WO2022184148A1 - Composition pharmaceutique de protéine de fusion d'anticorps à domaine unique il-21-anti-albumine et son utilisation - Google Patents

Composition pharmaceutique de protéine de fusion d'anticorps à domaine unique il-21-anti-albumine et son utilisation 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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Chinese (zh)
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刘洪川
刘辉
张静
孟琴
刘沛想
李秀鹏
赵文亭
冯辉
姚盛
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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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    • 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
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    • 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
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    • 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
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • 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
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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

L'invention concerne une composition pharmaceutique de protéine de fusion d'anticorps à domaine unique d'IL-21-anti-albumine et son utilisation. La composition pharmaceutique comprend une protéine de fusion d'anticorps à domaine unique d'IL-21-anti-albumine et une solution tampon, peut en outre comprendre au moins un stabilisant, et peut éventuellement comprendre en outre un tensioactif. La protéine de fusion d'anticorps à domaine unique d'IL-21-anti-albumine a une stabilité élevée, et le médicament unique associé et l'utilisation de celui-ci en combinaison avec l'anticorps monoclonal PD-1 exerce une activité antitumorale supérieure.
PCT/CN2022/079127 2021-03-03 2022-03-03 Composition pharmaceutique de protéine de fusion d'anticorps à domaine unique il-21-anti-albumine et son utilisation WO2022184148A1 (fr)

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