WO2022100684A1 - 一种包含人白细胞介素2变体或其衍生物的药物组合物及其用途 - Google Patents

一种包含人白细胞介素2变体或其衍生物的药物组合物及其用途 Download PDF

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WO2022100684A1
WO2022100684A1 PCT/CN2021/130246 CN2021130246W WO2022100684A1 WO 2022100684 A1 WO2022100684 A1 WO 2022100684A1 CN 2021130246 W CN2021130246 W CN 2021130246W WO 2022100684 A1 WO2022100684 A1 WO 2022100684A1
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pharmaceutical composition
variant
mannitol
trehalose
polysorbate
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PCT/CN2021/130246
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English (en)
French (fr)
Inventor
叶林茂
范迎方
孙燕燕
于淑香
陈昊
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
上海盛迪医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司, 上海盛迪医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN202180064837.0A priority Critical patent/CN116234912A/zh
Priority to EP21891204.6A priority patent/EP4245768A4/en
Priority to AU2021379816A priority patent/AU2021379816A1/en
Priority to MX2023005458A priority patent/MX2023005458A/es
Priority to CA3201446A priority patent/CA3201446A1/en
Priority to US18/036,587 priority patent/US20230398183A1/en
Priority to JP2023528086A priority patent/JP2023549191A/ja
Priority to KR1020237018510A priority patent/KR20230107598A/ko
Publication of WO2022100684A1 publication Critical patent/WO2022100684A1/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]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin

Definitions

  • the present disclosure relates to the field of pharmaceutical preparations, and more particularly, to a pharmaceutical composition comprising a variant of human interleukin 2 or a derivative thereof.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • TCGF T cell growth factor
  • IL-2 variants At present, many pharmaceutical companies in various countries are developing IL-2 variants, and related patent applications include WO2012062228, CN201280017730.1, US8906356, US9732134, US7371371, US7514073, US8124066, US7803361, WO2016014428 and so on.
  • WO2020125743 relates to a new class of IL-2 variants and derivatives thereof, with higher stability and improved properties for use as immunotherapeutics.
  • IL-2 variants and their derivatives are protein drugs, which are easily degraded when taken orally, and are thermally unstable and easily hydrolyzed. It is of great significance to obtain stable formulations with good appearance.
  • US4604377 describes freeze-dried IL-2 formulations comprising the stabilizer mannitol and the solubilizer sodium dodecyl sulfate (SDS) or sodium deoxycholate sulfate.
  • SDS sodium dodecyl sulfate
  • US5417970 describes lyophilized formulations of IL-2 comprising hydrolyzed gelatin or human serum albumin and alanine.
  • SDS may bind to proteins and be difficult to remove, which is not conducive to renaturation; serum albumin has a large market demand, is expensive, and has a complicated preparation process.
  • ZL01814445.4 discloses IL-2 formulations stabilized by histidine, sucrose and glycine, and it was found that Tween80 promotes the formation of soluble IL-2 aggregates, with significant amounts of soluble aggregates prior to lyophilization. (Wei We et al. Dual effects of Tween 80 on protein stability[J].
  • appearance is one of the important quality attributes of freeze-dried products.
  • the appearance of qualified freeze-dried products should be loose and porous, uniform in color and fine in texture.
  • the cakes of freeze-dried products sometimes have unqualified appearances such as shrinkage and cracking, thereby causing great economic losses, and the auxiliary ingredients can affect the appearance of freeze-dried products.
  • the pH, buffer, excipients and pH of the pharmaceutical composition are determined by indicators such as appearance, RP-HPLC ⁇ SE-HPLC ⁇ IE-HPLC purity and stability. Excipients, surfactants and other aspects were screened, and a pharmaceutical composition containing IL-2 was obtained.
  • the pharmaceutical composition has improved high temperature, freeze-thaw and normal temperature stability, appearance preparation reproducibility, and is suitable for actual production. and clinical applications provide products with better performance.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising IL-2, mannitol and trehalose.
  • the IL-2 concentration in the above pharmaceutical composition is about 0.1 mg/mL to 100 mg/mL, and in some specific embodiments, the IL-2 concentration is about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 10 mg/mL, about 2 mg/mL to about 5 mg/mL.
  • the concentration of IL-2 is about 0.1 mg/mL, 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1.0 mg/mL, about 1.1 mg/mL, about 1.2 mg/mL, about 1.3 mg/mL, about 1.4 mg/mL, about 1.5 mg/mL, About 1.6 mg/mL, about 1.7 mg/mL, about 1.8 mg/mL, about 1.9 mg/mL, about 2.0 mg/mL, about 2.1 mg/mL, about 2.2 mg/mL, about 2.3 mg/mL, about 2.4 mg/mL, about 2.5 mg/mL, about 2.6 mg/mL, about 2.7 mg/mL, about 2.8 mg/mL, about 2.9 mg/mL, about 3.0 mg/mL, about 3.1 mg
  • the concentration of IL-2 is about 2 mg/mL.
  • a saccharide is included in the pharmaceutical compositions of the present disclosure.
  • the "sugar” includes conventional compositions (CH2O ) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like.
  • mannitol, trehalose are included in the pharmaceutical compositions of the present disclosure.
  • the concentration of mannitol is from about 5 mg/mL to about 100 mg/mL. In some embodiments, the concentration of mannitol is about 10 mg/mL to about 50 mg/mL, about 15 mg/mL to 40 mg/mL, about 20 mg/mL to 35 mg/mL, about 25 mg/mL to 40 mg/mL, about 25 mg /mL to 35mg/mL.
  • the concentration of mannitol is about 30 mg/mL.
  • the trehalose in the pharmaceutical compositions of the present disclosure includes trehalose or a hydrate thereof, and in some embodiments, the trehalose is trehalose dihydrate.
  • trehalose is used in a concentration of about 10 mg/mL to about 100 mg/mL, about 20 mg/mL to about 80 mg/mL, about 30 mg/mL to about 70 mg/mL, about 30 mg/mL to about 70 mg/mL, based on trehalose dihydrate. 30 mg/mL to about 50 mg/mL, about 30 mg/mL to about 45 mg/mL, about 35 mg/mL to about 40 mg/mL.
  • trehalose is used at a concentration of about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL, about 21 mg/mL, about 22 mg/mL, about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26 mg/mL, about 27 mg/mL, about 28 mg/mL, about 29 mg/mL, about 30 mg/mL, about 31 mg/mL, about 32 mg/mL, about 33 mg/mL, about 34 mg/mL, about 35 mg/mL, about 36 mg/mL, about 37 mg/mL, about 38 mg/mL, about 39 mg/mL, about 40 mg/mL, about 41 mg/mL, about 42 mg/mL, about 43 mg/mL, about
  • the amount of trehalose used is about 40 mg/mL as trehalose dihydrate.
  • the ratio of mannitol to trehalose is 1:10 to 10:1, 1:7 to 7:1, 1:6 to 6:1, 1 :5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:7 to 3:4, 1:7 to 6:7, 1:7 to 1:1, 1:6 to 3:4, 1:6 to 6:7, 1:6 to 1:1, 1:5 to 3:4, 1:5 to 6:7, 1:5 to 1 :1, 1:4 to 3:4, 1:4 to 6:7, 1:4 to 1:1, 1:3 to 3:4, 1:3 to 6:7, 1:3 to 1:1 , 1:2 to 3:4, 1:2 to 6:7, 1:2 to 1:1, 6:8 to 6:7.
  • the above ratio of mannitol and trehalose is a mass ratio, which can be obtained by actual conversion according to the concentrations of mannitol and trehalose, and this conversion method is well known in the art.
  • the mass ratio of IL-2 to mannitol or trehalose can also be converted according to the concentration.
  • the mass ratio of IL-2 to mannitol ranges from 1:1 to 1:50, eg, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1 :7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50.
  • the mass ratio of IL-2 to trehalose ranges from 4:50 to 4:150, such as 4:50, 4:60, 4:70, 4:80, 4:90, 4:105, 4:120, 4:140.
  • the pharmaceutical compositions of the present disclosure further comprise a surfactant.
  • a surfactant Can be selected from polysorbate 20, polysorbate 80, polyhydroxyalkene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-, myristyl- , linoleyl-, stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, stearyl-sarcosine, linoleyl-, myristyl-, cetyl - Betaine, Lauramidopropyl-, Cocalamidopropyl-, Linoleamidopropyl-, Myristamidopropyl-, Palmamidopropyl-, Isostearamidopropyl- Betaine, Myristamidopropyl-, Palmamidopropyl-, Isostearamido
  • the pharmaceutical compositions of the present disclosure comprise a polysorbate (eg, polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80).
  • polysorbate 80 is included in the pharmaceutical compositions of the present disclosure.
  • polysorbate may act as a stabilizer by reducing the agglomeration of protein drugs.
  • Polysorbate eg, polysorbate 80 at a concentration of about 0.01 mg/mL to about 0.2 mg/mL, about 0.02 mg/mL to about 0.15 mg/mL, about 0.02 mg/mL to about 0.1 mg/mL, about 0.03 mg/mL to about 0.1 mg/mL, about 0.04 mg/mL to about 0.1 mg/mL, 0.02 mg/mL to about 0.05 mg/mL, about 0.05 mg/mL to about 0.1 mg/mL, such as about 0.01 mg/mL mL, about 0.02 mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, About 0.1 mg/mL, about 0.15 mg/mL, about 0.2 mg/mL.
  • the polysorbate (eg, polysorbate 80) concentration is about 0.05 mg/mL.
  • the pharmaceutical compositions of the present disclosure further comprise a buffer.
  • the buffer salts include common hydrates of salts, such as citrate buffer, acetate buffer, histidine buffer, phosphate buffer, carbonate buffer, and succinate buffer.
  • the buffer is a histidine buffer system, wherein the histidine buffer is selected from the group consisting of histidine-hydrochloric acid, histidine-acetate, histidine-phosphate, histidine - Buffers such as sulfate.
  • the buffer is a histidine-HCl buffer.
  • the buffer concentration in the pharmaceutical composition is about 2 mM to about 50 mM, about 5 mM to about 40 mM, about 5 mM to about 30 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 10 mM , for example about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, About 18 mM, about 19 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM.
  • the buffer concentration in the pharmaceutical composition is about 10 mM.
  • the buffer pH in the pharmaceutical composition is about 4.5 to about 6.0, about 5.0 to about 6.0, about 5.0 to about 5.6, about 5.3 to about 5.5, eg, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0.
  • the pH of the buffer in the pharmaceutical composition is about 5.3.
  • compositions of the present disclosure further comprise a solvent.
  • the solvent in the pharmaceutical composition is selected from but not limited to non-toxic physiologically acceptable liquid carriers, such as physiological saline, water for injection, glucose solution (eg 5% glucose injection, glucose and sodium chloride injection) and the like.
  • physiologically acceptable liquid carriers such as physiological saline, water for injection, glucose solution (eg 5% glucose injection, glucose and sodium chloride injection) and the like.
  • compositions comprising IL-2, including but not limited to:
  • a pharmaceutical composition comprising: IL-2, about 30 mg/mL of mannitol, about 40 mg/mL of trehalose, and about 0.05 mg/mL of polysorbate, at a pH of about 5.3, wherein the IL -2 at a concentration of about 1 mg/mL, about 2 mg/mL, about 5 mg/mL, about 10 mg/mL, or about 100 mg/mL;
  • a pharmaceutical composition comprising: IL-2, about 30 mg/mL of mannitol, about 40 mg/mL of trehalose, and 0.1 mg/mL of polysorbate, at a pH of about 5.3, wherein the IL -2 at a concentration of about 1 mg/mL, about 2 mg/mL, about 5 mg/mL, about 10 mg/mL, or about 100 mg/mL;
  • a pharmaceutical composition comprising: about 2 mg/mL of IL-2, mannitol, about 40 mg/mL of trehalose, and about 0.1 mg/mL of polysorbate, with a pH of about 5.3, wherein the mannitol the alcohol is at a concentration of about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, or about 50 mg/mL;
  • a pharmaceutical composition comprising: about 2 mg/mL of IL-2, mannitol, about 40 mg/mL of trehalose, and about 0.05 mg/mL of polysorbate, with a pH of about 5.3, wherein the mannitol the alcohol is at a concentration of about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, or about 50 mg/mL;
  • a pharmaceutical composition comprising: about 2 mg/mL of IL-2, about 30 mg/mL of mannitol, trehalose, and about 0.1 mg/mL of polysorbate, with a pH of about 5.3, wherein the seaweed the sugar is at a concentration of about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, or about 50 mg/mL;
  • a pharmaceutical composition comprising: about 2 mg/mL of IL-2, about 30 mg/mL of mannitol, trehalose, and about 0.05 mg/mL of polysorbate, with a pH of about 5.3, wherein the seaweed
  • concentration of sugar is about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, or about 50 mg/mL.
  • the above-mentioned pharmaceutical compositions (1) to (6) further comprise about 10 mM histidine buffer, eg, histidine-HCl buffer.
  • the IL-2 disclosed in the above (1) to (6) pharmaceutical compositions is an IL-2 variant or a derivative thereof, the IL-2 variant or a derivative thereof comprising N26Q, N29S and N88R Site mutations, which may or may not contain C125A site mutations, for example IL-2 variants or derivatives thereof comprise the amino acid sequence shown in SEQ ID NO:2.
  • the IL-2 is an IL-2 variant or derivative. Described IL-2 includes IL-2 variant or its derivative, in optional embodiment, the IL-2 variant or its derivative in the aforementioned pharmaceutical composition is in wild-type human IL-2. 11, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 70, 71, 72, 78, 82, 88 , 125, 132 have one or more amino acid mutations.
  • the expression for mutation in the present disclosure is abc, wherein a is the amino acid type before mutation, b is the mutation site, and c is the amino acid type after mutation.
  • N26S that is, the 26th position is mutated from asparagine (N) to serine (S); N26 is the mutation of the 26th asparagine (N); 26S is the 26th position mutated to serine (S).
  • the IL-2 variants or derivatives thereof provided by the present disclosure contain one or more amino acid mutations or any combination thereof at the following positions: 26, 29, 30, 71, 11, 132, 70, 82, 27 , 78 bits.
  • the amino acid prior to mutation eg, in wild-type human IL-2
  • 71 is asparagine (N)
  • 11 is glutamine (Q)
  • 132 is leucine (L)
  • 70 is leucine (L)
  • 82 is proline ( P)
  • G glycine
  • F phenylalanine
  • the IL-2 variant or derivative thereof comprises a first type of mutation, the first type of mutation being set forth in any of (1)-(7) below, or any combination thereof:
  • the IL-2 variants or derivatives thereof described above have increased stability, eg, increased deamination stability and/or thermal stability; in particular, the first type of mutation provided by the present disclosure is An IL-2 variant or derivative thereof comprising the mutation has increased stability, including, but not limited to, increased deamination stability and/or thermostability compared to wild-type IL-2.
  • the IL-2 variant or derivative thereof comprises a second type of mutation comprising one or more amino acid mutations at the following positions, or any combination thereof: 20, 88, 126 .
  • the amino acid prior to mutation eg, in wild-type human IL-2
  • the IL-2 variant or derivative thereof further comprises a second type of mutation selected from any one or any combination comprising: N88 mutation to A, R, E , L, F, G, I, M, S, Y, V or D; D20 is mutated to A, H, I, M, E, S, V, W or Y, and Q126 is mutated to N, L, P, F, G, I, M, R, S, T, Y, V.
  • the second type of mutation is selected from the mutations set forth in any one of (8)-(10) or any combination thereof:
  • the second type of mutation can retain the function of IL-2 to induce proliferation and activation of Treg, but eliminate or reduce the function of IL-2 to induce proliferation and activation of effector cells (such as NK and T cells).
  • the IL-2 variant or derivative thereof contains both the first type of mutation and the second type of mutation as described above, and optionally, may or may not contain a C125A site mutation, the first type of mutation being selected From any of (11)-(13):
  • the second type of mutation is N88R or N88G or N88I or N88D.
  • the IL-2 variant or derivative thereof comprises the mutations shown in (14) N26Q, N29S and N88R.
  • the IL-2 variants or derivatives thereof described in the present disclosure have any combination of mutation sites and mutation types in (1)-(13), including but not limited to the IL-2 variants disclosed in WO2020125743A.
  • the above-mentioned mutation is a mutation relative to wild-type IL-2, the amino acid sequence of which is shown in SEQ ID NO: 1.
  • the site numbering of the mutations of the present disclosure is counted from amino acid A at position 2 according to the amino acid sequence shown in SEQ ID NO: 1, and any IL-2 variant of the present disclosure comprises the first amino acid according to SEQ ID NO: 1 methionine (M) at position 1 or excluding methionine (M) at position 1.
  • the IL-2 variant or derivative thereof comprises a compound selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 , SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.
  • the amino acids and corresponding nucleotide sequence numbers of the polypeptides are shown in Table 1 (mutated amino acids are underlined):
  • the number of the above-mentioned mutation sites is calculated according to the number of the human IL-2 mature protein shown in SEQ ID NO: 1.
  • the human IL-2 mature protein does not contain the amino acid M at position 1, so the numbering starts from the amino acid at position 2.
  • A starts counting.
  • "/" indicates that in the same IL-2 variant, the mutations are present simultaneously. All variants may contain or not contain C125A, which is to avoid dimerization.
  • the IL-2 variant or derivative thereof comprises the amino acid sequence shown in SEQ ID NO:2.
  • the IL-2 variant or derivative thereof has the structure shown in formula I, and the relative molecular weight of the structure shown in formula I is about 36kD, wherein the structure shown in formula I comprises the structure shown in SEQ IL-2 variant amino acid sequence shown in ID NO: 2.
  • the structure shown in the formula I is prepared from the IL-2 variant shown in SEQ ID NO: 2 and a PEG molecule with a relative molecular weight of about 20KD, and the total relative molecular weight of the structure shown in the obtained formula I is obtained. is about 36kD.
  • derivatives of IL-2 variants include full-length, partial proteins related to the IL-2 variants of the present disclosure or muteins, functional proteins obtained by further mutation on the basis of the IL-2 variants of the present disclosure Derivatives, functional fragments, biologically active peptides, fusion proteins, isoforms or salts thereof.
  • fusion proteins comprising IL-2 variants, monomers or dimers or trimers or multimers of said IL-2 variants, various modified forms of said IL-2 variants (eg, PEG) ylation, glycosylation, albumin conjugation or fusion, Fc fusion or conjugation, hydroxyethylation, de-O-glycosylation, etc.), as well as homologues of the IL-2 variants in each species.
  • modified forms of said IL-2 variants eg, PEG
  • glycosylation e.g, albumin conjugation or fusion
  • Fc fusion or conjugation e.g., Fc fusion or conjugation
  • hydroxyethylation hydroxyethylation
  • de-O-glycosylation etc.
  • the modification of IL-2 does not result in adverse effects on treatment-related immunogenicity.
  • the IL-2 variant or derivative is PEGylated (may be denoted PEG-IL-2), eg, a mono- or di-PEGylated IL-2 variant or derivative.
  • PEG-IL-2 variants or derivatives include SC-PEG linkers.
  • the PEG-IL-2 variant or derivative includes a methoxy-PEG-aldehyde (mPEG-ALD) linker.
  • the average molecular weight of the PEG moiety is from about 5KD to about 50KD, specifically about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18 , about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50KD; or about 5KD to about 40KD, or about 10KD to about 30KD , or between about 10KD and about 30KD, or between about 15KD and about 20KD.
  • the mPEG-ALD linker comprises a PEG molecule having an average molecular weight selected from about 5 KDa, about 12 KDa, or about 20 KDa (20 ⁇ 2 KDa for quality control).
  • the aldehyde group of mPEG-ALD can be acetaldehyde, propionaldehyde, butyraldehyde, and the like.
  • the IL-2 variant or derivative thereof has an increased serum half-life compared to wild-type IL-2 or a derivative thereof.
  • the IL-2 variant or derivative thereof comprises a second type of mutation
  • the IL-2 variant or derivative thereof is capable of reducing the effect of IL-2 on high affinity receptors (IL-2R ⁇ / ⁇ / ⁇ ) and intermediate-affinity receptors (IL-2R ⁇ / ⁇ ), but the affinity for high-affinity receptors is more reduced than that for intermediate-affinity receptors.
  • IL-2 variants or derivatives thereof are capable of retaining the proliferation-inducing and activating functions of IL-2 on Tregs, but eliminating or reducing the proliferation-inducing and activation of effector cells (eg, NK and T cells) by IL-2 Features.
  • IL-2 variants or derivatives thereof, related sequences, and methods of preparation thereof in WO2020/125743 are incorporated herein in their entirety.
  • the present disclosure also provides a method for preparing a lyophilized formulation of a pharmaceutical composition comprising IL-2, the method comprising the step of lyophilizing the aforementioned pharmaceutical composition.
  • the present disclosure also provides a freeze-dried preparation of the pharmaceutical composition comprising IL-2, the freeze-dried preparation is obtained by freeze-drying any of the aforementioned IL-2 pharmaceutical compositions.
  • the process steps of the described preparation of freeze-dried preparations include:
  • the fill volume of the lyophilized formulation prior to lyophilization is about 1.10 to about 1.20 mL, eg, about 1.15 mL.
  • the lyophilized formulation is stable for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 18 months when stored in the dark at 2-8°C 30 months.
  • the lyophilized formulation is stable at 25°C for at least 1 month, at least 3 months, at least 6 months, at least 12 months.
  • the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 30 days.
  • the present disclosure also provides a reconstituted solution comprising IL-2, which is prepared by reconstituting the aforementioned lyophilized preparation.
  • the present disclosure also provides a method for preparing the above-mentioned reconstituted solution, which includes the step of reconstituting the aforementioned freeze-dried preparation, and the solution used for the reconstitution is selected from, but not limited to, water for injection, physiological saline or glucose solution such as 5% glucose injection , Glucose and Sodium Chloride Injection, etc.
  • the present disclosure also provides an article of manufacture comprising a container containing the aforementioned pharmaceutical composition, lyophilized formulation, or reconstitution solution.
  • the labeled volume of the container can be adjusted as needed, and can be selected from 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.0mL, 1.1mL, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL, 2.0mL, 2.1mL, 2.2mL, 2.3mL, 2.4mL, 2.5mL, 5.0mL, 10mL.
  • the filling volume of this product should be appropriately increased during actual production, each about 1mL.
  • the target filling volume of the pharmaceutical composition during actual filling and production is set to be about 1.15 mL, and the filling volume is controlled in the range of about 1.10 to about 1.20 mL.
  • the inner packaging material of the container is a medium borosilicate glass vial for injection, a bromobutyl rubber stopper for freeze-drying for injection, and an aluminum-plastic composite cap for antibiotic bottle.
  • the preparation of the pharmaceutical composition of the present disclosure may adopt a common method in the art, including the step of mixing an appropriate amount of IL-2 with the aforementioned pharmaceutical excipients.
  • a pharmaceutical formulation containing it is prepared, including adding appropriate amounts of trehalose, mannitol, and optionally other adjuvant ingredients to the pharmaceutical formulation.
  • the present disclosure provides pharmaceutical uses of the above-mentioned pharmaceutical compositions, lyophilized preparations or reconstituted solutions comprising IL-2 for treating autoimmune diseases or alleviating/treating/preventing autoimmune reactions after organ transplantation.
  • the autoimmune disease may be selected from the group consisting of any of the following: type I diabetes mellitus, rheumatoid arthritis, multiple sclerosis, chronic gastritis gastritis), Crohn's disease, Basedow disease, Bechterew disease, psoriasis, myasthenia gravis, autoimmune hepatitis ( autoimmune hepatitis), APECED, allergic granulomatous vasculitis (Chrug-Strauss syndrome), ulcerative colitis (ulcerative colitis), glomerulonephritis (glomerulonephritis), Guillain-Barré syndrome, Hashimoto thyroiditis, lichen sclerosus, systemic lupus erythematodes, PANDAS, rheumatic
  • the IL-2 variant or derivative thereof can be used in combination with an immunosuppressive agent.
  • the immunosuppressant is selected from the group consisting of: glucocorticoids, including decortin, prednisol; azathioprine; cyclosporin A ; mycophenolatemofetil; tacrolimus; anti-T lymphocyte globulin, anti-CD3 antibodies, including muromonab; anti-CD25 antibodies, including basiliximab ( basiliximab and daclizumab; anti-TNF-alpha antibodies, including infliximab and adalimumab; azathioprine; methotrexate; cyclosporine (cyclosporin); sirolimus; everolimus; fingolimod; CellCept; myfortic; and cyclophosphamide ( cyclophosphamide).
  • the pharmaceutical compositions, lyophilized formulations or reconstitution solutions of the present disclosure provide no cure but only partial benefit.
  • physiological changes with some benefit are also considered therapeutically beneficial.
  • the amount of the IL-2 variant, or derivative, immunoconjugate thereof, that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount.”
  • the subject, patient or individual in need of treatment is usually a mammal, more specifically a human.
  • the pharmaceutical composition, lyophilized formulation, or reconstituted solution can be administered by any effective route, for example, by injection, in a form suitable for injection, such as by bolus injection or continuous infusion, into the bloodstream of a patient, or Intravenous injection, subcutaneous injection, intradermal injection, intraperitoneal injection, etc.
  • local administration can be performed at the tumor site.
  • administration can be via any route, eg, by topical administration, regional administration, topical administration, systemic administration, convective enhanced delivery, or a combination thereof.
  • Injectable forms including sterile aqueous solutions or dispersions.
  • the above-mentioned pharmaceutical compositions can be prepared in sterile powder form for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the final injectable form must be sterile and, for ease of injection, must be readily flowable.
  • the pharmaceutical compositions must be stable during manufacture and storage. Therefore, preferably, the pharmaceutical composition is to be preserved under conditions that are resistant to contamination by microorganisms such as bacteria and fungi.
  • Interleukin-2 refers to any native IL-2 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in a cell.
  • the term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants.
  • the amino acid sequence of an exemplary wild-type human IL-2 is set forth in SEQ ID NO:1.
  • Unprocessed human IL-2 additionally contains an N-terminal 20 amino acid signal peptide (as shown in SEQ ID NO. 272 in WO2012107417), which is absent in the mature IL-2 molecule.
  • Amino acid mutation includes amino acid substitutions, deletions, insertions, modifications, and any combination thereof, to achieve a final construct such that the final construct possesses desired properties, such as enhanced stability.
  • Amino acid sequence deletions and insertions include amino- and/or carboxy-terminal deletions and amino acid insertions.
  • An example of a terminal deletion is the deletion of an alanine residue at position 1 of full-length human IL-2.
  • amino acid mutations are amino acid substitutions.
  • non-conservative amino acid substitutions ie replacing one amino acid with another amino acid with different structural and/or chemical properties, can be made.
  • amino acid substitutions include the replacement of hydrophobic amino acids with hydrophilic amino acids.
  • Amino acid substitutions include substitutions from non-naturally occurring amino acids or from naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) amino acid) replacement.
  • Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.
  • a "wild-type IL-2” is a form of IL-2 that is otherwise identical to a variant IL-2 polypeptide, except that the variant IL-2 polypeptide has a wild-type amino acid at each amino acid position. For example, if the IL-2 variant is full-length IL-2 (ie, IL-2 that is not fused or conjugated to any other molecule), then the wild-type form corresponding to this variant is full-length native IL-2. If the IL-2 variant is a fusion between IL-2 and another polypeptide encoded downstream (eg, an antibody chain), then the corresponding wild-type form of the IL-2 variant is fused to a polypeptide having the same downstream polypeptide as the The wild-type amino acid sequence of IL-2.
  • the IL-2 variant is a truncated form of IL-2 (a mutated or modified sequence within a non-truncated portion of IL-2)
  • the wild-type form of this IL-2 variant is one with wild-type A similar truncated IL-2 of the type sequence.
  • wild-type encompasses comparisons to naturally occurring, native IL-2 2.
  • a form of IL-2 comprising one or more amino acid mutations that do not affect binding to the IL-2 receptor, such as a cysteine substitution to alanine at a position corresponding to residue 125 of human IL-2 Acid C125A.
  • wild-type IL-2 comprises the amino acid sequence set forth in SEQ ID NO:1.
  • “Derivative” is intended to be construed broadly to include any IL-2 related product. Including but not limited to human and non-human IL-2 homologs, fragments or truncations, fusion proteins (such as fusions with signal peptides or other active or inactive ingredients, such as antibodies or antigen-binding fragments thereof), Modified forms (eg, PEGylation, glycosylation, albumin conjugation/fusion, Fc conjugation and/fusion, hydroxyethylation, etc.) and conservatively modified proteins, etc.
  • fusion proteins such as fusions with signal peptides or other active or inactive ingredients, such as antibodies or antigen-binding fragments thereof
  • Modified forms eg, PEGylation, glycosylation, albumin conjugation/fusion, Fc conjugation and/fusion, hydroxyethylation, etc.
  • High-affinity IL-2 receptor refers to the heterotrimeric form of the IL-2 receptor consisting of the receptor gamma subunit (also known as the universal cytokine receptor gamma subunit, gammac or CD132), the receptor beta subunit (also known as CD122 or p70) and receptor alpha subunit (also known as CD25 or p55).
  • a “medium affinity IL-2 receptor” refers to an IL-2 receptor that contains only gamma and beta subunits and no alpha subunit (see, eg, Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008) ).
  • Treg means a specialized CD4+ T cell type capable of suppressing the response of other T cells.
  • Tregs are characterized by expressing the alpha subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3), and are involved in the induction and maintenance of peripheral autotolerance to antigens, including those expressed by tumors plays a key role.
  • Tregs require IL-2 for their function and development as well as the induction of their inhibitory characteristics.
  • Effector cells refer to the population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8+ cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages/monocytes.
  • effector T cells such as CD8+ cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells and macrophages/monocytes.
  • Constant modifications apply to amino acid and nucleotide sequences. With respect to a particular nucleotide sequence, conservative modifications refer to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleotides do not encode an amino acid sequence, to substantially the same nucleotide sequence. With respect to amino acid sequences, “conservative modifications” refer to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent changes can be made without Does not alter the biological activity of the protein.
  • PEGylation refers to the attachment of at least one PEG molecule to another molecule (eg, a therapeutic protein).
  • Adagen a PEGylated formulation of adenosine deaminase
  • Linking of polyethylene glycol has been shown to prevent proteolysis (see, eg, Sada et al. (1991) J. Fermentation Bioengineering 71:137-139).
  • PEG is a linear or branched polyether linked at one end to a hydroxyl group and has the following general structure: HO-(CH2CH2O)n-CH2CH2-OH.
  • PEG can be activated by preparing derivatives of PEG with functional groups on some or both ends.
  • a common approach to PEG conjugation of proteins is to activate PEG with functional groups suitable for reaction with lysine and N-terminal amino acid groups.
  • a common reactive group involved in conjugation is the alpha or epsilon amino group of lysine.
  • Reaction of the PEGylated linker with the protein can result in attachment of the PEG moiety primarily at the following sites: the alpha amino group at the N-terminus of the protein, the epsilon amino group on the side chain of a lysine residue, or a histidine residue imidazolyl on the side chain. Since most recombinant proteins have a single alpha and many epsilon amino and imidazole groups, many positional isomers can be generated depending on the chemical nature of the linking group.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • administering when applied to human, veterinary or research subjects refer to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a subject an internal or external therapeutic agent, such as a composition comprising any of the IL-2 variants of the present disclosure and derivatives thereof, or compositions comprising the variants or derivatives, to the subject A person diagnosed with, suspected of having, or susceptible to one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population being treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically unmeasurable extent.
  • an “effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Buffer refers to a buffer that tolerates changes in pH through the action of its acid-base conjugated component.
  • buffers that control pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumaric acid salt, glycylglycine and other organic acid buffers.
  • a “histidine buffer” is a buffer containing histidine ions.
  • histidine buffers include, but are not limited to, histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, and the like buffers.
  • histidine-acetate buffer or histidine-hydrochloric acid buffer histidine-acetate buffer is prepared from histidine and acetic acid
  • histidine-hydrochloric acid buffer is histidine and acetic acid prepared with hydrochloric acid.
  • 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.
  • the citrate buffer is citrate-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, and the like.
  • succinate buffer is succinate-sodium succinate.
  • Phosphate buffer refers to a buffer containing phosphate ions.
  • the phosphate buffer can be selected from any phosphate buffer known to those skilled in the art that is suitable for the system of the present disclosure.
  • the phosphate buffer preferably contains a Mixtures of one or more phosphate buffers, eg, monobasic phosphate, dibasic phosphate, and the like.
  • Particularly useful phosphate buffers are selected from alkali metal and/or alkaline earth metal phosphates.
  • phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, Tris-HCl buffer, sodium phosphate-phosphoric acid solution, hydrogen phosphate Disodium-phosphoric acid solution, sodium phosphate-sodium dihydrogen phosphate solution, etc.
  • the phosphate buffer is disodium hydrogen phosphate-sodium hydrogen phosphate.
  • sodium dihydrogen phosphate and disodium hydrogen phosphate can be anhydrous or hydrated, such as anhydrous disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate heptahydrate, decahydrate Disodium hydrogen phosphate dihydrate, etc.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, acetate-histidine, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like.
  • the acetate buffer is acetic acid-sodium acetate.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as a physiologically/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, promote the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • “Lyophilized formulation” means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.
  • the terms “about”, “approximately” refer to an index value within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation. Alternatively, “about” or “consisting essentially of” may mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% ranging from 0.5% to 1%, in this disclosure, each instance of a number or range of values preceded by the term “about” also includes embodiments of the given number. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about” or “substantially comprising” should be assumed to be within an acceptable error range for the specific value.
  • the lyophilized formulation described in the present disclosure can achieve a stable effect: a pharmaceutical composition in which the protein substantially retains its physical stability, chemical stability and/or biological activity after storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • There are various analytical techniques for measuring protein stability including RP-HPLC, SE-HPLC, IE-HPLC, which measure stability after storage at selected temperatures for selected periods of time.
  • Typical acceptable criteria for stability are as follows: typically no more than about 10%, eg no more than about 5% protein (eg IL-2 variants or derivatives, as measured by RP-HPLC, SE-HPLC or IE-HPLC) substances) aggregate or degrade.
  • the formulation was a pale yellow near colorless clear liquid or colorless, or clear to slightly opalescent.
  • the formulations had no more than ⁇ 10% variation in concentration, pH and osmolality. A reduction of no more than about 10%, eg, no more than about 5%, is generally observed.
  • the protein does not show a significant increase in aggregation, precipitation and/or denaturation if after visual inspection of color and/or clarity, or by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS) , then the protein "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An IL-2 variant or derivative "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit a significant chemical change.
  • Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (as assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by peptide mapping such as combined with mass spectrometry or MALDI/TOF/MS, etc.) methods), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.), and isomerization (by measuring isoaspartic acid content, Peptide Mapping, etc.).
  • An IL-2 variant or derivative "retains it” in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation biological activity”.
  • the biological activity of IL-2 can be determined, for example, by assaying activated cells or downstream signaling pathways.
  • IL-2 variant and “IL-2 analog” are used interchangeably.
  • the “relative molecular weight” of PEG mentioned in the present disclosure refers to the relative molecular weight detected by MALDI-TOF, and the number of PEG repeating units can be obtained by inversely deducing the relative molecular weight of PEG.
  • Example 1 Study on the stability of IL-2 as a bulk drug
  • IL-2 is a PEGylated recombinant human interleukin-2 analog with structural information (Formula I) shown below.
  • the structure shown in the formula I is prepared from the IL-2 variant shown in SEQ ID NO: 2 and a PEG molecule with a molecular weight of about 20KD.
  • Relative molecular weight of formula I about 36 kD.
  • the inspection results of the accelerated test show that the product quality does not change significantly for 1 month under accelerated conditions.
  • 3 batches of bulk drug stock solution (the solution system where the stock solution is located is the same as the solution system obtained by screening in the following example) was placed under accelerated (5°C ⁇ 3°C) conditions for 1 month, and there was no significant change in each detection index. within quality standards.
  • the solution system of the IL-2 stock solution includes 4 ⁇ 0.5g/L bulk drug and pH 5.30 ⁇ 0.05 10mM histidine-hydrochloric acid buffer.
  • the present disclosure adopts the SDS-polyacrylamide gel electrophoresis method (reduction type) to detect the molecular weight as 35.5 ⁇ 3.6KD.
  • the microbial and bacterial endotoxin levels of the API will affect the quality of the finished product. Therefore, it is necessary to control the microbial and bacterial endotoxin levels of the API, which are in line with the provisions of the 2015 edition of the Chinese Pharmacopoeia.
  • a suitable buffer system under the premise of keeping the IL-2 variant (shown in formula I) at a concentration of 2 mg/mL, 5% mannitol as an excipient, and 0.05 mg/mL polysorbate 80 as a stabilizer,
  • the buffer systems were prepared as 10mM citric acid-sodium hydroxide (pH 4.0-5.5), 10mM succinic acid-sodium hydroxide (pH 5.0), 10mM histidine-hydrochloric acid (pH 5.5 and 6.0), 10mM phosphate (pH 6.0) -8.0) solution. Then, the above preparations with different buffer systems were placed at 40°C for 14d, and the relevant indicators were investigated at 0d, 7d, and 14d. The investigation results of each buffer system sample are shown in Table 2.
  • the SEC-HPLC and RP-HPLC data show that the histidine-hydrochloric acid buffer system (pH 5.5 and 6.0) is relatively stable, and the succinate, citrate and phosphate systems all show different degrees of degradation, and the most The best is histidine-hydrochloric acid 5.5 system. Therefore, the histidine-HCl buffer system was chosen.
  • Example 2 It can be seen from Example 2 that the histidine-hydrochloric acid buffer system has good stability, and pH 5.5 is better than 6.0. It is speculated that low pH is beneficial to stabilize the protein, so histidine pH 5.0 was added on the basis of pH 5.5 and 6.0. In the phosphate system, in the solution state, the protein stability of pH 6.0 and 7.0 is higher than that of other pH groups in the same system. In this example, the effects of these two pH systems on protein stability were investigated in the freeze-drying system.
  • mannitol and trehalose were selected as excipients, and the dosage of excipients was investigated. Specifically, under the premise that the buffer system is kept at pH 5.3, 10 mM histidine-hydrochloric acid, and the concentration of the IL-2 variant (shown in formula I) is 2 mg/mL, 1% mannitol+7% trehalose and 3% trehalose were prepared respectively. % mannitol + 4% trehalose samples, and the effects of different concentrations of polysorbate 80 on protein stability were investigated under the conditions of 3% mannitol + 4% trehalose. Then, the samples with different prescriptions mentioned above were placed at 40°C for 30d, and the relevant indicators were investigated at 0d, 14d, and 30 days. The results of the investigation of IL-2 variants (shown in formula I) are shown in Table 4.
  • the stability of 1% mannitol + 7% trehalose and 3% mannitol + 4% trehalose is basically the same when the polysorbate is 0.05mg/mL after being placed at a high temperature of 40 °C for 30 days; Under 3% mannitol + 4% trehalose, when the polysorbate 80 is 0.05mg/mL and 0.1mg/mL, there is no significant difference in stability, but considering the safety, the dosage of polysorbate 80 should be less. Therefore, the polysorbate 80 concentration was chosen to be 0.05 mg/mL.
  • the dosage of the excipient composition in the IL-2 variant (shown in formula I) formulation was finally determined to be 3% mannitol + 4% trehalose, that is, the concentration of mannitol was 30 mg /mL, the concentration of trehalose was 40 mg/mL, and the polysorbate was 0.05 mg/mL.
  • the molecular formula of trehalose is C 12 H 22 O 11 ⁇ 2H 2 O; the molecular formula of histidine is C 6 H 9 N 3 O 2 ; the molecular formula of histidine-hydrochloric acid is C 6 H 9 N 3 O 2 . HCl.H 2 O; water for injection is removed in the freeze-drying process; when the product's declared filling volume is 1 mL, the target filling volume in actual production can be set to 1.15 mL.

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Abstract

提供了一种包含人白细胞介素2变体或其衍生物的药物组合物及其用途。具体而言，提供了一种包含人白细胞介素2(IL-2)变体或其衍生物以及药学上可接受的赋形剂的药物组合物。所述药物组合物具有改进的高温、冻融及常温稳定性、外观制剂重现性。

Description

一种包含人白细胞介素2变体或其衍生物的药物组合物及其用途 技术领域
本公开涉及药物制剂领域，更具体地涉及到一种包含人白细胞介素2变体或其衍生物的药物组合物。
背景技术
人白细胞介素-2(interleukin-2，IL-2)，也称为T细胞生长因子(TCGF)，由133个氨基酸组成，分子量约15kD，其编码基因位于4号染色体(4q27)，包括共7kb的序列。1976年和1977年，Doris Morgan，Francis Ruscetti，Robert Gallo和Steven Gillis，Kendal Smith等分别发现活化后的T细胞培养液可以促进T细胞增殖。之后培养液中的刺激因子被纯化并被鉴定为单一的蛋白质即IL-2。IL-2在体内扩充淋巴细胞群体和提高这些细胞的效应器功能的能力使得其具有抗肿瘤效果，IL-2免疫疗法成为某些转移性癌症患者的治疗选择，目前，高剂量IL-2已被批准用于治疗转移性肾细胞癌和恶性黑素瘤。
目前各国已有多家制药公司正在研发IL-2变体，相关专利申请包括WO2012062228、CN201280017730.1、US8906356、US9732134、US7371371、US7514073、US8124066、US7803361、WO2016014428等。例如，WO2020125743涉及一类新的IL-2变体及其衍生物，具有更高稳定性，并具有用作免疫治疗剂的改善特性。
IL-2变体及其衍生物为蛋白药，口服易降解，具有热不稳定、容易水解的特性。获得稳定、外观优良的制剂具有重要意义。US4604377描述了冷冻干燥的IL-2制剂，它包含稳定剂甘露醇和增溶剂十二烷基硫酸钠(SDS)或者脱氧胆酸硫酸钠。US5417970描述了包含水解明胶或人血清白蛋白和丙氨酸的IL-2冻干制剂。然而，SDS可能与蛋白结合而难以除去，不利于复性；血清白蛋白市场需求量大，价格昂贵、制备工艺复杂。ZL01814445.4公开了组氨酸、蔗糖和甘氨酸稳定的IL-2制剂，且发现Tween80促进可溶性IL-2凝聚物形成，在冻干前就有明显量的可溶性凝聚物。Wang等(Wei W et al.Dual effects of Tween 80 on protein stability[J].Int J Pharm,2008,347(1-2):31-38.)也发现，虽然Tween 80显著抑制摇动诱导的IL-2突变蛋白的聚集，但Tween 80影响IL-2存储稳定性，这种影响与温度有关：在5℃下存储22个月，Tween 80存在几乎无法检测到聚集；在40℃下储存2个月期间，添加0.1％Tween 80可以显著提高储存期间IL-2突变蛋白的聚集速率。
此外，外观是冻干制品的重要质量属性之一，合格的冻干制品外观应是疏松多孔、色泽均匀、质地细腻的固体。在大规模生产中，冻干制品的饼块有时会出现收缩、裂化等不合格外观，从而造成极大的经济损失，而辅料成分会影响冻干 制品的外观。
本公开根据IL-2的药物特性和剂型特点，通过大量的筛选工作，以外观、RP-HPLC\SE-HPLC\IE-HPLC纯度、稳定性等指标对药物组合物的pH、缓冲液、赋形剂、表面活性剂等多方面进行了筛选，获得了包含IL-2的药物组合物，所述药物组合物具有改进的高温、冻融及常温稳定性、外观制剂重现性，为实际生产和临床应用提供了具有更优性能的产品。
发明内容
本公开提供一种药物组合物，其包含IL-2、甘露醇和海藻糖。
一些实施方案中，上述药物组合物中所述IL-2浓度为约0.1mg/mL至100mg/mL，一些具体实施方案中，IL-2的浓度为约1mg/mL至约50mg/mL、约1mg/mL至约10mg/mL、约2mg/mL至约5mg/mL。
一些实施方案中，IL-2的浓度为约0.1mg/mL、0.2mg/mL、约0.3mg/mL、约0.4mg/mL、约0.5mg/mL、约0.6mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL、约1.0mg/mL、约1.1mg/mL、约1.2mg/mL、约1.3mg/mL、约1.4mg/mL、约1.5mg/mL、约1.6mg/mL、约1.7mg/mL、约1.8mg/mL、约1.9mg/mL、约2.0mg/mL、约2.1mg/mL、约2.2mg/mL、约2.3mg/mL、约2.4mg/mL、约2.5mg/mL、约2.6mg/mL、约2.7mg/mL、约2.8mg/mL、约2.9mg/mL、约3.0mg/mL、约3.1mg/mL、约3.2mg/mL、约3.3mg/mL、约3.4mg/mL、约3.5mg/mL、约3.6mg/mL、约3.7mg/mL、约3.8mg/mL、约3.9mg/mL、约4.0mg/mL、约4.1mg/mL、约4.2mg/mL、约4.3mg/mL、约4.4mg/mL、约4.5mg/mL、约4.6mg/mL、约4.7mg/mL、约4.8mg/mL、约4.9mg/mL、约5.0mg/mL、约6.0mg/mL、约7.0mg/mL、约8.0mg/mL、约9.0mg/mL、约10mg/mL、约20mg/mL、约30mg/mL、约40mg/mL、约50mg/mL、约60mg/mL、约70mg/mL、约80mg/mL、约90mg/mL、约100mg/mL。
一些具体实施方案中，IL-2的浓度为约2mg/mL。
甘露醇、海藻糖：
一些实施方案中，本公开的药物组合物中包含糖。所述“糖”包含常规组合物(CH 2O) n及其衍生物，包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。可选自葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、sylitol、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等等。
一些实施方案中，本公开的药物组合物中包括甘露醇、海藻糖。
一些实施方案中，甘露醇的浓度为约5mg/mL至约100mg/mL。一些具体实施方案中，甘露醇的浓度为约10mg/mL至约50mg/mL、约15mg/mL至40mg/mL、约20mg/mL至35mg/mL、约25mg/mL至40mg/mL、约25mg/mL至35mg/mL。例 如约10mg/mL、约20mg/mL、约30mg/mL、约40mg/mL、约50mg/mL、约60mg/mL、约70mg/mL、约80mg/mL、约90mg/mL或约100mg/mL，或约为5mg/mL、约6mg/mL、约7mg/mL、约8mg/mL、约9mg/mL、约10mg/mL、约11mg/mL、约12mg/mL、约13mg/mL、约14mg/mL、约15mg/mL、约16mg/mL、约17mg/mL、约18mg/mL、约19mg/mL、约20mg/mL、约21mg/mL、约22mg/mL、约23mg/mL、约24mg/mL、约25mg/mL、约26mg/mL、约27mg/mL、约28mg/mL、约29mg/mL、约30mg/mL、约31mg/mL、约32mg/mL、约33mg/mL、约34mg/mL、约35mg/mL、约36mg/mL、约37mg/mL、约38mg/mL、约39mg/mL、约40mg/mL、约41mg/mL、约42mg/mL、约43mg/mL、约44mg/mL、约45mg/mL、约46mg/mL、约47mg/mL、约48mg/mL、约49mg/mL、约50mg/mL。
一些具体实施方案中，甘露醇的浓度为约30mg/mL。
一些实施方案中，本公开药物组合物中的海藻糖包括海藻糖或其水合物，一些具体实施方案中，海藻糖为海藻糖二水合物。
一些实施方案中，海藻糖的用量以海藻糖二水合物计，浓度为约10mg/mL至约100mg/mL、约20mg/mL至约80mg/mL、约30mg/mL至约70mg/mL、约30mg/mL至约50mg/mL、约30mg/mL至约45mg/mL、约35mg/mL至约40mg/mL。例如约10mg/mL、约20mg/mL、约30mg/mL、约40mg/mL、约50mg/mL、约60mg/mL、约70mg/mL、约80mg/mL、约90mg/mL、约100mg/mL。
一些实施方案中，海藻糖的用量以海藻糖二水合物计，浓度为约10mg/mL、约11mg/mL、约12mg/mL、约13mg/mL、约14mg/mL、约15mg/mL、约16mg/mL、约17mg/mL、约18mg/mL、约19mg/mL、约20mg/mL、约21mg/mL、约22mg/mL、约23mg/mL、约24mg/mL、约25mg/mL、约26mg/mL、约27mg/mL、约28mg/mL、约29mg/mL、约30mg/mL、约31mg/mL、约32mg/mL、约33mg/mL、约34mg/mL、约35mg/mL、约36mg/mL、约37mg/mL、约38mg/mL、约39mg/mL、约40mg/mL、约41mg/mL、约42mg/mL、约43mg/mL、约44mg/mL、约45mg/mL、约46mg/mL、约47mg/mL、约48mg/mL、约49mg/mL、约50mg/mL、约51mg/mL、约52mg/mL、约52.5mg/mL、约53mg/mL、约54mg/mL、约55mg/mL、约56mg/mL、约57mg/mL、约58mg/mL、约59mg/mL、约60mg/mL、约61mg/mL、约62mg/mL、约63mg/mL、约64mg/mL、约65mg/mL、约66mg/mL、约67mg/mL、约68mg/mL、约69mg/mL、约70mg/mL、约71mg/mL、约72mg/mL、约73mg/mL、约74mg/mL、约75mg/mL、约76mg/mL、约77mg/mL、约78mg/mL、约79mg/mL、约80mg/mL。
一些具体实施方案中，海藻糖的用量以海藻糖二水合物计，浓度为约40mg/mL。
一些实施方案中，甘露醇和海藻糖(以海藻糖计以海藻糖二水合物计)的比例为1:10至10:1、1:7至7:1、1:6至6:1、1:5至5:1、1:4至4:1、1:3至3:1、1:2至2:1、1:7至3:4、1:7至6:7、1:7至1:1、1:6至3:4、1:6至6:7、1:6至1:1、1:5 至3:4、1:5至6:7、1:5至1:1、1:4至3:4、1:4至6:7、1:4至1:1、1:3至3:4、1:3至6:7、1:3至1:1、1:2至3:4、1:2至6:7、1:2至1:1、6:8至6:7。例如1:10、1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:9、2:8、2:7、2:6、2:5、2:4、2:3、2:2、2:1、3:9、3:8、3:7、3:6、3:5、3:4、3:3、3:2、3:1、4:9、4:8、4:7、4:6、4:5、4:4、4:3、4:2、4:1、5:9、5:8、5:7、5:6、5:5、5:4、5:3、5:2、5:1、6:9、6:8、6:7、6:6、6:5、6:4、6:3、6:2、6:1、7:9、7:8、7:7、7:6、7:5、7:4、7:3、7:2、7:1、8:9、8:8、8:7、8:6、8:5、8:4、8:3、8:2、8:1、9:9、9:8、9:7、9:6、9:5、9:4、9:3、9:2、9:1、10:1、10:9、10:8、10:7、10:6、10:5、10:4、10:3、10:2、10:1。
上述甘露醇和海藻糖的比例是质量比，可根据甘露醇和海藻糖的浓度实际换算得到，这种换算方法是本领域公知的。
同样，本公开所述的药物组合物中，IL-2与甘露醇或海藻糖的质量比也可根据浓度换算得到。
一些实施方案中，IL-2与甘露醇质量比的范围是1:1至1:50，例如1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:15、1:20、1:25、1:30、1:35、1:40、1:45、1:50。
一些实施方案中，IL-2与与海藻糖质量比的范围是4:50至4:150，例如4:50、4:60、4:70、4:80、4:90、4:105、4:120、4:140。
表面活性剂：
一些实施方案中，本公开的药物组合物还包含表面活性剂。可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-、肉豆蔻基-、亚油基-、硬脂基-磺基甜菜碱、月桂基-、肉豆蔻基-、亚油基-、硬脂基-肌氨酸、亚油基-、肉豆蔻基-、鲸蜡基-甜菜碱、月桂酰胺基丙基-、柯卡酰胺基丙基-、亚油酰胺基丙基-、肉豆蔻酰胺基丙基-、棕榈酰胺基丙基-、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-、棕榈酰胺基丙基-、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。
一些实施方案中，本公开的药物组合物包含聚山梨酯(例如聚山梨酯20、聚山梨酯40、聚山梨酯60或聚山梨酯80)。一些具体实施方案中，本公开的药物组合物中包含聚山梨酯80。本公开发现，聚山梨酯可能通过减少蛋白药物的团聚现象的产生，进而起到稳定剂的作用。聚山梨酯(例如聚山梨酯80)浓度为约0.01mg/mL至约0.2mg/mL、约0.02mg/mL至约0.15mg/mL、约0.02mg/mL至约0.1mg/mL、约0.03mg/mL至约0.1mg/mL、约0.04mg/mL至约0.1mg/mL，0.02mg/mL至约0.05mg/mL、约0.05mg/mL至约0.1mg/mL，例如约0.01mg/mL、约0.02mg/mL、约0.03mg/mL、约0.04mg/mL、约0.05mg/mL、约0.06mg/mL、约0.07mg/mL、约0.08mg/mL、约0.09mg/mL、约0.1mg/mL、约0.15mg/mL、约0.2mg/mL。
一些具体实施方案中，聚山梨酯(例如聚山梨酯80)浓度为约0.05mg/mL。
缓冲体系：
一些实施方案中，本公开的药物组合物还包含缓冲液。所述的缓冲盐包括盐的常见水合物，例如柠檬酸盐缓冲液、醋酸盐缓冲液、组氨酸盐缓冲液、磷酸盐缓冲液、碳酸缓冲液、琥珀酸缓冲液。
一些实施方案中，所述缓冲液为组氨酸盐缓冲体系，其中组氨酸盐缓冲液选自组氨酸-盐酸、组氨酸-醋酸盐、组氨酸-磷酸盐、组氨酸-硫酸盐等缓冲液。
一些具体实施方案中，所述缓冲液为组氨酸-盐酸缓冲液。
在一些方案中，药物组合物中所述缓冲液浓度为约2mM至约50mM、约5mM至约40mM、约5mM至约30mM、约5mM至约20mM、约5mM至约15mM、约5mM至约10mM，例如约2mM、约3mM、约4mM、约5mM、约6mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、约15mM、约16mM、约17mM、约18mM、约19mM、约20mM、约25mM、约30mM、约40mM、约50mM。
一些具体实施方案中，药物组合物中所述缓冲液浓度为约10mM。
在一些方案中，药物组合物中所述缓冲液pH为约4.5至约6.0、约5.0至约6.0、约5.0至约5.6、约5.3至约5.5，例如约4.5、约4.6、约4.7、约4.8、约4.9、约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0。
一些具体实施方案中，药物组合物中所述缓冲液pH为约5.3。
溶剂：
一些实施方案中，本公开的药物组合物还包含溶剂。
药物组合物中溶剂选自但不限于无毒性的生理学可接受的液体载体，如生理盐水、注射用水、葡萄糖溶液(如5％葡萄糖注射液、葡萄糖氯化钠注射液)等。
此外，本公开还提供包含IL-2的药物组合物，包含但不限于：
(1)药物组合物，其中包含：IL-2、约30mg/mL的甘露醇、约40mg/mL的海藻糖，和约0.05mg/mL的聚山梨醇酯，pH为约5.3，其中所述IL-2的浓度为约1mg/mL、约2mg/mL、约5mg/mL、约10mg/mL或约100mg/mL；
(2)药物组合物，其中包含：IL-2、约30mg/mL的甘露醇、约40mg/mL的海藻糖，和0.1mg/mL的聚山梨醇酯，pH为约5.3，其中所述IL-2的浓度为约1mg/mL、约2mg/mL、约5mg/mL、约10mg/mL或约100mg/mL；
(3)药物组合物，其中包含：约2mg/mL的IL-2、甘露醇、约40mg/mL的海藻糖，和约0.1mg/mL的聚山梨醇酯，pH为约5.3，其中所述甘露醇的浓度为约10mg/mL、约20mg/mL、约30mg/mL、约35mg/mL或约50mg/mL；
(4)药物组合物，其中包含：约2mg/mL的IL-2、甘露醇、约40mg/mL的海藻糖，和约0.05mg/mL的聚山梨醇酯，pH为约5.3，其中所述甘露醇的浓度为约10mg/mL、约20mg/mL、约30mg/mL、约35mg/mL或约50mg/mL；
(5)药物组合物，其中包含：约2mg/mL的IL-2、约30mg/mL的甘露醇、海藻糖，和约0.1mg/mL的聚山梨醇酯，pH为约5.3，其中所述海藻糖的浓度为约10mg/mL、 约20mg/mL、约30mg/mL、约35mg/mL、约40mg/mL或约50mg/mL；
(6)药物组合物，其中包含：约2mg/mL的IL-2、约30mg/mL的甘露醇、海藻糖，和约0.05mg/mL的聚山梨醇酯，pH为约5.3，其中所述海藻糖的浓度为约10mg/mL、约20mg/mL、约30mg/mL、约35mg/mL、约40mg/mL或约50mg/mL。
一些具体实施方案中，上述(1)至(6)药物组合物还包含约10mM组氨酸盐缓冲液，例如组氨酸-盐酸缓冲液。
一些具体实施方式中，上述(1)至(6)药物组合物公开的IL-2是IL-2变体或其衍生物，所述IL-2变体或其衍生物包含N26Q、N29S和N88R位点突变，可以含有或不含有C125A位点突变，例如IL-2变体或其衍生物包含SEQ ID NO：2所示的氨基酸序列。
一些实施方案中，所述IL-2是IL-2变体或衍生物。所述的IL-2包括IL-2变体或其衍生物，在可选的实施方案中，前述的药物组合物中的IL-2变体或其衍生物是在野生型人IL-2的第11、26、27、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、70、71、72、78、82、88、125、132位具有一个或多个氨基酸突变。本公开对于突变的表示方式为abc，其中a为突变前的氨基酸类型，b为突变位点，c为突变后的氨基酸类型。例如，N26S，即第26位由天冬酰胺(N)突变为丝氨酸(S)；N26为第26位的天冬酰胺(N)发生突变；26S为第26位突变为丝氨酸(S)。
具体地，本公开提供的IL-2变体或其衍生物含有以下位点上的一个或多个氨基酸突变或其任意组合：26、29、30、71、11、132、70、82、27、78位。在一些实施方案中，突变前的氨基酸(例如野生型人IL-2中的)为：26位为天冬酰胺(N)、29位为天冬酰胺(N)、30位为天冬酰胺(N)、71位为天冬酰胺(N)、11位为谷氨酰胺(Q)、132位为亮氨酸(L)、70位为亮氨酸(L)、82位为脯氨酸(P)、27位为甘氨酸(G)、78位为苯丙氨酸(F)。
在一些具体实施方案中，IL-2变体或其衍生物包含第一类突变，所述第一类突变为如下(1)-(7)任一所示或其任意组合：
(1)N26Q，
(2)N29S，
(3)N30S，
(4)N71Q，
(5)Q11C和L132C，
(6)L70C和P82C，和
(7)G27C和F78C。
在一些具体实施方案中，上述IL-2变体或其衍生物具有增加的稳定性，例如，增加的脱氨稳定性和/或热稳定性；具体地，本公开提供的第一类突变是包含所述突变的IL-2变体或其衍生物与野生型IL-2相比具有增加的稳定性，包括但不限于， 具有增加的脱氨稳定性和/或热稳定性。
在一些具体实施方案中，IL-2变体或其衍生物包含第二类突变，所述第二类突变含有如下位点上的一个或多个氨基酸突变或其任意组合：20、88、126。一些实施方案中，突变前的氨基酸(例如野生型人IL-2中的)为：20位为天冬氨酸(D)、88位为天冬酰胺(N)、126位为谷氨酰胺(Q)。在一些具体实施方案中，IL-2变体或其衍生物还包含第二类突变，所述第二类突变选自包含以下的任一项或其任意组合：N88突变为A、R、E、L、F、G、I、M、S、Y、V或D；D20突变为A、H、I、M、E、S、V、W或Y，和Q126突变为N、L、P、F、G、I、M、R、S、T、Y、V。
在一些具体实施方案中，第二类突变选自(8)-(10)任一项所示的突变或其任意组合：
(8)N88R或N88G或N88I或N88D，
(9)D20H或D20Y，和
(10)Q126L。
所述第二类突变能够保留IL-2对Treg的诱导增殖和激活功能，但消除或降低IL-2对效应细胞(如NK和T细胞)的诱导增殖和激活功能。
一些实施方案中，IL-2变体或其衍生物同时含有如上所述的第一类突变和第二类突变，以及任选地，可以含有或不含有C125A位点突变，第一类突变选自(11)-(13)中的任一项：
(11)N26Q和N29S，
(12)N26Q、N29S和N71Q，和
(13)N26Q和N30S；
第二类突变为N88R或N88G或N88I或N88D。
一些实施方案中，IL-2变体或其衍生物包含(14)N26Q、N29S和N88R所示的突变。
本公开所述IL-2变体或其衍生物，其具有(1)-(13)中突变位点、突变类型的任意组合方式，包括但不限于WO2020125743A中所公开的IL-2变体。
一些实施方案中，上述突变是相对于野生型IL-2发生的突变，所述野生型IL-2的氨基酸序列如SEQ ID NO：1所示。本公开的突变的位点编号根据SEQ ID NO：1所示的氨基酸序列从第2位的氨基酸A开始计数，本公开的任意IL-2变体包含根据SEQ ID NO：1所示的第1位的甲硫氨酸(M)或不包含第1位的甲硫氨酸(M)。
在一些实施方式中，所述的IL-2变体或其衍生物包含选自SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11或SEQ ID NO：12任一项所示的氨基酸。所述多肽的氨基酸和对应的核苷酸序列号如表1所示(下划线为突变氨基酸)：
表1.IL-2变体氨基酸序列和核酸序列
Figure PCTCN2021130246-appb-000001
Figure PCTCN2021130246-appb-000002
(注：上述的突变位点编号按SEQ ID NO：1所示的人IL-2成熟蛋白编号计算，人IL-2成熟蛋白不含有第1位的氨基酸M，因此编号从第2位的氨基酸A开始计数。其中，“/”表示在同一个IL-2变体中，所述突变是同时存在的。所有变体均可以含有或不含有C125A，含有C125A是避免形成二聚体。)
在一些具体实施方式中，所述的IL-2变体或其衍生物包含SEQ ID NO：2所示的氨基酸序列。
一些具体实施方案中，所述IL-2变体或其衍生物具有如式I所示的结构，式I所示的结构的相对分子量为约36kD，其中，式I所示的结构包含如SEQ ID NO：2所示的IL-2变体氨基酸序列。
Figure PCTCN2021130246-appb-000003
(注：加粗位点为与野生型相比突变的位点为：N26Q、N29S、N88R、C125A，相连的Cys58-Cys105为二硫键位置)
一些具体实施方案中，所述式I所示的结构是由SEQ ID NO：2所示的IL-2变体与相对分子量约20KD的PEG分子制备所得，所得式I所示的结构总相对分子量为约36kD。
在一些实施方案中，IL-2变体的衍生物包括涉及本公开IL-2变体的全长、部分蛋白或在本公开IL-2变体的基础上进一步突变获得的突变蛋白、功能性衍生物、功能性片段、生物活性肽、融合蛋白、同种型或其盐。例如，包含IL-2变体的融合蛋白，所述IL-2变体的单体或二聚物或三聚物或多聚物，所述IL-2变体的各种修饰形式(如PEG化、糖基化、白蛋白缀合或融合、Fc融合或缀和、羟乙基化、去除O-糖基化等)，以及所述IL-2变体在各物种中的同源物。所述IL-2的修饰不会导致对治疗相关的免疫原性的不利影响。
在一些实施方案中，IL-2变体或衍生物是PEG化的(可以表示为PEG-IL-2)， 例如是单或双PEG化的IL-2变体或衍生物。PEG-IL-2变体或衍生物包括SC-PEG连接基。在另一些实施方案中，PEG-IL-2变体或衍生物包括甲氧基-PEG-醛(mPEG-ALD)连接基。在某些实施方案中，PEG部分的平均分子量在约5KD至约50KD，具体地约5、约10、约11、约12、约13、约14、约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25、约30、约35、约40、约45、约50KD；或约5KD至约40KD，或约10KD至约30KD，或约10KD至约30KD之间，或约15KD至约20KD之间。某些具体实施方案中，mPEG-ALD连接基包括具有选自下列平均分子量的PEG分子：约5KDa、约12KDa或约20KDa(质控标准为20±2KDa)。在某些实施方案中，mPEG-ALD的醛基可以是乙醛、丙醛或丁醛等。在一个实施方案中，IL-2变体或其衍生物相比于野生型IL-2或其衍生物具有延长的血清半衰期。
当IL-2变体或其衍生物包含第二类突变时，一些实施方案中，IL-2变体或其衍生物能够降低IL-2对高亲和力受体(IL-2Rα/β/γ)和中等亲和力受体(IL-2Rβ/γ)的亲和力，但对高亲和力受体的亲和力比对中等亲和力受体的亲和力降低得更多。一些实施方案中，IL-2变体或其衍生物能够保留IL-2对Treg的诱导增殖和激活功能，但消除或降低IL-2对效应细胞(如NK和T细胞)的诱导增殖和激活功能。
此处全文引入WO2020/125743中的IL-2变体或其衍生物、相关序列、以及制备方法。
冻干制剂：
本公开还提供一种制备包含IL-2的药物组合物的冻干制剂的方法，所述方法包括将前述药物组合物经冷冻干燥的步骤。
本公开还提供一种包含IL-2的药物组合物的冻干制剂，所述冻干制剂通过将前面任一所述的IL-2药物组合物经冷冻干燥获得。所述制备冻干制剂的工艺步骤包括：
①组氨酸盐缓冲液配制、辅料母液配制；
②式I所示IL-2蛋白溶液转移至配制相；
③加入辅料母液；
④加入缓冲液调节蛋白浓度；
⑤除菌预过滤；
⑥除菌过滤到储液袋；
⑦灌装，半压塞；
⑧冻干；
⑨压塞，出料，轧盖。
在一些实施方案中，冻干制剂在冻干前的灌装装量为约1.10至约1.20mL，例如为约1.15mL。
在一些实施方案中，冻干制剂于2-8℃避光保存，稳定至少1个月，至少3个月， 至少6个月，至少12个月，至少18个月，至少24个月，至少30个月。
在一些实施方案中，该冻干制剂于25℃稳定至少1个月，至少3个月，至少6个月，至少12个月。
在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少30天。
本公开还提供一种包含IL-2的复溶溶液，所述复溶溶液是通过将前述冻干制剂经复溶制备获得。
本公开还提供制备上述复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液选自但不限于：注射用水、生理盐水或葡萄糖溶液如5％葡萄糖注射液、葡萄糖氯化钠注射液等。
本公开还提供一种制品，其包括容器，该容器中装有前述的药物组合物、冻干制剂或复溶溶液。容器标示装量根据需要调整，可选自0.5mL、0.6mL、0.7mL、0.8mL、0.9mL、1.0mL、1.1mL、1.2mL、1.3mL、1.4mL、1.5mL、1.6mL、1.7mL、1.8mL、1.9mL、2.0mL、2.1mL、2.2mL、2.3mL、2.4mL、2.5mL、5.0mL、10mL。注射剂在临床给药过程中可能会发生药液的残留和损失，故为了保证标示装量，根据中国药典2020版四部注射剂通则中的要求，实际生产时应对本品适当增加装量，每约1mL药物组合物实际灌装生产时的目标装量设定为约1.15mL，装量范围控制在约1.10至约1.20mL。
在一些实施方案中，该容器内包材为中硼硅玻璃管制注射剂瓶、注射用冷冻干燥用溴化丁基橡胶塞和抗生素瓶用铝塑组合盖。
制备方法：
制备本公开药物组合物可采用本领域常见的方法，包括将适宜量IL-2与前述药用辅料混合的步骤。
在一些实施方案中，制备IL-2之后，制备包含它的药物制剂，包括将适宜量的海藻糖、甘露醇和可选的其他辅料成分添加到药物制剂中。
用途：
本公开提供上述包含IL-2的药物组合物、冻干制剂或复溶溶液的制药用途，用于治疗自身免疫性疾病或缓解/治疗/预防器官移植之后的自身免疫反应。自身免疫性疾病可以是选自以下或由以下任意组成的组：I型糖尿病(type I diabetes mellitus)、类风湿性关节炎(rheumatoid arthritis)、多发性硬化症(multiple sclerosis)、慢性胃炎(chronic gastritis)、克罗恩病(Crohn’s disease)、巴塞多病(Basedow disease)、别赫捷列夫病(Bechterew disease)、银屑病(psoriasis)、重症肌无力(myasthenia gravis)、自身免疫肝炎(autoimmune hepatitis)、APECED、变应性肉芽肿血管炎(Chrug-Strauss syndrome)、溃疡性结肠炎(ulcerative colitis)、肾小球肾炎(glomerulonephritis)、吉兰-巴雷综合征(Guillain-Barrésyndrome)、桥本甲状腺炎(Hashimoto thyroiditis)、硬化萎缩性苔藓(lichen sclerosus)、系统性红斑狼疮(systemic lupus erythematodes)、PANDAS、风湿热(rheumatic fever)、结节病(sarcoidosis)、舍 格伦综合征(syndrome)、僵人综合症(Stiff-Man syndrome)、硬皮病(scleroderma)、韦格纳肉芽肿病(Wegener’s granulomatosis)、白斑(vitiligo)、自身免疫性肠病(autoimmune enteropathy)、肺出血肾炎综合征(Goodpasturesyndrome)、皮肌炎(dermatomyositis)、多发性肌炎(polymyositis)、自身免疫性过敏症(autoimmune allergy)、哮喘(asthma)。
在一些实施方案中，IL-2变体或其衍生物可以与免疫抑制剂联用。一些实施方案中，免疫抑制剂选自由以下各项组成的组：糖皮质激素(glucocorticoid)，包括去氧皮质酮(decortin)、prednisol；硫唑嘌呤(azathioprine)；环孢素A(cyclosporin A)；吗替麦考酚酯(mycophenolatemofetil)；他克莫司(tacrolimus)；抗T淋巴细胞球蛋白，抗CD3抗体，包括莫罗单抗(muromonab)；抗CD25抗体，包括巴利昔单抗(basiliximab)和达克珠单抗(daclizumab)；抗TNF-α抗体，包括英利昔单抗(infliximab)和阿达木单抗(adalimumab)；硫唑嘌呤；甲氨蝶呤(methotrexate)；环孢素(cyclosporin)；西罗莫司(sirolimus)；依维莫司(everolimus)；芬戈莫德(fingolimod)；骁悉(CellCept)；麦考酚酸钠肠溶剂(myfortic)；以及环磷酰胺(cyclophosphamide)。
一些实施方案中，本公开的药物组合物、冻干制剂或复溶溶液不能提供治愈而仅能提供部分益处。在一些实施方案中，具有一些益处的生理学变化也被视为治疗有益的。如此，在一些实施方案中，提供生理学变化的IL-2变体或其衍生物、免疫缀合物的量被视为“有效量”或“治疗有效量”。需要治疗的受试者、患者或个体通常为哺乳动物，更特定地为人。
可通过任何有效途径施用药物组合物、冻干制剂或复溶溶液，例如，通过注射，可以是适用于注射的形式例如通过弹丸注射法或者持续输注法而施用到患者的血流中，或静脉注射、皮下注射、皮内注射、腹腔注射等。作为选择，可以在肿瘤部位局部施用(肿瘤内或肿瘤周围施用)。在肿瘤内或肿瘤周围施用的情况中，可以经由任意途径施用，例如通过局部施用、区域施用、局地施用、系统性地施用、对流增强输送或者它们的组合进行施用。
注射的形式，包括无菌水溶液或分散体系。进一步地，上述药物组合物可以制备成无菌粉末形式以用于即时配制无菌注射液或分散液。无论如何，最终的注射形式必须是无菌的，且为了易于注射，必须是易于流动的。此外，所述药物组合物在制备和储存过程中必须稳定。因此，优选地，所述药物组合物要在抗微生物如细菌和真菌污染的条件下保存。
发明详述
术语
为了更容易理解本申请，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本申请所属领域的一般技术人员通常理解的含义。
本公开将已公开专利文本WO2020/125743A1的内容全部引入。
本申请所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。
“白介素-2”或“IL-2”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)的任何天然的IL-2。该术语涵盖未加工的IL-2以及源自细胞中的加工的任何形式的IL-2。该术语还涵盖天然存在的IL-2变体，例如剪接变体或等位变体。例示性野生型人IL-2的氨基酸序列如SEQ ID NO：1所示。未加工的人IL-2额外包含N端20个氨基酸的信号肽(如WO2012107417中的SEQ ID NO.272所示)，所述信号肽在成熟的IL-2分子中是缺乏的。
“氨基酸突变”包括氨基酸取代、缺失、插入、修饰及其任意组合，以实现最终构建体，使得最终构建体拥有期望的特性，例如增强的稳定性。氨基酸序列缺失和插入包括氨基和/或羧基端缺失和氨基酸插入。末端缺失的例子是在全长人IL-2的位置1缺失丙氨酸残基。优选地氨基酸突变是氨基酸取代。为了改变例如IL-2多肽的结合特性，可以将非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。优选地氨基酸取代包括用亲水性氨基酸替换疏水性氨基酸。氨基酸取代包括由非天然存在的氨基酸或由20种标准氨基酸的天然存在的氨基酸衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变，包括定点诱变、PCR、基因合成、化学修饰等方法。
“野生型IL-2”是在其它方面与变体IL-2多肽相同，只是在变体IL-2多肽的每个氨基酸位置具有野生型氨基酸的IL-2形式。例如，如果IL-2变体是全长IL-2(即未与任何其它分子融合或缀合的IL-2)，那么此变体所对应的野生型形式是全长天然的IL-2。如果IL-2变体是IL-2与其下游编码的另一种多肽(例如抗体链)之间的融合物，那么此IL-2变体所对应的野生型形式是融合至具有与相同下游多肽的野生型氨基酸序列的IL-2。此外，如果IL-2变体是IL-2的截短形式(在IL-2的非截短部分内的经突变或修饰的序列)，那么此IL-2变体的野生型形式是具有野生型序列的类似截短的IL-2。为了对各种形式的IL-2变体与相应的IL-2野生型形式比较IL-2受体结合亲和力或生物学活性，术语“野生型”涵盖相比于天然存在的、天然的IL-2，包含一处或多处不影响对IL-2受体结合的氨基酸突变的IL-2形式，例如在与人IL-2的残基125对应的位置处的半胱氨酸取代为丙氨酸C125A。在一些实施方案中，野生型IL-2包含SEQ ID NO：1所示的氨基酸序列。
“衍生物”旨在被广义地解释，包括任意IL-2相关的产品。包括但不限于人和非人的IL-2同系物、片段或截短体、融合蛋白(如与信号肽融合或其他活性、非活性成份融合，活性成份例如是抗体或其抗原结合片段)、修饰形式(如PEG化、糖基化、白蛋白缀合/融合、Fc缀和/融合、羟乙基化等)和保守修饰的蛋白等。
“高亲和力IL-2受体”指IL-2受体的异型三聚体形式，其由受体γ亚基(也称 为通用细胞因子受体γ亚基、γc或CD132)、受体β亚基(也称为CD122或p70)和受体α亚基(也称为CD25或p55)组成。比较而言，“中等亲和力IL-2受体”指仅包含γ亚基和β亚基而无α亚基的IL-2受体(参见例如Olejniczak和Kasprzak，MedSci Monit14，RA179-189(2008))。
“调节性T细胞”或“T调节细胞”或“Treg”意指一种能抑制其它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受体的α亚基(CD25)和转录因子叉头框P3(FOXP3)，并在诱导和维持对抗原(包括那些由肿瘤表达的抗原)的外周自体耐受性中起着关键作用。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。
“效应细胞”指介导IL-2的细胞毒性效果的淋巴细胞群体。效应细胞包括效应T细胞如CD8+细胞毒性T细胞、NK细胞、淋巴因子激活的杀伤(LAK)细胞和巨噬细胞/单核细胞。
“保守修饰”适用于氨基酸和核苷酸序列。对于特定的核苷酸序列，保守改性是指编码相同或基本相同的氨基酸序列的那些核酸，或在核苷酸不编码氨基酸序列的情况下，是指基本上相同的核苷酸序列。对于氨基酸序列，“保守修饰”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecμlar Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。
“PEG化”是指至少一个PEG分子与另一个分子(例如治疗性蛋白)连接。例如，批准Adagen(腺苷脱氨酶的PEG化制剂)用于治疗严重联合免疫缺陷病。已经显示，聚乙二醇的连接可以防止蛋白水解作用(参见，例如，Sada等人，(1991)J.Fermentation Bioengineering 71：137-139)。在最常见形式中，PEG是在一端与羟基连接的直链或支链聚醚，并且具有下列常规结构：HO-(CH2CH2O)n-CH2CH2-OH。为了使PEG与分子(多肽、多糖、多核苷酸和小的有机分子)偶联，可以通过制备一些或两个末端具有官能团的PEG的衍生物来活化PEG。蛋白的PEG缀合的常见途径是用官能团活化PEG，该官能团适合与赖氨酸和N-末端氨基酸基团的反应。尤其是，参与缀合的常见反应基团是赖氨酸的α或ε氨基。聚乙二醇化连接基与蛋白的反应可导致PEG部分主要在下列位点处的连接：蛋白的N-末端的α氨基、赖氨酸残基侧链上的ε氨基、或组氨酸残基侧链上的咪唑基。由于大部分重组蛋白质具有单个α和许多ε氨基和咪唑基，可以根据连接基团的化学性质，产生许多位置异构体。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、 药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“施用”、“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种IL-2变体及其衍生物或包含所述变体或衍生物的组合物，所述受试者确诊患有、疑似患有、易感于一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床不可测量的程度。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“缓冲液”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲液。将pH控制在适当范围中的缓冲液的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲液。
“组氨酸盐缓冲液”是包含组氨酸根离子的缓冲液。组氨酸盐缓冲液的实例包括但不限于组氨酸-盐酸、组氨酸-醋酸盐、组氨酸-磷酸盐、组氨酸-硫酸盐等缓冲液。优选组氨酸-醋酸盐缓冲液或组氨酸-盐酸缓冲液，组氨酸-醋酸盐缓冲液是组氨酸与醋酸配制而成，组氨酸-盐酸缓冲液是组氨酸与盐酸配制而成。
“枸橼酸盐缓冲液“是包括枸橼酸根离子的缓冲液。枸橼酸盐缓冲液的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选地枸橼酸盐缓冲液是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲液“是包括琥珀酸根离子的缓冲液。琥珀酸盐缓冲液的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选地琥珀酸盐缓冲液是琥珀酸-琥珀酸钠。
“磷酸盐缓冲液”是包括磷酸根离子的缓冲液，磷酸盐缓冲液选自本领域技术人员知晓的任一种适用于本公开体系的磷酸盐缓冲液即可，磷酸盐缓冲成分优选含有一种或多种磷酸盐缓冲液，例如，一碱价磷酸盐、二碱价磷酸盐等等的混合。特别有用的磷酸盐缓冲液选自碱金属和/或碱土金属的磷酸盐。磷酸盐缓冲液的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸、Tris-HCl缓冲液、磷酸钠-磷酸溶液、磷酸氢二钠-磷酸溶液、磷酸钠-磷酸二氢钠溶液等。优选地磷酸盐缓冲液是磷酸氢二钠-磷酸二氢钠。根据需要，磷酸二氢钠和 磷酸氢二钠可为无水或水合物，如无水磷酸氢二钠、一水磷酸二氢钠、二水磷酸二氢钠、七水磷酸氢二钠、十二水磷酸氢二钠等。
“醋酸盐缓冲液“是包括醋酸根离子的缓冲液。醋酸盐缓冲液的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。优选地醋酸盐缓冲液是醋酸-醋酸钠。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持抗体活性成分的稳定性，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。
本文中，“药物组合物”和“制剂”并不互相排斥。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
本文所用术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围，例如1％至15％之间、在1％至10％之间、在1％至5％之间、在0.5％至5％之间、在0.5％至1％之间变化，本公开中，数字或数值范围之前有术语“约”的每种情况也包括给定数的实施方案。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开所述的冻干制剂能够达到一种稳定的效果：其中的蛋白在贮藏后基本上保留其物理稳定性、化学稳定性和/或生物学活性的药物组合物。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，包括RP-HPLC、SE-HPLC、IE-HPLC，可测量在选定温度贮藏选定时间段后的稳定性。稳定性的典型的可接受的标准如下：通过RP-HPLC、SE-HPLC或IE-HPLC测得，通常不超过约10％，例如不超过约5％的蛋白(例如IL-2变体或衍生物)发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、例如不超过约5％的减少。如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，蛋白没有显示出显著的聚集增加、沉淀和/或变性，那么所述蛋白在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果IL-2变体或衍生物没有显示出显著的化学改变，那么所述IL-2变体或衍生物在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通 过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果IL-2变体或衍生物在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述IL-2变体或衍生物在药物制剂中“保留它的生物活性”。IL-2的生物活性可以例如通过激活细胞或下游信号通路测试来确定。
本公开中，“IL-2变体”和“IL-2类似物”通用。
本公开所述的PEG的“相对分子量”，是指通过MALDI-TOF检测的相对分子质量，PEG重复单元次数可以通过PEG的相对分子量逆推获得。
具体实施方式
以下结合实施例用于进一步描述，但这些实施例并非旨在限制本公开的保护范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.IL-2作为原料药的稳定性研究
IL-2为如下所示结构信息(式I)的聚乙二醇化重组人白介素-2类似物。
Figure PCTCN2021130246-appb-000004
(注：加粗位点为与野生型相比突变的位点为：N26Q、N29S、N88R、C125A，相连的Cys58-Cys105为二硫键位置)
其中，所述式I所示的结构是由SEQ ID NO：2所示的IL-2变体与分子量约20KD的PEG分子制备所得。
式I相对分子量：约36kD。
外观：无色或微黄色澄明液体。
pH：4.5～6.0。
加速试验考察结果显示：加速条件下1个月，产品质量无明显变化。3批原料药原液(原液所处溶液体系同在后实施例筛选获得的溶液体系)在加速(5℃±3℃) 条件下放置1个月，各项检测指标均无显著变化，所有结果均在质量标准范围之内。所述IL-2原液的溶液体系包括4±0.5g/L的原料药和pH 5.30±0.05的10mM组氨酸-盐酸缓冲液。
本公开采用SDS-聚丙烯酰胺凝胶电泳法(还原型)检测分子量为35.5±3.6KD。
原料药的微生物与细菌内毒素水平会对制剂成品的质量产生影响，故需对原料药的微生物与细菌内毒素水平进行控制，均符合中国药典2015年版四部通则的规定。
实施例2.IL-2制剂中缓冲体系和pH筛选
为了筛选适宜的缓冲体系，在保持IL-2变体(式I所示)浓度为2mg/mL、5％甘露醇作为赋形剂、0.05mg/mL聚山梨酯80为稳定剂的前提下，分别制备缓冲体系为10mM柠檬酸-氢氧化钠(pH 4.0-5.5)、10mM琥珀酸-氢氧化钠(pH 5.0)、10mM组氨酸-盐酸(pH 5.5和6.0)、10mM磷酸盐(pH 6.0-8.0)的溶液。然后将上述不同缓冲体系的制剂于40℃放置14d，并于0d、7d、14d考察相关指标。各缓冲体系样品的考察结果见表2。
表2.不同缓冲体系的IL-2变体(式I所示)制剂40℃14d考察结果
Figure PCTCN2021130246-appb-000005
(注：表中“/”或空白表示未检测)
根据表2考察结果可知，SEC-HPLC和RP-HPLC数据显示组氨酸-盐酸缓冲体系(pH 5.5和6.0)比较稳定，琥珀酸盐、柠檬酸盐和磷酸盐体系均呈现不同程度降解，最优是组氨酸-盐酸5.5体系。因此，选择使用组氨酸-盐酸缓冲体系。
实施例3.IL-2制剂中冻干保护剂和pH值的筛选
由实施例2可知，组氨酸-盐酸缓冲体系稳定性较好，且pH5.5优于6.0。推测低pH有利于稳定蛋白，所以在pH 5.5和6.0的基础上增加了组氨酸pH 5.0。磷酸 盐体系中，在溶液状态下，pH6.0和7.0蛋白稳定性高于同体系其他pH组，本实施例在冻干体系中考察这2个pH体系对蛋白稳定性影响。
在保持IL-2变体(式I所示)浓度为2mg/mL、0.05mg/mL聚山梨酯80为稳定剂的前提下，分别将5％甘露醇、1％甘露醇+7％蔗糖、1％甘露醇+7％海藻糖组合的方式为赋形剂，来考察对IL-2蛋白的保护作用。将上述不同处方的制剂于40℃放置30d，并于0d、7d、14d、30d考察相关指标，考察结果见表3。
表3.不同pH和冻干保护剂的IL-2变体(式I所示)制剂40℃30d考察结果
Figure PCTCN2021130246-appb-000006
根据上述考察结果可知，通过40℃0d、7d、14d、30d实验发现，磷酸盐pH 7.0体系在RP和SE上表现均不好，磷酸盐pH 6.0体系在前期溶液中稳定性弱于组氨酸-盐酸体系且处于缓冲极限，因此排除磷酸盐体系；在组氨酸-盐酸pH5.5条件下，7％蔗糖+1％甘露醇和7％海藻糖+1％甘露醇在30天的SE结果均优于5％甘露醇；组氨酸-盐酸pH 6.0体系在30天RP和SE值低于组氨酸-盐酸pH 5.5和5.0体系，稳定性较差，故确定pH适宜范围为5.0-5.5，例如以5.3为目标pH。
实施例4.IL-2制剂中赋形剂比例和聚山梨酯80浓度的筛选
不同赋形剂比例不但会影响产品外观也会影响蛋白的稳定性。初步选择使用 甘露醇和海藻糖作为赋形剂，并对赋形剂的用量进行了考察。具体地，在保持缓冲体系为pH 5.3 10mM组氨酸-盐酸，IL-2变体(式I所示)浓度为2mg/mL的前提下，分别制备1％甘露醇+7％海藻糖和3％甘露醇+4％海藻糖样品，同时在3％甘露醇+4％海藻糖条件下考察不同浓度聚山梨酯80对蛋白稳定性影响。然后将上述不同处方的样品于40℃放置30d，并于0d、14d、30天考察相关指标。IL-2变体(式I所示)考察结果见表4。
表4.不同赋形剂比例和聚山梨酯80浓度的IL-2变体(式I所示)制剂40℃30d考察结果
Figure PCTCN2021130246-appb-000007
根据试验结果可知，高温40℃放置30d后，在聚山梨酯为0.05mg/mL时，1％甘露醇+7％海藻糖和3％甘露醇+4％海藻糖处方稳定性基本无差别；在3％甘露醇+4％海藻糖下，当聚山梨酯80为0.05mg/mL和0.1mg/mL时，稳定性无明显差别，但考虑到安全性，聚山梨酯80用量以少为宜，因此选择聚山梨酯80浓度为0.05mg/mL。综合考虑样品的稳定性和外观，最终将IL-2变体(式I所示)制剂中赋形剂组合物的用量确定为3％甘露醇+4％海藻糖，即甘露醇的浓度为30mg/mL，海藻糖的浓度为40mg/mL，聚山梨酯为0.05mg/mL。
本公开中，海藻糖分子式为C 12H 22O 11·2H 2O；组氨酸分子式为C 6H 9N 3O 2；组氨酸-盐酸，分子式为C 6H 9N 3O 2.HCl.H 2O；注射用水在冷冻干燥工序去除；当产品标示装量为1mL，实际生产时的目标装量可设定为1.15mL。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本公开的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (18)

  1. 一种药物组合物，其包含IL-2、甘露醇、海藻糖和组氨酸盐缓冲液，其中，甘露醇和海藻糖的质量比为1:7至3:4，优选为3:4。
  2. 根据权利要求1所述的药物组合物，所述IL-2为IL-2变体或其衍生物，
    所述IL-2变体或其衍生物包含N26Q和N29S突变，优选地，还进一步包含选自N88R、N88G、N88I或N88D的突变。
  3. 根据前述任一项权利要求所述的药物组合物，其中所述甘露醇浓度为约10mg/mL至约100mg/mL，优选为约10mg/mL至约50mg/mL，更优选为约30mg/mL。
  4. 根据前述任一项权利要求所述的药物组合物，其中所述海藻糖浓度为约10mg/mL至约100mg/mL，优选为约30mg/mL至约70mg/mL，更优选为约40mg/mL。
  5. 根据前述任一项权利要求所述的药物组合物，其中所述IL-2的浓度为约0.1mg/mL至约100mg/mL，优选为约1mg/mL至约10mg/mL，更优选为约2mg/mL。
  6. 根据前述任一项权利要求所述的药物组合物，其还包含表面活性剂，优选为聚山梨酯，更优选为聚山梨酯80。
  7. 根据权利要求6所述的药物组合物，其中所述表面活性剂的浓度为约0.01mg/mL至约0.2mg/mL，优选为约0.05mg/mL至约0.1mg/mL，更优选为约0.05mg/mL。
  8. 根据前述任一项权利要求所述的药物组合物，所述组氨酸盐缓冲液浓度为约2mM至约50mM，优选为约5mM至约20mM，更优选为约10mM。
  9. 根据前述任一项权利要求所述的药物组合物，所述药物组合物的pH为约4.5至约6.0，优选为约5.0至约5.6，更优选为约5.3。
  10. 根据前述任一项权利要求所述的药物组合物，其包含：
    (a)约0.1mg/mL至约100mg/mL的IL-2，
    (b)约10mg/mL至约100mg/mL的甘露醇，
    (c)约10mg/mL至约100mg/mL的海藻糖，
    (d)约0.01mg/mL至约0.2mg/mL的聚山梨醇酯，和
    (e)约2mM至约50mM的组氨酸盐缓冲液，
    所述药物组合物的pH约为约4.5至约6.0；
    优选地，所述药物组合物中包含：
    (a)约1mg/mL至约10mg/mL的IL-2，
    (b)约10mg/mL至约50mg/mL的甘露醇，
    (c)约30mg/mL至约70mg/mL的海藻糖，
    (d)约0.02mg/mL至约0.1mg/mL的聚山梨醇酯80，和
    (e)约5mM至约20mM的组氨酸盐缓冲液，
    所述药物组合物的pH为约5.0至约5.5；
    更优选地，所述药物组合物中包含：
    (a)约2mg/mL的IL-2，
    (b)约30mg/mL的甘露醇，
    (c)约40mg/mL的海藻糖，
    (d)约0.05mg/mL的聚山梨醇酯80，和
    (e)约10mM的组氨酸盐缓冲液，
    所述药物组合物的pH为约5.3。
  11. 根据前述任一项权利要求所述的药物组合物，其中所述药物组合物进一步包含生理学可接受的溶剂，优选生理盐水、注射用水或葡萄糖溶液。
  12. 根据前述任一项权利要求所述的药物组合物，其中所述IL-2是IL-2变体或其衍生物，包含选自SEQ ID NO：2和8-12中任一项所示的氨基酸序列，并且所述SEQ ID NO：2和8-12中第1位的甲硫氨酸(M)可以存在或缺失。
  13. 根据前述任一项权利要求所述的药物组合物，所述IL-2是单体、和/或PEG化的、和/或糖基化的、和/或白蛋白缀合或融合的、和/或Fc融合的、和/或羟乙基化的、和/或去除O-糖基化的；
    优选地，PEG连接至IL-2变体的N端；
    更优选地，PEG的相对分子量为约5KD至约50KD；
    最优选地，PEG的相对分子量为约20KD。
  14. 根据前述任一项权利要求所述的药物组合物，所述IL-2包含如式I所示的结构，其中所述PEG的相对分子量为约20KD，所示的氨基酸序列与SEQ ID NO:2所示的氨基酸序列相同，
    Figure PCTCN2021130246-appb-100001
  15. 一种冻干制剂，所述冻干制剂通过将权利要求1-14任一项所述的药物组合物经冷冻干燥获得，或所述冻干制剂经复溶可形成如权利要求1-14任一项所述的药物组合物。
  16. 一种复溶溶液，所述复溶溶液通过将权利要求15所述的冻干制剂经复溶制备获得。
  17. 根据权利要求1-14任一项所述的药物组合物、或如权利要求15所述的冻干制剂、或如权利要求16所述的复溶溶液在制备药物中的用途，所述药物用于治疗和/或预防自身免疫性疾病或器官移植之后的自身免疫反应，优选地，所述自身免疫疾病选自I型糖尿病、类风湿性关节炎、多发性硬化、系统性红斑狼疮(SLE)、湿疹、哮喘。
  18. 制备如权利要求1-14任一项所述的药物组合物的方法，包括将IL-2与各药用辅料混合的步骤。
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