WO2023165553A1 - IL2突变体-抗体Fc嵌段融合蛋白的药物组合物及其应用 - Google Patents

IL2突变体-抗体Fc嵌段融合蛋白的药物组合物及其应用 Download PDF

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WO2023165553A1
WO2023165553A1 PCT/CN2023/079252 CN2023079252W WO2023165553A1 WO 2023165553 A1 WO2023165553 A1 WO 2023165553A1 CN 2023079252 W CN2023079252 W CN 2023079252W WO 2023165553 A1 WO2023165553 A1 WO 2023165553A1
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pharmaceutical composition
fusion protein
mutant
disease
cancer
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PCT/CN2023/079252
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English (en)
French (fr)
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李胜男
王帅
李鑫鑫
严玉玺
胡建中
唐任宏
任晋生
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海南先声药业有限公司
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Publication of WO2023165553A1 publication Critical patent/WO2023165553A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to the field of pharmaceutical preparations, in particular to the pharmaceutical composition of IL2 mutant-antibody Fc block fusion protein and its application.
  • Interleukin 2 was originally identified as T cell growth factor (TCGF). Studies have found that IL-2 can bind to its receptor to activate the proliferation and activation of immune cells such as T cells and NK cells.
  • IL-2 receptors include IL-2R ⁇ subunit (CD25), IL-2R ⁇ subunit (CD122) and IL-2R ⁇ subunit (CD132). Different subunits can form receptor complexes with different affinity, including High affinity receptor IL-2R ⁇ , intermediate affinity receptor IL-2R ⁇ , low affinity receptor IL-2R ⁇ or IL-2R ⁇ . The types of IL-2R subunits expressed by different cells are different.
  • the surface of traditional T cells CD4 + T and CD8 + T cells under resting conditions generally express IL-2 receptor ⁇ (IL-2R ⁇ , CD122), IL- 2 receptor ⁇ (IL-2R ⁇ , CD132), almost no expression of IL-2 receptor ⁇ (IL-2R ⁇ , CD25), and regulatory T cells (Treg) in addition to expressing IL-2R ⁇ , IL-2R ⁇ , composed of High expression of IL-2R ⁇ .
  • IL2 is a macromolecular protein, which is prone to stability problems such as particles and aggregation in solution. Therefore, after obtaining a new type of IL2 mutant, it is of great significance to develop a preparation system suitable for the new type of IL2 mutant and ensure the stability of the new type of IL2 mutant in solution.
  • the invention provides a pharmaceutical composition and a preparation method thereof, a freeze-dried preparation and a preparation or reconstitution method thereof, a reconstitution solution obtained according to the reconstitution method, corresponding pharmaceutical applications, a method for treating autoimmune diseases and products.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody Fc block fusion protein comprising an IL2 mutant, a buffer, an osmotic pressure regulator and a surfactant;
  • the IL2 mutant at least includes Y31V, A73L and H79Q mutations;
  • the IL2 mutant further includes one or more mutations among V91R mutation, H16E mutation and D20A mutation, for example, further includes H16E mutation and V91R mutation;
  • the IL2 mutant has an amino acid sequence that is at least 80% identical to any of the sequences shown in SEQ ID NO: 3, 8-11, such as at least 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • amino acid sequence of the wild-type IL2 is shown in SEQ ID NO: 2.
  • the fusion protein includes, from the N-terminus to the C-terminus, in order:
  • the fusion protein forms a homodimer through dimerization of antibody Fc block
  • the linker has an amino acid sequence of (G 4 S) n , where n is selected from 1, 2, 3, 4, 5 or 6;
  • the linker has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 12, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% identity;
  • the antibody Fc block includes a N297G mutation
  • the antibody Fc block has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 13, such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;
  • the fusion protein has an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 14-18, such as at least 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the wild-type IL2 has the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • the concentration of the fusion protein is about 1-50 mg/mL
  • the concentration of the fusion protein is about 1-40 mg/mL, 1-30 mg/mL, 1-20 mg/mL, 1-15 mg/mL, 1-10 mg/mL, 5-50 mg/mL, 5-40 mg/mL mL, 5 ⁇ 30mg/mL, 5 ⁇ 20mg/mL, 5 ⁇ 19mg/mL, 5 ⁇ 18mg/mL, 5 ⁇ 17mg/mL, 5 ⁇ 16mg/mL, 5 ⁇ 15mg/mL, 5 ⁇ 14mg/mL, 5 ⁇ 13mg/mL, 5 ⁇ 12mg/mL, 5 ⁇ 11mg/mL, 5 ⁇ 10mg/mL, 5 ⁇ 9mg/mL, 5 ⁇ 8mg/mL, 5 ⁇ 6mg/mL, 10 ⁇ 50mg/mL, 10 ⁇ 40mg/mL, 10 ⁇ 30mg/mL, 10 ⁇ 20mg/mL, 10 ⁇ 15mg/mL, 20 ⁇ 50mg/mL, 20 ⁇
  • the concentration of the fusion protein is about 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 23mg/mL mL, 24mg/mL, 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, 36mg/mL,
  • the concentration of the fusion protein is about 2 mg/mL or 5 mg/mL.
  • the buffer is selected from acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, histidine-histidine hydrochloride, succinic acid-sodium succinate, or hydrogen phosphate Disodium-sodium dihydrogen phosphate buffer, preferably acetic acid-sodium acetate buffer; and/or,
  • the concentration of the buffer is about 1-100mM
  • the concentration of the buffer is about 1-90mM, 1-80mM, 1-70mM, 1-60mM, 1-50mM, 1-40mM, 1-30mM, 1-20mM, 1-10mM, 10-90mM, 10 ⁇ 80mM, 10 ⁇ 70mM, 10 ⁇ 60mM, 10 ⁇ 50mM, 10 ⁇ 40mM, 10 ⁇ 30mM, 10 ⁇ 20mM, 20 ⁇ 90mM, 20 ⁇ 80mM, 20 ⁇ 70mM, 20 ⁇ 60mM, 20 ⁇ 50mM, 20 ⁇ 40mM, 20 ⁇ 30mM, 30 ⁇ 90mM, 30 ⁇ 80mM, 30 ⁇ 70mM, 30 ⁇ 60mM, 30 ⁇ 50mM, 30 ⁇ 40mM, 40 ⁇ 90mM, 40 ⁇ 80mM, 40 ⁇ 70mM, 40 ⁇ 60mM, 40 ⁇ 50mM, 50 ⁇ 90mM, 50 ⁇ 80mM, 50 ⁇ 70mM, 50 ⁇ 60mM, 60 ⁇ 90mM, 60 ⁇ 80mM, 60 ⁇ 70mM, 70 ⁇ 90
  • the concentration of the buffer is about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM; Or the concentration range value formed between the above concentration values;
  • the buffer has a concentration of about 20 mM; and/or,
  • the pH value of the buffer is about 4.5-8.0
  • the pH value of the buffer solution is about 4.5-7.5, 4.5-7.0, 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, 5.0-8.0, 5.0-7.5, 5.0-7.0, 5.0-6.5, 5.0 ⁇ 6.0, 5.0 ⁇ 5.5, 5.5 ⁇ 8.0, 6 ⁇ 7.5, 6.5 ⁇ 7.5, 7.0 ⁇ 7.5 or 7.5 ⁇ 8.0;
  • the buffer solution has a pH value of about, about 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0, or a concentration range value formed between the above pH values;
  • the buffer has a pH of about 5.5.
  • the osmotic pressure regulator is selected from salts, amino acids, sugars or sugar alcohols or combinations thereof; preferably, the salts are selected from sodium chloride, potassium chloride or arginine hydrochloride; preferably , the amino acid is selected from glycine, arginine, histidine, glutamic acid or methionine; preferably, the sugar or sugar alcohol is selected from sucrose, trehalose, mannitol or sorbitol;
  • the osmotic pressure regulator is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol or sorbitol, more preferably sucrose or arginine hydrochloride;
  • the concentration of the sugar or sugar alcohol is about 1-15%w/v, and the concentration of the salt is about 50-200mM; for example, the concentration of the sugar or sugar alcohol is about 1-10%w /v, 1-5% w/v or 5-10% w/v; for example, the concentration of said sugar or sugar alcohol is about 1% w/v, 1.5% w/v, 2% w/v, 2.5 %w/v, 3%w/v, 3.5%w/v, 4%w/v, 4.5%w/v, 5%w/v, 5.5%w/v, 6%w/v, 6.5%w /v, 7%w/v, 7.5%w/v, 8%w/v, 8.5%w/v, 9%w/v, 9.5%w/v, 10%w/v, 10.5%w/v , 11%w/v, 11.5%w/v, 12%w/v, 12.5%w/v, 13%w/v, 13.5%w/v, 14%w/v, 14.5%w/v, 10%
  • the amino acid concentration is about 1-15% w/v, for example, the amino acid concentration is about 1-10% w/v, 1-5% w/v or 5-10% w/v ; for example, the concentration of the amino acid is about 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/ v, 4.5%w/v, 5%w/v, 5.5%w/v, 6%w/v, 6.5%w/v, 7%w/v, 7.5%w/v, 8%w/v, 8.5%w/v, 9%w/v, 9.5%w/v, 10%w/v, 10.5%w/v, 11%w/v, 11.5%w/v, 12%w/v, 12.5% w/v, 13% w/v, 13.5% w/v, 14% w/v, 14.5% w/v, 15% w/v, or the concentration range value composed between
  • the concentration of the salt is about 50-200mM; for example, the concentration of the salt is about 50-150mM, 50-100mM or 100-150mM; for example, the concentration of the salt is about 50mM, 60mM, 70mM . Preferably 140 mM.
  • the osmotic pressure regulator is sucrose or arginine hydrochloride.
  • the surfactant is polysorbate-80 (PS-80), polysorbate 20 (PS-20) or poloxamer; and/or,
  • the concentration of the surfactant is about 0.01-0.1% w/v, for example, 0.01% w/v-0.05% w/v, 0.05% w/v-0.1% w/v, 0.02% w/v-0.08 %w/v, for example about 0.01%w/v, 0.02%w/v, 0.03%w/v, 0.04%w/v, 0.05%w/v, 0.06%w/v, 0.07%w/v, 0.08% w/v, 0.09% w/v or 1% w/v, or a concentration range value composed between the above concentration values.
  • the pharmaceutical composition comprises the fusion protein, acetic acid-sodium acetate buffer, sucrose and polysorbate-80; preferably, the pharmaceutical composition comprises about 1-50 mg/mL of the Fusion protein, about 1-100 mM acetic acid-sodium acetate buffer, about 1-15% w/v sucrose and about 0.01-0.1% w/v polysorbate-80, the pH value is about 4.5-6.0;
  • the pharmaceutical composition comprises about 1-30 mg/mL, 5-30 mg/mL, 10-30 mg/mL, 10-25 mg/mL, 10-20 mg/mL, 10-15 mg/mL, 15-20 mg /mL, 15-30 mg/mL or 20-30 mg/mL of the fusion protein, about 10-50 mM or 10-30 mM acetic acid-sodium acetate buffer, about 1-15% w/v, 1-10% w/v , 5-15% w/v, 5-10% w/v or 10-15% w/v sucrose and about 0.01-0.1% w/v, 0.02-0.06% w/v polysorbate-80, the pH value is about 4.5 ⁇ 6.0 or 4.5 ⁇ 5.5;
  • the pharmaceutical composition comprises about 1-10 mg/mL of the fusion protein, about 10-30 mM acetic acid-sodium acetate buffer, about 5-10% w/v sucrose and about 0.01-0.1% w/v poly Sorbitate-80, the pH value is about 4.5-6.0;
  • the pharmaceutical composition comprises about 2 mg/mL or 5 mg/mL of the fusion protein, about 20 mM acetic acid-sodium acetate buffer, about 8% w/v sucrose and about 0.04% w/v polysorbate-80 , pH is about 5.5.
  • the pharmaceutical composition includes fusion protein, acetic acid-sodium acetate buffer, arginine hydrochloride and polysorbate-80; preferably, the pharmaceutical composition includes about 1-50 mg/mL of the said fusion protein, about 1-100 mM acetic acid-sodium acetate buffer, about 50-200 mM arginine hydrochloride and about 0.01-0.1% w/v polysorbate-80, with a pH value of about 4.5-6.0;
  • the pharmaceutical composition comprises about 1-30 mg/mL, 5-30 mg/mL, 10-30 mg/mL, 10-25 mg/mL, 10-20 mg/mL, 10-15 mg/mL, 15-20 mg /mL, 15-30mg/mL or 20-30mg/mL fusion protein, about 10-50mM or 10-30mM acetic acid-sodium acetate buffer, about 50-200mM, 50-150mM, 100-150mM arginine hydrochloride and about 0.01-0.1% w/v, 0.02-0.06% w/v polysorbate-80, with a pH of about 4.5-6.0 or 4.5-6.0;
  • the pharmaceutical composition comprises about 1-10 mg/mL of the fusion protein, about 10-30 mM acetic acid-sodium acetate buffer, about 120-150 mM arginine hydrochloride and about 0.01-0.1% w/v polysorbate Ester-80, the pH value is about 4.5-6.0.
  • the pharmaceutical composition comprises about 2 mg/mL or 5 mg/mL of the fusion protein, about 20 mM acetic acid-sodium acetate buffer, about 140 mM arginine hydrochloride and about 0.04% w/v or 0.06% w/v Polysorbate-80 with a pH of approximately 5.5.
  • the pharmaceutical composition is intravenous, intramuscular or subcutaneous injection, preferably subcutaneous injection.
  • the present invention also provides the preparation method of the aforementioned pharmaceutical composition, the method includes the step of mixing the fusion protein with the buffer, the osmotic pressure regulator and the surfactant; preferably, the methods include The stock solution of the fusion protein is concentrated and replaced into the buffer by ultrafiltration.
  • the present invention also provides a freeze-dried preparation, wherein the freeze-dried preparation is formed by freeze-drying the aforementioned pharmaceutical composition.
  • the present invention also provides a method for preparing the aforementioned freeze-dried preparation, wherein the method includes the step of freeze-drying the aforementioned pharmaceutical composition.
  • the present invention also provides a method for preparing a reconstitution solution of an antibody Fc block fusion protein containing an IL2 mutant, which includes reconstituting the aforementioned lyophilized preparation with a solvent, preferably, the solvent is water for injection.
  • the present invention also provides the reconstitution solution of the antibody Fc block fusion protein containing IL2 mutant prepared according to the aforementioned method.
  • the present invention also provides the application of the aforementioned pharmaceutical composition, freeze-dried preparation or reconstitution solution in the preparation of drugs for treating autoimmune diseases or proliferative diseases;
  • the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, sclerosis Skin disease, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ Transplant rejection, autoimmune hepatitis, type 1 diabetes, autoimmune vasculitis, eczema, or asthma;
  • the proliferative disease is selected from neoplasm, solid tumor, hematoma, malignant ascites or malignant pleural effusion; wherein, the solid tumor can be benign or malignant, primary or metastatic, and the malignant solid tumor can be Carcinoma or sarcoma, for example, epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung neoplasm, papillomavirus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, angiosarcoma, neuroblastoma tumor, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer;
  • the hematological tumor may be selected from leukemia, lymphoma, multiple myeloma, eg B cell lymphoma, T cell lymphom
  • the present invention also provides the aforementioned pharmaceutical composition, lyophilized preparation or reconstitution solution for treating autoimmune or proliferative diseases;
  • the autoimmune disease is selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, sclerosis Skin disease, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ Transplant rejection, autoimmune hepatitis, type 1 diabetes, autoimmune vasculitis, eczema, or asthma;
  • the proliferative disease is selected from neoplasm, solid tumor, hematoma, malignant ascites or malignant pleural effusion; wherein, the solid tumor can be benign or malignant, primary or metastatic, and the malignant solid tumor can be Carcinoma or sarcoma, for example, epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung neoplasm, papillomavirus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, angiosarcoma, neuroblastoma tumor, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer;
  • the hematological tumor may be selected from leukemia, lymphoma, multiple myeloma, eg B cell lymphoma, T cell lymphom
  • the present invention also provides a method for treating an autoimmune disease, wherein the method comprises administering to the subject an effective amount of the aforementioned pharmaceutical composition or reconstitution solution; preferably, the autoimmune disease Selected from rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft versus host disease, organ transplant rejection, autoimmune hepatitis, type I Diabetes, autoimmune vasculitis, eczema, or asthma.
  • the autoimmune disease
  • the effective amount is 0.001-10 mpk, such as 0.001 mpk, 0.002 mpk, 0.003 mpk, 0.004 mpk, 0.005 mpk, 0.006 mpk, 0.007 mpk, 0.008 mpk, 0.009 mpk, 0.01 mpk, 0.02 mpk ,0.03mpk,0.04mpk,0.05mpk,0.06mpk,0.07mpk,0.08mpk,0.09mpk,0.1mpk,0.2mpk,0.3mpk,0.4mpk,0.5mpk,0.6mpk,0.7mpk,0.8mpk,0.9mpk,1mpk , 2mpk, 3mpk, 4mpk, 5mpk, 6mpk, 7mpk, 8mpk, 9mpk or 10mpk, or the effective amount range value formed between the above effective amounts.
  • the present invention also provides a method for treating proliferative diseases, wherein the method comprises administering to the subject an effective amount of the aforementioned pharmaceutical composition or reconstitution solution; preferably, the proliferative diseases are selected from Biological, solid tumor, hematological tumor, malignant ascites or malignant pleural effusion; wherein, the solid tumor can be benign or malignant, primary or metastatic, and the malignant solid tumor can be carcinoma or sarcoma, for example, epithelial cell carcinoma, Endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, papillomavirus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, angiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, Small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle invasive bladder cancer; said hematological
  • the present invention also provides a product comprising a container filled with the aforementioned pharmaceutical composition, freeze-dried preparation or reconstitution solution.
  • IL2 refers to any vertebrate source, including mammals such as primates (such as humans) and rodents (such as mice and rats) and any native or recombinant IL-2 of a domesticated or farmed mammal.
  • the "IL2” or “IL-2” in the present invention includes unprocessed mature IL-2 (such as IL-2 containing N-terminal signal peptide) and any form of IL-2 that is processed and matured in cells.
  • the "IL2” or “IL-2” in the present invention also includes natural variants and fragments of IL-2, such as splice variants or allelic variants.
  • the "IL2" or “IL-2” mentioned in the present invention also includes non-naturally occurring mutants, such as IL-2 mutants artificially modified by genetic engineering.
  • a “wild-type IL-2” is a form of IL-2 that is otherwise identical to an IL-2 mutant except that a wild-type amino acid is maintained at each mutated amino acid position of the IL-2 mutant.
  • the IL-2 mutant is unprocessed mature IL-2, then the wild-type form of the mutant is unprocessed mature IL-2; if the IL-2 mutant is processed mature IL-2, The wild-type form of the mutant is then processed mature IL-2; if the IL-2 mutant is a truncated form of IL-2, then the wild-type form of the mutant is the corresponding IL-2 with the wild-type sequence truncated form.
  • the "wild-type IL-2" of the present invention may have the following amino acid sequence: Wherein the 125th amino acid residue "X" represents C, S, A or V.
  • mutation in the present invention includes amino acid substitution, deletion, insertion or any combination thereof.
  • the “mutation” in the present invention can use genetic or chemical methods known in the art to generate amino acid mutations, including but not limited to site-directed mutagenesis, PCR, gene synthesis and other methods.
  • the "mutation site” numbering of the IL-2 mutant of the present invention is counted from the first amino acid residue A of the "wild-type IL-2" shown in SEQ ID NO:1.
  • the "Y31 mutation” of the present invention refers to the IL-2 mutant having a mutation at the 31st amino acid residue (Tyr, Y) of wild-type IL-2 shown in SEQ ID NO:1.
  • the "Y31V mutation” of the present invention means that the amino acid residue at position 31 of wild-type IL-2 shown in SEQ ID NO: 1 is mutated from Y (Tyr) to V (Val) in the IL-2 mutant.
  • Y31/A73/H79 refers to mutations at the Y31 site, A73 site and H79 site at the same time in the same IL-2 mutant
  • Y31V/A73L/H79Q refers to mutations in the same IL-2 mutant.
  • the -2 mutant has Y31V, A73L and H79Q mutations at the same time.
  • fusion protein in the present invention refers to the protein product obtained by linking the coding regions of two or more genes by gene recombination methods, chemical methods or other appropriate methods, and expressing gene recombination under the control of the same regulatory sequence.
  • the coding regions of two or more genes may be fused at one or several positions by a sequence encoding a connecting peptide. Connecting peptides can be used to construct fusion proteins of the invention.
  • Linker in the present invention refers to a peptide used to link IL-2 with another protein molecule or protein fragment in the present invention to ensure correct folding and stability of the protein.
  • the other molecule includes, but is not limited to, an antibody Fc block.
  • the "linker” of the present invention is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5 or 6.
  • antibody Fc block in the present invention refers to the constant region of the immunoglobulin chain, especially the carboxy-terminal or part of the constant region of the heavy chain of the immunoglobulin, which has no antigen-binding activity and is the key to the interaction between antibody molecules and effector molecules or cells.
  • the "Fc” of the present invention may be any Fc or its variants, derived from human or non-human mammals.
  • an immunoglobulin Fc may comprise two or more domains of heavy chains CH1, CH2, CH3, CH4 in combination with an immunoglobulin hinge region.
  • Fc can be derived from different species, preferably human immunoglobulins.
  • immunoglobulins can be divided into different classes, and there are mainly 5 classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. Some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2.
  • "Fc" preferably includes at least one immunoglobulin hinge region, and the CH2 and CH3 domains of IgG. More preferably, it includes a CH2 domain of IgG1, a CH3 domain and an immunoglobulin hinge region, and the initial amino acid position of the hinge region can be changed.
  • amino acid residues in the Fc, constant region or antibody of the present invention are determined according to Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991
  • the EU numbering system described in also known as the EU Index, is numbered.
  • composition refers to a mixture containing one or more IL-2 mutants, fusion proteins, nucleic acid fragments, vectors or host cells of the present invention, said mixture also contains other components, said Other components include, but are not limited to, pharmaceutically acceptable carriers, diluents or adjuvants thereof.
  • pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable carriers, diluents or adjuvants thereof.
  • the purpose of the pharmaceutical composition of the present invention is to promote the The administration of organisms is beneficial to the absorption of active ingredients and then exerts their biological activity.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) unwanted physiological changes or lesions in the subject of treatment, such as autoimmune diseases (such as systemic erythema lupus) progression.
  • autoimmune diseases such as systemic erythema lupus
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject of the present invention refers to an organism receiving treatment for a particular disease or disorder (such as an autoimmune disease) as described herein.
  • a particular disease or disorder such as an autoimmune disease
  • subjects and patients include mammals, such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, cows or other members of the bovid family, sheep, horses, and the like, receiving treatment for a disease or condition.
  • an effective amount in the present invention refers to the amount of a therapeutic agent that can effectively prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or object.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • autoimmune disease refers to a condition in a subject characterized by cell, tissue and/or organ damage caused by the subject's immune response to its own cells, tissues and/or organs.
  • autoimmune diseases include but are not limited to rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, sclerosis Skin disease, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ Transplant rejection, autoimmune hepatitis, type 1 diabetes, autoimmune vasculitis, ec
  • proliferative disease refers to unregulated and/or abnormal growth of cells or tissues that can lead to the development of an unwanted condition or disease, which may or may not be cancerous, including but not limited to neoplastic Biological, solid tumor, hematological tumor, malignant ascites or malignant pleural effusion.
  • the "solid tumors" of the present invention can be benign or malignant, primary or metastatic; malignant solid tumors can be carcinomas or sarcomas.
  • the "solid tumor” of the present invention includes, but is not limited to, epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, teratoma, lung tumor, carcinoma caused by papillomavirus, adenocarcinoma, carcinoma, melanoma , angiosarcoma, neuroblastoma, metastatic lung cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, Merkel cell carcinoma, ovarian cancer, renal cell carcinoma, metastatic kidney cancer, head and neck cancer, bladder cancer, non-muscle layer Invasive bladder cancer.
  • hematological tumor in the present invention includes, but is not limited to, leukemia, lymphoma, multiple myeloma, such as B-cell lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell large granular lymphocytic leukemia.
  • IL-2 receptor alpha subunit also known as “CD25” in the present invention, refers to any and rat) or mutants thereof, including "full-length” unprocessed IL-2 receptor alpha subunits as well as any form of IL-2 receptor derived from processing in cells 2 Receptor ⁇ subunits, also including naturally occurring variants of IL-2 receptor ⁇ subunits, such as splice variants or allelic variants, and also artificially modified on the basis of natural IL-2 receptor ⁇ subunits mutants.
  • IL-2 receptor beta subunit (IL-2R ⁇ ), also known as “CD122” in the present invention, refers to any and rat) or mutants thereof, including "full-length” unprocessed IL-2 receptor beta subunits as well as any form of IL-2 receptor derived from processing in cells 2 Receptor ⁇ subunits, also including naturally occurring variants of IL-2 receptor ⁇ subunits, such as splice variants or allelic variants, also including artificially modified IL-2 receptor ⁇ subunits based on natural mutants.
  • IL-2 receptor gamma subunit (IL-2R ⁇ ), also known as “CD132” in the present invention, refers to any and rat) of any native IL-2 receptor gamma subunit or mutants thereof, including "full-length” unprocessed IL-2 receptor gamma subunit as well as any form of IL-2 receptor gamma derived from processing in cells 2 Receptor gamma subunits, also including naturally occurring IL-2 receptor gamma subunit variants, such as splice variants or allelic variants, also including artificially modified IL-2 receptor gamma subunits mutants.
  • Treg in the present invention, also known as "regulatory T cell” or “T regulatory cell”, refers to a specialized CD4 + T cell type that can suppress the response of other T cells.
  • Treg is characterized by the expression of the IL-2 receptor ⁇ subunit (CD25) and the transcription factor Forkheadbox protein P3 (FOXP3), and plays a key role in the induction and maintenance of peripheral self-tolerance to antigens. Tregs require IL-2 for their function and development as well as the induction of their suppressive features.
  • percent (%) sequence identity and “percent (%) sequence identity” are interchangeable and refer to the sequence after aligning sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be ignored for comparison purposes), after the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be achieved in a number of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% to 100% sequence identity.
  • the length of a candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence .
  • FIG. 1 Body weight change curve of mice The body weight change curve of DTH model mice given CsA, AMG592 and IL2-1-2 injections, 10 mice in each group. Data points represent the mean body weight of the animals within the group and error bars represent the standard error (SEM).
  • Figure 2 Mouse body weight change rate curve The body weight change rate of DTH model mice after administration of CsA, AMG592 and IL2-1-2 injections. 10 mice per group. Data points represent mean rate of change in body weight of animals within a group, and error bars represent standard deviation (SEM).
  • FIG. 3 DTH model mouse ear piece thickness change curve The ear piece thickness change curve of DTH model mice after being given CsA, AMG592 and IL2-1-2 injections. 10 mice per group. The data points represent the mean variation in ear thickness within the group, and the error bars represent the standard error (SEM). ****P ⁇ 0.0001, compared with vehicle control group.
  • Fig. 4 The change rate curve of ear piece thickness in DTH model mice after administration of CsA, AMG592 and IL2-1-2 injection. 10 mice per group. The data points represent the mean variation in ear thickness within the group, and the error bars represent the standard error (SEM). ****P ⁇ 0.0001, compared with vehicle control group.
  • the ultrafiltration concentration replacement in the following examples refers to: adding the protein stock solution into the corresponding ultrafiltration tube, using a centrifuge for ultrafiltration concentration, the protein is retained, and the buffer of the protein stock solution is obtained from the ultrafiltration tube. Membrane flow through to achieve the purpose of protein concentration, then add the target solution to be replaced, the buffer of the original protein stock solution is diluted, continue to concentrate by ultrafiltration, after concentration, continue to add the target solution to be replaced, continue to concentrate by ultrafiltration, repeat the operation until the liquid exchange is complete.
  • Blocking wash the plate, then add 300 ⁇ l/well of PBS containing 3% BSA, block at 37° C. for 1 hour, and then wash the plate.
  • mouse anti-human IgG Fc ⁇ -HRP Jackson Immuno, 209-035-098
  • diluted 1:10000 (v/v)
  • 100 ⁇ l/well 100 ⁇ l/well, and incubate at 37°C for 40-80min.
  • thermostability mutations and functional mutations comprising the following combination mutations:
  • IL2 thermostable mutants or combined mutants were further connected to Fc through a linker to form a fusion protein (hereinafter referred to as IL2 mutant fusion protein).
  • IL2 mutant fusion protein Each element and its overall sequence are shown in Table 3.
  • the IL2 mutant fusion protein of the present invention has the following characteristics:
  • the Tm value of the IL2 mutant fusion protein containing a thermostability mutation was increased: the Tm value of the IL2 mutant 1-linker-Fc increased by about 8 ⁇ 9°C, the Tm value of IL2 mutant 1-2-linker-Fc increased by more than 12°C, the Tm value of IL2 mutant 1-3-linker-Fc increased by 8-9°C, and the Tm value of IL2 mutant 1-4
  • the Tm value of -linker-Fc increased by about 9-10°C
  • the Tm value of IL2 mutant 1-5-linker-Fc increased by about 11-12°C.
  • IL2 mutant 1-2-linker-Fc showed better drug efficacy (DTH model mice and GVHD mouse models).
  • Good curative effect showing higher exposure and bioavailability in terms of pharmacokinetics, and longer half-life, among which subcutaneous administration is more obvious.
  • the IL2 mutant fusion protein is expressed using CHO as the host cell, and cultured in OPM-CHO CD063 medium (manufacturer: OPM, article number: C483260), the culture period does not exceed 14 days, and the reactor control parameters are pH 6.8-7.2 , dissolved oxygen (DO) is 20% to 80%, the rotation speed is 75RPM to 80RPM, and the initial culture temperature is 36.0°C to 37.0°C; when the culture reaches the 6th day, reduce the culture temperature to 34.0 ⁇ 0.5°C until harvest.
  • the IL2 mutant fusion protein was purified sequentially by multi-step chromatography, concentration and filtration unit operations: the supernatant of the fermentation harvest liquid was captured by ProteinA affinity chromatography (AT ProteinADiamondPlus). The captured IL2 mutant fusion protein solution was incubated with low pH to inactivate potential viruses. After the IL2 mutant fusion protein solution was neutralized, the precipitate was removed by deep filtration. Then carry out cation exchange chromatography (Diamond S) in order to remove impurities such as HCP and aggregates, and anion exchange chromatography (POROS 50HQ) to remove impurities such as HCD, HCP and shedding Protein A. Filter through nanofiltration membrane cassettes to remove potential endogenous virus.
  • cation exchange chromatography Diamond S
  • POROS 50HQ anion exchange chromatography
  • the IL2 mutant fusion protein solution was then concentrated and buffer exchanged using an ultrafiltration membrane bag. Finally, add auxiliary materials and adjust the concentration, and filter to obtain the IL2 mutant fusion protein stock solution. In subsequent examples, the stock solution was replaced with the corresponding preparation system through ultrafiltration concentration and liquid replacement treatment.
  • Examples 2 to 5 develop a formulation to maintain the stability of the IL2 mutant 1-2-linker-Fc (V91R/Y31V/A73L/H79Q, SEQ ID NO: 15) in Example 1
  • X appearance, pH, protein concentration, DLS, SEC-HPLC, CE-SDS (NR&R).
  • CE-SDS(R) ranking F8, F9>F3, F6>F2, F7, F10>F1, F4, F5, F11, F12.
  • Example 2 On the basis of the optimal buffer system (20mM acetic acid-sodium acetate, pH5.5) screened in Example 2, 7 prescriptions were designed, and the addition of sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, and manna was independently investigated.
  • the stability of IL2 mutant fusion protein (concentration of IL2 mutant fusion protein is 2 mg/mL) after alcohol or sorbitol, and an optimal formulation was screened for subsequent surfactant screening.
  • the inspection indicators include: appearance, pH value, concentration, SEC-HPLC, DLS, CE-SDS (NR&R). See Table 15 for the specific prescription composition and inspection plan.
  • X appearance, pH value, concentration, DLS, SEC-HPLC, CE-SDS (NR&R).
  • CE-SDS(R) ranking F3-1, F3-3, F3-4, F3-5, F3-6>F3-7>F3-2.
  • the groups F3-4, F3-5, F3-6 and F3-7 that appear particles in the appearance of long-term stability at 2-8°C are excluded first.
  • the oligomer content detected by CE-SDS(NR) was higher than that of F3-2 and F3-3, indicating that IL-2 protein was in the F3-1 formulation, easier to degrade.
  • SEC-HPLC of F3-2 There is little difference between the detection index and other groups.
  • CE-SDS(R) has a higher proportion of other peaks at 40°C-4W, but there is no significant difference with other groups in the long-term stability of 2-8°C and 25°C.
  • F3-2 and F3-3 prescriptions are superior to other prescriptions, showing good stability.
  • X appearance, pH, protein concentration, DLS, SEC, CE-SDS (NR&R), microparticles insoluble (MFI).
  • Freeze-thaw test (-80 ⁇ 25°C): continuous multiple rounds of freeze-thaw, each round of freeze-thaw: place the sample at -80°C until it freezes, then place it At 25°C, until completely thawed, 1 round of freezing and thawing is formed.
  • This example investigates the protective effect of surfactant polysorbate 80 (PS80) on protein in pH5.5 acetic acid system.
  • PS80 surfactant polysorbate 80
  • the concentration of 2mg/mL and 5mg/mL protein is stable for high temperature and repeated freezing and thawing. and shaking are well tolerated;
  • the protein concentration of 2mg/mL is good for high temperature, repeated Freeze-thawing and shaking are also well tolerated.
  • formulation F3-3-1 showed better results in terms of CE-SDS(R) compared to other formulations, and considering that both 2mg/mL and 5mg/mL belong to low concentration protein , and 5mg/mL IL2 mutation
  • the body fusion protein showed good stability in the druggability study of a similar prescription to F3-3-1. Therefore, 5mg/mL, 20mM acetic acid-acetate, pH5.5, 8% sucrose (w/v), 0.04% PS80 (w/v) was used as the target prescription for the next step of prescription confirmation stability study.
  • Embodiment 5 prescription confirms stability
  • X Appearance, pH value, protein concentration, icIEF, SEC-HPLC, CE-SDS(NR), CE-SDS(R), insoluble particles (MFI), binding activity test.
  • IL2-1-2 KLH-induced delayed hypersensitivity (DTH) model mice
  • the IL2 mutant fusion protein is IL2 mutant 1-2-linker -Fc(V91R/Y31V/A73L/H79Q), hereinafter referred to as IL2-1-2
  • the sequence is shown in SEQ ID NO:15.
  • the positive control adopts Fc-linker-IL2 mutant 6 (V91K), i.e. AMG592, the sequence of which is shown in SEQ ID NO:20.
  • the injection prescription of described IL2-1-2 and AMG592 is 5mg/mL IL2-1-2 or AMG592, 20mM acetic acid-sodium acetate, pH5.5, 8% sucrose (w/v), 0.04% PS80 (w /v).
  • mice Use 6-8 week-old BALB/c mice, weigh the weight of each mouse, and randomly divide the mice into 8 experimental groups of G0-G7 according to their weight, with 10 mice in each group, and start administration on the day of grouping , recorded as Day 0, each dose group of IL2-1-2 and AMG592 was injected subcutaneously once every three days, and CsA was administered intraperitoneally once a day, and the administration continued until the end of the experiment.
  • KLH emulsion The volume ratio of KLH (3mg/ml): IFA: CFA is 1:1:1, and the antigen is emulsified by the connecting tube syringe method, which can fully emulsify the antigen to form a viscous emulsion.
  • mice were taken and sensitized by injecting a total of 100 ⁇ l of KLH emulsion (KLH, IFA, CFA volume ratio 1:1:1 emulsification) at two points on the right side of the back on day 0; the normal control group Inject an equal volume of KLH-free emulsion; on the fifth day, inject 10 ⁇ l of 1 mg/ml KLH solution intradermally into the right ear of each mouse to stimulate the mice, causing delayed hypersensitivity symptoms in the local skin tissue of the model mice.
  • KLH KLH, IFA, CFA volume ratio 1:1:1 emulsification
  • the thickness of the right ear of the mice was measured with a micrometer to evaluate the degree of local delayed hypersensitivity of the mice in different treatment groups.
  • Body weight detection Weigh the weight of the animals before grouping, and weigh the weight of the mice every three days after the animals are grouped. The unit is g, and one decimal place is reserved.
  • the thickness of the mouse ears was measured at 24h, 48h, 72h, and 96h.
  • mice were euthanized by suffocation with excessive CO 2 when the experiment ended 96 hours after stimulation or when the humane end point was implemented.
  • the body weight of experimental animals was used as a reference index for indirect determination of drug toxicity.
  • the body weight and body weight change rate of the mice are shown in Figures 1 and 2 and Table 36.
  • DTH model mice were subcutaneously injected with various therapeutic drugs once every three days. During the observation of the experimental period, the weight of the mice fluctuated. Except for the hormone administration CsA group, the weight of the mice decreased, but no mice lost more than 10%, the body weights of mice in other administration groups all showed an upward trend. The state of all mice was normal, and there was no other morbidity or death phenomenon.
  • CsA was at 10mg/kg
  • AMG592 was at 1mg/kg
  • IL2 mutant 1-2 was at 0.03mg/kg, 0.1mg/kg, 0.3mg/kg and At a dose of 1 mg/kg, treatment was well tolerated.
  • the changes in the ear piece thickness of the DTH model mice in each treatment group are shown in Figure 3-4.
  • the onset of the DTH model reached its peak at 48 hours after KLH stimulation.
  • the thickness of the ears in the model group was 15.7mm ⁇ 10 -2 at the peak of the onset, which was 74.1% higher than the initial ear thickness of the mice.
  • the model was successfully established.
  • IL2- 1-2 Injection at 1, 0.3, 0.1 and 0.03mg/kg treatment groups increased the thickness of mouse ears by 2.45, 4.01, 9.09 and 12.26mm ⁇ 10 -2 , which was 10.2% higher than that before KLH stimulation. 22.0%, 41.3% and 53.6%.
  • IL2 mutant fusion protein is IL2 mutant 1-2-linker-Fc (V91R/Y31V/A73L/ H79Q), hereinafter referred to as IL2-1-2, the sequence is as shown in SEQ ID NO:15.
  • the positive control adopts Fc-linker-IL2 mutant 6 (V91K), i.e. AMG592, the sequence of which is shown in SEQ ID NO:20.
  • the injection prescription of described IL2-1-2 and AMG592 is 5mg/mL IL2-1-2 or AMG592, 20mM acetic acid-sodium acetate, pH5.5, 8% sucrose (w/v), 0.04% PS80 (w /v).
  • mice After the 6-8 week-old BALB/c mice were adaptively fed, the weight of each mouse was weighed, and the weight of the mice was randomly assigned to the 5 experimental groups of G1-G5 groups based on random numbers using EXCEL. There were 5 rats in each group, and the administration began on the day of grouping, which was recorded as Day 1.
  • mice On the 4th day after administration, the mice were euthanized and subjected to cardiac puncture, and peripheral blood was collected. Immune cell surface markers (CD3+, CD4+, CD25+) and nuclear factor (Foxp3+, Ki-67+) fluorescent label antibodies for labeling. Immune cell counting and grouping analysis were carried out by the following FACS methods:
  • step (3) to lyse red blood cells again.
  • step (3) Resuspend the cells with 4ml washing solution (PBS). Transfer the cell suspension to a new flow tube after filtering through a membrane filter.
  • (6) Add 500 ⁇ l of fixative dropwise to the flow tube while shaking, and incubate overnight at 4° C. in the dark.
  • Step (7) was repeated, and the cells were treated again with 3 ml of membrane-penetrating solution.
  • Vortex and count beads for at least 30 seconds at room temperature Add 50 ⁇ l counting microspheres to the flow cytometer, and perform FACS detection on the flow cytometer. Each flow cytometer needs to be fully shaken before being put on the machine. (12) Analyze the types of immune cells with FlowJo software according to the FACS results, and calculate the number of cells according to the instructions for counting microspheres.
  • the animal body weight is lower than 81% of the body weight at the beginning of the drug treatment, stop the administration, and return to 90% of the body weight at the beginning of the drug treatment to continue the administration;
  • mice After BALB/c mice were treated with different doses of IL2-1-2, the changes in peripheral blood immune cell counts compared with those in the vehicle control group were shown in Table 38: subcutaneous injection of IL2-1-2 could be observed Significant promotion of mouse Treg cell proliferation. Compared with the vehicle control group, Treg cells were significantly stimulated after administration of IL2-1-2 or AMG592 for 3 days. After subcutaneous administration of 0.1, 0.3, and 1mg/kg IL2-1-2, the multiples of changes in peripheral blood Treg cell counts of mice were 13.73, 35.27, and 12.05, respectively. 1mg/kg IL2-1-2 administration group mice, but lower than 0.3mg/kg administration group mice, the results show that IL2-1-2 can have a relatively low dose on Treg cells Good facilitation. The fold change in Treg/CD4+ ratio is presented in Table 39. observed in the experiment CD4+Foxp3 cells or CD3+CD4- cells were not activated or slightly activated.
  • IL2-1-2 IL2 mutant 1-2-linker-Fc (V91R/Y31V/A73L/H79Q) , hereinafter referred to as IL2-1-2, the sequence is shown in SEQ ID NO:15.
  • the injection prescriptions of IL2-1-2 are all 5mg/mL IL2-1-2, 20mM acetic acid-sodium acetate, pH5.5, 8% sucrose (w/v), 0.04% PS80 (w/v).
  • SD rats aged 6-8 weeks were adaptively fed and randomly assigned to 6 experimental groups, with 3 males and 3 females in each group.
  • Administration started on the day of grouping (referred to as day 1), and IL2-1-2 injection was administered subcutaneously or through tail vein four times on day 1, day 8, day 15 and day 22 respectively.
  • the body weight of the animals was weighed after grouping and before each administration, and recorded in g.
  • Immune cell surface markers CD3+, CD4+, CD25+
  • nuclear factors Foxp3+, Ki-67+
  • Step (7) Add 2ml of transmembrane and nuclei penetrating solution into the flow tube and shake, centrifuge the flow tube at 500g/rcf at 4°C for 5 minutes, and discard the supernatant.
  • Step (7) was repeated, and the cells were treated again with 3 ml of membrane-penetrating solution.
  • (9) Add the antibody mixture of nuclear factor (Foxp3+, Ki-67+) into the flow tube, incubate at 4°C in the dark for 40 minutes, and shake the flow tube once every 20 minutes.
  • the administration should be stopped when the body weight of the animal is lower than 81% of the body weight before drug treatment, and the administration can be continued when it returns to 90% of the body weight before drug treatment;
  • the decision to continue treatment or experimentation is based on an assessment of the animal's health status, or whether to euthanize if any one or more of the following occurs:
  • the body weight of the animal is lower than 20% of the body weight before grouping
  • the animal has hypothermia and is in a dying state.
  • Table 41 shows the changes in the proportion of immune cells in the peripheral blood of SD rats after IL2-1-2 administration.
  • Both subcutaneous and tail vein injection of IL2-1-2 into SD rats can cause the expansion of Treg cells.
  • low-dose IL2-1-2 (0.005 mg/kg subcutaneous injection or 0.002 mg/kg tail vein injection) showed a slight effect on Treg cell expansion from day 4 to day 25 after administration. effect.
  • a moderate dose of IL2-1-2 0.05 mg/kg
  • the number of Treg cells in the peripheral blood of rats increased to 9.55 times the baseline value on day 4 and remained at 9.5 times the baseline value on day 25. 3.16 times.
  • the number of Treg cells in the peripheral blood of rats peaked at day 11 (13.8 times the baseline value) and decreased to the baseline value at day 25 3.18 times.
  • the number of Treg cells in the peripheral blood of rats was significantly increased on the 4th day and the 11th day, which were 21.25 times and 30.13 times the baseline value, respectively. times. But similar to other treatment groups, the number of Treg cells also decreased to 3.26 times the baseline value at day 25. This phenomenon may be caused by the anti-drug antibody (ADA) produced in rats after repeated administration.
  • ADA anti-drug antibody

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Abstract

一种IL2突变体-抗体Fc嵌段融合蛋白的药物组合物及其制备方法、冻干制剂及其制备或复溶方法以及获得的复溶溶液、相应的制品、对自身免疫性疾病和增生性疾病的治疗方法以及相应的制药用途，药物组合物包括：包含IL2突变体和抗体Fc嵌段的融合蛋白、缓冲液、渗透压调节剂和表面活性剂，具有较好的稳定性。

Description

IL2突变体-抗体Fc嵌段融合蛋白的药物组合物及其应用 技术领域
本发明涉及药物制剂领域，具体而言，涉及IL2突变体-抗体Fc嵌段融合蛋白的药物组合物及其应用。
背景技术
白细胞介素2(interleukin-2，IL2)，最初被鉴定为T细胞生长因子(TCGF)，研究发现IL-2可结合其受体，激活T细胞、NK细胞等免疫细胞的增殖和活化。IL-2受体包括IL-2Rα亚基(CD25)、IL-2Rβ亚基(CD122)和IL-2Rγ亚基(CD132)，不同的亚基之间可形成亲和力不同的受体复合物，包括高亲和力受体IL-2Rαβγ，中间亲和力受体IL-2Rβγ，低亲和力受体IL-2Rα或IL-2Rαβ。不同细胞表达的IL-2R亚基类型有所不同，例如，静息条件下的传统T细胞CD4+T、CD8+T表面一般表达IL-2受体β(IL-2Rβ，CD122)、IL-2受体γ(IL-2Rγ，CD132)，几乎不表达IL-2受体α(IL-2Rα，CD25)，而调节性T细胞(Treg)除了表达IL-2Rβ、IL-2Rγ之外，组成性高表达IL-2Rα。
目前，研究人员通过IL2或其突变体激活免疫细胞或某个亚群的免疫细胞，治疗肿瘤或自身免疫性疾病。例如，高剂量的IL2被批准用于治疗恶性黑色素瘤或转移性肾细胞癌，IL2聚乙二醇偶联药物NKTR-358被批准开展自身免疫性疾病的临床试验。进一步开发新的IL2突变体，提高其稳定性、产量和/或改变其与某类受体复合物的结合能力，对IL-2类型药物的开发具有重要意义。同时，IL2属于大分子蛋白，在溶液中容易出现颗粒和聚集等稳定性方面的问题。因此，在获得新型IL2突变体后，开发获得适合该新型IL2突变体的制剂体系，确保新型IL2突变体在溶液中的稳定性具有重要意义。
发明内容
本发明提供一种药物组合物及其制备方法、冻干制剂及其制备或复溶方法以及根据所述复溶方法获得的复溶溶液、相应的制药用途、治疗自身免疫性疾病方法和制品。
在第一方面，本发明提供一种药物组合物，所述药物组合物包括包含IL2突变体的抗体Fc嵌段融合蛋白、缓冲液、渗透压调节剂和表面活性剂；
优选地，所述IL2突变体与野生型IL2相比，至少包括Y31V、A73L和H79Q突变；
优选地，所述IL2突变体还进一步包括V91R突变、H16E突变和D20A突变中的一个或多个突变，例如还进一步包括H16E突变和V91R突变；
更优选地，所述IL2突变体具有与SEQ ID NO:3、8～11任一项所示序列相比至少80％同一性的氨基酸序列，例如至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
所述野生型IL2的氨基酸序列如SEQ ID NO：2所示。
在一些具体实施方式中，所述融合蛋白从N端到C端，依次包括：
(1)IL2突变体、连接子(linker)和抗体Fc嵌段，或
(2)抗体Fc嵌段、连接子和IL2突变体；
优选的，所述融合蛋白通过抗体Fc嵌段二聚化形成同源二聚体；
优选地，所述连接子具有(G4S)n的氨基酸序列，n选自1，2，3，4，5或6；
优选地，所述连接子具有与SEQ ID NO:12所示序列相比至少80％同一性的氨基酸序列，例如至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性；
优选地，所述抗体Fc嵌段包括N297G突变；
优选地，所述抗体Fc嵌段具有与SEQ ID NO:13所示序列相比至少80％同一性的氨基酸序列，例如至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性；
更优选地，所述融合蛋白具有与SEQ ID NO:14～18所示序列相比至少80％同一性的氨基酸序列，例如至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
在一些具体的实施方式中，所述野生型IL2具有入SEQ ID NO:1或2所示的氨基酸序列。
在一些具体实施方式中，所述融合蛋白的浓度约为1～50mg/mL；
例如，所述融合蛋白的浓度约为1～40mg/mL、1～30mg/mL、1～20mg/mL、1～15mg/mL、1～10mg/mL、5～50mg/mL、5～40mg/mL、5～30mg/mL、5～20mg/mL、5～19mg/mL、5～18mg/mL、5～17mg/mL、5～16mg/mL、5～15mg/mL、5～14mg/mL、5～13mg/mL、5～12mg/mL、5～11mg/mL、5～10mg/mL、5～9mg/mL、5～8mg/mL、5～6mg/mL、10～50mg/mL、10～40mg/mL、10～30mg/mL、10～20mg/mL、10～15mg/mL、20～50mg/mL、20～40mg/mL、20～30mg/mL、30～50mg/mL、30～40mg/mL、40～50mg/mL；
例如，所述融合蛋白的浓度约为1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、31mg/mL、32mg/mL、33mg/mL、34mg/mL、35mg/mL、36mg/mL、37mg/mL、38mg/mL、39mg/mL、40mg/mL、41mg/mL、42mg/mL、43mg/mL、44mg/mL、45mg/mL、46mg/mL、47mg/mL、48mg/mL、49mg/mL或50mg/mL；或者上述浓度值之间组成的浓度范围值。
优选地，所述融合蛋白的浓度约为2mg/mL或5mg/mL。
在一些具体实施方式中，所述缓冲液选自醋酸-醋酸钠缓冲液、枸橼酸-枸橼酸钠缓冲液、组氨酸-盐酸组氨酸、琥珀酸-琥珀酸钠、或磷酸氢二钠-磷酸二氢钠缓冲液，优选为醋酸-醋酸钠缓冲液；和/或，
所述缓冲液的浓度约为1～100mM；
例如，缓冲液的浓度约为1～90mM、1～80mM、1～70mM、1～60mM、1～50mM、1～40mM、1～30mM、1～20mM、1～10mM、10～90mM、10～80mM、10～70mM、10～60mM、10～50mM、10～40mM、10～30mM、10～20mM、20～90mM、20～80mM、20～70mM、20～60mM、20～50mM、20～40mM、20～30mM、30～90mM、30～80mM、30～70mM、30～60mM、30～50mM、30～40mM、 40～90mM、40～80mM、40～70mM、40～60mM、40～50mM、50～90mM、50～80mM、50～70mM、50～60mM、60～90mM、60～80mM、60～70mM、70～90mM、80～90mM；
例如，缓冲液的浓度约为1mM、5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM、55mM、60mM、65mM、70mM、75mM、80mM、85mM、90mM、95mM或100mM；或者上述浓度值之间组成的浓度范围值；
优选地，所述缓冲液的浓度约为20mM；和/或，
所述缓冲液的pH值约为4.5～8.0；
例如所述缓冲液的pH值约为4.5～7.5、4.5～7.0、4.5～6.5、4.5～6.0、4.5～5.5、4.5～5.0、5.0～8.0、5.0～7.5、5.0～7.0、5.0～6.5、5.0～6.0、5.0～5.5、5.5～8.0、6～7.5、6.5～7.5、7.0～7.5或7.5～8.0；
例如所述缓冲液的pH值约为，约为4.5、5.0、5.5、6.0、6.5、7.0、7.5或8.0，或者上述pH值之间组成的浓度范围值；
优选地，所述缓冲液的pH值约为5.5。
在一些具体实施方式中，所述渗透压调节剂选自盐、氨基酸、糖或糖醇或其组合；优选地，所述盐选自氯化钠、氯化钾或盐酸精氨酸；优选地，所述氨基酸选自甘氨酸、精氨酸、组氨酸、谷氨酸或甲硫氨酸；优选地，所述糖或糖醇选自蔗糖、海藻糖、甘露醇或山梨醇；
优选地，所述渗透压调节剂选自氯化钠、甘氨酸、盐酸精氨酸、蔗糖、海藻糖、甘露醇或山梨醇，更优选为蔗糖或盐酸精氨酸；
更优选地，所述糖或糖醇的浓度约为1～15％w/v，所述盐的浓度约为50～200mM；例如，所述糖或糖醇的浓度约为1～10％w/v、1～5％w/v或5～10％w/v；例如，所述糖或糖醇的浓度约为1％w/v、1.5％w/v、2％w/v、2.5％w/v、3％w/v、3.5％w/v、4％w/v、4.5％w/v、5％w/v、5.5％w/v、6％w/v、6.5％w/v、7％w/v、7.5％w/v、8％w/v、8.5％w/v、9％w/v、9.5％w/v、10％w/v、10.5％w/v、11％w/v、11.5％w/v、12％w/v、12.5％w/v、13％w/v、13.5％w/v、14％w/v、14.5％w/v、15％w/v，或者上述浓度值之间组成的浓度范围值。优选为4.5％w/v或8％w/v。
更优选地，所述氨基酸的浓度约为1～15％w/v，例如所述氨基酸的浓度约为1～10％w/v、1～5％w/v或5～10％w/v；例如，所述氨基酸的浓度约为1％w/v、1.5％w/v、2％w/v、2.5％w/v、3％w/v、3.5％w/v、4％w/v、4.5％w/v、5％w/v、5.5％w/v、6％w/v、6.5％w/v、7％w/v、7.5％w/v、8％w/v、8.5％w/v、9％w/v、9.5％w/v、10％w/v、10.5％w/v、11％w/v、11.5％w/v、12％w/v、12.5％w/v、13％w/v、13.5％w/v、14％w/v、14.5％w/v、15％w/v，或者上述浓度值之间组成的浓度范围值。优选为2％w/v。
更优选地，所述盐的浓度约为50～200mM；例如，所述盐的浓度约为50～150mM、50～100mM或100～150mM；例如，所述盐的浓度约为50mM、60mM、70mM、80mM、90mM、100mM、110mM、120mM、130mM、140mM、150mM、160mM、170mM、180mM、190mM或200mM，或者上述浓度值之间组成的浓度范围值。优选为140mM。
更优选地，所述渗透压调节剂为蔗糖或盐酸精氨酸。
在一些具体实施方式中，所述表面活性剂为聚山梨酯-80(PS-80)、聚山梨酯20(PS-20)或泊洛沙姆；和/或，
所述表面活性剂的浓度约为0.01～0.1％w/v，例如，0.01％w/v-0.05％w/v、0.05％w/v-0.1％w/v、0.02％w/v-0.08％w/v，例如约为0.01％w/v、0.02％w/v、0.03％w/v、0.04％w/v、0.05％w/v、0.06％w/v、0.07％w/v、0.08％w/v、0.09％w/v或1％w/v，或者上述浓度值之间组成的浓度范围值。优选约为0.04％w/v。
在一些具体实施方式中，所述药物组合物包括所述融合蛋白、醋酸-醋酸钠缓冲液、蔗糖和聚山梨酯-80；优选地，所述药物组合物包括约1～50mg/mL所述融合蛋白、约1～100mM醋酸-醋酸钠缓冲液、约1～15％w/v蔗糖和约0.01～0.1％w/v聚山梨酯-80，pH值约为4.5～6.0；
更优选地，所述药物组合物包括约1～30mg/mL、5～30mg/mL、10～30mg/mL、10～25mg/mL、10～20mg/mL、10～15mg/mL、15～20mg/mL、15～30mg/mL或20～30mg/mL所述融合蛋白，约10～50mM或10～30mM醋酸-醋酸钠缓冲液，约1～15％w/v、1～10％w/v、5～15％w/v、5～10％w/v或10～15％w/v蔗糖和约0.01～0.1％w/v、0.02～0.06％w/v聚山梨酯-80，pH值约为4.5～6.0或4.5～5.5；
更优选地，所述药物组合物包括约1～10mg/mL所述融合蛋白、约10～30mM醋酸-醋酸钠缓冲液、约5～10％w/v蔗糖和约0.01～0.1％w/v聚山梨酯-80，pH值约为4.5～6.0；
更优选地，所述药物组合物包括约2mg/mL或5mg/mL所述融合蛋白，约20mM醋酸-醋酸钠缓冲液、约8％w/v蔗糖和约0.04％w/v聚山梨酯-80，pH约为5.5。
在一些具体实施方式中，所述药物组合物包括融合蛋白、醋酸-醋酸钠缓冲液、盐酸精氨酸和聚山梨酯-80；优选地，所述药物组合物包括约1～50mg/mL所述融合蛋白、约1～100mM醋酸-醋酸钠缓冲液、约50～200mM盐酸精氨酸和约0.01～0.1％w/v聚山梨酯-80，pH值约为4.5～6.0；
更优选地，所述药物组合物包括约1～30mg/mL、5～30mg/mL、10～30mg/mL、10～25mg/mL、10～20mg/mL、10～15mg/mL、15～20mg/mL、15～30mg/mL或20～30mg/mL所述融合蛋白，约10～50mM或10～30mM醋酸-醋酸钠缓冲液，约50～200mM、50～150mM、100～150mM盐酸精氨酸和约0.01～0.1％w/v、0.02～0.06％w/v聚山梨酯-80，pH值约为4.5～6.0或4.5～6.0；
更优选地，所述药物组合物包括约1～10mg/mL所述融合蛋白、约10～30mM醋酸-醋酸钠缓冲液、约120～150mM盐酸精氨酸和约0.01～0.1％w/v聚山梨酯-80，pH值约为4.5～6.0。
更优选地，所述药物组合物包括约2mg/mL或5mg/mL所述融合蛋白、约20mM醋酸-醋酸钠缓冲液、约140mM盐酸精氨酸和约0.04％w/v或0.06％w/v聚山梨酯-80，pH值约为5.5。
在一些具体实施方式中，所述药物组合物为静脉、肌内或皮下注射液，优选为皮下注射液。
在第二方面，本发明还提供前述药物组合物的制备方法，所述方法包括将所述融合蛋白与所述缓冲液、渗透压调节剂和表面活性剂混合配置的步骤；优选地，所述方法包括将 所述融合蛋白的原液通过超滤浓缩置换到所述缓冲液中的步骤。
在第三方面，本发明还提供一种冻干制剂，其中，所述冻干制剂由前述药物组合物冻干后形成。
在第四方面，本发明还提供制备前述冻干制剂的方法，其中，所述方法包括冷冻干燥前述药物组合物的步骤。
在第五方面，本发明还提供制备含IL2突变体的抗体Fc嵌段融合蛋白的复溶溶液的方法，其中包括将前述冻干制剂用溶剂复溶，优选地，所述溶剂为注射用水。
在第六方面，本发明还提供根据前述方法制备得到的含IL2突变体的抗体Fc嵌段融合蛋白的复溶溶液。
在第七方面，本发明还提供前述药物组合物、冻干制剂或复溶溶液在制备用于治疗自身免疫性疾病或增生性疾病的药物中的应用；
优选地，所述自身免疫性疾病选自类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘；
优选地，所述增生性疾病选自赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水；其中，所述实体瘤可为良性或恶性，原发性或转移性，所述恶性实体瘤可为癌或肉瘤，例如，上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌；所述血液瘤可选自白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
在第八方面，本发明还提供前述药物组合物、冻干制剂或复溶溶液，用于治疗自身免疫性疾病或增生性疾病；
优选地，所述自身免疫性疾病选自类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘；
优选地，所述增生性疾病选自赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水；其中，所述实体瘤可为良性或恶性，原发性或转移性，所述恶性实体瘤可为癌或肉瘤，例如，上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌；所述血液瘤可选自白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
在第九方面，本发明还提供一种治疗自身免疫性疾病的方法，其中，所述方法包括给予受试者有效量的前述药物组合物或复溶溶液；优选地，所述自身免疫性疾病选自类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘。
在一些具体的实施方式中，所述有效量为0.001～10mpk，例如0.001mpk、0.002mpk、0.003mpk、0.004mpk、0.005mpk、0.006mpk、0.007mpk、0.008mpk、0.009mpk、0.01mpk、0.02mpk、0.03mpk、0.04mpk、0.05mpk、0.06mpk、0.07mpk、0.08mpk、0.09mpk、0.1mpk、0.2mpk、0.3mpk、0.4mpk、0.5mpk、0.6mpk、0.7mpk、0.8mpk、0.9mpk、1mpk、2mpk、3mpk、4mpk、5mpk、6mpk、7mpk、8mpk、9mpk或10mpk，或者上述有效量之间组成的有效量范围值。
在第十方面，本发明还提供一种治疗增生性疾病地方法，其中所述方法包括给予受试者有效量的前述药物组合物或复溶溶液；优选地，所述增生性疾病选自赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水；其中，所述实体瘤可为良性或恶性，原发性或转移性，所述恶性实体瘤可为癌或肉瘤，例如，上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌；所述血液瘤可选自白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
在第十一方面，本发明还提供一种制品，其包括容器，所述容器装有前述药物组合物、冻干制剂或复溶溶液。
术语定义和说明
除非本发明另外定义，与本发明相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
除非另有说明，本发明所述术语“IL2”或“IL-2”，是指来自任何脊椎动物源，包括哺乳动物例如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)和驯养或农业哺乳动物的任何天然或重组IL-2。本发明所述“IL2”或“IL-2”包括未加工成熟的IL-2(例如包含N端信号肽的IL-2)以及产生在细胞中加工成熟的IL-2的任何形式。本发明所述“IL2”或“IL-2”还包括IL-2的天然变体和片段，例如剪切变体或等位基因变体。本发明所述“IL2”或“IL-2”还包括非天然存在的突变体，例如通过基因工程人工改造的IL-2突变体。
“野生型IL-2”是在其他方面与IL-2突变体相同，只是在IL-2突变体的每个突变氨基酸位置保持野生型氨基酸的IL-2形式。示例性地，如果IL-2突变体是未加工成熟的IL-2，那么该突变体的野生型形式是未加工成熟的IL-2；如果IL-2突变体是加工成熟的IL-2，那么该突变体的野生型形式是加工成熟的IL-2；如果IL-2突变体是IL-2的截短形式，那么该突变体的野生型形式是具有野生型序列的相应的IL-2截短形式。示例性地，本发明所述“野生型IL-2”可具有如下所示氨基酸序列：
其中第125位氨基酸残基“X”代表C、S、A或V。
本发明术语“突变”包含氨基酸取代、缺失、插入或其任意组合。本发明所述“突变”可以使用本领域公知的遗传学或化学方法生成氨基酸突变，包括但不限于定点诱变、PCR、基因合成等方法。
本发明所述IL-2突变体的“突变位点”编号从SEQ ID NO：1所示“野生型IL-2”的第1位氨基酸残基A开始计数。示例性地，本发明“Y31突变”是指IL-2突变体在SEQ ID NO：1所示野生型IL-2第31位氨基酸残基(Tyr，Y)处发生突变。示例性地，本发明“Y31V突变”是指IL-2突变体在SEQ ID NO：1所示野生型IL-2第31位处氨基酸残基由Y(Tyr)突变位V(Val)。
本发明用于突变位点间的术语“/”表示“和”的意思，是指“/”前后的突变在同一个IL-2突变体中同时存在。示例性地，“Y31/A73/H79”是指在同一个IL-2突变体同时在Y31位点、A73位点和H79位点发生突变，“Y31V/A73L/H79Q”是指在同一个IL-2突变体同时存在Y31V、A73L和H79Q突变。
本发明术语“融合蛋白”(fusionprotein)是指，通过基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物。本发明的融合蛋白中，两个或多个基因的编码区之间可由编码连接肽的序列于一个或数个位置融合。连接肽可用于构建本发明的融合蛋白。
本发明术语“连接子(Linker)”是指，在本发明中用于连接IL-2与另一蛋白分子或蛋白片段，以保证蛋白的正确折叠和稳定性的肽。所述另一分子包括但不限于抗体Fc嵌段。本发明的“连接子”优选为(GGGGS)n，其中n可以为0、1、2、3、4、5或6。
本发明术语“抗体Fc嵌段”是指，免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，无抗原结合活性，是抗体分子与效应分子或细胞相互作用的部位。本发明所述“Fc”可以是任何Fc或其变体，来源于人或非人哺乳动物。例如，免疫球蛋白Fc可包括重链CH1、CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。Fc可以来源于不同的种属，优选人的免疫球蛋白。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM。其中一些还可进一步分成亚类(同种型)，如IgG-1、IgG-2、IgG-3、IgG-4；IgA-1和IgA-2。“Fc”优选包括至少一个免疫球蛋白铰链区，以及IgG的CH2和CH3结构域。更优选包括IgG1的一个CH2结构域，一个CH3结构域和一个免疫球蛋白铰链区，铰链区起始氨基酸位置可以变动。除非另外说明，否则本发明所述Fc、恒定区或抗体中的氨基酸残基根据如Kabat等人,Sequences of Proteins of Immunological Interes,第5版,Public Health Service,National Institutes ofHealth,Bethesda,MD,1991中所述的EU编号体系，也称作EU索引，进行编号。
本发明术语“药物组合物”是指含有一种或多种本发明所述IL-2突变体、融合蛋白、核酸片段、载体或宿主细胞的混合物，所述混合物还含有其他组分，所述其他组分包括但不限于其在药学上可接受的运载体、稀释剂或助剂。本发明所述药物组合物的目的是促进对 生物体的给药，利于活性成分的吸收进而发挥生物活性。
本发明术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如自身免疫性疾病(如系统性红斑狼疮)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本发明术语“受试者”是指接受对如本发明所述的特定疾病或病症(如自身免疫性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛或其他牛科家族成员、绵羊和马等。
本发明术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本发明术语“自身免疫病”指特征是对象对其自身的细胞、组织和/或器官产生免疫反应而引起的细胞、组织和/或器官损伤的对象病症。示例性地，自身免疫病包括但不限于类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘。
本发明术语“增生性疾病”指细胞或组织的生长不受调节和/或异常生长，可导致发展不需要的病况或疾病的病况，可为或可不为癌性的，其包括但不限于赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水。本发明“实体瘤”可为良性(benign)或恶性(maligant)，原发性(Primary)或转移性(metastatic)；恶性实体瘤可为癌(carcinomas)或肉瘤(sarcomas)。示例性地，本发明“实体瘤”包括但不限于上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌。示例性地，本发明“血液瘤”包括但不限于白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
本发明术语“IL-2受体α亚基”(IL-2Rα)，又称“CD25”，指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然IL-2受体α亚基或其突变体，包括“全长”的未加工的IL-2受体α亚基以及源自细胞中的加工的任何形式的IL-2受体α亚基，还包括天然存在的IL-2受体α亚基变体，例如剪接变体或等位变体，还包括在天然IL-2受体α亚基基础上经过人工改造的突变体。
本发明术语“IL-2受体β亚基”(IL-2Rβ)，又称“CD122”，指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然IL-2受体β亚基或其突变体，包括“全长”的未加工的IL-2受体β亚基以及源自细胞中的加工的任何形式的IL-2受体β亚基，还包括天然存在的IL-2受体β亚基变体，例如剪接变体或等位变体，还包括在天然IL-2受体β亚基基础上经过人工改造的突变体。
本发明术语“IL-2受体γ亚基”(IL-2Rγ)，又称“CD132”，指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然IL-2受体γ亚基或其突变体，包括“全长”的未加工的IL-2受体γ亚基以及源自细胞中的加工的任何形式的IL-2受体γ亚基，还包括天然存在的IL-2受体γ亚基变体，例如剪接变体或等位变体，还包括在天然IL-2受体γ亚基基础上经过人工改造的突变体。
本发明术语“Treg”，又称“调节性T细胞”或“T调节细胞”，是指一种能抑制其它T细胞的应答的特殊化CD4+T细胞类型。Treg的特征在于表达IL-2受体α亚基(CD25)和转录因子叉头框P3(Forkheadboxprotein P3，FOXP3)，并在诱导和维持对抗原的外周自体耐受中起着关键作用。Treg需要IL-2来实现其功能和发育以及其抑制性特征的诱导。
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
附图说明
图1小鼠体重变化曲线DTH模型小鼠给予CsA，AMG592和IL2-1-2注射液后的体重变化曲线，每组10只小鼠。数据点代表组内动物平均体重，误差线代表标准误(SEM)。
图2小鼠体重变化率曲线DTH模型小鼠在给予CsA，AMG592和IL2-1-2注射液后的体重变化率。每组10只小鼠。数据点代表组内动物平均体重变化率，误差线代表标准差(SEM)。
图3DTH模型小鼠耳片厚度变化曲线DTH模型小鼠在给予CsA，AMG592和IL2-1-2注射液后的耳片厚度变化曲线。每组10只小鼠。数据点代表组内平均耳片厚度变化值，误差线代表标准误(SEM)。****P<0.0001，与vehicle control组相比。
图4DTH模型小鼠在给予CsA，AMG592和IL2-1-2注射液后的耳片厚度变化率曲线。 每组10只小鼠。数据点代表组内平均耳片厚度变化值，误差线代表标准误(SEM)。****P<0.0001，与vehicle control组相比。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
关于实施例1-5的说明
1.超滤浓缩换液
如无特别说明，下述实施例中的超滤浓缩换液是指：将蛋白原液加入到对应超滤管中，使用离心机进行超滤浓缩，蛋白被截留，蛋白原液的缓冲液从超滤膜流穿，达到蛋白浓缩的目的，然后加入需替换的目标溶液，原蛋白原液的缓冲液被稀释，继续超滤浓缩，浓缩完毕后继续加入需替换的目标溶液，继续超滤浓缩，重复操作，直到换液完毕。
2.英文缩写及其含义
如无特别说明，下述英文缩写具有如下表所示的含义：
表1缩写说明
3.实施例5涉及的IL2突变体融合蛋白的结合活性检测方法
(1)包被：选择pH9.6的碳酸盐缓冲液(50mM Na2CO3-NaHCO3,pH9.6)作为包被液，将人IL-2R alpha蛋白(Acro，ILA-H52H9)用包被液稀释至0.5μg/mL，100μl/孔加入酶标板，2～8℃包被过夜(>16h)。
(2)封闭：洗板，之后，按300μl/孔加入含3％BSA的PBS，37℃封闭1h，之后洗板。
(3)样品稀释：IL2突变体融合蛋白以200ng/mL作为起始浓度进行稀释(详见表2)。
表2 IL2突变融合蛋白稀释方法
注：取120μL配置好的样品1，加入280μL样品稀释液，混匀后，即得样品2；取120μL配置好的样品2，加入280μL样品稀释液，混匀后，即得样品3。样品4～8的稀释依次类推。
(4)按100μl/孔加入IL2突变体融合蛋白的稀释样品，37℃孵育40～80min。
(5)洗涤后，加入按1:10000(v/v)进行稀释的鼠抗人IgG Fcγ-HRP(Jackson Immuno，209-035-098)，100μl/孔，37℃孵育40～80min。
(6)洗涤后，显色液TMB(Thermo，34029)以100μl/孔加至酶标板中，室温避光反应5～9min后100μl/孔加入1M硫酸终止反应，2min内使用酶标仪进行读板，测量波长为450nm，参比波长为630nm。
(7)用Molecular Devices酶标仪自带的软件SoftMax Pro或GraphPad Prism或同类型分析软件，采用四参数回归分别计算供试品和对照品的EC50。按以下公式计算供试品相对活性：供试品相对结合活性(％)＝对照品EC50/供试品EC50×100，供试品相对活性在70％～130％范围之间时，判为合格。
实施例1 IL2突变体融合蛋白的制备和纯化
1.IL2突变体融合蛋白的设计
(1)使用多种算法，设计提高IL2热稳定性的突变位点Y31V/A73L/H79Q。
(2)使用多种算法，设计减弱IL2与IL2受体βγ亚基复合物相互作用的功能性突变位点V91R、H16E、D20A或H16E/V91R。
(3)组合上述热稳定性突变和功能性突变，获得分别包含以下组合突变的IL2突变体：
Y31V/A73L/H79Q/V91R；
Y31V/A73L/H79Q/H16E；
Y31V/A73L/H79Q/D20A；
Y31V/A73L/H79Q/H16E/V91R。
为延长半衰期，进一步通过连接子将IL2热稳定突变体或组合突变体与Fc连接，构成融合蛋白(下称IL2突变体融合蛋白)，各元件及其整体序列如表3所示。
与野生型IL2融合蛋白(SEQ ID NO:19)相比，本发明所述IL2突变体融合蛋白具有以下特点：
(1)经DSF法分析，与野生型IL2融合蛋白相比，含热稳定性突变的IL2突变体融合蛋白的Tm值均升高：IL2突变体1-连接子-Fc的Tm值上升约8～9℃，IL2突变体1-2-连接子-Fc的Tm值升高12℃以上，IL2突变体1-3-连接子-Fc的Tm值上升8～9℃，IL2突变体1-4-连接子-Fc的Tm值上升约9～10℃，IL2突变体1-5-连接子-Fc的Tm值上升约11～12℃。
(2)与野生型IL2融合蛋白相比，含功能性突变的IL2突变体融合蛋白与细胞表面的人源IL2受体βγ二聚体的结合活性被抑制，CD4+CD25-FoxP3-T细胞或者CD8+T细胞内的STAT5磷酸化水平的激活显著降低，对Treg细胞增殖的影响与野生型IL2融合蛋白相似，对NK细胞的增殖水平显著降低。
(3)与野生型IL2融合蛋白或Fc-连接子-IL2突变体6相比，IL2突变体1-2-连接子-Fc在药效(DTH模型小鼠和GVHD小鼠模型)方面显示更好的疗效，在药代方面显示更高的暴露量和生物利用度，以及更长的半衰期，其中皮下给药表现更为明显。
(4)选择IL2突变体1-2-连接子-Fc(Y31V/A73L/H79Q/V91R，SEQ ID NO:15)进行后续的工艺开发。
表3 IL2突变体融合蛋白序列信息


2.IL2突变体融合蛋白的表达
IL2突变体融合蛋白利用CHO作为宿主细胞进行表达，在OPM-CHO CD063培养基(厂家：奥浦迈,货号：C483260)中进行培养，培养周期不超过14天，反应器控制参数为pH 6.8～7.2，溶解氧(DO)为20％～80％，转速75RPM～80RPM，初始培养温度36.0℃～37.0℃；当培养至第6天时，降低培养温度至34.0±0.5℃直到收获。
3.IL2突变体融合蛋白的纯化
IL2突变体融合蛋白的纯化采用多步骤层析、浓缩和过滤单元操作依次对IL2突变体融合蛋白进行纯化：发酵收获液上清经过ProteinA亲和层析(AT ProteinADiamondPlus)进行捕获。捕获后的IL2突变体融合蛋白溶液采用低pH孵育处理以灭活潜在的病毒。IL2突变体融合蛋白溶液中和后通过深层过滤除去沉淀。然后依次进行阳离子交换层析(Diamond S)以去除HCP和聚集体等杂质，以及阴离子交换层析(POROS 50HQ)以去除HCD,HCP和脱落ProteinA等杂质。通过纳滤膜包过滤以去除潜在的内外源病毒。然后用超滤膜包对IL2突变体融合蛋白溶液进行浓缩和缓冲液置换。最后添加辅料并调节浓度，过滤，获得IL2突变体融合蛋白原液。在后续实施例中，通过超滤浓缩换液处理，将原液置换至对应的制剂体系。
实施例2～5针对实施例1中的IL2突变体1-2-连接子-Fc(V91R/Y31V/A73L/H79Q,SEQ ID NO:15)开发维持其稳定性的制剂处方
实施例2缓冲体系的筛选
1.筛选方案
(1)设计12种不同的缓冲液处方体系F1-F12，具体处方体系如表4所示。(2)通过超滤浓缩换液处理，将实施例1获得的IL2突变体融合蛋白原液置换至F1-F12中，调整蛋白浓度为2mg/mL。(3)通过25℃加速稳定性试验和40℃加速稳定性试验，综合考察IL2突变体融合蛋白在12种缓冲体系中的稳定性，筛选出最优的缓冲体系，用于后续辅料和表面活性剂的筛选。考察指标包括外观、pH值、蛋白浓度、纯度(SEC-HPLC)、CE-SDS(NR)、CE-SDS(R)、动态光散射(DLS)。考察方案详见表4。
表4制剂缓冲体系和筛选考察方案
注：X＝外观，pH，蛋白浓度，DLS，SEC-HPLC，CE-SDS(NR&R)。
2.缓冲体系的筛选结果
缓冲体系筛选主要结果汇总如下所示：
表5制剂处方在25℃或40℃加速稳定性试验中的外观检测结果

表6制剂处方在25℃或40℃加速稳定性试验中的pH检测结果
表7制剂处方在25℃或40℃加速稳定性试验中的浓度(mg/mL)检测结果

表8制剂处方在25℃或40℃加速稳定性试验中的DLS(半径,nm)检测结果
表9制剂处方在25℃加速稳定性试验中的SEC-HPLC检测结果

表10制剂处方在40℃加速稳定性试验中的SEC-HPLC检测结果
表11制剂处方在25℃加速稳定性试验中的CE-SDS(NR)检测结果

表12制剂处方在40℃加速稳定性试验中的CE-SDS(NR)检测结果
表13制剂处方在25℃加速稳定性试验中的CE-SDS(R)检测结果
表14制剂处方在40℃加速稳定性试验中的CE-SDS(R)检测结果

3.考察结果
(1)外观优劣排序：F1、F3>F2、F7、F8、F9、F11>F10、F12>F4、F5、F6。
(2)pH值：12种缓冲体系的pH值稳定，无显著性变化。
(3)蛋白浓度优劣排序：F1、F2、F3、F6、F7、F8、F9、F11、F12>F5>F4、F10。
(4)DLS优劣排序：F1、F5、F9>F6、F7、F8>F3、F4、F10、F11、F12>F2。
(5)SEC-HPLC优劣排序：F2、F3、F5、F6、F7、F8、F9>F4、F10>F1>F11、F12。
(6)CE-SDS(NR)优劣排序：F2、F3、F6>F1、F5、F7、F8、F9>F4、F10>F11、F12。
(7)CE-SDS(R)优劣排序：F8、F9>F3、F6>F2、F7、F10>F1、F4、F5、F11、F12。
总结：经过综合考量25℃加速稳定性试验和40℃加速稳定性试验中检测到的各指标，处方F3(20mM醋酸-醋酸钠，pH5.5)优于其他处方，显示出更好的稳定性，因此选择F3进行下一步开发。
实施例3辅料的筛选
1.筛选方案
在实施例2筛选得到的最优缓冲体系(20mM醋酸-醋酸钠，pH5.5)基础上，设计7个处方，独立考察加入氯化钠、甘氨酸、盐酸精氨酸、蔗糖、海藻糖、甘露醇或山梨醇后，IL2突变体融合蛋白(IL2突变体融合蛋白浓度为2mg/mL)的稳定性，筛选出1个最优制剂处方进行后续的表面活性剂筛选。考察指标包括：外观、pH值、浓度、SEC-HPLC、DLS、CE-SDS(NR&R)，具体处方组成和考察方案见表15。
表15辅料筛选方案

注：X＝外观、pH值、浓度、DLS、SEC-HPLC、CE-SDS(NR&R)。
2.辅料的筛选结果
辅料筛选主要结果汇总如下所示。
表16制剂处方在不同温度/时间点的外观检测结果
表17制剂处方在不同温度/时间点的pH值或蛋白浓度检测结果
表18制剂处方在不同温度/时间点的DLS(直径，nm)检测结果
表19-1制剂处方在不同温度/时间点的SEC-HPLC检测结果
表19-2制剂处方在不同温度/时间点的SEC-HPLC检测结果
表20-1制剂处方在不同温度/时间点的CE-SDS(R)检测结果
表20-2制剂处方在不同温度/时间点的的CE-SDS(R)检测结果
表21-1制剂处方在不同温度/时间点的CE-SDS(NR)检测结果
表21-2制剂处方在不同温度/时间点的CE-SDS(NR)检测结果
3.考察结果
(1)外观优劣排序：F3-2＞F3-1＞F3-3＞F3-5＞F3-4、F3-6＞F3-7。
(2)pH值优劣排序：各组之间数据无显著差异。
(3)浓度优劣排序：各组之间数据无显著差异。
(4)DLS优劣排序：F3-2＞F3-1＞F3-3、F3-4、F3-5、F3-6＞F3-7。
(5)SEC-HPLC优劣排序：F3-1、F3-3、F3-4、F3-5、F3-6＞F3-7＞F3-2。
(6)CE-SDS(R)优劣排序：F3-1、F3-3、F3-4、F3-5、F3-6＞F3-7＞F3-2。
(7)CE-SDS(NR)优劣排序：F3-2、F3-3＞F3-4＞F3-5＞F3-7＞F3-6＞F3-1。
总结：根据外观的表现，首先排除在长期稳定性2～8℃中外观出现颗粒的组别F3-4、F3-5、F3-6和F3-7。含有甘氨酸的F3-1处方，在40℃-4W时，CE-SDS(NR)检测到的低聚体含量高于F3-2和F3-3，说明IL-2蛋白在F3-1处方中，更易于降解。F3-2的SEC-HPLC 检测指标与其他组别差异不大，CE-SDS(R)在40℃-4W时其他峰比例较高，但在长期稳定性2～8℃和25℃中与其他组别无显著差异。因此，综合考虑稳定性考察数据，F3-2处方和F3-3处方优于其他处方，表现出良好的稳定性。选择F3-2处方(含140mM盐酸精氨酸)和F3-3处方(含8％w/v蔗糖)进一步考察加入表面活性剂后处方的稳定性。
实施例4表面活性剂的考察
1.考察方案
在F3-2处方和F3-3处方的基础上，考察加入表面活性剂PS-80后处方的稳定性。处方情况详见表22。考察方案详见表23。考察指标包括外观、pH、蛋白浓度、DLS、SEC-HPLC、CE-SDS(NR&R)、不溶性微粒(MFI)。根据考察结果选择合适的制剂处方，用于处方稳定性确认。
表22制剂处方
表23考察方案
注：
X＝外观，pH，蛋白浓度，DLS，SEC，CE-SDS(NR&R)，不溶性微粒(MFI)。
振摇试验(300rpm，25℃)：25℃，300rpm，持续振摇。
冻融试验(-80～25℃)：连续多轮冻融，每轮冻融：将样品放置到-80℃下，到冻结为止，之后放置 到25℃下，到完全融化，形成1轮冻融。
2.筛选结果
主要结果汇总如下所示：
表24不同温度/时间点的外观检测结果
表25不同温度/时间点的pH检测结果

表26不同温度/时间点的蛋白浓度(mg/mL)检测结果
表27不同温度/时间点的DLS(直径，nm)检测结果

表28不同温度/时间点的不溶性微粒(MFI，个微粒/mL)检测结果
表29不同温度/时间点的SEC-HPLC检测结果

表30不同温度/时间点的CE-SDS(NR)检测结果

表31不同温度/时间点的CE-SDS(R)检测结果
3.考察结果
本实施例考察表面活性剂聚山梨酯80(PS80)对蛋白在pH5.5醋酸体系中的保护作用。根据结果显示，蛋白在20mM醋酸-醋酸钠，140mM精氨酸盐酸盐，0.04％或0.06％PS80(w/v)条件下，2mg/mL及5mg/mL的浓度，对于高温、反复冻融和振摇均有良好的耐受性；蛋白在20mM醋酸-醋酸钠，8％蔗糖(w/v)，0.04％PS80(w/v)条件下，2mg/mL的蛋白浓度，对于高温、反复冻融和振摇也有良好的耐受性。各处方之间在外观、pH、浓度、蛋白粒径(DLS)、SEC-HPLC、CE(NR)无明显差异；处方F3-3-1使用8％蔗糖作为辅料，与其他使用盐酸精氨酸的处方相比，40℃-4W时，在蛋白纯度方面CE-SDS(R)的结果更优，其他峰中占比为2.68％，低于其他处方的4.25％～5.05％。综上所述，PS80可有效保护蛋白耐受环境和生产过程中的剪切力，防止蛋白聚集。
4.成药性研究
考察5mg/mL IL-2突变体融合蛋白在20mM醋酸-醋酸钠、pH5.2、8％蔗糖(w/v)、0.02％PS80(w/v)体系中的稳定性，结果显示，5mg/mL IL2突变体融合蛋白在成药性研究的处方中表现出的良好的成药性，详见表32。
表32成药性研究结果
5.结论
考虑到在筛选表面活性剂时，与其他处方相比，处方F3-3-1在CE-SDS(R)方面显示更佳的结果，同时考虑到2mg/mL和5mg/mL均属于低浓度蛋白，且5mg/mL IL2突变 体融合蛋白在与F3-3-1相似处方的成药性研究中显示良好的稳定性，因此，选取5mg/mL，20mM醋酸-醋酸盐、pH5.5、8％蔗糖(w/v)、0.04％PS80(w/v)作为下一步处方确认稳定性研究的目标处方。
实施例5处方确认稳定性
1.处方确认稳定性方案
选择目标处方(5mg/mL IL2突变体融合蛋白、20mM醋酸-醋酸钠、pH5.5、8％蔗糖(w/v)、0.04％PS80(w/v))进行处方确认稳定性研究：将1mL液体制剂分装至2mL西林瓶中，使用胶塞和铝塑盖组成密闭的包装系统，分别正置和倒置，考察其稳定性，考察方案见表33。
表33处方确认稳定性方案
备注：X＝外观、pH值、蛋白浓度、icIEF、SEC-HPLC、CE-SDS(NR)、CE-SDS(R)、不溶性颗粒(MFI)、结合活性试验。
2.处方确认稳定性结果
处方确认稳定性主要结果汇总见下表。
表34-1处方确认稳定性结果

表34-2处方确认稳定性结果
3处方确认稳定性研究结论
2-8℃低温放置3个月和25℃加速稳定性考察3个月，正置和倒置样品在常规检测指标、不溶性微粒、纯度、电荷异质体、活性等方面均没有显著改变，且正置和倒置样品之间无明显差异。40℃加速稳定性考察4周，电荷变体的主峰下降9.2％，主要趋于酸性峰值，纯度和电荷变体发生显著变化。
综上，在低温(2-8℃)实时稳定性和25℃加速试验的考察过程中，样品的常规检测指标、不溶性微粒、纯度、电荷异质体、活性等均没有显著改变，各指标均在质量标准范围内，说明IL2突变体融合蛋白在本处方中具有较好的稳定性。在高温40℃加速稳定性考察实验中，纯度、电荷异质体方面有下降趋势，说明本发明所示制剂对高温敏感。
实施例6 IL2突变体融合蛋白注射液在DTH模型小鼠单药药效学研究
本实施例在KLH诱导的迟发性超敏反应(DTH)模型小鼠上评价IL2突变体融合蛋白注射液的单药药效，其中IL2突变体融合蛋白为IL2突变体1-2-连接子-Fc(V91R/Y31V/A73L/H79Q)，下称IL2-1-2，序列如SEQ ID NO:15所示。阳性对照采用Fc-连接子-IL2突变体6(V91K)，即AMG592，序列如SEQ ID NO:20所示。所述IL2-1-2和AMG592的注射液处方均为5mg/mL IL2-1-2或AMG592、20mM醋酸-醋酸钠、pH5.5、8％蔗糖(w/v)、0.04％PS80(w/v)。
6.1动物分组
使用6-8周龄的BALB/c小鼠，称量每只小鼠的体重，以小鼠体重随机分组分配到G0-G7组8个实验组中，每组10只，分组当天开始给药，记为Day 0，IL2-1-2和AMG592各剂量组均每三天皮下注射给一次药，CsA每天一次腹腔注射给药，给药持续到实验终点。
6.2动物模型构建方案
6.2.1试剂配制
(1)3mg/ml KLH：称取冻干粉，以PBS配置成3mg/ml KLH溶液。
(2)1mg/ml KLH乳剂：将KLH(3mg/ml)：IFA：CFA体积比为1：1：1，用连通管注射器法将抗原乳化，可使抗原充分乳化形成粘稠的乳剂。
(3)1mg/ml KLH溶液：3mg/ml KLH用PBS 3倍稀释为1mg/ml。
6.2.2 DTH模型造模方法
取BALB/c小鼠，第0天在偏右侧背部两点注射总量100μl浓度为1mg/ml的KLH乳剂(KLH、IFA、CFA体积比1：1：1乳化)致敏；正常对照组注射等体积不含KLH的乳剂；第5天，每只小鼠右耳皮内注射10μl浓度为1mg/ml KLH溶液刺激小鼠，使模型小鼠局部皮肤组织产生迟发性超敏反应症状。在右耳局部注射KLH前及注射后24h、48h、72h、96h，使用千分尺测量小鼠右耳厚度，以评价不同处理组小鼠局部迟发性超敏反应程度。
6.3分组给药方案如表35所示
表35 IL2突变体融合蛋白注射液在DTH模型小鼠中的给药方案
6.4检测指标
6.4.1体重检测：分组前称取动物体重，动物分组后每三天一次称取小鼠体重，以g为单位，保留小数点后一位。
6.4.2小鼠耳片厚度测量：
(1)刺激前，用数显外径千分尺测量每只小鼠右耳片厚度，作为本底值
(2)刺激后，在24h、48h、72h、96h测定小鼠耳片厚度。
6.4.3一般临床观察：适应性饲养期和实验期间每周最少观察2次，观察内容包括但不限于动物精神状态、饮食情况等，如有意外情况，必须在实验记录本(纸)上进行记录。
6.5实验终止
6.5.1给药间隔时间应注意观察动物健康状态，若出现以下任何一项或多项情况时，应暂停给药，直至动物恢复正常状态：
(1)动物体重低于开始药物处理时体重的81％停止给药，恢复至开始药物处理时体重的90％继续给
(2)给药后，动物行动迟缓或异常，或有急性应激现象的发生；
6.5.2实验动物人道终点，在实验过程中，当动物状态出现异常时，对动物的健康状态进行评估，决定是否进行治疗，是否可以继续进行实验，或是否实施安乐死。
6.5.3安乐死，小鼠刺激后96h结束实验或实施人道终点时，使用过量CO2将动物窒息安乐死。
6.6统计学分析方法
测定和观察的原始数据必须进行记录。基于原始数据进行分析处理，结果分析用平均数和标准误表示(Mean±SEM)。同时，对数据进行统计学分析，对小鼠耳朵厚度变化的分析采用Two-wayANOVA进行分析，对小鼠在第10天时的体重分析采用One-wayANOVA分析，P<0.05认为有统计学意义。
6.7结果与讨论
6.7.1 IL2-1-2注射液在DTH模型上对小鼠体重的影响
实验动物的体重作为间接测定药物毒性的参考指标。给予CsA、AMG592和不同剂量的IL2-1-2后小鼠体重及体重变化率见图1、2及表36。三天一次皮下注射给予DTH模型小鼠各治疗药物，在实验周期内的观察中，小鼠体重有所波动，除激素给药CsA组小鼠体重有所下降，但是未有小鼠体重下降超过10％，其他给药组小鼠体重均呈上升趋势。所有小鼠状态均无异常，无其他发病或死亡现象，可见CsA在10mg/kg、AMG592在1mg/kg以及IL2突变体1-2在0.03mg/kg，0.1mg/kg，0.3mg/kg和1mg/kg剂量下，治疗均可耐受。
表36突变体1-2注射液对DTH模型小鼠体重的影响

a平均值±标准误(SEM),**P<0.005。
6.7.2 IL2-1-2注射液对DTH模型小鼠耳片厚度变化的影响
各治疗组DTH模型小鼠耳片厚度的变化见图3-4。DTH模型发病在KLH刺激后48h达到顶峰，模型组小鼠在发病高峰时耳片厚度变化值为15.7mm×10-2，比小鼠初始耳片厚度增加了74.1％，模型构建成功，IL2-1-2注射液在1、0.3、0.1和0.03mg/kg治疗组小鼠耳片厚度增加2.45，4.01，9.09和12.26mm×10-2，比KLH刺激前小鼠耳片变化了10.2％，22.0％，41.3％和53.6％。阳性药物CsA 10mg/kg和AMG5921mg/kg对该模型也具有明显的治疗作用，与溶媒对照组相比，显示出一定的治疗效果(P<0.0001)，但两者的治疗效果不如0.3和1mg/kg IL2-1-2。综上所述，IL2-1-2在KLH诱导的DTH模型小鼠上具有明显的治疗效果。
实施例7 IL2突变体融合蛋白注射液在BALB/c小鼠上药效动力学研究
本实施例在BALB/c小鼠上评价IL2突变体融合蛋白注射液的药效动力学特性，其中IL2突变体融合蛋白为IL2突变体1-2-连接子-Fc(V91R/Y31V/A73L/H79Q)，下称IL2-1-2，序列如SEQ ID NO:15所示。阳性对照采用Fc-连接子-IL2突变体6(V91K)，即AMG592，序列如SEQ ID NO:20所示。所述IL2-1-2和AMG592的注射液处方均为5mg/mL IL2-1-2或AMG592、20mM醋酸-醋酸钠、pH5.5、8％蔗糖(w/v)、0.04％PS80(w/v)。
7.1动物分组
将6-8周龄的BALB/c小鼠适应性饲养后，称量每只小鼠的体重，使用EXCEL以小鼠体重基于随机数字的随机分组分配到G1-G5组5个实验组中，每组5只，分组当天开始给药，记为Day 1。
7.2分组给药方案，如表37所示。
表37 IL2突变体融合蛋白注射液在BALB/c小鼠中的给药方案
7.3检测指标
7.3.1体重检测：分组前称取动物体重，动物分组后，取血前称量体重，以g为单位。
7.3.2流式细胞术对小鼠外周血进行免疫细胞分型
在给药后第4天，安乐死小鼠后心脏穿刺，收集外周血。免疫细胞表面标志物(CD3+、 CD4+、CD25+)及核内因子(Foxp3+、Ki-67+)荧光标签抗体进行标记。通过如下FACS方法进行免疫细胞计数及分群分析：
(1)终点安乐死小鼠后，心脏穿刺，收集小鼠外周血至EDTA-2K抗凝管内。轻柔倒转抗凝管，使血液与抗凝剂充分混合以防止其凝固。(2)将300μl未凝结的全血转移到流式管内。向流式管内加入细胞表面标志物(CD3+、CD4+、CD25+)的抗体混合物，室温避光孵育20分钟。(3)向流式管内加入1ml裂解液来裂解红细胞，室温避光孵育5分钟。20℃400g/rcf离心6分钟，弃去上清。(4)重复步骤(3)，再次裂解红细胞。(5)用4ml清洗液(PBS)重悬细胞。将细胞悬液经滤膜过滤后转移到新的流式管内。(6)边振荡边向流式管内逐滴滴加500μl固定液，4℃避光孵育过夜。(7)向流式管内加入2ml穿膜穿核液并振荡，4℃500g/rcf离心流式管5分钟，弃去上清。(8)重复步骤(7)，用3ml穿膜穿核液再次处理细胞。(9)向流式管内加入核内因子(Foxp3+、Ki-67+)的抗体混合物，4℃避光孵育40分钟，每隔20分钟将流式管振荡一次。(10)向流式管内加入4ml清洗液并振荡，4℃500g/rcf离心流式管5分钟，弃去上清并用200μl PBS重悬细胞。(11)
室温下涡旋平衡计数微球至少30秒。向流式管内加入50μl计数微球，在流式细胞仪上进行FACS检测，每个流式管在上机前都需要充分振荡。(12)根据FACS结果用FlowJo软件分析免疫细胞的类型，根据计数微球的说明计算细胞数量。
7.3.3一般临床观察：适应性饲养期和实验期间每周最少观察2次，观察内容包括但不限于动物精神状态、饮食情况等，如有意外情况，必须在实验记录本(纸)上进行记录。
7.4实验终止
7.4.1给药间隔时间应注意观察动物健康状态，若出现以下任何一项或多项情况时，应暂停给药，直至动物恢复正常状态：
①动物体重低于开始药物处理时体重的81％停止给药，恢复至开始药物处理时体重的90％继续给药；
②给药后，动物行动迟缓或异常，或有急性应激现象的发生；
7.4.2实验动物人道终点，在实验过程中，当动物状态出现异常时，对动物的健康状态进行评估，决定是否进行治疗，是否可以继续进行实验，或是否实施安乐死。
7.4.3安乐死，致敏后第26天实验终点时，使用过量CO2将动物窒息安乐死。
7.5统计学分析方法
测定和观察的原始数据必须进行记录。基于原始数据进行分析处理，结果分析用平均数和标准误表示(Mean±SEM)。
7.6结果与讨论
7.6.1 IL2-1-2注射液对BALB/c小鼠外周血免疫细胞的影响
BALB/c小鼠接受不同剂量IL2-1-2给药处理后，外周血免疫细胞计数相比于溶媒对照组小鼠变化倍数如表38所示：皮下注射IL2-1-2可观察到对小鼠Treg细胞增殖的显著促进作用。相比于溶媒对照组，IL2-1-2或AMG592给药3天后，显著刺激了Treg细胞。0.1，0.3，1mg/kg IL2-1-2皮下给药后，小鼠外周血Treg细胞计数变化的倍数分别为13.73，35.27和12.05。1mg/kgAMG592给药组小鼠Treg细胞计数增高程度高于1mg/kg IL2-1-2给药组小鼠，但低于0.3mg/kg给药组小鼠，该结果表明IL2-1-2只需在相对较低的剂量即可对Treg细胞有较好促进作用。Treg/CD4+比例变化倍数展现在表39中。实验中观察到 CD4+Foxp3细胞或CD3+CD4-细胞未被激活或轻度活化。
表38.外周血免疫细胞计数变化倍数
表39 Treg/CD4+比例变化倍数
7.7结论
结果表明皮下注射IL2-1-2注射液可显著促进BALB/c小鼠外周血中Treg细胞增殖，且小鼠对受试分子给药剂量明显耐受。
实施例8 IL2突变体融合蛋白注射液在SD大鼠上药效动力学研究
本实施例在SD大鼠上评价IL2突变体融合蛋白注射液的药效动力学特性，其中IL2突变体融合蛋白为IL2突变体1-2-连接子-Fc(V91R/Y31V/A73L/H79Q)，下称IL2-1-2，序列如SEQ ID NO:15所示。所述IL2-1-2的注射液处方均为5mg/mL IL2-1-2、20mM醋酸-醋酸钠、pH5.5、8％蔗糖(w/v)、0.04％PS80(w/v)。
8.1动物分组
将6-8周龄的SD大鼠适应性饲养后，随机分配到6个实验组中，每组3只雄性和3只雌性。分组当天开始给药(记为第1天)，IL2-1-2注射液分别于第1天、第8天、第15天和第22天经皮下或尾静脉注射给药4次。
8.2分组给药方案如表40所示。
表40 IL2突变体融合蛋白注射液在SD大鼠中的给药方案

注：s.c.：皮下注射，i.v.：静脉注射
8.3观测指标
8.3.1体重监测
分组后及每次给药前称取动物体重，以g为单位记录。
8.3.2大鼠外周血内免疫细胞表型流式分析
分别于第1天给药前及给药后第4天、第11天和第25天收取大鼠外周血。免疫细胞表面标志物(CD3+、CD4+、CD25+)及核内因子(Foxp3+、Ki-67+)用荧光标签抗体进行标记。免疫细胞数量通过下面的荧光激活细胞分选方法(FACS)进行计算：
(1)从大鼠颈静脉收集全血到含有EDTA-2K的抗凝管内。轻柔倒转抗凝管，使血液与抗凝剂充分混合以防止其凝固。(2)将300μl未凝结的全血转移到流式管内。向流式管内加入细胞表面标志物(CD3+、CD4+、CD25+)的抗体混合物，室温避光孵育20分钟。(3)
向流式管内加入1ml裂解液来裂解红细胞，室温避光孵育5分钟。20℃400g/rcf离心流式管，弃去上清。(4)重复步骤(3)，再次裂解红细胞。(5)用4ml清洗液(PBS)重悬细胞。将细胞悬液经滤膜过滤后转移到新的流式管内。(6)边振荡边向流式管内逐滴滴加500μl固定液，4℃避光孵育过夜。(7)向流式管内加入2ml穿膜穿核液并振荡，4℃500g/rcf离心流式管5分钟，弃去上清。(8)重复步骤(7)，用3ml穿膜穿核液再次处理细胞。(9)向流式管内加入核内因子(Foxp3+、Ki-67+)的抗体混合物，4℃避光孵育40分钟，每隔20分钟将流式管振荡一次。(10)向流式管内加入4ml清洗液并振荡，4℃500g/rcf离心流式管5分钟，弃去上清并用200μl PBS重悬细胞。(11)室温下涡旋平衡计数微球至少30秒。向流式管内加入50μl计数微球，在流式细胞仪上进行FACS检测，每个流式管在上机前都需要充分振荡。(12)根据FACS结果用FlowJo软件分析免疫细胞的类型，根据计数微球的说明计算细胞数量。
8.3.3一般临床观察
适应性饲养期和实验期间每周最少进行2次临床观察，观察内容包括但不仅限于动物的健康状态、表现、日常活动、精神状态、饮食情况等。如有意外情况，必须在实验记录本(纸)上进行记录。
8.4实验终止
8.4.1实验过程中及给药间隔内应注意观察动物的健康状态。若出现以下任何一项或多项情况时，应暂停给药，直至动物恢复正常状态：
(1)动物体重低于药物处理前体重的81％时应停止给药，恢复至药物处理前体重的90％时可以继续给药；
(2)给药后，动物行动迟缓或异常，或有急性应激现象发生。
8.4.2人道终点
通过对动物的健康状况进行评估来决定治疗或实验是否继续进行，或是出现以下任何一项或多项情况时是否实施安乐死：
(1)动物行动异常或瘫痪；
(2)动物体重低于分组前体重的20％；
(3)动物体温过低，处于濒死状态等。
8.4.3安乐死
人道终点或实验结束时，用过量CO2使动物安乐死。
8.5.统计学分析
结果分析用平均数和标准误表示(Mean±SEM)。
8.6结果
8.6.1 IL2-1-2对大鼠PBMC内免疫细胞数量和比例的影响
SD大鼠在IL2-1-2给药处理后外周血内免疫细胞的数量比例变化见表41。IL2-1-2经皮下和尾静脉注射到SD大鼠体内后均能引起Treg细胞的扩增。与对照相比，低剂量的IL2-1-2(0.005mg/kg皮下注射或0.002mg/kg尾静脉注射)在给药后第4天到第25天内表现出轻微的促进Treg细胞扩增的作用。皮下注射中等剂量的IL2-1-2(0.05mg/kg)后，大鼠外周血中的Treg细胞的数量在第4天时增加到了基线值的9.55倍，并在第25天时维持在基线值的3.16倍。皮下注射高剂量的IL2-1-2(1mg/kg)后，大鼠外周血内Treg细胞的数量在第11天时达到峰值(基线值的13.8倍)，并在第25天时降低至基线值的3.18倍。尾静脉注射高剂量的IL2-1-2(1mg/kg)后，大鼠外周血内Treg细胞的数量在第4天和第11天时均有显著升高，分别为基线值的21.25倍和30.13倍。但与其他处理组相似的是，Treg细胞的数量在第25天时同样降低到了基线值的3.26倍。这一现象有可能是大鼠经多次给药后体内产生了抗药抗体(ADA)所引起。Treg细胞中Ki-67+细胞的扩增模式也与Treg细胞数量增加的趋势一致。
Treg/CD4+细胞的数量比例变化见表42。低剂量(0.005mg/kg皮下注射或0.002mg/kg尾静脉注射)和中等剂量(0.05mg/kg皮下注射)的IL2-1-2对大鼠外周血内CD4+Foxp3-细胞和CD3+CD4-细胞数量不具有或有轻微的激活作用，但观察到了高剂量(1mg/kg皮下注射或1mg/kg尾静脉注射)的IL2-1-2能够轻微激活上述细胞的扩增。
表41.细胞数量比例变化(以基线值为标准)

注：结果以平均值±标准误表示
表42 Treg/CD4+细胞数量比例变化
注：结果以平均值±标准误表示
8.7结论
以上实验结果表明，IL2-1-2经皮下和尾静脉注射到SD大鼠体内均能够引起Treg细胞的增殖，并且SD大鼠对各个剂量的IL2-1-2均表现出良好的耐受性。

Claims (19)

  1. 一种药物组合物，所述药物组合物包括：包含IL2突变体和抗体Fc嵌段的融合蛋白、缓冲液、渗透压调节剂和表面活性剂；
    所述融合蛋白从N端到C端依次包括：
    (1)IL2突变体、连接子(linker)和抗体Fc嵌段，或
    (2)抗体Fc嵌段、连接子(linker)和IL2突变体；
    优选地，所述IL2突变体与野生型IL2相比，至少包括Y31V、A73L和H79Q突变；
    更优选地，所述IL2突变体还进一步包括V91R突变、H16E突变和D20A突变中的一个或多个突变，例如进一步包括H16E突变和V91R突变；
    所述野生型IL2的氨基酸序列如SEQ ID NO：2所示；
    更优选地，所述IL2突变体具有与SEQ ID NO:3、8～11任一项所示序列相比至少80％同一性的氨基酸序列。
  2. 根据权利要求1所述的药物组合物，其中，
    所述连接子具有与SEQ ID NO:12所示序列相比至少80％同一性的氨基酸序列，或所述连接子具有(G4S)n所示序列，其中，n选自1，2，3，4，5或6；
    所述抗体Fc嵌段具有N297G突变，或所述抗体Fc嵌段具有与SEQ ID NO:13所示序列相比至少80％同一性的氨基酸序列；
    优选地，所述融合蛋白具有与SEQ ID NO:14～18任一项所示序列相比至少80％同一性的氨基酸序列；
    更优选的，所述融合蛋白通过抗体Fc嵌段二聚化形成同源二聚体。
  3. 根据权利要求1～2任一项所述的药物组合物，其中，所述融合蛋白的浓度为1～50mg/mL。
  4. 根据权利要求1～3任一项所述的药物组合物，其中，所述缓冲液选自：醋酸-醋酸钠缓冲液、枸橼酸-枸橼酸钠缓冲液、组氨酸-盐酸组氨酸、琥珀酸-琥珀酸钠、磷酸氢二钠-磷酸二氢钠缓冲液，优选为醋酸-醋酸钠缓冲液；
    所述缓冲液的浓度为1～100mM；
    所述缓冲液的pH值为4.5～8.0。
  5. 根据权利要求1-4任一项所述的药物组合物，其中，所述渗透压调节剂选自：盐、氨基酸、糖或糖醇或其组合；优选地，所述盐选自氯化钠、氯化钾或盐酸精氨酸；优选地，所述氨基酸选自甘氨酸、精氨酸、组氨酸、谷氨酸或甲硫氨酸；优选地，所述糖或糖醇选自蔗糖、海藻糖、甘露醇或山梨醇；
    更优选地，所述渗透压调节剂选自氯化钠、甘氨酸、盐酸精氨酸、蔗糖、海藻糖、甘露醇或山梨醇，最优选为蔗糖或盐酸精氨酸；
    所述糖或糖醇的浓度为1～15％w/v，所述盐的浓度为50～200mM。
  6. 根据权利要求1-5任一项所述的药物组合物，其中，所述表面活性剂选自：聚山梨 酯80(PS-80)、聚山梨酯20(PS-20)或泊洛沙姆；所述表面活性剂的浓度为0.01～0.1％w/v。
  7. 根据权利要求1～6任一项所述的药物组合物，其中，所述药物组合物包括：所述融合蛋白、醋酸-醋酸钠缓冲液、蔗糖和聚山梨酯-80；优选地，所述药物组合物包括：1～50mg/mL所述融合蛋白、1～100mM醋酸-醋酸钠缓冲液、1～15％w/v蔗糖和0.01～0.1％w/v聚山梨酯-80，pH值为4.5～6.0。
  8. 根据权利要求1～6任一项所述的药物组合物，其中，所述药物组合物包括：融合蛋白、醋酸-醋酸钠缓冲液、盐酸精氨酸和聚山梨酯-80；优选地，所述药物组合物包括1～50mg/mL所述融合蛋白、1～100mM醋酸-醋酸钠缓冲液、50～200mM盐酸精氨酸和0.01～0.1％w/v聚山梨酯-80，pH值为4.5～6.0。
  9. 根据权利要求1～8任一项所述的药物组合物，其中，所述药物组合物为静脉、肌内或皮下注射液，优选为皮下注射液。
  10. 制备权利要求1～9任一项所述药物组合物的方法，其中，所述方法包括将所述融合蛋白与所述缓冲液、渗透压调节剂和表面活性剂混合配置的步骤；优选地，所述方法包括将所述融合蛋白的原液通过超滤浓缩置换到所述缓冲液中的步骤。
  11. 一种冻干制剂，其中，所述冻干制剂由权利要求1～9任一项所述药物组合物冻干后形成。
  12. 制备权利要求11所述冻干制剂的方法，其中，所述方法包括冷冻干燥权利要求1～9任一项所述的药物组合物的步骤。
  13. 制备权利要求11所述的冻干制剂的复溶溶液的方法，其中包括将权利要求12所述冻干制剂用溶剂复溶，优选地，所述溶剂为注射用水。
  14. 根据权利要求13所述方法制备得到的含IL2突变体的Fc融合蛋白的复溶溶液。
  15. 权利要求1～9任一项所述药物组合物、权利要求11所述冻干制剂或权利要求14所述复溶溶液在制备用于治疗自身免疫性疾病或增生性疾病的药物中的应用；
    优选地，所述自身免疫性疾病选自类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘；
    优选地，所述增生性疾病选自赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水；其中，所述实体瘤可为良性或恶性，原发性或转移性，所述恶性实体瘤可为癌或肉瘤，例如，上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌；所述血液瘤可选自白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
  16. 权利要求1～9任一项所述药物组合物、权利要求11所述冻干制剂或权利要求14所述复溶溶液，用于治疗自身免疫性疾病或增生性疾病；
    优选地，所述自身免疫性疾病选自类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘；
    优选地，所述增生性疾病选自赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水；其中，所述实体瘤可为良性或恶性，原发性或转移性，所述恶性实体瘤可为癌或肉瘤，例如，上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌；所述血液瘤可选自白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
  17. 一种治疗自身免疫性疾病的方法，其中，所述方法包括给予受试者有效量的如权利要求1～9任一项所述药物组合物或权利要求14所述复溶溶液；优选地，所述自身免疫性疾病选自类风湿关节炎、强直性脊柱炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘；优选地，所述有效量为0.001～10mpk。
  18. 一种治疗增生性疾病地方法，其中所述方法包括给予受试者有效量的如权利要求1～9任一项所述药物组合物或权利要求14所述复溶溶液；优选地，所述增生性疾病选自赘生物、实体瘤、血液瘤、恶性腹水或恶性胸水；其中，所述实体瘤可为良性或恶性，原发性或转移性，所述恶性实体瘤可为癌或肉瘤，例如，上皮细胞癌、内皮细胞癌、鳞状细胞癌、畸胎瘤、肺部肿瘤、乳头瘤病毒引起的癌、腺癌、癌肿、黑色素瘤、血管肉瘤、神经母细胞瘤、转移性肺癌、非小细胞肺癌、小细胞肺癌、乳腺癌、Merkel细胞癌、卵巢癌、肾细胞癌、转移性肾癌、头颈癌、膀胱癌、非肌层浸润性膀胱癌；所述血液瘤可选自白血病、淋巴瘤、多发性骨髓瘤，例如B细胞淋巴瘤、T细胞淋巴瘤、皮肤T细胞淋巴瘤、T细胞大颗粒淋巴细胞白血病。
  19. 一种制品，其包括容器，所述容器装有权利要求1～9任一项所述药物组合物、权利要求11所述冻干制剂或权利要求14所述复溶溶液。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109071623A (zh) * 2016-05-04 2018-12-21 美国安进公司 用于扩增t调节性细胞的白细胞介素-2突变蛋白
CN111432831A (zh) * 2017-12-06 2020-07-17 潘迪恩治疗公司 Il-2突变蛋白及其用途
CN113166220A (zh) * 2018-03-09 2021-07-23 奥美药业有限公司 创新细胞因子前药
WO2022048640A1 (zh) * 2020-09-04 2022-03-10 江苏先声药业有限公司 Il-2突变体及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109071623A (zh) * 2016-05-04 2018-12-21 美国安进公司 用于扩增t调节性细胞的白细胞介素-2突变蛋白
CN111432831A (zh) * 2017-12-06 2020-07-17 潘迪恩治疗公司 Il-2突变蛋白及其用途
CN113166220A (zh) * 2018-03-09 2021-07-23 奥美药业有限公司 创新细胞因子前药
WO2022048640A1 (zh) * 2020-09-04 2022-03-10 江苏先声药业有限公司 Il-2突变体及其应用

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