
(6)诱导的大部分显著富集的KEGG信号通路由IL-21融合蛋白介导。
在又一个方面，本发明提供了一种药物组合，包含：
(1)免疫检查点调节剂；和
(2)IL-21-抗白蛋白单域抗体融合蛋白或如上述药物组合物；
在又一个方面，本发明提供了一种试剂盒，其包含：
(1)一个或多个单次药物剂量单元的IL-21融合蛋白和一个或多个单次药物剂量单元的免疫检查点调节剂，其中IL-21融合蛋白如本文所公开；优选地，其中免疫检查点调节剂如本文所公开；或
(2)一个或多个单次药物剂量单元的如本文所公开的药物组合。
在一个或多个实施方案中，本发明所公开的试剂盒，其还包含用于指示其中药物组合物的使用方法的说明书。
附图说明
图1：本发明融合蛋白JS-EC21对MC38肿瘤的抑制作用。
图2：低剂量、中剂量和高剂量的JS-EC21在食蟹猴中的药代动力学曲线。
图3：本发明融合蛋白JS-EC21联合PD-1单抗对MC38肿瘤的抑制作 用。
图4：本发明融合蛋白JS-EC21与特瑞普利单抗单独给药或联用后小鼠肿瘤体积变化。
图5：本发明融合蛋白JS-EC21与特瑞普利单抗单独给药或联用后小鼠肿瘤重量变化。
图6：本发明融合蛋白JS-EC21与特瑞普利单抗单独给药或联用后小鼠肿瘤内免疫细胞变化。
图4-6中，两组间通过two-tailed unpaired Student’s t test进行差异分析，*表示P<0.05；**表示P<0.01，***表示P<0.001，ns表示无显著性差异。
具体实施方式
定义和说明
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。应理解本发明不限于具体的方法、试剂、化合物、组合物或生物系统，当然可以对以上进行变化。还应理解本申请所用术语仅为了描述具体的实施方式，并不旨在进行限制。
除非该内容被另外明确说明，否则本说明书以及所附权利要求中所用的单数形式"一个"、"一种"和"该"包括复数指代。因此，例如，提及"一种多肽"包括了两种或更多种多肽等的组合。
术语"药物组合物"或“制剂”表示含有一种或多种本文所述抗体与其他组分的混合物，上述其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“液体制剂”是指处于液体状态下的制剂，且不意图指称重悬浮的冻干制剂。本发明的液体制剂在储存时稳定，并且其稳定性不依赖于冻干(或其他状态改变方法，例如喷雾干燥)。
术语“水性液体制剂”是指使用水作为溶剂的液体制剂。在一些方案中，水性液体制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。
术语“赋形剂”是指可以向制剂添加以提供所需特性(例如稠度、提高的稳定性) 和/或调节渗透压的试剂。常用赋形剂的实例包括但不限于糖类、多元醇、氨基酸、表面活性剂和聚合物。
本申请所用的"约"在指代可测量数值(如量、持续时间等)时意在涵盖相对于具体数值±20％或±10％的变化，包括±5％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语"缓冲液pH约为4.0-6.5"是指这样的试剂，通过其酸/碱共轭组分的作用使得包含该试剂的溶液能抵抗pH变化。本发明的制剂中使用的缓冲液可具有约4.0至约6.5范围内的pH、或约4.5至约6.5范围内的pH、或约4.5至约5.5范围内的pH。
在本文中，将pH控制在该范围内的“缓冲液”实例包括乙酸盐(例如乙酸钠)、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸、组氨酸盐酸盐、甲硫氨酸、柠檬酸盐、磷酸盐、柠檬酸盐/磷酸盐、咪唑、醋酸、醋酸盐、枸橼酸盐、其组合和其他有机酸缓冲剂。
"组氨酸缓冲液"为包含组氨酸离子的缓冲液。组氨酸缓冲液的实例包括组氨酸和组氨酸的盐，如组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐和组氨酸硫酸盐等，如含有组氨酸与组氨酸盐酸盐的组氨酸缓冲液；本发明的组氨酸缓冲液也包括含有组氨酸和醋酸盐(如钠盐或钾盐)或冰醋酸的组氨酸缓冲液。
“柠檬酸盐缓冲液”是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的实例包括柠檬酸-柠檬酸钠、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选的柠檬酸盐缓冲液为柠檬酸-柠檬酸钠缓冲液。
“醋酸盐缓冲液”是包括醋酸根离子的缓冲液。醋酸盐缓冲液的实例包括醋酸-醋酸钠、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲液为醋酸-醋酸钠缓冲液。
术语“稳定剂”表示药学上可接受的赋形剂，其在制造，储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。稳定剂包括但不限于如以下定义的糖，氨基酸，盐，多元醇和它们的代谢产物，例如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖、海藻糖、精氨酸或其盐(如盐酸精氨酸)、甘氨酸、丙氨酸(α-丙氨酸、β-丙氨酸)、甜菜碱、亮氨酸、赖氨酸、谷氨酸、天冬氨酸、脯氨酸、4-羟基脯氨酸、肌氨酸、γ-氨基丁酸(GABA)、奥品类(opines)、丙氨奥品、章鱼碱、 甘氨奥品(strombine))和三甲胺的N-氧化物(TMAO)、人血清白蛋白(hsa)、牛血清白蛋白(bsa)、α-酪蛋白、球蛋白、α-乳白蛋白、LDH、溶菌酶、肌红蛋白、卵清蛋白和RNAaseA。部分稳定剂，如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖等也可起到控制渗透压的作用。在本发明中具体地使用的稳定剂选自多元醇、氨基酸、盐、糖中的一种或一种以上。优选的盐为氯化钠，优选的糖为蔗糖和海藻糖，优选的多元醇为山梨醇和甘露醇。优选的氨基酸为精氨酸或其盐(如盐酸精氨酸)、甘氨酸、脯氨酸。优选的稳定剂为氯化钠、甘露醇、山梨醇、蔗糖、海藻糖、盐酸精氨酸、甘氨酸、脯氨酸、氯化钠-山梨醇、氯化钠-甘露醇、氯化钠-蔗糖、氯化钠-海藻糖、盐酸精氨酸-甘露醇、盐酸精氨酸-蔗糖，更优选为盐酸精氨酸、氯化钠-蔗糖、盐酸精氨酸-甘露醇、盐酸精氨酸-蔗糖，更优选为蔗糖或海藻糖。
术语“表面活性剂”一般包括保护蛋白质例如抗体免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂。示例性的表面活性剂包括但不限于非离子型表面活性剂例如聚氧乙烯脱水山梨醇脂肪酸酯(如聚山梨醇酯20和聚山梨醇酯80)、聚乙烯-聚丙烯共聚物、聚乙烯-聚丙烯二醇、聚氧乙烯-硬脂酸酯、聚氧乙烯烷基醚、例如聚氧乙烯单月桂基醚、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆，Pluronic)、十二烷基硫酸钠(SDS)。本文中，如无特别说明，术语“聚山梨酯20的浓度”和“聚山梨酯80的浓度”均是指质量体积浓度(w/v)，如“约0.02％聚山梨酯80”中“0.02％”即指“100mL液体中含有0.02g的聚山梨酯80”。
术语"等渗"是指该制剂具有与人血液基本相同的渗透压。等渗制剂一般具有约250至350mOsm的渗透压。可使用蒸汽压或冰点下降式的渗透压计测量等渗性。
术语“稳定的”制剂是其中的抗体在制造过程期间和/或储存时基本上保持其物理稳定性和/或化学稳定性和/或生物活性的制剂。即使所含的抗体在经过一定时间储存之后未能保持其100％的化学结构或生物功能，医药制剂也可以是稳定的。在某些情况下，在经过一定时间储存之后，能维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，也可被认为是“稳定的”。用于测量蛋白质稳定性的各种分析技术在本技术领域中是可得的，并综述在《肽和蛋白质药物递送》(Peptide and Protein Drug Delivery)247-301，Vincent Lee主编，Marcel Dekker，Inc.，New York，N.Y.，Pubs.(1991))，和Jones，A.(1993)Adv.Drug Delivery Rev.10：29- 90中(二者引入作为参考)。
制剂在一定温度下经过一定时间的储存之后，通过测定其中剩余的天然抗体的百分比(及其它方法)，可以测量其稳定性。除其它方法外，天然抗体的百分比可以通过尺寸排阻色谱法(例如尺寸排阻高效液相色谱法[SEC-HPLC])来测量，“天然的”指未聚集的和未降解的。在一些方案中，蛋白质的稳定性按照具有低百分比的降解(例如片段化)和/或聚集蛋白质的溶液中单体蛋白质的百分数来确定。在一些方案中，制剂可以在室温、约25-30℃或40℃下稳定储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长，最多不超过约6％、5％、4％、3％、2％、1％、0.5％，或0.1％聚集形式的抗体。
通过测定在离子交换期间在此抗体主馏分(“主要荷电形式”)较为酸性的馏分中迁移的抗体(“酸性形式”)的百分比(及其它方法)，可以测量稳定性，其中稳定性与酸性形式抗体的百分比成反比。除其它方法外，“酸化”抗体的百分比可以通过离子交换色谱法(例如阳离子交换高效液相色谱法[CEX-HPLC])来测量。在一些实施方式中，可接受程度的稳定性意为当制剂在一定温度下经过一定时间的储存之后，其中可检测出的酸性形式的抗体最多不超过约49％、45％、40％、35％、30％、25％、20％、15％、10％、5％、4％、3％、2％、1％、0.5％或0.1％。在测量稳定性之前储存的一定时间可以是至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长。当评估稳定性时，容许储存医药制剂的一定温度可以是约-80℃至约45℃范围内的任何温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约2-8℃、约5℃、约25℃，或约40℃。
如果抗体在颜色和/或澄清度目测检查时或通过UV光散射或通过孔径排阻层析测量时基本上不显示出例如聚集、沉淀和/或变性的迹象，则上述抗体在该药物组合物中“保持其物理稳定性”。聚集是单个分子或复合物共价或非共价缔合以形成聚集体的过程。聚集可以进行到形成可见沉淀物的程度。
制剂的稳定性例如物理稳定性可以通过本技术领域中公知的方法来评估，包括 测量样品的表观消光度(吸光度或光密度)。这样的消光测量与制剂的浊度相关。制剂的浊度部分地是溶解在溶液中的蛋白质的固有性质，并且通常通过比浊法来测量，并用比浊法浊度单位(NTU)来量度。
随着例如溶液中一种或多种组分的浓度(例如蛋白质和/或盐浓度)而变化的浊度水平也被称为制剂的“乳浊”或“乳浊外观”。浊度水平可以参照使用已知浊度的悬液产生的标准曲线来计算。用于测定药物组合物的浊度水平的参比标准品可以基于《欧洲药典》标准(《欧洲药典》(European Pharmacopoeia)，第四版，“欧洲药品质量委员会指令”(Directorate for the Quality of Medicine of the Council of Europe)(EDQM)，Strasbourg，France)。根据《欧洲药典》标准，澄清溶液被定义为浊度低于或等于按照《欧洲药典》标准具有约3的参比悬液的浊度的溶液。比浊法的浊度测量可以检测在不存在缔合或非理想效应的情况下的瑞利散射，其通常随浓度线性变化。用于评估物理稳定性的其他方法在本技术领域中是公知的。
如果抗体在给定时间点的化学稳定性使得抗体被认为仍保持如下文中所定义的其生物活性，则上述抗体在药物组合物中“保持其化学稳定性”。可以通过例如检测或定量抗体的化学改变的形式来评估化学稳定性。化学改变可以包括尺寸改变(例如剪短)，其可以使用例如孔径排阻层析、SDS-PAGE和/或基质辅助的激光解吸电离/飞行时间质谱(MALDI/TOF MS)来评估。其他类型的化学改变包括电荷改变(例如作为脱酰胺或氧化的结果而发生)，其可以通过例如离子交换层析来评估。
如果药物组合物中的抗体对于其预期目的来说是生物活性的，则上述抗体在药物组合物中“保持其生物活性”。例如，如果制剂于例如5℃、25℃、45℃等温度下储存一定时间(例如1至12个月)之后，该制剂所含IL-21融合蛋白与IL-17A结合的亲和力为上述储存之前抗体结合亲和力的至少90％、95％或以上，则可认为本发明之制剂是稳定的。结合亲和力也可用例如ELISA或等离子共振技术测定。
在本发明的情形中，在药理学意义上，抗体的“治疗有效量”或“有效量”是指在抗体可以有效治疗的障碍的症状的预防或治疗或减轻方面有效的量。
本发明中，药物或治疗剂的“治疗有效量”或“治疗有效剂量”或“有效量”是当单独使用或与另一种治疗剂组合使用时保护受试者免于疾病发作或促进疾病消退的任何量的药物，所述疾病消退通过疾病症状的严重性的降低，疾病无症状期的频率和持续时间的增加，或由疾病痛苦引起的损伤或失能的预防来证明。药物或治疗剂 促进疾病消退的能力可以使用本领域技术人员已知的多种方法来评价，比如在临床试验期间的人受试者中，在预测人类功效的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。
药物治疗有效量包括“预防有效量”，即当单独或如与其它治疗药物(如抗肿瘤剂)组合给与处于患病风险(如发展癌症风险)的受试者或患病复发(如患有癌症复发)的受试者时，抑制疾病(如癌症)的发展或复发的任何量的药物。
术语“受试者”、“病人”或“患者”意图包括哺乳动物生物体。受试者/患者的实例包括人类和非人类哺乳动物，例如非人类灵长动物、狗、奶牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因非人类动物。在本发明的特定实施方式中，受试者是人类。
术语“施用”、“给与”及“处理”是指采用本领域技术人员已知的各种方法或递送系统中的任意一种将包含治疗剂的组合物引入受试者。抗PD-1抗体的给药途径包括静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径，比如注射或输注。“胃肠外给药”是指除了肠内或局部给药以外的通常通过注射的给药方式，包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、框内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜内和胸骨内注射和输注以及经体内电穿孔。
术语“抗体”以其最广泛的含义使用，并且涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、全长抗体及其抗原结合片段，只要它们表现出期望的抗原结合活性。术语“抗体部分”指全长抗体或其抗原结合片段。
本文所用的术语“全长抗体”或“完整抗体分子”指包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个 CDR和4个FR组成。通常是根据如下的定义将氨基酸分配至每一个结构域的：Sequences of Proteins of Immunological Interest,Kabat等人；National Institutes of Health,Bethesda,Md.；第5版；NIH出版号91-3242(1991)：Kabat(1978)Adv.Prot.Chem.32:1-75；Kabat等人，(1977)J.Biol.Chem.252:6609-6616；Chothia等人，(1987)J Mol.Biol.196:901-917或Chothia等人，(1989)Nature 341:878-883。
重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
当在本文中使用时，术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。这些CDR的准确边界按照不同的系统有不同的定义。本文公开的抗体和抗原结合片段的CDR边界可以按照Kabat、Chothia或Al-Lazikani的惯例来定义或划分(Al-Lazikani 1997；Chothia 1985；Chothia 1987；Chothia 1989；Kabat 1987；Kabat 1991)。重链或轻链的三个CDR插入侧翼延伸段(称为框架区(FR))之间，框架区比CDR更加高度保守，并形成支架以支撑超变环。重链和轻链的恒定区不参与抗原结合，但表现出各种效应子功能。抗体基于其重链恒定区的氨基酸序列被分类。抗体的五种主要类别或同种型为IgA、IgD、IgE、IgG和IgM，其特征分别在于存在α、δ、ε、γ和μ重链。几种主要抗体类别被分成亚类，例如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)。
本文所使用的“抗原结合片段”包括抗体的片段或其衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗原结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗体的结合活性在摩尔浓度基础上表示时，结合片段或其衍生物通常保持亲代抗体抗原结合活性的至少10％。优选结合片段或其衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
除非另有说明，否则本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
本文所述的“框架区”或“FR”是指不包括CDR区的免疫球蛋白可变区。
“嵌合抗体”是指如下的抗体以及其片段：其中重链和/或轻链的一部分与源自特定物种(如人)或属于特定抗体种类或亚类的抗体中相应序列相同或同源，而链的其余部分与源自另一物种(如小鼠)或属于另一抗体种类或亚类的抗体中相应序列相同或同源，只要其表现出期望的生物活性即可。
“人抗体”是指仅包含人免疫球蛋白序列的抗体。若人抗体是在小鼠、小鼠细胞或源自小鼠细胞的杂交瘤中产生，则其可含有鼠类碳水化合物链。类似的，“小鼠抗体”或“大鼠抗体”是指仅分别包含小鼠或大鼠免疫球蛋白序列的抗体。
“人源化抗体”是指含有来自非人(如鼠类)抗体以及人抗体的序列的抗体形式。此类抗体含有源自非人免疫球单边的最小序列。通常，人源化抗体将包含实质上全部的至少一个且通常两个可变结构域，其中全部或实质上全部超变环对应于非人免疫球蛋白的超变环，且全部或实质上全部FR区为人免疫球蛋白的FR区。人源化抗体任选还包括免疫球蛋白恒定区(Fc)(通常为人免疫球蛋白恒定区)的至少一部分。
“Fv”是含有完整抗原识别位点和抗原结合位点的最小抗体片段。该片段由紧密、非共价结合的一个重链可变区结构域和一个轻链可变区结构域的二聚体组成。这两个结构域折叠后产生六个超变环(重链和轻链各3个环)，为抗原结合贡献氨基酸残基并为抗体赋予抗原结合特异性。然而，即使是单个可变结构域(或只包括特异性针对某抗原的三个CDR的半个Fv)也具有识别和结合抗原的能力，但亲和力低于整个结合位点。
术语“重链抗体”或“HCAb”是指包括重链但缺乏通常在4链抗体中常见的轻链的功能性抗体。已知骆驼科动物(诸如骆驼、美洲驼或羊驼)产生HCAb。
术语“单域抗体”或“sdAb”指具有三个互补决定区(CDR)的单一抗原结合多肽。sdAb能够单独与抗原结合而不与对应的含CDR的多肽配对。在一些情况下，单结构域抗体改造自骆驼科HCAb，并且其重链可变结构域在本文中被称为“VHH”(重链抗体的重链的可变结构域)。骆驼科动物sdAb是已知最小的抗原结合抗体片段之一(参见例如Hammers-Casterman等人，Nature，第363卷：第446-448页，1993 年；Greenberg等人，Nature，第374卷：第168-173页，1995年；Hassanzadeh-Ghassaeh等人，Nanomedicine(Lond)，第8卷：第1013-1026页，2013年)。碱性VHH从N末端到C末端具有以下结构：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，其中FR1至FR4分别指框架区1至4，并且其中CDR1至CDR3指互补决定区1至3。
除非另有说明，本文中免疫球蛋白重链中残基的编号参照Kabat等人的EU索引编号。“Kabat的EU索引”指人IgG1EU抗体的残基编号。
“治疗性抗PD-1单克隆抗体”是指特异性结合至在某些哺乳动物细胞表面上表达的特定PD-1的成熟形式的抗体。成熟的PD-1无前分泌前导序列，或叫前导肽。术语“PD-1”及“成熟的PD-1”在本文中可互换使用，且除非另有明确定义，或明确能从上下文看出，否则应理解为相同分子。
如本文所述，治疗性抗人PD-1抗体或抗hPD-1抗体是指特异性结合至成熟人PD-1的单克隆抗体。
“分离的抗体或其抗原结合片段”是指纯化状态且在该情况下所指定的分子实质上不含有其他生物分子，诸如核酸、蛋白质、脂质、碳水化合物或其他材料(诸如细胞碎片或生长培养基)。
如本文所用，术语“细胞因子”应理解为是指任何蛋白质或肽、其类似物或功能性片段，它们能够在哺乳动物中刺激或诱导针对预选细胞类型(例如，癌细胞或病毒感染的细胞)的杀细胞免疫应答。因此，预期可将多种细胞因子纳入本申请中。有用的细胞因子包括，例如，肿瘤坏死因子(TNF)、白细胞介素(IL)、淋巴因子(L)、集落刺激因子(CSF)、干扰素(IFN)，包括能够刺激或诱导这种杀细胞免疫应答的其物种变体、截短类似物。有用的肿瘤坏死因子包括，例如，TNFα。有用的淋巴因子包括，例如，LT。有用的集落刺激因子包括，例如，GM-CSF和M-CSF。有用的白细胞介素包括，例如，IL-2、IL-4、IL-5、IL-7、IL-12、IL-15、IL-18、IL-21、IL22和IL-33。有用的干扰素包括，例如，IFN-α、IFN-β和IFN-γ。术语“细胞因子”也应理解为包括野生型细胞因子的任何变体(诸如IL-21、IL-7、IL-15等)，其包括修饰并保持其任何期望功能的至少重要部分(诸如至少约50％)。
“免疫检查点”是指在免疫细胞上表达、能调节免疫激活程度的一系列分子，它们对防止自身免疫(免疫功能发生异常，对正常细胞发动攻击)作用的发生起着重 要作用。常见的免疫检查点包括但不限于PD-1、PD-L1、PD-L2、CTLA-4、4-1BB、CD47、TIGIT、GITR、CD112R、BTLA、TIM3、LAG3、CD27和B7H4。本文所述的免疫检查点调节剂指能特异性结合这些免疫检查点并增强、减弱或阻断其生物学活性的试剂，如抑制剂。该类试剂尤其包括特异性结合这些免疫检查点的抗体或其抗原结合片段。本文所述的免疫检查点相关肿瘤指可通过结合免疫检查点而减弱或阻断其生物学活性，从而实现肿瘤的预防和治疗的肿瘤。在特别优选的实施方案中，本文所述的免疫检查点相关肿瘤是PD-1或PD-L1或TIGIT相关的肿瘤。
本文所述的“不良反应”(AE)是与使用医学治疗相关的任何不利的和通常无意的或不期望的迹象、症状或疾病。例如，不良反应可能与在响应治疗时免疫系统的激活或免疫系统细胞的扩增相关。医学治疗可以具有一种或多种相关的AE，并且每种AE可以具有相同或不同的严重性水平。
“肿瘤负荷”是指分布于整个体内的肿瘤物质的总量。肿瘤负荷是指整个体内的癌细胞的总数目或肿瘤的总大小。肿瘤负荷可通过现有技术中已知的多种方法测定，比如在肿瘤自受试者移除后使用卡尺、或在体内时使用成像技术(比如超声、骨扫描、计算层析X射线照相术(CT)或磁共振成像(MRI)扫描)测量其尺寸。
术语“肿瘤大小”是指肿瘤的总大小，其可测量为肿瘤的长度及宽度。肿瘤大小可通过现有技术中已知的多种方法测定，例如在肿瘤自受试者移除后使用卡尺、或在体内时使用成像技术(比如骨扫描、超声、CT或MRI扫描)测量其尺寸。
术语“受试者”、“个体”、“对象”包括任何生物体，优选动物，更优选哺乳动物(例如大鼠、小鼠、狗、猫、兔等)，且最优选的是人。术语“受试者”和“患者”在本文中可以互换使用。
本文所述的“治疗”癌症是指向患有癌症或经诊断患有癌症的受试者采用本文所述治疗方案(如施用抗PD-1抗体)以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。癌症中的阳性治疗效果可以多种方式测量(参见W.A.Weber,J.Nucl.Med.,50:1S-10S(2009))。比如，关于肿瘤生长抑制，根据NCI标准，T/C≤42％是抗肿瘤活性的最小水平。T/C(％)＝经治疗肿瘤体积中值/对照肿瘤体积中值×100。PFS(也叫“至肿瘤进展的时间”)是指治疗期间及之后癌症不生长的时间长度，且包括患者经历CR或PR的时间量以及患者经历SD的时间量。DFS是指治疗期间 及之后患者仍无疾病的时间长度。OS是指与初始或未经治疗的个体或患者相比预期寿命的延长。有效治疗癌症患者的本发明组合的治疗方案可根据多种因素(比如患者的疾病状态、年龄、体重及疗法激发受试者的抗癌反应的能力)而变。尽管本发明的实施方式可不在每个受试者中达到有效的阳性治疗效果，但在统计学上显著数目的受试者中应有效并达到了阳性治疗效果。
术语“给药方式”、“给药方案”可互换使用，是指本发明组合中每一治疗剂的使用剂量及时间。
本文中，术语“癌症”或“恶性肿瘤”是指以身体中异常细胞不受控制的生长为特征的广泛的各种疾病。不受调节的细胞分裂、生长分裂和生长导致恶性肿瘤的形成，其侵入邻近组织并还可以通过淋巴系统或血流转移至身体的远端部分。
IL-21与其受体结合后能调节B细胞的增殖，分化成浆细胞来进行免疫应答，促进CD8 +T细胞和NK细胞的增殖、分化，提高NK细胞的杀伤活性，从而实现肿瘤治疗或预防。因此，本文所述的“与IL-21相关的肿瘤”指在肿瘤的治疗或预防中受益于所给予的IL-21或IL-21融合蛋白的肿瘤。
适合采用本发明的方法、药物和试剂盒来治疗或预防的肿瘤或癌症的实例尤其指受益于IL-21的肿瘤或癌症，或免疫检查点相关肿瘤，尤其是PD-1或PD-L1或TIGIT相关的肿瘤，包括但不限于间皮瘤、肺癌、乳腺癌、卵巢癌、黑色素瘤、肾癌、胰腺癌、淋巴瘤、白血病、头颈癌、肝癌、非霍奇金淋巴瘤、食管癌、胃癌和结肠直肠癌。在特别优选的实施方案中，本发明公开的肿瘤/癌症指结肠直肠癌。
在以下段落中，进一步详细描述本发明的各个方面。
IL-21融合蛋白
本发明公开的IL-21-抗白蛋白单域抗体融合蛋白(IL-21融合蛋白)包括公开号为WO2019246004中描述的任意一个IL-21融合蛋白，本文将其所公开的全部内容以引入的方式纳入本文。在一些方案中，在本发明的方法和组合物中使用的抗体的氨基酸序列包括来自于WO2019246004中描述的IL-21融合蛋白P798的氨基酸序列。
在本文实施例中所用的非限制性、示范性抗体选自WO2019246004中描述的IL-21-抗白蛋白单域抗体融合蛋白P798(JS-EC21)，其氨基酸序列如下所示：
IL-21的氨基酸序列：
Figure PCTCN2022079127-appb-000001
单域抗体的氨基酸序列：
Figure PCTCN2022079127-appb-000002
HCDR1：GSTWSINT(SEQ ID NO:2)
HCDR2：ISSGGST(SEQ ID NO:3)
HCDR3：YAQSTWYPPS(SEQ ID NO:4)
IL-21-抗白蛋白单域抗体融合蛋白JS-EC21的全长氨基酸序列：
Figure PCTCN2022079127-appb-000003
抗PD-1抗体
本文中，“PD-1抗体”是指结合PD-1受体，阻断表达于癌细胞上的PD-L1与表达于免疫细胞(T、B、NK细胞)上的PD-1结合且优选也能阻断表达于癌细胞上的PD-L2与表达于免疫细胞上的PD-1结合的任何化学化合物或生物分子。在治疗人个体的任何本发明治疗方法、药物及用途中，PD-1抗体阻断人PD-L1与人PD-1的结合，且优选阻断人PD-L1和PD-L2二者与人PD1结合。人PD-1氨基酸序列可见于NCBI基因座编号：NP_005009。人PD-L1及PD-L2氨基酸序列可分别见于NCBI基因座编号：NP_054862及NP_079515。
本文中，当提及“抗PD-1抗体”时，除非另有说明或描述，否则该术语包括其抗原结合片段。
适用于本发明所述的任意用途、疗法、药物及试剂盒的抗PD-1抗体或其抗原 结合片段以高特异性和高亲和力结合PD-1，阻断PD-L1/2与PD-1的结合，并抑制PD-1信号转导，从而达到免疫抑制效果。本文所公开的任意用途、疗法、药物及试剂盒中，抗PD-1抗体包括全长抗体本身，以及结合PD-1受体并在抑制配体结合和上调免疫系统方面表现出类似完整Ab的功能特性的抗原结合部分或片段。在一些实施方式中，抗PD-1抗体为与特瑞普利单抗交叉竞争结合人PD-1的抗PD-1抗体。在其他的实施方式中，抗PD-1抗体是鼠源、嵌合、人源化或人Ab或其抗原结合片段。在用于治疗人个体的某些实施方式中，所述的Ab为人源化Ab。
在一些实施方式中，恒定区是选自人IgG1、IgG2、IgG3及IgG4恒定区组成的组；优选地，适用于本发明所述的任何用途、疗法、药物及试剂盒的抗PD-1抗体包含人IgG1或IgG4同种型的重链恒定区，更优选是人IgG4恒定区。在一些实施方式中，抗PD-1抗体的IgG4重链恒定区的序列包含S228P突变，其用IgG1同种型抗体的相应位置处通常存在的脯氨酸残基替代铰链区中的丝氨酸残基。
在一些实施方式中，本发明公开的抗PD-1抗体包括公开号为WO2014206107中描述的任意一个抗PD-1抗体或其抗原结合片段，本文将其所公开的全部内容以引入的方式纳入本文。在一些实施方式中，可用于本发明公开的任一用途、疗法、药物及试剂盒的抗PD-1抗体选自WO2014206107中描述的人源化抗体38、39、41和48。在优选的实施方案中，可用于本发明公开的任一用途、疗法、药物及试剂盒的抗PD-1抗体为特瑞普利单抗(Toripalimab)(一种具有WHO Drug Information(第32卷，第2期，第372-373页(2018))。
在本文实施例中所用的非限制性、示范性抗体选自WO2014206107中描述的人源化抗体特瑞普利单抗，其氨基酸CDR序列，可变区序列及全长氨基酸序列如下(KABAT)：
特瑞普利单抗轻链可变区(SEQ ID NO:13)
Figure PCTCN2022079127-appb-000004
LCDR1：RSSQSIVHSNGNTYLE(SEQ ID NO:7)
LCDR2：KVSNRFS(SEQ ID NO:8)
LCDR3：FQGSHVPLT(SEQ ID NO:9)
特瑞普利单抗重链可变区(SEQ ID NO:14)
Figure PCTCN2022079127-appb-000005
Figure PCTCN2022079127-appb-000006
HCDR1：DYEMH(SEQ ID NO:10)
HCDR2：VIESETGGTAYNQKFKG(SEQ ID NO:11)
HCDR3：EGITTVATTYYWYFDV(SEQ ID NO:12)
特瑞普利单抗轻链(SEQ ID NO:15)
Figure PCTCN2022079127-appb-000007
特瑞普利单抗重链(SEQ ID NO:16)
Figure PCTCN2022079127-appb-000008
优选地，在本发明公开的用途、疗法、药物及试剂盒的任意一个实施方式中，抗PD-1抗体是单克隆抗体或其抗原结合片段，其轻链CDR为SEQ ID NO：7、8和9所示的氨基酸，重链CDR为SEQ ID NO：10、11和12所示的氨基酸。
更优选地，在本发明公开的用途、疗法、药物及试剂盒的任意一个实施方式中，抗PD-1抗体包含：(a)包含SEQ ID NO：13的轻链可变区，及(b)包含SEQ ID NO：14的重链可变区的单克隆抗体。
进一步优选地，在本发明公开的用途、疗法、药物及试剂盒的任意一个实施方式中，抗PD-1抗体是包含：(a)包含SEQ ID NO：15的轻链，及(b)包含SEQ ID NO：16的重链的单克隆抗体。
可用于本发明公开的任一用途、疗法、药物及试剂盒的抗PD-1抗体还包括FDA已经批准的Nivolumab和Pembrolizumab。
在某些实施方案中，可用于本发明公开的任一用途、疗法、药物及试剂盒的抗PD-1抗体也包括特异性结合PD-L1以阻断PD-L1与PD-1结合的抗PD-L1单克隆 抗体，如nivolumab、pembrolizumab、toripalimab、Sintilimab、Camrelizumab、Tislelizumab、Cemiplimab。
抗TIGIT抗体
术语“TIGIT”，全称为T细胞免疫球蛋白和ITIM结构域蛋白(T cell Ig and ITIM domain)，是指来自任何脊椎动物(包括哺乳动物如灵长类动物(例如人)和啮齿类动物(例如，小鼠和大鼠)的任何天然TIGIT，除非另有说明。该术语涵盖“全长”未加工的TIGIT以及由细胞内加工产生的任何形式的TIGIT或其任何片段。该术语还包括天然存在的TIGIT的变体，例如，剪接变体或等位变体。在一个实施方案中，TIGIT是指来自人和食蟹猴TIGIT全长或其片段(诸如其缺乏信号肽的成熟片段)。在一个实施方案中，人TIGIT是指与Genbank登录号NP_776160.2氨基酸残基22-244序列一致的成熟TIGIT(氨基酸残基1-21为前导肽)。在一个实施方案中，人TIGIT是指与Genbank登录号NP_776160.2氨基酸残基22-141序列一致的TIGIT胞外结构域。在一个实施方案中，食蟹猴(Wacaca fascicuiaris)TIGIT指与Genbank登录号XP_005548158.1氨基酸残基22-245序列一致的成熟TIGIT。
术语“抗TIGIT抗体”、“抗TIGIT”、“TIGIT抗体”或“结合TIGIT的抗体”是指能够以足够的亲合力结合TIGIT蛋白或其片段以致所述抗体可以用作靶向TIGIT的诊断剂和/或治疗剂。
本文中，当提及“抗TIGIT抗体”时，除非另有说明或描述，否则该术语包括其抗原结合片段。
在一些实施方案中，本发明公开的抗TIGIT抗体或其抗原结合片段包括申请号为PCT/CN2020/101883(WO2021/008523)中描述的任意一个抗TIGIT抗体或其抗原结合片段，本文将其所公开的全部内容以引入的方式纳入本文。在一些方案中，在本发明的方法、用途和组合物中使用的抗TIGIT抗体的CDR序列包括来自于PCT/CN2020/101883中描述的抗体hu20或hu3的CDR序列。在一些方案中，在本发明的方法、用途和组合物中使用的抗TIGIT抗体的轻链可变区和重链可变区包括来自于PCT/CN2020/101883中描述的抗体hu20或hu3的的轻链可变区和重链可变区。在一些方案中，在本发明的方法、用途和组合物中使用的抗TIGIT抗体为PCT/CN2020/101883中描述的抗体hu20或hu3。
在一些实施方案中，本发明公开的抗TIGIT抗体选自：Tiragolumab、Etigilimab、Vibostolimab、Domvanalimab、EOS-884448或BMS-986207。
医药制剂
本发明公开的药物组合物是一种含有IL-21-抗白蛋白单域抗体融合蛋白的高稳定性药物组合物。本发明发现海藻糖的组合能明显提高药物组合物的稳定性。
本发明公开的药物组合物对pH较敏感，pH范围控制4.5-5.1时，本发明公开的药物组合物具有较高的稳定性。
本发明提供了一种药物组合物，包含：(1)缓冲液；(2)IL-21-抗白蛋白单域抗体融合蛋白。
本发明药物组合物中IL-21融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，上述IL-21包括如SEQ ID NO:1所示的氨基酸序列，上述单域抗体包含氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的HCDR1、HCDR2和HCDR3。优选地，本发明药物组合物中的融合蛋白包含氨基酸序列如SEQ ID NO:1所示的细胞因子IL-21，和氨基酸序列如SEQ ID NO:5所示的sdAb；更优选地，本发明药物组合物中的融合蛋白包含如SEQ ID NO:6所示的氨基酸序列或由其组成。
本发明的药物组合物中，IL-21-抗白蛋白单域抗体融合蛋白浓度约为0.1-100mg/mL，优选约为0.2-20mg/mL，优选约为0.2-10mg/mL，优选约为0.5-5mg/mL，更优选1-5mg/mL；更优选地，上述IL-21-抗白蛋白单域抗体融合蛋白浓度约为0.5mg/mL，0.8mg/mL，1mg/mL，1.2mg/mL，1.5mg/mL，1.8mg/mL，2mg/mL，2.5mg/mL，3mg/mL，3.5mg/mL，4mg/mL，4.5mg/mL，5mg/mL，6mg/mL，7mg/mL，8mg/mL或10mg/mL，优选约为1mg/mL。
本发明药物组合物中的缓冲液可选自醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液，用以为本发明的药物组合物提供4.0到6.5、优选4.5到5.5，更优选4.8±0.3，更优选为约4.8的pH。另一方面，用于本发明药物组合物中的缓冲液的pH可为4.0-6.5，优选为4.5-5.5，更优选4.8±0.3，更优选为约4.8。
本发明药物组合物中特别优选的缓冲液是醋酸缓冲液，优选地，上述醋酸缓冲液为醋酸-醋酸钠缓冲液或醋酸-醋酸钾缓冲液，优选醋酸-醋酸钠缓冲液。在一些方案中，醋酸缓冲液由1-30mM的醋酸和1-30mM的醋酸钠制成。在一些方案中，醋酸缓冲液由摩尔比为约2:3的醋酸和醋酸钠制成。在一些方案中，醋酸缓冲液由摩尔比为约1:2.1的醋酸和醋酸钠制成。在一些方案中，醋酸缓冲液由摩尔比为约1:5.7 的醋酸和醋酸钠制成。在一些方案中，醋酸缓冲液为：由约8mM的醋酸和约12mM的醋酸钠制成的pH约为4.8的醋酸缓冲液。在一些方案中，醋酸缓冲液为：由约6.5mM的醋酸和约13.5mM的醋酸钠制成的pH约为5.0的醋酸缓冲液。在一些方案中，醋酸缓冲液为：由约3mM的醋酸和约17mM的醋酸钠制成的pH约为5.5的醋酸缓冲液。
因此，本发明的药物组合物可含有：pH为4.5-5.5(优选4.5-5.0)的醋酸-醋酸钠缓冲液，其在药物组合物中的浓度为10-30mM；和0.5-5mg/mL的前文任一实施方案的IL-21-抗白蛋白单域抗体融合蛋白，优选为本文公开的JS-EC21。
在一些方案中，本发明的药物组合物还含有稳定剂。优选地，上述稳定剂选自氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或一种以上，优选地，上述稳定剂为海藻糖。本发明的药物组合物中稳定剂的浓度为约10mM-400mM，优选20mM-300mM，更优选30mM-200mM。在一些方案中，上述稳定剂为浓度约100-300mM、优选200-300mM的甘露醇；或上述稳定剂为浓度约100-300mM、优选200-300mM的蔗糖；或上述稳定剂为浓度约100-300mM、优选200-300mM的海藻糖；或上述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合；或上述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖的组合。优选地，上述稳定剂为约100-300mM的海藻糖或100-300mM的蔗糖；更优选地，上述稳定剂为约200-280mM的海藻糖。
因此，在一些实施方案中，本发明的药物组合物含有：pH为4.5-5.5(优选4.5-5.0)的醋酸-醋酸钠缓冲液，其在药物组合物中的浓度为10-30mM；0.5-5mg/mL的前文任一实施方案的IL-21-抗白蛋白单域抗体融合蛋白，优选为本文公开的JS-EC21；以及20mM-300mM的稳定剂，优选地，上述稳定剂包括氯化钠、甘露醇、蔗糖和海藻糖中的一种或一种以上，优选为100-300mM的海藻糖或100-300mM的蔗糖，更优选为200-280mM的海藻糖。
在一些方案中，本发明的药物组合物还包括表面活性剂。优选的表面活性剂选自聚山梨醇酯80、聚山梨醇酯20和泊洛沙姆188。最优选的表面活性剂是聚山梨醇酯80。以w/v计，本发明药物组合物中表面活性剂的浓度约为0.001％-0.1％，优选约为0.02％-0.08％，优选约为0.02％-0.04％。作为非限制性实施例，本发明药物组合物中表面活性剂的浓度约为0.02％，0.04％或0.08％，优选0.02％。
因此，在一些实施方案中，本发明的药物组合物含有：pH为4.5-5.5(优选4.5-5.0)的醋酸-醋酸钠缓冲液，其在药物组合物中的浓度为10-30mM；0.5-5mg/mL的前文任一实施方案的IL-21-抗白蛋白单域抗体融合蛋白，优选为本文公开的JS-EC21；以及20mM-300mM的稳定剂，优选地，上述稳定剂包括氯化钠、甘露醇、蔗糖和海藻糖中的一种或一种以上，优选为100-300mM的海藻糖或100-300mM的蔗糖，更优选为200-280mM的海藻糖；以及以w/v计0.02％-0.04％的聚山梨醇酯80。
本发明的药物组合物可以是液体制剂，或者是冻干制剂。
药物组合
本发明还提供了一种药物组合，其含有本文所述IL-21融合蛋白和免疫检查点调节剂。本发明公开的免疫检查点调节剂可如前述任一实施方案所述；优选地，所述免疫检查点调节剂为抗PD-1抗体或抗TIGIT抗体。该药物组合中，上述抗IL-21融合蛋白和免疫检查点调节剂可以混合物的形式提供(即为药物组合物的形式)，或者各自均以独立的制剂的形式提供。当以独立的制剂的形式存在时，每一制剂除所述活性成分外，还含有药学上可接受的载体。当以药物组合物的形式提供时，药物组合物中通常除含有所述活性成分外，还含有药学上可接受的载体。
本发明的药物组合亦可包含一种或多种额外治疗剂。额外治疗剂可为(例如)化学治疗剂、生物治疗剂、免疫原性剂(例如，减毒癌细胞、肿瘤抗原、抗原呈递细胞(诸如经肿瘤衍生的抗原或核酸脉冲的树突状细胞)、免疫刺激细胞因子(例如，IL-2、IFN-瘤、GM-CSF)及经编码免疫刺激细胞因子(诸如但不限于GM-CSF)的基因转染的细胞)。
治疗方法和用途
本发明提供了IL-21融合蛋白、或上述药物组合物、或IL-21融合蛋白和免疫检查点调节剂的组合或上述药物组合物和免疫检查点调节剂的组合在制备用于预防或治疗肿瘤的药物中的用途。本发明还提供上述药物组合物或IL-21融合蛋白在增强或改善免疫检查点调节剂的抗肿瘤活性的药物中的用途。
本发明提供了预防或治疗肿瘤的方法，其包含向有需要的个体施用有效量的IL-21融合蛋白，或上述药物组合物，或IL-21融合蛋白和免疫检查点调节剂的组合或上述药物组合物和免疫检查点调节剂的组合。所述有效量包括预防有效量和治 疗有效量。
本发明提供IL-21融合蛋白，或上述药物组合物，或IL-21融合蛋白与免疫检查点调节剂的组合或上述药物组合物和免疫检查点调节剂的组合，其用于治疗或预防肿瘤。
本发明公开的肿瘤可如前述任一实施方案所述。优选地，本文所述的肿瘤是受益于IL-21治疗的肿瘤，或者为PD-1或TIGIT相关的肿瘤，优选选自间皮瘤、肺癌、乳腺癌、卵巢癌、黑色素瘤、肾癌、胰腺癌、淋巴瘤、白血病、头颈癌、肝癌、非霍奇金淋巴瘤、食管癌、胃癌和结肠直肠癌；优选地，所述肿瘤为转移性黑色素瘤、肾细胞癌、结肠直肠癌和非霍奇金淋巴瘤，更优选为结肠直肠癌。
本发明公开的免疫检查点调节剂可如前述任一实施方案所述；优选地，所述免疫检查点调节剂为抗PD-1抗体或抗TIGIT抗体。
在一个或多个实施方案中，本发明两组间通过two-tailed unpaired Student’s t test进行差异分析，*表示P<0.05；**表示P<0.01,***表示P<0.001，ns表示无显著性差异；P<0.05表示显著提高或显著增加。
在一个或多个实施方案中，相比IL-21融合蛋白或抗PD-1抗体单独使用，本发明公开的IL-21融合蛋白与抗PD-1抗体组合使用具有选自以下的一种或多种性质：
(1)免疫细胞中CD3 +T细胞的比例显著提高；
(2)T细胞中CD8 +T细胞的比例显著提高；
(3)增殖状态(Ki 67 +)的CD8 +T细胞的比例显著提高；和
(4)免疫细胞中NK细胞的比例显著提高。
因此，在一些实施方案中，本申请提供本文公开的IL-21融合蛋白与抗PD-1抗体的组合在制备用于下述一个或多个用途的试剂中的用途：
(1)提高免疫细胞中CD3 +T细胞的比例；
(2)提高T细胞中CD8 +T细胞的比例；
(3)提高增殖状态(Ki 67 +)的CD8 +T细胞的比例；和
(4)提高免疫细胞中NK细胞的比例。
在优选的实施方案中，所述试剂用于本文公开的肿瘤的治疗或预防。
在一个或多个实施方案中，相比IL-21融合蛋白或抗TIGIT抗体单独使用，本发明IL-21融合蛋白与抗TIGIT抗体组合使用具有选自以下的一种或多种性质：
(1)显著增加了干扰素γ的表达水平；
(2)显著增加了颗粒酶A的表达水平；
(3)显著增加了颗粒酶B的表达水平；
(4)显著增加了穿孔素1的表达水平；
(5)富集更多具有差异基因表达的KEGG信号通路；和
(6)诱导的大部分显著富集的KEGG信号通路由IL-21融合蛋白介导。
因此，在一些实施方案中，本申请提供本文公开的IL-21融合蛋白与上述抗TIGIT抗体的组合在制备用于下述一个或多个用途的试剂中的用途：
(1)增加对象干扰素γ的表达水平；
(2)增加对象颗粒酶A的表达水平；
(3)增加对象颗粒酶B的表达水平；和
(4)增加对象穿孔素1的表达水平。
在优选的实施方案中，所述试剂用于本文公开的肿瘤的治疗或预防。
优选的用于本发明公开的肿瘤的IL-21融合蛋白可如本文任一实施方案所述，优选为包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，细胞因子IL-21包括如SEQ ID NO:1所示的氨基酸序列，单域抗体(sdAb)包含氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的HCDR1、HCDR2和HCDR3；更优选为包含如SEQ ID NO:1所示的氨基酸序列，和如SEQ ID NO:5所示的氨基酸序列；更优选为包含如SEQ ID NO:6所示的氨基酸序列。
优选的用于本发明公开的肿瘤的抗PD-1抗体可如本文任一实施方案所述，更优选为包含轻链CDR为SEQ ID NO：7、8和9所示的氨基酸，和重链CDR为SEQ ID NO：10、11和12所示的氨基酸的抗体，更优选为包含SEQ ID NO：13所示轻链可变区和SEQ ID NO：14所示的重链可变区的单克隆抗体，更优选为包含SEQ ID NO：15所示的轻链和SEQ ID NO：16所示的重链的单克隆抗体，更优选为WO2014206107中描述的人源化抗体38、39、41和48，最优选为特瑞普利单抗。
在特别优选的实施方案中，本发明提供预防或治疗肿瘤的方法，该方法包括给予肿瘤患者治疗有效量的IL-21融合蛋白，或上述药物组合物，或IL-21融合蛋白和抗PD-1抗体的组合或上述药物组合物和抗PD-1抗体的组合；优选地，该肿瘤患者为结肠直肠癌患者；优选地，IL-21融合蛋白包含如SEQ ID NO:6所示的氨基酸序列；优选地，抗PD-1抗体为特瑞普利单抗。
在特别优选的实施方案中，本发明提供IL-21融合蛋白，或上述药物组合物，或IL-21融合蛋白和抗PD-1抗体的组合或上述药物组合物和抗PD-1抗体的组合在制备用于预防或治疗肿瘤的药物的用途；优选地，该肿瘤患者为结肠直肠癌患者；优选地，IL-21融合蛋白包含如SEQ ID NO:6所示的氨基酸序列；优选地，抗PD- 1抗体为特瑞普利单抗。
试剂盒
本发明提供了一种试剂盒，其包含：
(1)一个或多个单次药物剂量单元的IL-21融合蛋白和一个或多个单次药物剂量单元的免疫检查点调节剂，其中IL-21融合蛋白如本文所述；优选地，免疫检查点调节剂如本文所述；或
(2)一个或多个单次药物剂量单元的如本文所述的药物组合物。
在一个或多个实施方案中，本发明公开的试剂盒，其还包含用于指示所述药物组合物的使用方法的说明书。
医药用途和方法
本发明还提供了用于治疗或预防癌症的本发明任一实施方案公开的药物组合物，本发明任一实施方案公开的药物组合物在制备治疗或预防癌症的药物中的用途，以及给予需要的个体或患者治疗有效量的本发明任一实施方案公开的药物组合物以治疗或预防癌症的方法。
本发明中，适用于本发明药物组合物进行治疗和预防的疾病包括但不限于间皮瘤、肺癌、乳腺癌、卵巢癌、胰腺癌、淋巴瘤、白血病、头颈癌、肝癌、食管癌、胃癌和结肠直肠癌。
下面会通过实施例具体描述本发明，这些实施例并不意味着对本发明的任何限制。实施例中所用到的方法和材料，除非另有说明，否则为本领域的常规方法和材料。
本发明采用下述缩略词：
his-tag代表组氨酸标签；
HRP代表辣根过氧化物酶；
TMB代表3,3',5,5'-四甲基联苯胺；
HBSS代表Hank's Balanced Salt Solution，即Hank's平衡盐溶液；
HEPES代表N-2-羟乙基哌嗪-N'-2-乙磺酸，Good’s缓冲试剂。
BID代表一个剂量，每日2次
CDR代表互补决定区
DFS代表无疾病生存
FR代表框架区
IgG代表免疫球蛋白G
Q2W代表每两周一个剂量
QD代表每天一个剂量
本发明毛细管微量差示扫描荧光法(DSF)
使用毛细管自动吸满样品放入对应编号位置，扫描设置温度20℃-95℃，升温速率1℃/min。仪器运行结束处理和分析数据。
本发明外观检测方法
外观采用目检法来检测。确保澄明度检测仪的光照强度保持在1000lx-1500lx之间。将样品保持在眼睛的同一水平面，轻轻摇晃或颠倒以避免产生气泡。分别在黑色背景和白色背景前进行目检。观察颜色，乳光和可见异物。
本发明蛋白含量检测方法
蛋白浓度使用紫外分光光度计来检测。将百分比消光系数(E1％)设定在1.527(g/ml) -1cm -1。使用BIO MATE 3S仪器，用超纯水清洗比色皿后加入150μL超纯水，点击测量，以超纯水做空白校正。每个样品平行测定2份溶液并且每份溶液重复测定3次，取平均值。通过消光系数及OD值计算出对应检品的蛋白浓度。
本发明尺寸排阻色谱(SEC-HPLC)检测方法
SEC-HPLC纯度采用安装了SEC色谱柱(Waters Xbrige BEH 125A 7.8*300mm,3.5μm)的HPLC(Waters e2695仪器)进行检测。流动相组成为50mM磷酸盐缓冲液，300mM氯化钠，300mM精氨酸，pH 6.8±0.2。采用面积归一化法计算主峰(单体)，聚体和片段的相对百分比。详细见下表：
色谱条件 色谱参数
检测器波长 280nm
自动进样器温度 25±3℃
柱温 5±3℃
流速 0.50ml/min
进样体积 25μl
运行模式 等度模式
运行时间 30.00min
本发明非还原毛细管电泳(NR-CE-SDS)检测方法
NR-CE-SDS纯度采用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)进行检测。取稀释液(1×样品缓冲液Maurice CE-SDS)50μl，加入0.8M碘乙酰胺5.0μl混合均匀，后加入50μl的样品(1mg/ml)，涡旋混匀；同时取稀释液50μl，加入0.8M碘乙酰胺5.0μl混合均匀，70℃孵育5min，按面积归一化法计算，主峰占所有修正峰面积之和的百分比为主峰纯度。
本发明还原毛细管电泳(R-CE-SDS)检测方法
R-CE-SDS纯度采用安装了CE-SDS卡盒的毛细管电泳仪(Maurice仪器)进行检测。取稀释液(1×样品缓冲液Maurice CE-SDS)50μl，加入2-巯基乙醇5.0μl混合均匀，后加入50μl的样品(1mg/ml)，涡旋混匀；同时取稀释液50μl，加入2-巯基乙醇5.0μl混合均匀，70℃孵育15min，按面积归一化法计算，主峰占所有修正峰面积之和的百分比为主峰纯度。
本发明成像毛细管等电聚焦(iCIEF)检测方法
iCIEF采用安装了iCIEF卡盒的毛细管电泳仪(Maurice仪器)进行检测。样品经iCIEF混合液稀释处理后，使用毛细管电泳仪(Maurice)检测。不同等电点组分被聚焦在不同位置而达到聚焦和分离的效果。
iCIEF混合液配制如下表：
试剂名称 每个样品所需体积(μL)
1％MC(甲基纤维素) 35
Pharmalyte Mix(两性电解质) 4
pI Marker Mix(等电点标志物) 1
500mM Arg(精氨酸) 2
8M尿素 50
8μl样品溶液(1mg/mL)与92μL的iCIEF混合液混匀，离心去除气泡，仪器分析检测。
本发明结合活性检测方法
将重组人IL-21R蛋白His tag(Acro公司，货号ILR-H5226)稀释至4.0μg/ml包被，在37±2℃恒温培养箱中静置孵育90min。洗板并用2％脱脂牛奶封闭。加入不同浓度的待测样品(从4μg/mL起始，3倍梯度稀释)，孵育1小时并洗板。再与 1:5000稀释的HRP偶联的兔抗骆驼VHH抗体(GenScript公司，货号A01861-200)孵育1小时，然后与HRP底物TMB(Sigma，货号T2885)孵育30分钟显色，检测待测样品的结合信号。使用对数(激动剂)对响应变量的斜率曲线拟合(GraphPad Prism)EC 50。
本发明细胞活性检测方法
第一天用待测样品分析缓冲液(HBSS+10nM HEPES)稀释待测样品至30μg/ml(6×分析浓度)，3倍梯度稀释，12个浓度。将Pfeiffer细胞密度调整为4.0×10 6个细胞/ml，25μl/孔加入到96孔圆底板中，再将预先稀释的待测样品5μl/孔加入到96孔圆底板中，置于37.0℃，5.0％二氧化碳培养箱中孵育30min。每孔加入10μl裂解液(STAT3phospho-Y705kit)室温裂解30min后，每孔吸取16μl细胞裂解混合液到HTRF 96孔小体积平板中，再加入4μl混合抗体(STAT3phospho-Y705kit)，室温避光孵育18±2h。第二天用多功能酶标仪读板和GraphPad Prism软件进行数据分析处理。计算公式为：Ratio 665/620＝OD 665nm/OD 620nm*10000。
具体实施方式
为了更好的理解本发明的内容，下面结合具体实施例来做进一步的说明，但具体的实施方式并不是对本发明的内容所做的限制。整个申请中引用的所有参考文献的内容通过引用的方式明确并入本文。
实施例1：蛋白浓度筛选实验
液体型药物组合物中，蛋白浓度密切影响抗体的稳定性，每种具有独特理化性质的融合蛋白都具有最适宜的浓度。本实施例旨在筛选一种最佳蛋白浓度，使本发明公开的IL-21融合蛋白具有最佳的稳定性以适宜临床应用。
本实施例以pH4.5和pH5.0的组氨酸缓冲液(组氨酸-醋酸)来考察不同浓度IL-21-融合蛋白的稳定性。将浓度为4.0mg/ml IL-21融合蛋白(编号JS-EC21)透析换液至相应缓冲液，换液三次，每次换液时间大于4小时，调整至目标浓度后加吐温80，过滤灌装到2R西林瓶，规格为0.5ml/瓶，进行稳定性放样和检测。分别筛选了1mg/ml、3mg/ml和5mg/ml的IL-21-融合蛋白，具体如表1所示。
将样品分别放置在加速条件(25±2℃)和长期条件(5±3℃)下，在第0周、第2 周、第4周取出进行分析检测。蛋白降解的主要途径是聚集物、裂解产品和带电变体的形成。评价指标包括：1.尺寸排阻色谱法(SEC-HPLC)测定蛋白单体与聚集形式所占的百分比；2.CE-SDS(十二烷基硫酸钠毛细管电泳)法，在还原和非还原条件下，检测蛋白的纯度；3.ELISA法检测抗体结合活性和细胞活性。考察不同浓度对IL-21-融合蛋白稳定性的影响，结果见表2-表6。
表1蛋白浓度和pH筛选实验中的处方信息
Figure PCTCN2022079127-appb-000009
1.1 SEC-HPLC筛选结果
根据表2中的SEC-HPLC结果，经加速条件放置4周，处方FS1-3和处方FS1-4中蛋白聚体含量增加，处方FS1-1和处方FS1-2则无明显变化；经长期条件下放置4周，SEC-HPLC蛋白单体纯度未见明显变化。
表2蛋白浓度和pH筛选实验中SEC-HPLC数据
Figure PCTCN2022079127-appb-000010
Figure PCTCN2022079127-appb-000011
1.2 NR-CE-SDS筛选结果
根据表3中的NR-CE-SDS结果，经加速条件放置4周，所有处方中的蛋白纯度略有下降；经长期条件下放置4周，所有样品均未出现明显的纯度变化。
表3蛋白浓度和pH筛选实验中CE-SDS数据
Figure PCTCN2022079127-appb-000012
1.3 R-CE-SDS筛选结果
根据表4中的R-CE-SDS结果，经加速条件放置4周，处方FS1-3和处方FS1-4中蛋白R-CE-SDS纯度下降；经长期条件下放置4周，所有样品均未出现明显纯度变化。
表4蛋白浓度和pH筛选实验中CE-SDS数据
Figure PCTCN2022079127-appb-000013
1.4生物学活性筛选结果
根据表5中的活性筛选结果，经加速和长期条件下放置4周，所有样品结合活性和细胞活性均无明显变化。
表5蛋白浓度和pH筛选实验中活性数据
Figure PCTCN2022079127-appb-000014
Figure PCTCN2022079127-appb-000015
从SEC-HPLC结果来看，在pH5.0的组氨酸缓冲液中，高蛋白浓度3mg/ml和5mg/ml的样品(处方FS1-3和FS1-4)经加速条件放置4周，SEC-HPLC单体纯度出现明显下降，而低蛋白浓度1mg/ml无显著变化。不同处方在R-CE-SDS纯度和NR-CE-SDS纯度的表现无明显差异。从活性结果来看，各处方样品结合活性和细胞活性均未明显变化。上述结果显示该产品对蛋白浓度比较敏感，因此选择1mg/ml的蛋白浓度进行下一轮处方筛选。
实施例2：缓冲液体系和pH筛选
为了进一步探究不同缓冲液体系和pH对本发明的IL-21-融合蛋白JS-EC21稳定性的影响，筛选了醋酸缓冲液、组氨酸缓冲液和柠檬酸缓冲液，pH范围从4.7到6.5。将IL-21-融合蛋白(编号JS-EC21)透析袋进行对应缓冲液置换，换液后样品处于相应的处方中，最终蛋白浓度约1mg/ml，无菌灌装到2R西林瓶，规格为2.5ml/瓶，进行稳定性检测(如表6)。
将样品分别放置在加速条件(25±2℃)和长期条件(5±3℃)中，在第0周、第1周、第2周、第4周、第8周分别取出进行分析检测。考察不同缓冲液和pH对IL-21-融合蛋白稳定性的影响，结果见表7-表13。
评价指标包括：1.DSC(毛细管微量差示扫描荧光法)测定蛋白的Tm值(熔融温度)；2.目测外观；3.紫外分光光度法测定蛋白含量；4.尺寸排阻色谱法(SEC-HPLC)测定蛋白单体、聚集和碎片的百分比；5.iCIEF(成像毛细管等电聚焦)法检测蛋白主要电荷、酸性电荷或碱性电荷的百分比；6.CE-SDS(十二烷基硫酸钠毛细管电泳)法，在还原和非还原条件下，检测蛋白的纯度；7.ELISA法检测抗体结合活性和细胞活性。
表6缓冲液体系和pH筛选实验方案
Figure PCTCN2022079127-appb-000016
Figure PCTCN2022079127-appb-000017
2.1 DSF筛选结果
根据表7中的DSF结果，8组处方随着pH上升，Tm值有下降趋势，其中FS2-1、FS2-2、FS2-3和FS2-4略高于其余4组。
表7缓冲液体系和pH筛选中DSF数据
Figure PCTCN2022079127-appb-000018
2.2外观和蛋白浓度筛选结果
根据表8中的结果，FS2-7和FS2-8两组在T0时即发现白点析出；经加速条件或长期条件下放置8周，仅FS2-1和FS2-3两组外观未见异常。
根据表9中的结果，FS2-4，FS2-5，FS2-6，FS2-7和FS2-8五组由于外观异常在稳定性放样过程中终止检测；其余三组经加速条件或长期条件下放置8周，未见蛋白含量变化。
表8缓冲液体系和pH筛选中外观结果
Figure PCTCN2022079127-appb-000019
注：“/”表示未测定。
表9缓冲液体系和pH筛选中蛋白含量结果
Figure PCTCN2022079127-appb-000020
注：“/”表示未测定。
2.3 SEC-HPLC纯度筛选结果
根据表10中的SEC-HPLC结果，FS2-4、FS2-6、FS2-7和FS2-8四组由于外观异常终止检测；其余四组经加速条件或长期条件下放置8周，SEC-HPLC蛋白单体纯度未见明显变化。
表10缓冲液体系和pH筛选中SEC-HPLC数据
Figure PCTCN2022079127-appb-000021
Figure PCTCN2022079127-appb-000022
注：“/”表示未测定。
2.4 NR/R-CE-SDS纯度筛选结果
根据表11中的NR/R-CE-SDS纯度结果，其中样品FS2-4、FS2-6、FS2-7和FS2-8四组由于外观异常终止检测；其余四组经加速条件和长期条件下放置8周，所有样品均未出现蛋白纯度明显变化。
表11缓冲液体系和pH筛选中CE-SDS数据
Figure PCTCN2022079127-appb-000023
注：“/”表示未测定。
2.5 iCIEF筛选结果
根据表12中的iCIEF结果，其中样品FS2-4、FS2-6、FS2-7和FS2-8四组由于外观异常终止检测；其余四组经加速条件下放置8周，均出现酸性峰增加，组间未见差异；经长期条件下放置8周，所有样品均未出现显著变化。
表12缓冲液体系和pH筛选中iCIEF结果
Figure PCTCN2022079127-appb-000024
注：“/”表示未测定。
2.6生物学活性筛选结果
根据表13中的活性结果，经加速和长期条件下放置8周，其中样品FS2-6、FS2-7和FS2-8三组由于外观异常终止检测；其余5组结合活性和细胞活性均未出现明显变化。
表13缓冲液体系和pH筛选中活性结果
Figure PCTCN2022079127-appb-000025
Figure PCTCN2022079127-appb-000026
注：“/”表示未测定。
综合各项数据分析，从DSF结果来看，FS2-1、FS2-2、FS2-3、FS2-4略高于其余4组；从外观结果来看，经加速条件放置8周，仅FS2-1和FS2-3两组处方未出现外观变化；从SEC-HPLC纯度结果、NR/R-CE-SDS纯度结果、iCIEF结果和活性结果来看，FS2-1和FS2-3两组处方之间未见显著差异。基于上述结果，本品对于pH较敏感，整体来看缓冲液pH在4.5-5.0范围内表现最优。因此，选择20mM醋酸-醋酸钠缓冲液，pH 4.5-5.0开展下一轮处方筛选。
实施例3：稳定剂和表面活性剂筛选实验
为了进一步探究不同稳定剂(辅料)和表面活性剂对本发明的IL-21-融合蛋白JS-EC21稳定性的影响，选取蔗糖、海藻糖或甘露醇做为稳定剂进行了比较测试。
将IL-21-融合蛋白使用Millipore Pellicon3 0.11m 2膜进行UF/DF换液，稀释，使样品处于相应的处方中，蛋白浓度约1mg/ml，具体处方信息如表14所示。样品灌装到2R西林瓶和6R西林瓶，规格分别为2.0ml/瓶和3.0ml/瓶，进行稳定性放样和检测。
各处方制剂分别放置于加速(25±2℃)和长期(5±3℃)条件，分别在第0周、第2周、第4周取出进行分析检测；冻融(-40℃/RT)条件，检测反复冻融3次及5次；振摇(250rpm,RT)条件，分别在第1天及第3天进行分析检测；光照(5000lux,RT)条件下，分别在第5天及第10天进行分析检测。考察不同稳定剂和表面活性剂对IL-21-融合蛋白稳定性的影响，结果见表15-表19。
评价指标包括：1.目测外观；2.紫外分光光度法检测蛋白含量；3.尺寸排阻色谱法(SEC-HPLC)测定蛋白单体、聚集和碎片的百分比；4.阳离子交换色谱法(CEX- HPLC)测量抗体主要电荷、酸性电荷或碱性电荷含量；5.CE-SDS(十二烷基硫酸钠毛细管电泳)法，在还原和非还原条件下，检测蛋白的纯度；6.ELISA法检测抗体结合活性。
表14稳定剂和表面活性剂筛选实验方案
Figure PCTCN2022079127-appb-000027
3.1外观和蛋白含量筛选结果
根据表15中的结果，经加速条件和长期条件下放置4周，所有样品的蛋白含量均未出现显著变化；未见外观异常；经振摇条件3天，未见外观异常；经白光照射10天，未见外观异常；经3次冻融循环，FS3-1处方出现少许白点。
表15稳定剂和表面活性剂筛选中蛋白含量和外观结果
Figure PCTCN2022079127-appb-000028
注：“/”表示未测定。
3.2 SEC-HPLC筛选结果
根据表16中的SEC纯度结果，经加速、长期、冻融、振摇和光照条件，均未出现单体含量的明显变化。
表16稳定剂和表面活性剂筛选中SEC-HPLC纯度
Figure PCTCN2022079127-appb-000029
3.3 NR/R-CE-SDS筛选结果
根据表17中的NR-CE-SDS纯度结果，3组样品经加速条件下放置4周，NR-CE-SDS和R-CE-SDS蛋白纯度均出现下降，但未表现出组间差异；经长期条件下放置4周，5次冻融循环，3天振摇或10天光照，均未见NR-CE-SDS或R-CE-SDS蛋白纯度的明显变化。
表17稳定剂和表面活性剂筛选-NR/R-CE-SDS纯度结果
Figure PCTCN2022079127-appb-000030
3.4 CEX-HPLC筛选结果
根据表18中的CEX-HPLC结果，3组样品经加速条件下放置4周，均出现酸性峰的增加，但未表现出组间差异。在光照条件下，FS3-2处方酸性峰的增加相对明显。经长期条件下放置4周，5次冻融循环，3天振摇或10天光照，所有样品 CEX-HPLC结果均未见显著变化。
表18稳定剂和表面活性剂筛选-CEX-HPLC数据
Figure PCTCN2022079127-appb-000031
3.5生物学活性筛选结果
根据表19中的生物学活性测定结果，经高温，加速或长期条件下放置4周，以及5次冻融循环，3天振摇或10天光照，所有样品的结合活性均未出现显著变化。
表19稳定剂和表面活性剂筛选-结合活性结果
Figure PCTCN2022079127-appb-000032
从外观结果来看，FS3-1在冻融条件下出现少许白点析出；从蛋白含量、SEC-HPLC纯度、NR/R-CE纯度和CEX以及活性结果来看，三组处方未表现出显著差异。基于上述结果，本发明IL-21融合蛋白在醋酸-醋酸钠缓冲液(pH 4.5-5.0)，海藻糖或蔗糖以及聚山梨酯80作为稳定剂的条件下表现出了良好的稳定性。
实施例4：IL-21融合蛋白的药代动力学
1、测试目的
小鼠腹腔给药评价本发明IL-21-抗白蛋白单域抗体融合蛋白JS-EC21与重组 人IL-21的药代动力学性质的差异。本实施例IL-21-抗白蛋白单域抗体融合蛋白JS-EC21按处方FS3-3配制。
2、给药及血样准备
选取12只Balb/c雌性小鼠，随机分为2组，每组6只动物，分别腹腔注射0.15mg/kg的JS-EC21组和0.15mg/kg的重组人IL-21。在给药前，给药后0.5h，2h，6h，24h，48h，72h和96h，采集小鼠血液于EDTA-K2抗凝管中，3500rpm离心10分钟，收集血浆保存在-80℃冰箱待测。
3、PK检测过程
a.将抗人IL-21,250×Cap.Ab(invitrogen，88-8218-88)为抗原稀释到1×，按100μl/孔包被96孔板，2-8℃孵育过夜。
b.按照200μL/孔加入1×ELISA稀释液，室温封闭1小时。按照300μl/孔使用1×PBST洗板4次。
c.用空白小鼠血浆配置JS-EC21标准曲线样品，范围为312.5ng/mL-20μg/mL；用空白小鼠血浆配置重组人IL-21标准曲线样品，范围为156.3ng/mL-20μg/ml；用空白小鼠血浆稀释待检样品至标准曲线范围内。最后，使用1×ELISA稀释液将上述标准曲线样品和待检样品统一稀释5倍，100μL/孔进行加样。室温孵育2小时后洗板。
d.按照100μL/孔加入稀释到1×的抗人IL-21 250x Det.Ab(invitrogen，88-8218-88)，室温孵育1小时后洗板。
e.按照100μL/孔加入稀释到1×的avidin-HRP(invitrogen，88-8218-88)，室温孵育0.5小时后洗板。
f.按照100μL/孔加入1×TMB显色底物(invitrogen，88-8218-88)，室温避光孵育15分钟后，按照100μL/孔加入2M盐酸溶液终止反应。
g.酶标仪上检测450nm/570nm测吸收值，用SoftMax Pro5.4.1的4参数模型分析检品的血药浓度。
h.使用DAS 3.0软件，选择非房室模型统计矩，对g步骤所得数据进行PK参数拟合。
4、测试结果
如表20所示，本发明小鼠体内给药剂量为0.15mg/kg时，IL-21-融合蛋白JS- EC21的半衰期为15.5h，Cmax为185.2μg/L，AUC (0-t)为3368.1μg/L*h；重组人IL-21的半衰期2.7h，Cmax为3.1μg/L，AUC (0-t)为11.5μg/L*h。本发明IL-21-融合蛋白JS-EC21的药物暴露量更大，半衰期更长，显著优于重组人IL-21。
表20本发明的融合蛋白JS-EC21与重组人IL-21的小鼠腹腔单次给药PK分析表
Figure PCTCN2022079127-appb-000033
注：AUC (0-t)：药物浓度-时间曲线下面积；t 1/2z：末端消除半衰期；C max：药峰浓度。
实施例5：IL-21融合蛋白单用对小鼠MC38皮下移植瘤生长的抑制作用
1、测试目的
评价本发明JS-EC21在小鼠MC38皮下移植模型中的抗肿瘤作用，并与重组人IL-21(rhIL-21)进行比较。本实施例IL-21-抗白蛋白单域抗体融合蛋白JS-EC21按处方FS3-3配制。
2、测试过程
6-8周龄雌性C57BL/6WT小鼠(昭衍(苏州)新药研究中心有限公司)，于右侧背部皮下接种1x 10 6(个/0.1mL)MC38细胞。当肿瘤大小约为91mm 3时，将小鼠随机分为5组(n＝8/组)，分别为：
溶剂对照组：生理盐水；
治疗组：
JS-EC21，0.45mg/kg；
JS-EC21，1.25mg/kg；
JS-EC21，3.75mg/kg；
重组人IL-21，0.625mg/kg。
所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI。TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％，其中，Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤 体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值。
3、测试结果
如图1所示，给药后第21天，生理盐水组平均肿瘤体积为2446mm 3。治疗组中，JS-EC21(0.42mg/kg)组的平均肿瘤体积为1482mm 3，TGI为41.0％；JS-EC21(1.25mg/kg)组的平均肿瘤体积为599mm 3，TGI为78.4％；JS-EC21(3.75mg/kg)组的平均肿瘤体积为390mm 3，TGI为87.3％。重组人IL-21(0.625mg/kg)组的平均肿瘤体积为1698mm 3，TGI为31.8％。与生理盐水组相比，给予1.25mg/kg和3.75mg/kg JS-EC21显著性抑制了小鼠肿瘤生长，且呈现良好的剂量效应。1.25mg/kg的JS-EC21抑瘤作用显著优于等摩尔质量的重组人IL-21(0.625mg/kg)。
实施例6：IL-21融合蛋白JS-EC21在食蟹猴中的药代动力学
1.测试目的
比较本发明JS-EC21融合蛋白与rhIL-21在食蟹猴中的药代动力学，包括半衰期(t 1/2)和AUC等。
2.测试过程
将总共18只食蟹猴(每组6只，雌雄各半，猴龄3.8-4.3岁)随机分配到融合蛋白JS-EC21低剂量、中剂量和高剂量组。通过静脉输注约10分钟，动物在低剂量组静脉注射0.05mg/kg，中剂量组0.15mg/kg，高剂量组0.5mg/kg。给药前，给药后5分钟，输注完成后，以及给药开始后的0.5小时，1小时，2小时，4小时，6小时，8小时，12小时，24小时，36小时，48小时，72小时，120小时(D6)和168小时(D8)，采集3个剂量组的血液样品。所有血液样品均分离为血清。使用经过验证的ELISA方法测定血清中的JS-EC21浓度，定量下限为2ng/mL。使用WinNonlin 8.0中的非房室模型分析(NCA)确定药代动力学参数。
食蟹猴由华珍实验动物繁育中心(中国广州)提供。昭衍实验室(苏州)公司机构动物护理和使用委员会(IACUC)IACUC序列号：ACU20-2031。
3、测试结果
已有文献报道，在评估的剂量范围0.1-0.5mg/kg内rhIL-21在食蟹猴中的药代动力学似乎是剂量依赖性的，并且显示出短的消除半衰期(0.4-0.8h)。为了与rhIL-21进行比较，接下来确定了食蟹猴在低剂量(0.05mg/kg)、中剂量(0.15mg/kg)和高剂量(0.5mg/kg)单次静脉输注JS-EC21后的药代动力学特征。如图2所示，低剂量组、中剂量组和高剂量组的JS-EC21血清浓度呈剂量比例关系。如表21所示，剂量为0.5mg/kg时，JS-EC21的半衰期约为7.08小时，远长于rIL-21的半衰期(0.81小时)。此外，JS-EC21在食蟹猴体内的暴露量显著改善，Cmax和AUC分别比rhIL-21高出近6倍和50倍。这些数据表明与rhIL-21相比，经工程改造的融合蛋白JS-EC21的半衰期显著延长，暴露量显著增加。
表21食蟹猴静脉给与JS-EC21的药代动力学参数
Figure PCTCN2022079127-appb-000034
备注:t 1/2,末端消除半衰期；T max,最大浓度时间；C max,药峰浓度；AUC last,从给药时间到最后一次样本采集的药物浓度-时间曲线下面积；V d,表观分布容积；CL,清除率；MRT,平均停留时间。
a数据来自已发表的论文Waggie KS,Holdren MS,Byrnes-Blake K等，Preclinical safety,pharmacokinetics,and pharmacodynamics of recombinant human interleukin-21 in cynomolgus macaques(Macaca fascicularis)，International journal of toxicology，2012；31(4):303-316。
b无数据。
实施例7：IL-21融合蛋白与PD-1单抗联用对小鼠MC38皮下移植瘤生长的抑制作用
1、测试目的
评价本发明JS-EC21联合PD-1单抗在小鼠MC38皮下移植模型中的抗肿瘤作用。
2、测试过程
6-8周龄雌性hPD-1人源化小鼠(百奥赛图江苏基因生物技术有限公司)，于右侧背部皮下接种1×10 6(个/0.1mL)MC38细胞。当肿瘤大小约为91mm 3时，将小鼠随机分为5组(n＝7/组)，分别为：
溶剂对照组：抗-KLH hIgG4，0.3mg/kg；
治疗组：
PD-1单抗，0.3mg/kg；
JS-EC21，1mg/kg；
PD-1单抗+JS-EC21，0.3mg/kg+1mg/kg；
抗-KLH hIgG4+JS-EC21，0.3mg/kg+1mg/kg。
本实施例IL-21-抗白蛋白单域抗体融合蛋白JS-EC21按处方FS3-3配制；PD-1单抗为特瑞普利单抗(Toripalimab)，其为具有WHO Drug Information(第32卷，第2期，第372-373页(2018))中公开的结构且包含序列SEQ ID NO：15和16所示的轻链及重链氨基酸序列的人源化IgG4mAb。
所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI。TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％，其中Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值。
3、测试结果
如图3所示，给药后第21天，抗-KLH hIgG4组平均肿瘤体积为2008mm 3。PD-1单抗组的平均肿瘤体积为1229mm 3，TGI为41.3％。JS-EC21组的平均肿 瘤体积为1216mm 3，TGI为42.0％。PD-1单抗联合JS-EC21组的平均肿瘤体积为673mm 3，TGI为70.8％。实验结果表明本发明IL-21-融合蛋白JS-EC21与PD-1联用，具有显著的肿瘤抑制作用，且药效优于PD-1抗体(p＜0.001)或JS-EC21单药治疗(p＜0.05)，具有一定的协同效应。
实施例8：IL-21融合蛋白与特瑞普利单抗(Toripalimab)联用对hPD-1人源化小鼠MC38皮下移植瘤生长的抑制作用及其作用机制
1.测试目的
评价本发明JS-EC21联合特瑞普利单抗在小鼠MC38皮下移植瘤模型中的抗肿瘤作用及其联用的药效机制。
2.测试过程
6-8周龄雌性hPD-1人源化小鼠(百奥赛图江苏基因生物技术有限公司)，于右侧背部皮下接种1x 10 6(个/0.1mL)MC38细胞。当肿瘤大小约为91mm 3时，将小鼠随机分为5组，每组7只小鼠(见表22)，开始给药(见表23)。特瑞普利单抗为上海君实生物自制。所有组给药途径均为腹腔注射，每周给药2次，连续给药6次，末次给药3天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI。TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％，其中Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值。其中，表22中组别1为溶剂对照组，组别2、3和4为治疗组。
表22小鼠分组及给药参数
组别 药物 给药剂量 动物数量 给药方法
1 hIgG4同型对照 0.3mg/kg 7 ip
2 JS-EC21 1mg/kg 7 ip
3 特瑞普利单抗 0.3mg/kg 7 ip
4 JS-EC21+特瑞普利单抗 1+0.3mg/kg 7 ip
表23给药时间安排
Figure PCTCN2022079127-appb-000035
Figure PCTCN2022079127-appb-000036
给药后第21天，取出小鼠肿瘤后立即将其转入在冰上放置的50mL离心管中，加入预冷PBS溶液至没过肿瘤。单细胞的制备：取等量(约0.5g)的肿瘤组织，将肿瘤剪碎为2-4mm的小块，通过Tumor Dissociation Kit(鼠：130-096-730)将肿瘤组织制备成单细胞悬液；然后使用CD45(TIL)MicroBeads(鼠：130-110-618)富集TIL细胞。取适量上述细胞，每管加入100微升检测抗体应用液(将PE抗鼠CD8a/FITC抗鼠CD3e/Pacific Blue抗鼠NK1.1母液以1:200的比例用PBS进行稀释，得到检测抗体应用液，现用现配。)，4℃孵育30min；到达作用时间后，以1mL的PBS溶液，500g，5min离心洗涤1次；每管加入400微升1×FOXP3Fixtion/Perm Buffer固定破膜，室温避光20-60min，600g，5min离心洗涤1次；加入400微升1×FOXP3Perm Buffer，600g，5min离心洗涤1次；每管加入100微升检测抗体应用液(将APC抗鼠Ki-67母液以1:200的比例用1×FOXP3Perm Buffe进行稀释，得到检测抗体应用液，现用现配。)，4℃孵育1-2h；到达作用时间后，以1mL的PBS溶液，600g，5min离心洗涤2次；BD流式细胞仪上机检测CD8 +/Ki-67 +T细胞比例。
3、测试结果
如图4所示，给药后第21天，hIgG4同型对照组平均肿瘤体积为2008mm 3。特瑞普利单抗组的平均肿瘤体积为1229mm 3，TGI为41.3％。JS-EC21组的平均肿瘤体积为1216mm 3，TGI为42.0％。特瑞普利单抗联合JS-EC21组的平均肿瘤体积为673mm 3，显著低于特瑞普利单抗组(p＜0.001)以及JS-EC21组(p＜0.05)，TGI为70.8％。如图5所示，称量给药后第21天的肿瘤重量，特瑞普利单抗联合JS-EC21组显著低于特瑞普利单抗组(p＜0.001)以及JS-EC21组(p＜0.05)。因此，这些实验结果表明IL-21-融合蛋白JS-EC21与特瑞普利单抗联用，具有显著的肿瘤抑制作用，且药效优于特瑞普利单抗或JS-EC21单药治疗，具有一定的协同效应。
使用流式细胞仪对肿瘤组织进行免疫细胞分型研究，对于免疫细胞(CD45 +)中CD3 +T细胞的比例，如图6(A)所示，联合给药组显著高于特瑞普利单抗 给药组(p＜0.01)或JS-EC21给药组(p＜0.01)；对于CD3 +T细胞中CD8 +T细胞的比例，如图6(B)所示，联合给药组显著高于特瑞普利单抗给药组(p＜0.05)或JS-EC21给药组(p＜0.01)；对于处于增殖状态(Ki 67 +)的CD8 +T细胞的比例，如图6(C)所示，联合给药组显著高于特瑞普利单抗给药组(p＜0.05)或JS-EC21给药组(p＜0.05)；对于免疫细胞(CD45 +)中NK细胞的比例，如图6(D)所示，联合给药组显著高于特瑞普利单抗给药组(p＜0.05)或JS-EC21给药组(p＜0.05)。这些研究结果表明特瑞普利单抗与JS-EC21联用可以显著增加肿瘤浸润的CD8 +T细胞和NK细胞的比例，进而增强抗肿瘤免疫应答。

Claims (16)

  1. 药物组合物，包含：
    (1)缓冲液；和
    (2)IL-21-抗白蛋白单域抗体融合蛋白；
    其中所述IL-21-抗白蛋白单域抗体融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，所述IL-21包括如SEQ ID NO:1所示的氨基酸序列，所述单域抗体(sdAb)包含氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的HCDR1、HCDR2和HCDR3。
  2. 如权利要求1所述的药物组合物，其中所述单域抗体融合至所述细胞因子的C末端，且所述细胞因子和所述单域抗体直接相连；
    优选地，所述单域抗体(sdAb)包含如SEQ ID NO:5所示的氨基酸序列。
  3. 如权利要求1或2所述的药物组合物，其中所述缓冲液选自醋酸缓冲液、柠檬酸缓冲液和组氨酸缓冲液中的一种或多种；优选地，所述缓冲液选自醋酸-醋酸钠缓冲液、醋酸-醋酸钾缓冲液、柠檬酸-柠檬酸钠缓冲液、组氨酸-醋酸缓冲液和组氨酸-盐酸盐缓冲液中的一种或多种；更优选地，所述缓冲液为组氨酸-醋酸缓冲液和醋酸-醋酸钠缓冲液，优选为醋酸-醋酸钠缓冲液。
  4. 如权利要求1-3任一项所述的药物组合物，其中所述缓冲液的浓度约为1-200mM，优选约为5-200mM，优选约为10-50mM，优选约为10-30mM，优选约为10-20mM；优选地，所述缓冲液的pH约为4.0-6.5，优选约为4.5-5.5，优选4.5-5.1，优选约为4.5-5.0，优选约为4.7-5.0，更优选为约4.7或4.8。
  5. 如权利要求1或2所述的药物组合物，其中所述药物组合物还包括稳定剂，所述稳定剂选自氯化钠、甘露醇、山梨醇、蔗糖、和海藻糖中的一种或多种；优选地，所述稳定剂为海藻糖。
  6. 如权利要求5所述的药物组合物，其中所述稳定剂的浓度为约10mM-400mM，优选20mM-300mM，更优选30mM-200mM；优选地，所述稳定剂为浓度约100-300mM的甘露醇，所述甘露醇的浓度优选约为150-300mM，优选约为200-280mM，优选为约200mM，210mM，220mM，230mM，240mM，250mM，260mM，270mM或280mM，更优选为240mM；或所述稳定剂为浓度约100-300mM的蔗糖， 所述蔗糖的浓度优选约为150-300mM，优选约为200-280mM，优选为约200mM，210mM，220mM，230mM，235mM，240mM，250mM，260mM，270mM，280mM，更优选为235mM；或所述稳定剂为浓度约100-300mM的海藻糖，所述海藻糖的浓度优选约为150-300mM，优选约为200-280mM，优选为约180mM，200mM，210mM，220mM，230mM，235mM，240mM，250mM，260mM，270mM，280mM，更优选为235mM；或所述稳定剂为约30-200mM的氯化钠与约30-200mM的甘露醇的组合；或所述稳定剂为约30-200mM的氯化钠与约30-200mM的蔗糖的组合；优选地，所述稳定剂为约100-300mM的海藻糖；更优选地，所述稳定剂为约200-280mM的海藻糖。
  7. 如权利要求1-6任一项所述的药物组合物，其中所述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨醇酯80、聚山梨醇酯20或泊洛沙姆188，优选地，所述表面活性剂为聚山梨醇酯80。
  8. 如权利要求7所述的药物组合物，其中所述表面活性剂浓度约为0.001％-0.1％，优选约为0.01％-0.1％，优选约为0.02％-0.08％，优选约为0.01％-0.05％。
  9. 如权利要求1-8任一项所述的药物组合物，其中，所述IL-21-抗白蛋白单域抗体融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，其中所述IL-21包括如SEQ ID NO:1所示的氨基酸序列，且所述单域抗体(sdAb)包含如SEQ ID NO:5所示的氨基酸序列。
  10. 如权利要求1-9任一项所述的药物组合物，其中所述IL-21-抗白蛋白单域抗体融合蛋白包含如SEQ ID NO:6所示的氨基酸序列。
  11. 如权利要求1-10任一项所述的药物组合物，其中所述IL-21-抗白蛋白单域抗体融合蛋白浓度约为0.1-100mg/mL，优选约为0.2-20mg/mL，优选约为0.2-10mg/mL，优选约为0.5-5mg/mL，优选约为1-5mg/mL，更优选约为0.5-1mg/mL；更优选地，所述IL-21-抗白蛋白单域抗体融合蛋白浓度约为0.5mg/mL，0.8mg/mL，1mg/mL，1.2mg/mL，1.5mg/mL，1.8mg/mL，2mg/mL，2.5mg/mL，3mg/mL，3.5mg/mL，4mg/mL，4.5mg/mL，5mg/mL，6mg/mL，7mg/mL，8mg/mL或10mg/mL，优选约为1mg/mL。
  12. 如权利要求1-11任一项所述的药物组合物，其包含如下(1)-(6)任一项所示的组分：
    (1)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM醋酸-醋酸钠缓冲液，pH约为4.5-5.5；(c)约100-300mM的海藻糖；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
    (2)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM醋酸-醋酸钠缓冲液，pH约为4.5-5.5；(c)约100-300mM的蔗糖；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
    (3)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM醋酸-醋酸钠缓冲液，pH约为4.5-5.5；(c)约100-300mM的甘露醇；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
    (4)(a)约0.5mg/mL-5mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约10-30mM组氨酸缓冲液，pH约为4.5-5.5；(c)约30-200mM的氯化钠和约30-200mM的甘露醇；(d)以及约0.01％-0.1％的聚山梨醇酯80；或
    (5)(a)约1mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约20mM醋酸-醋酸钠缓冲液，pH约为4.8；(c)约235mM的海藻糖；(d)以及约0.02％的聚山梨醇酯80；或
    (6)(a)约1mg/mL的IL-21-抗白蛋白单域抗体融合蛋白；(b)约20mM醋酸-醋酸钠缓冲液，pH约为4.8；(c)约235mM的蔗糖；(d)以及约0.02％的聚山梨醇酯80。
  13. IL-21-抗白蛋白单域抗体融合蛋白、或如权利要求1-12任一项所述的药物组合物、或IL-21-抗白蛋白单域抗体融合蛋白和免疫检查点调节剂的组合或如权利要求1-12任一项所述的药物组合物和免疫检查点调节剂的组合在制备用于预防或治疗肿瘤的药物中的用途，或IL-21-抗白蛋白单域抗体融合蛋白或如权利要求1-12任一项所述的药物组合物在制备增强免疫检查点调节剂的抗肿瘤活性的药物中的用途；
    优选地，所述IL-21-抗白蛋白单域抗体融合蛋白包含：(a)细胞因子IL-21，和(b)特异性结合白蛋白的单域抗体(sdAb)，所述IL-21包括如SEQ ID NO:1所示的氨基酸序列，所述单域抗体(sdAb)包含氨基酸序列分别如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的HCDR1、HCDR2和HCDR3；
    优选地，所述单域抗体(sdAb)包含如SEQ ID NO:5所示的氨基酸序列；
    优选地，所述单域抗体融合至所述细胞因子的C末端，且所述细胞因子和所述单域抗体直接相连；
    优选地，所述IL-21-抗白蛋白单域抗体融合蛋白包含如SEQ ID NO:6所示的氨基酸序列；
    优选地，所述IL-21-抗白蛋白单域抗体融合蛋白与免疫检查点调节剂组合使用；
    优选地，所述免疫检查点调节剂选自PD-1、PD-L1、PD-L2、CTLA-4、4-1BB、CD47、TIGIT、GITR、CD112R、BTLA、TIM3、LAG3、CD27和B7H4免疫检查点抑制剂；
    优选地，所述免疫检查点调节剂选自抗PD-1抗体或抗TIGIT抗体；
    优选地，所述抗PD-1抗体选自nivolumab、pembrolizumab、toripalimab、Sintilimab、Camrelizumab、Tislelizumab、Cemiplimab中的一种或一种以上；优选为toripalimab；
    优选地，所述抗PD-1抗体包含：
    (1)氨基酸序列如SEQ ID NO：7、8和9所示的轻链互补决定区，和氨基酸序列如SEQ ID NO：10、11和12所示的重链互补决定区；或
    (2)氨基酸序列如SEQ ID NO:13所示的轻链可变区，和氨基酸序列如SEQ ID NO:14所示的重链可变区；或
    (3)氨基酸序列如SEQ ID NO:15所示的轻链，和氨基酸序列如SEQ ID NO:16所示的重链；
    优选地，所述抗TIGIT抗体选自：Tiragolumab、Etigilimab、Vibostolimab、Domvanalimab、EOS-884448和BMS-986207；
    优选地，相比IL-21-抗白蛋白单域抗体融合蛋白或抗PD-1抗体单独使用，所述IL-21-抗白蛋白单域抗体融合蛋白与抗PD-1抗体联合使用具有选自以下的一种或多种性质：
    (1)免疫细胞中CD3 +T细胞的比例显著提高；
    (2)T细胞中CD8 +T细胞的比例显著提高；
    (3)增殖状态(Ki 67 +)的CD8 +T细胞的比例显著提高；和
    (4)免疫细胞中NK细胞的比例显著提高；
    优选地，相比IL-21-抗白蛋白单域抗体融合蛋白或抗TIGIT抗体单独使用，所述IL-21-抗白蛋白单域抗体融合蛋白与抗TIGIT抗体联合使用具有选自以下的一种或多种性质：
    (1)显著增加干扰素γ的表达水平；
    (2)显著增加颗粒酶A的表达水平；
    (3)显著增加颗粒酶B的表达水平；
    (4)显著增加穿孔素1的表达水平；
    (5)富集更多具有差异基因表达的KEGG信号通路；和
    (6)诱导的大部分显著富集的KEGG信号通路由IL-21-抗白蛋白单域抗体融合蛋白介导；
    优选地，所述肿瘤为与IL-21相关的肿瘤，或为免疫检查点相关的肿瘤；
    优选地，所述肿瘤选自间皮瘤、肺癌、乳腺癌、卵巢癌、黑色素瘤、肾癌、胰腺癌、淋巴瘤、白血病、头颈癌、肝癌、非霍奇金淋巴瘤、食管癌、胃癌和结肠直肠癌；
    优选地，所述肿瘤为结肠直肠癌。
  14. 药物组合，包含：
    (1)免疫检查点调节剂；和
    (2)IL-21-抗白蛋白单域抗体融合蛋白或如权利要求1-12任一项所述的药物组合物；
    其中，所述IL-21-抗白蛋白单域抗体融合蛋白如权利要求13所述；
    优选地，所述免疫检查点调节剂如权利要求13所述。
  15. 预防或治疗肿瘤的方法，所述方法包含向有需要的个体施用有效量的如权利要求13所述IL-21-抗白蛋白单域抗体融合蛋白，或如权利要求13所述IL-21-抗白蛋白单域抗体融合蛋白以及如权利要求13所述的免疫检查点调节剂，或如权利要求1-12所述药物组合物以及如权利要求13所述的免疫检查点调节剂，或如权利要求14所述的药物组合。
  16. 试剂盒，其包含：
    (1)一个或多个单次药物剂量单元的IL-21-抗白蛋白单域抗体融合蛋白和一个或多个单次药物剂量单元的免疫检查点调节剂；其中所述IL-21-抗白蛋白单域抗 体融合蛋白如权利要求13所述；优选地，所述免疫检查点调节剂如权利要求13所述；或
    (2)一个或多个单次药物剂量单元的如权利要求14所述的药物组合。
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