WO2023025249A1 - 一种含融合蛋白的药物组合物 - Google Patents

一种含融合蛋白的药物组合物 Download PDF

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WO2023025249A1
WO2023025249A1 PCT/CN2022/114858 CN2022114858W WO2023025249A1 WO 2023025249 A1 WO2023025249 A1 WO 2023025249A1 CN 2022114858 W CN2022114858 W CN 2022114858W WO 2023025249 A1 WO2023025249 A1 WO 2023025249A1
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Prior art keywords
pharmaceutical composition
buffer
seq
histidine
antibody
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PCT/CN2022/114858
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English (en)
French (fr)
Inventor
马夏珍
田晨敏
李小菲
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Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
Original Assignee
Jiangsu Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Application filed by Jiangsu Hengrui Pharmaceutical Co Ltd, Shanghai Shengdi Pharmaceutical Co Ltd filed Critical Jiangsu Hengrui Pharmaceutical Co Ltd
Priority to EP22860595.2A priority Critical patent/EP4393949A4/en
Priority to CA3229503A priority patent/CA3229503A1/en
Priority to MX2024002203A priority patent/MX2024002203A/es
Priority to AU2022332510A priority patent/AU2022332510A1/en
Priority to CN202280048851.6A priority patent/CN117642424A/zh
Priority to KR1020247008409A priority patent/KR20240049304A/ko
Priority to JP2024512159A priority patent/JP2024530978A/ja
Publication of WO2023025249A1 publication Critical patent/WO2023025249A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
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    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
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    • 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
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07K2319/00—Fusion polypeptide

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and in particular, the present disclosure relates to a pharmaceutical composition comprising a fusion protein containing covalently bound IL-2 and an anti-IL-2 antibody, and its use as a medicine.
  • Interleukin-2 also known as T cell growth factor (TCGF)
  • TCGF T cell growth factor
  • the gene is located on chromosome 4 (4q27), including a total of 7kb sequence, consisting of 133 amino acids, with a molecular weight of about 15kD.
  • TCGF T cell growth factor
  • Doris Morgan, Francis Ruscetti, Robert Gallo, Steven Gillis, and Kendal Smith found that activated T cell culture medium could promote T cell proliferation.
  • the stimulatory factor in the culture was then purified and identified as a single protein, IL-2.
  • IL-2 acts through the IL-2R on the cell surface.
  • IL-2R includes three subunits, IL-2R ⁇ (ie CD25), IL-2R ⁇ (ie CD122) and IL-2R ⁇ (ie CD132).
  • Three subunits can form three receptor forms: high-binding receptors contain all three subunits IL-2R ⁇ / ⁇ / ⁇ , intermediate-binding receptors contain IL-2R ⁇ / ⁇ , and low-binding receptors are IL -2R ⁇ .
  • IL-2R ⁇ and IL-2R ⁇ are necessary for IL-2 to activate downstream signaling pathways.
  • IL-2 When IL-2 binds IL-2R ⁇ and IL-2R ⁇ simultaneously, the two receptor subunits form a heterodimer, and the phospho IL-2R ⁇ is not necessary for signal transduction, but it can enhance the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ .
  • Treg continues to express high-binding receptors IL-2R ⁇ / ⁇ / ⁇ , while unactivated CD8+ T cells and NK cells express only intermediate-binding receptors IL-2R ⁇ / ⁇ . Therefore, low-dose IL-2 preferentially binds and activates Tregs, while high-dose IL-2 can also activate immune effector cells such as CD8+ T cells and NK cells.
  • low-dose IL-2 can be used for a series of immune-related, such as the treatment of autoimmune diseases and inflammatory diseases, such as systemic systemic lupus erythematosus (SLE), type I diabetes (type I diabetes, T1D), chronic graft versus host disease (chronic Graft versus Host Disease, cGvHD), bone marrow transplantation (hematopoietic stem cell transplantation, HSCT), alopecia areata (alopecia areata), hepatitis C virus-induced vasculitis, chronic GvHD, asthma, dermatitis, alopecia areata, acute lung injury, chronic beryllium dust disease, sepsis, etc.
  • SLE systemic systemic lupus erythematosus
  • type I diabetes type I diabetes, T1D
  • chronic graft versus host disease chronic Graft versus Host Disease, cGvHD
  • bone marrow transplantation hematopoietic stem
  • IL-2 has a short half-life and requires frequent administration; on the other hand, IL-2 has a small window of administration. If the dose is slightly higher, it may activate immune effector cells and aggravate autoimmune diseases.
  • the present disclosure provides a pharmaceutical composition containing the fusion protein.
  • the composition has therapeutic activity.
  • the composition also has the advantages of good stability, suitable for freeze-drying and the like.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a fusion protein comprising covalently bound IL-2 and an anti-IL-2 antibody and a buffer, the buffer being histidine Buffer, Acetate Buffer or Succinate Buffer.
  • the buffer is a histidine buffer or an acetate buffer.
  • the buffer is a histidine-histidine hydrochloride buffer or an acetic acid-sodium acetate buffer.
  • the buffer is a histidine-histidine HCl buffer.
  • the pharmaceutical composition of any one of the above, wherein the pH of the buffer is 5.0-6.5.
  • the pH of the buffer is 5.0-6.0.
  • the buffer has a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.
  • the buffer has a pH of 5.5-6.0.
  • the buffer has a pH of 6.0.
  • the pH of the pharmaceutical composition differs by no more than ⁇ 0.3 from the pH of a buffer it contains.
  • the pH of the pharmaceutical composition is 6.0-6.5.
  • the pH of the pharmaceutical composition is 6.0-6.3.
  • the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.
  • the pharmaceutical composition according to any one of the above, wherein the concentration of the fusion protein is 5 mg/mL to 50 mg/mL. In some embodiments, the concentration of the fusion protein is 5 mg/mL to 30 mg/mL. In some embodiments, the concentration of the fusion protein is 10 mg/mL to 30 mg/mL. In some embodiments, the concentration of the fusion protein is 5 mg/mL, 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, 30 mg/mL. In some embodiments, the concentration of the fusion protein is 30 mg/mL. In some embodiments, the concentration of the fusion protein is 10 mg/mL.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from the group consisting of polysorbate, polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, octyl Sodium Glycoside, Lauryl-Sultaine, Myristyl-Sultaine, Linoleyl-Sultaine, Stearyl-Sultaine, Lauryl-Sarcosine, Myristyl- Sarcosine, Linoleyl-Sarcosine, Stearyl-Sarcosine, Linoleyl-Betaine, Myristyl-Betaine, Cetyl-Betaine, Lauramidopropyl-Betaine, Cocamidopropyl-Betaine, Linoleamidopropyl-Betaine, Myr
  • the surfactant is a polysorbate or a poloxamer. In some embodiments, the surfactant is polysorbate 80 (PS80), polysorbate 20 (PS20), or poloxamer 188 (PF68). In some embodiments, the surfactant is polysorbate 80 or poloxamer 188. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the surfactant is polysorbate 80.
  • the pharmaceutical composition of any one of the above, wherein the concentration of the surfactant is 0.05 mg/mL to 2 mg/mL. In some embodiments, the surfactant concentration is 0.05 mg/mL to 1.0 mg/mL. In some embodiments, the surfactant concentration is 0.1 mg/mL to 0.8 mg/mL. In some embodiments, the surfactant concentration is 0.1 mg/mL to 0.6 mg/mL. In some embodiments, the surfactant concentration is 0.2 mg/mL to 0.6 mg/mL.
  • the surfactant concentration is 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg /mL, 0.8mg/mL, 0.9mg/mL, 1.0mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, 1.6mg/mL, 1.7mg /mL, 1.8mg/mL, 1.9mg/mL or 2.0mg/mL. In some embodiments, the surfactant concentration is 1.6 mg/mL.
  • the surfactant concentration is 1.5 mg/mL. In some embodiments, the surfactant concentration is 0.8 mg/mL. In some embodiments, the surfactant concentration is 0.6 mg/mL. In some embodiments, the surfactant concentration is 0.4 mg/mL.
  • the pharmaceutical composition of any one of the above, wherein the pharmaceutical composition comprises a sugar comprises a sugar.
  • the sugar is selected from the conventional composition (CH 2 O) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. .
  • the sugar can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mili Disaccharide, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.
  • the sugar is selected from one or more of sucrose, mannitol and trehalose.
  • the sugar is sucrose.
  • the pharmaceutical composition as described in any one of the above in some embodiments, is 20 mg/mL to 100 mg/mL. In some embodiments, the concentration of the sugar is 30 mg/mL to 80 mg/mL. In some embodiments, the concentration of the sugar is 40 mg/mL to 80 mg/mL. In some embodiments, the sugar is present at a concentration of 30 mg/mL to 50 mg/mL.
  • the concentration of the sugar is 20 mg/mL, 30 mg/mL, 36 mg/mL, 40 mg/mL, 45 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 87 mg/mL mL, 90mg/mL or 100mg/mL.
  • the sugar is at a concentration of 87 mg/mL.
  • the sugar is at a concentration of 80 mg/mL.
  • the concentration of the sugar is 50 mg/mL.
  • the concentration of the sugar is 45 mg/mL.
  • the concentration of the sugar is 40 mg/mL.
  • the concentration of the sugar is 36 mg/mL.
  • the excipient is glycine, methionine, proline, histidine, phenylalanine, arginine hydrochloride, glutamic acid, aspartic acid, or EDTA sodium. In some embodiments, the excipient is glycine.
  • the pharmaceutical composition according to any one of the above wherein the concentration of the excipient is 1 mg/mL to 30 mg/mL; preferably, the concentration of the excipient is 5 mg/mL to 15 mg/mL. In some embodiments, the pharmaceutical composition according to any one of the above, wherein the concentration of the excipient is 12 mg/mL. In some embodiments, the pharmaceutical composition according to any one of the above, wherein the concentration of the excipient is 10 mg/mL. In some embodiments, the pharmaceutical composition according to any one of the above, wherein the concentration of the excipient is 9 mg/mL.
  • the concentration of the buffer is 5 mM to 100 mM. In some embodiments, the concentration of the buffer is 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the buffer is at a concentration of 5 mM to 30 mM. In some embodiments, the buffer is at a concentration of 10 mM to 30 mM. In some embodiments, the buffer is at a concentration of 5 mM to 10 mM. In some embodiments, the buffer is at a concentration of 10 mM. In some embodiments, the concentration of the buffer is 5 mM.
  • the KD value of the affinity of the anti-IL-2 antibody to IL-2 is ⁇ 5nM. In some other embodiments, the KD value of the affinity of the anti-IL-2 antibody to IL-2 is >5nM or ⁇ 10nM, and ⁇ 200nM or ⁇ 150nM or ⁇ 100nM or ⁇ 80nM or ⁇ 60nM.
  • the KD value is > 5 nM and ⁇ 60 nM, > 5 nM and ⁇ 50 nM, > 10 nM and ⁇ 50 nM, > 10 nM and ⁇ 60 nM, > 5 nM and ⁇ 80 nM, > 10 nM and ⁇ 80 nM, > 5 nM and ⁇ 100nM, ⁇ 10nM and ⁇ 100nM, >5nM and ⁇ 150nM, or ⁇ 10nM and ⁇ 150nM.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • HCDR1 set forth in SEQ ID NO: 13, 19, 25, 31, 37 or 43, and/or
  • HCDR2 set forth in SEQ ID NO: 14, 20, 26, 32, 38 or 44, and/or
  • HCDR3 set forth in SEQ ID NO: 15, 21, 27, 33, 39 or 45, and/or
  • LCDR1 shown in SEQ ID NO: 16, 22, 28, 34, 40 or 46, and/or
  • LCDR2 shown in SEQ ID NO: 17, 23, 29, 35, 41 or 47, and/or
  • LCDR3 shown in SEQ ID NO: 18, 24, 30, 36, 42 or 48.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • HCDR1 set forth in SEQ ID NO: 13, 25 or 37, and/or
  • HCDR2 shown in SEQ ID NO: 14, 26 or 38, and/or
  • HCDR3 set forth in SEQ ID NO: 15, 27 or 39, and/or
  • LCDR2 shown in SEQ ID NO: 17, 29 or 41, and/or
  • LCDR3 shown in SEQ ID NO: 18, 30 or 42.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • HCDR1 set forth in SEQ ID NO: 19, 31 or 43, and/or
  • HCDR2 set forth in SEQ ID NO: 20, 32 or 44, and/or
  • HCDR3 as set forth in SEQ ID NO: 21, 33 or 45, and/or
  • LCDR2 shown in SEQ ID NO: 23, 35 or 47, and/or
  • LCDR3 shown in SEQ ID NO: 24, 36 or 48.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • HCDR1-3 shown in SEQ ID NO: 43-45 and/or LCDR1-3 shown in SEQ ID NO: 46-48.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • a VH as set forth in or at least 90% identical to SEQ ID NO: 3 a VL as set forth in or at least 90% identical to SEQ ID NO: 4;
  • a VH as set forth in or at least 90% identical to SEQ ID NO: 5 a VL as set forth in or at least 90% identical to SEQ ID NO: 6;
  • a VH as set forth in or at least 90% identical to SEQ ID NO: 7 a VL as set forth in or at least 90% identical to SEQ ID NO: 8;
  • VH as set forth in or at least 90% identical to SEQ ID NO: 9
  • VL as set forth in or at least 90% identical to SEQ ID NO: 10
  • variable regions and CDRs of the anti-IL-2 antibody adopt the Kabat numbering system.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • HC as shown in one of SEQ ID NO: 157, 159, 161, 163, 165, 167 or having at least 80%, 90%, 95%, 98%, 99% identity thereto; and/or
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody comprises
  • HC as shown in or at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID NO: 159, or at least 80%, 90% as shown in SEQ ID NO: 160 , 95%, 98%, 99% identical LC;
  • HC as shown in or at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID NO: 161, or at least 80%, 90% as shown in SEQ ID NO: 162 , 95%, 98%, 99% identical LC;
  • HC as shown in or at least 90% identical to SEQ ID NO: 163, as shown in SEQ ID NO: 164 or at least 80%, 90%, 95%, 98%, 99% identical thereto LC;
  • HC as shown in or at least 90% identical to SEQ ID NO: 165, as shown in SEQ ID NO: 166 or at least 80%, 90%, 95%, 98%, 99% identical thereto LC; or
  • HC as shown in or at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID NO: 167, or at least 80%, 90% as shown in SEQ ID NO: 168 , 95%, 98%, 99% identity LC.
  • the pharmaceutical composition according to any one of the above, the anti-IL-2 antibody is Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, domain antibody and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), e.g. specifically scFv, Fv, Fab or Fab' fragments.
  • the pharmaceutical composition as described in any one of the above, the IL-2 in the fusion protein is connected with the light chain variable region or the heavy chain variable region of the anti-IL-2 antibody, for example, IL-2 2 is connected to the N-terminus of the light chain variable region or the heavy chain variable region of the anti-IL-2 antibody. In some embodiments, IL-2 is linked to the N-terminus of the light chain variable region of an anti-IL-2 antibody.
  • IL-2 in the fusion protein is connected to the anti-IL-2 antibody through a linker.
  • the linker is a flexible peptide linker.
  • the linker is 5-50, 10-40, 20-30 or 25 amino acids in length.
  • the linker is selected from the amino acid sequence shown as (G m S n ) x or (GGNGT) x or (YGNGT) x , wherein m and n are each independently selected from 1-8 Integer (for example, 1,2,3,4,5,6,7 or 8), x is independently selected from the integer of 1-20 (for example, 1,2,3,4,5,6,7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker has an amino acid sequence as represented by (G 4 S)x, where x is independently selected from an integer of 1-6 (eg, x is 4 or 5).
  • the linker may also be other linkers having similar flexibility and length to the amino acid sequence shown in (G m S n ) x or (GGNGT) x or (YGNGT) x above.
  • the amino acid sequence of the linker is shown in SEQ ID No:190.
  • the pharmaceutical composition as described in any one of the above selectively enhances the activity of cells that highly express IL-2Ra or promotes their proliferation, and the cells that highly express IL-2Ra are, for example, regulating Sexual T (Treg) cells.
  • IL-2 in the fusion protein has an amino acid substitution of the third amino acid (as an example, T3) or an N-terminal deletion.
  • the third amino acid substitution of IL-2 is Ala, Gln or Glu (as an example, T3A, T3Q, T3E), and the N-terminal deletion is the deletion of the first 3 or 7 amino acids at the N-terminal.
  • the pharmaceutical composition of any one of the above, the fusion protein comprises HC and LC selected from the group consisting of:
  • the pharmaceutical composition according to any one of the above selectively enhances the effect of IL-2 on Treg activity (such as pro-proliferation activity, STAT5 phosphorylation-promoting activity), and/or Selectively increasing the ability of IL-2 to promote the expression of one or more of FOXP3, CD25 and Icos in Treg cells.
  • the pharmaceutical composition as described in any one of the above, the fusion protein also has one or more of (i)-(iii):
  • Treg cells are promoted to a higher degree than the proliferation of CD8+T cells, CD4+T cells or NK cells.
  • the pharmaceutical composition as described in any one of the above, the anti-IL-2 antibody comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region has: comprising SEQ ID NO HCDR1 comprising the amino acid sequence of 37, HCDR2 comprising the amino acid sequence of SEQ ID NO: 38 and HCDR3 comprising the amino acid sequence of SEQ ID NO: 39
  • the light chain variable region has: comprising the amino acid sequence of SEQ ID NO: 40 LCDR1, LCDR2 comprising the amino acid sequence of SEQ ID NO: 41 and LCDR3 comprising the amino acid sequence of SEQ ID NO: 42.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:9 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:10.
  • the anti-IL-2 antibody comprises a heavy chain of the amino acid sequence of SEQ ID NO: 165 and a light chain of the amino acid sequence of SEQ ID NO: 166.
  • the pharmaceutical composition according to any one of the above the IL-2 is fused directly or through a linker to the N-terminus of the light chain of the anti-IL-2 antibody.
  • the amino acid residue at position 3 of the IL-2 is E.
  • the fusion protein comprises a heavy chain of the amino acid sequence of SEQ ID NO: 165 and a light chain of the amino acid sequence of SEQ ID NO: 189.
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • the acid buffer has a pH of 6.0.
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • said fusion protein of 10mg/mL to 30mg/mL, (b) polysorbate 80 of 0.1mg/mL to 0.8mg/mL, (c) sucrose of 30mg/mL to 50mg/mL, (d) 5 mg/mL to 15 mg/mL glycine, and (e) 5 mM to 10 mM histidine-histidine hydrochloride buffer having a pH of 6.0.
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • composition according to any one of the above, which comprises the following components:
  • the pharmaceutical composition of any one of the above, the pharmaceutical composition is a liquid formulation.
  • the solvent of the liquid formulation is water.
  • the route of administration of the composition is subcutaneous injection, intradermal injection, intravenous injection or intramuscular injection. In some embodiments, the route of administration of the composition is subcutaneous injection.
  • the present disclosure also provides a lyophilized preparation, which is characterized in that the lyophilized preparation can form the pharmaceutical composition as described above after reconstitution.
  • the present disclosure also provides a lyophilized preparation, which is a lyophilized form of the pharmaceutical composition described in any one of the above.
  • the present disclosure also provides a method for preparing a freeze-dried preparation, which includes the step of freeze-drying the pharmaceutical composition as described above.
  • the present disclosure also provides a freeze-dried preparation, which is obtained by freeze-drying the pharmaceutical composition as described in any one of the above.
  • the present disclosure also provides a reconstitution solution, which is characterized in that the reconstitution solution is prepared by reconstitution of the freeze-dried preparation as described in any one of the above items.
  • the present disclosure also provides a lyophilized preparation, which is a reconstituted form of any one of the lyophilized preparations described above.
  • the present disclosure also provides a product, which includes a container filled with the pharmaceutical composition according to any one of the above, the freeze-dried preparation according to any one of the above or the reconstitution solution according to any one of the above.
  • the present disclosure also provides a pharmaceutical composition as described in any of the above, a lyophilized formulation as described in any of the above, a reconstitution solution as described in any of the above, or a product as described in any of the above.
  • a pharmaceutical composition as described in any of the above, a lyophilized formulation as described in any of the above, a reconstitution solution as described in any of the above, or a product as described in any of the above.
  • the present disclosure also provides a method for treating autoimmune diseases or delaying the progress of autoimmune diseases, comprising administering to patients an effective amount of the pharmaceutical composition as described in any one of the above, the freeze-dried preparation of any of the above, or the lyophilized preparation of any of the above Reconstitution solution or preparation as described in any of the above.
  • the present disclosure also provides the pharmaceutical composition as described in any of the above, the lyophilized formulation as described in any of the above, the reconstitution solution as described in any of the above, or the product as described in any of the above, which can be used It is used to treat autoimmune diseases or delay the progression of autoimmune diseases.
  • the autoimmune disease is selected from any one or combination of lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, multiple sclerosis, inflammatory bowel disease , chronic GvHD, asthma, dermatitis, alopecia areata, acute lung injury, sepsis, reactive arthritis, rheumatoid arthritis.
  • Figure 1A and Figure 1B are the results of ELISA experiments in which anti-IL-2 antibodies block the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ , respectively;
  • Figure 1A shows that anti-IL-2 antibodies block the binding of IL-2 to IL-2R ⁇
  • Figure 1B is the ELISA result of anti-IL-2 antibody blocking the combination of IL-2 and IL-2R ⁇ .
  • Fig. 2 is the result of detecting the binding ability of anti-IL-2 antibody to IL-2 by ELISA experiment.
  • Figure 3A to Figure 3H are the experimental results of non-covalent complexes of anti-IL-2 antibody and IL-2 stimulating STAT5 phosphorylation of human peripheral blood Treg and CD8 + T cells.
  • Figure 4A to Figure 4D are the experimental results of the fusion protein stimulating STAT5 phosphorylation activity in human peripheral blood regulatory T cells Treg and CD8 + T cells.
  • Figure 5A to Figure 5F are the experimental results of the effect of the fusion protein on the proliferation of lymphocytes in the peripheral blood of Balb/c mice.
  • Figure 6 shows the experimental results of the effect of the fusion protein on the proliferation of lymphocytes in the spleen of mice immunized with OVA.
  • Fig. 7 is the pharmacodynamic result of the fusion protein in the mouse delayed hypersensitivity reaction model.
  • Figure 8A to Figure 8B are the pharmacodynamic results of the fusion protein in the mouse arthritis model.
  • Figure 9A to Figure 9F are the experimental results of the fusion proteins formed by anti-IL-2 antibody and different IL-2 variants stimulating the phosphorylation activity of STAT5 in human peripheral blood regulatory T cells Treg and CD8 + T cells.
  • Figure 11A and Figure 11B are the experimental results of the effect of D2-60-b-T3E on the content of dsDNA IgG and IgM in the serum of MRL/lpr mice (**P ⁇ 0.01 vs. model control group).
  • Interleukin-2 or "IL-2” is intended to be construed broadly, including any IL-2 related product. Including but not limited to human and non-human IL-2 homologues, fragments or truncations, fusion proteins (such as fusion with signal peptides or other active and inactive components, active components such as antibodies), modified forms (such as PEGylated , glycosylation, albumin conjugation/fusion, Fc conjugation/fusion, hydroxyethylation, etc.), and conservatively modified proteins, etc.
  • the term encompasses any native IL-2 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats).
  • the term also encompasses unprocessed IL-2 as well as any form of IL-2 derived from processing in cells, also naturally occurring variants of IL-2, such as splice variants or allelic variants, and IL-2 Conservatively modified variant of 2.
  • IL-2R ⁇ refers to those derived from any vertebrate source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats)
  • Any native CD25 including "full-length” unprocessed CD25 and any form of CD25 derived from processing in the cell, also includes naturally occurring CD25 variants, such as splice variants or allelic variants.
  • CD25 is human CD25, and an exemplary sequence is shown in SEQ ID NO.37.
  • High affinity IL-2 receptor refers to the heterotrimeric form of the IL-2 receptor, which consists of the receptor gamma subunit (also known as the general cytokine receptor gamma subunit, gammac, or CD132), the receptor ⁇ subunit (also known as CD122, p70 or IL-2R ⁇ ) and receptor ⁇ subunit (also known as CD25, p55, IL-2R ⁇ ).
  • an "intermediate affinity IL-2 receptor” refers to an IL-2 receptor comprising only a gamma subunit and a beta subunit without an alpha subunit (see e.g. Olejniczak and Kasprzak, MedSci Monit 14, RA179-189 (2008 )).
  • Constant modifications apply to both amino acid and nucleotide sequences. With respect to particular nucleotide sequences, conservatively modified refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleotides do not encode an amino acid sequence, to essentially identical nucleotide sequences. With respect to amino acid sequences, “conservative modification” refers to the replacement of amino acids in proteins by other amino acids with similar characteristics (e.g. charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) Does not change the biological activity of the protein.
  • amino acid mutations include 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, increased activity.
  • Amino acid sequence deletions and insertions include amino and/or carboxyl terminal deletions and amino acid insertions.
  • Preferred amino acid mutations are amino acid substitutions. To alter eg the binding properties of an anti-IL-2 antibody, non-conservative amino acid substitutions can be made, ie one amino acid is replaced with another amino acid having different structural and/or chemical properties.
  • Preferred amino acid substitutions include replacing hydrophobic amino acids with hydrophilic amino acids.
  • Amino acid substitutions include those made 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 site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.
  • Antibody is used in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigen-specificity is also different.
  • immunoglobulins can be divided into five classes, or isotypes, of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are mu, delta, and gamma chains, respectively , ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region).
  • the variable region includes 3 hypervariable regions (CDRs) and 4 framework regions (FRs) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • definitive delineation of the CDRs can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex, and identifying the residues involved in the antigen binding site. This can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • Various analytical methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformation definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see eg Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature , 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, e.g., Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and de-novo methods to model the tertiary structure of antibodies from the base sequence (see Samudrala et al., 1999, in “Proteins, Structure, Function and Genetics Suppl., 3: 194-198") Those described in "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach").
  • the positions of the CDRs can be identified as residues making enthalpy contributions to antigen binding (see eg Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).
  • CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental results that specific residues or groups of residues do not significantly affect antigen binding.
  • a CDR may refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, IMGT, contact and/or conformational definitions.
  • a “monoclonal antibody” or “mAb” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, unlike polyclonal antibodies, each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the characteristics of an antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies for use in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or by recombinant DNA methods such as described in US Pat. No. 4,816,567.
  • monoclonal antibodies can also be isolated from phage libraries generated using the technique described in McCafferty et al., 1990, Nature 348:552-554.
  • Human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, “human antibodies” do not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (ie, "humanized antibodies”).
  • Human antibody or “recombinant human antibody” includes human antibodies produced, expressed, created or isolated by recombinant methods, techniques and methods involved are well known in the art, such as:
  • Antibodies isolated from host cells transformed to express the antibodies such as transfectomas
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences.
  • Such recombinant human antibodies comprise variable and constant regions which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur, such as during antibody maturation.
  • Antigen (Ag) refers to a molecule used in immunization of immunocompetent vertebrates to produce antibodies (Ab) that recognize Ag; The molecule or mimic used.
  • Ag is defined more broadly and is generally intended to include target molecules recognized by Abs.
  • Ag includes molecules, or portions or mimetics thereof, used in immunization procedures for production of Abs or in library screening for selection of Abs.
  • full-length IL-2 from mammalian species e.g., human, monkey, mouse, and rat IL-2
  • monomers and multimers thereof e.g. Dimers, trimers, etc., as well as truncated and other variants of IL-2 are known as antigens.
  • Epitope refers to a site on an antigen to which an immunoglobulin (or antibody) specifically binds.
  • Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids that are juxtaposed by the tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally maintained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents.
  • An epitope typically comprises, for example, 3 to 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as x-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • Antibody fragment includes single chain antibodies; Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibody (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above. Methods for producing and preparing these antibody fragments are well known in the art (see eg Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).
  • Fab-Fv format was first disclosed in WO2009/040562, and its disulfide bond-stabilized form Fab-dsFv was first disclosed in WO2010/035012.
  • Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multiple specificities such as bispecifics or may be monospecific (see e.g. WO92/22583 and WO05/113605), an example of the latter being the Tri-Fab (or TFM).
  • Binding to IL-2 refers to being capable of interacting with IL-2 or its epitope, and said IL-2 or its epitope may be of human origin.
  • Antigen-binding site refers to a continuous or discontinuous three-dimensional site on an antigen recognized by an antibody or antigen-binding fragment of the present disclosure.
  • Treg means a specialized CD4+ T cell type capable of suppressing the response of other T cells.
  • Tregs are characterized by the expression of the alpha subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3 (FOXP3), and are important in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors plays a key role in.
  • Tregs require IL-2 for their function and development as well as the induction of their suppressive features.
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with the antibody isotype.
  • Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; body) downregulation; and B cell activation.
  • ADCC means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of antibodies. The ADCC effector function of the antibody can be reduced or eliminated by modifying the Fc segment of IgG.
  • the modification refers to mutation in the heavy chain constant region of the antibody, such as N297A, L234A, L235A, P329G selected from IgG1; IgG2/4chimera, F234A/L235A mutation of IgG4.
  • CDC refers to the cytotoxic form that activates the complement cascade by binding the complement component Clq to antibody Fc. Methods for detecting ADCC and CDC activities of antibodies are known in the art. For example, CDC can be evaluated by measuring the binding activity between the antibody to be tested and Fc receptors (such as C1q).
  • Fc receptor or “FcR” describes a receptor that binds the Fc region of an antibody.
  • a preferred FcR is a human FcR.
  • a preferred FcR is an IgG antibody binding FcR (gamma receptor), and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA ("activating receptor”) and FcyRIIB ("inhibiting receptor”), which have similar amino acid sequences that differ primarily in their cytoplasmic domains.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • FcR herein encompasses other FcRs, including those to be identified in the future.
  • the term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • an antibody is used as a ligand, when measured in an instrument by the surface plasmon resonance (SPR) technique, the antibody has a concentration below about 10 -7 M or even higher.
  • a small equilibrium dissociation constant (K D ) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is lower than that of the predetermined antigen (or its epitope) or closely related antigens At least twice the binding affinity of non-specific antigens (such as BSA, etc.).
  • the term "antibody that recognizes an antigen” is used interchangeably herein with the term “antibody that specifically binds”.
  • “Avidity” is used herein as a measure of the strength of a non-covalent interaction between two molecules, eg, an antibody or antigen-binding fragment thereof and an antigen.
  • the term “avidity” is used to describe monovalent interactions (intrinsic activity).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, the Surface Plasmon Resonance (SPR) method (Biacore).
  • SPR Surface Plasmon Resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be determined directly by well known methods and can be calculated even for complex mixtures by methods such as those described in Caceci et al. (1984, Byte 9:340-362).
  • KD can be determined using a double filtration nitrocellulose filter binding assay such as that disclosed in Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90:5428-5432).
  • Binding kinetics and binding affinity of antibodies can also be assessed by standard assays known in the art, such as surface plasmon resonance (SPR), for example by using the Biacore TM system or KinExA. Binding affinities associated with different molecular interactions can be compared by comparing the KD values of the individual antibodies/fusion proteins, eg, a comparison of the binding affinities of different antibodies for a given antigen.
  • SPR surface plasmon resonance
  • an anti-IL-2 antibody of the present disclosure is capable of binding its target with an affinity at least 2-fold, 10-fold, 50-fold, 100-fold, greater than the affinity with which it binds another non-IL-2 molecule. 200 times, 500 times, 1,000 times or 10,000 times are not limited herein.
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition and blocking are also intended to include any measurable IL-2 binding affinity, pro-cell proliferation (e.g. T cells) decreased activity.
  • pro-cell proliferation e.g. T cells
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both compared sequences is occupied by the same base or subunit of an amino acid monomer, for example if every position in two DNA molecules is occupied by an adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, two sequences are 60% homologous if there are 6 matches or homology at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when two sequences are aligned to yield the greatest percent homology.
  • “Pharmaceutical composition” means containing one or more of the fusion proteins described herein, together with other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • Effective amounts 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 dose of administration, and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline.
  • PBS phosphate buffered saline
  • Compositions containing such carriers are formulated by well known conventional methods.
  • Buffering agent refers to a buffering agent that is resistant to pH changes through the action of its acid-base conjugated component.
  • buffers to control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate , glycylglycine and other organic acid buffers.
  • a “histidine buffer” is a buffer comprising histidine.
  • histidine buffers include histidine-histidine hydrochloride, histidine-acetate histidine, histidine-phosphohistidine, histidine-histidine sulfate buffers, preferably histidine Acid-histidine hydrochloride buffer.
  • Histidine-HCl histidine buffers can be formulated with histidine and hydrochloric acid, or with histidine and histidine hydrochloride.
  • citrate buffer is a buffer comprising citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is citric acid-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-succinate sodium salt, succinate-succinate potassium salt, succinate-succinate calcium salt, and the like.
  • a preferred succinate buffer is succinic acid-succinic acid sodium salt.
  • the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • the phosphate buffer include disodium hydrogenphosphate-sodium dihydrogenphosphate, disodium hydrogenphosphate-potassium dihydrogenphosphate, disodium hydrogenphosphate-citric acid, and the like.
  • a preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetic acid-sodium acetate, histidine-acetic acid histidine, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • a preferred acetate buffer is acetic acid-sodium acetate.
  • “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 pharmaceutical composition described in the present disclosure can achieve a stable effect: the fusion protein therein substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, A pharmaceutical composition substantially retains its physical and chemical stability as well as its biological activity upon storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • a stable formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years .
  • stable liquid formulations include liquid formulations that exhibit desired characteristics after storage at a temperature including 25°C for a period of time including 1 month, 3 months, or 6 months.
  • Typical examples of stability Usually no more than about 10%, preferably no more than about 5%, of the fusion protein aggregates or degrades as measured by SEC-HPLC. By visual analysis, the formulation is a pale yellow to near colorless clear liquid or a colorless clear liquid, or clear to slightly milky white.
  • the concentration, pH and osmolality of the formulations have a variation of no more than ⁇ 10%, preferably no more than ⁇ 5%.
  • the formulations generally form no more than about 10%, preferably no more than about 5%, aggregates.
  • the fusion protein does not show significant increased aggregation, precipitation and/or Denatured, the fusion protein then "retains its physical stability" in pharmaceutical formulations. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • a fusion protein "retains its chemical stability" in a pharmaceutical formulation if the fusion protein exhibits no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter protein chemical structure include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS, etc.
  • deamidation evaluationated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.
  • isomerization by measuring isoaspartic acid content, Peptide mapping, etc. to evaluate).
  • a fusion protein "retains its biological activity" in a pharmaceutical formulation if the biological activity of the fusion protein at a given time is within a predetermined range of biological activity exhibited when the pharmaceutical formulation was prepared.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic, diagnostic or composition Contact with animals, 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 contacting the reagents with the cells, and contacting the reagents with a fluid, wherein the fluid contacts the cells.
  • administering also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic, binding composition or by another cell.
  • Treatment as it applies to human, veterinary or research subjects means therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treating means administering a therapeutic agent, internally or externally, such as a pharmaceutical composition comprising any of the present disclosure, to a patient having one or more disease symptoms for which the therapeutic agent is known to have therapeutic effect. effect.
  • a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measured extent.
  • the amount of a therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount”) will vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether disease symptoms have been alleviated can be assessed by any of the clinical tests commonly used by a physician or other professional health care practitioner to assess the severity or progression of such symptoms.
  • embodiments of the present disclosure e.g., methods of treatment or articles of manufacture
  • any statistical test known in the art such as Student's t-test, chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the target disease symptoms in a statistically significant number of patients.
  • PCT/CN2021/076806 (priority: CN202010107662.4) is incorporated into this disclosure in its entirety by reference.
  • the preserved natural library phage suspension was diluted and blocked with BSA, co-incubated with magnetic beads Dynabeads (M-280, invitrogen), and the phage after negative screening incubation were collected.
  • the magnetic beads were bound and washed according to the Kingfisher magnetic bead screening system method (Thermo Scientific), and 5% BSA was incubated with the magnetic beads.
  • the Dynabeads were coated with biotin-labeled human Fc-tagged IL-2 (Sanyou Biopharmaceutical, GenBank Accession No: P60568-1), the phage suspension collected after negative screening was incubated with the Dynabeads, and the Kingfisher magnetic bead Screening systems approach for binding and cleaning.
  • Phage were eluted with trypsin. After elution, the phage solution was fully mixed with Escherichia coli SS320 cells (Sanyou Biopharmaceutical) in logarithmic growth phase, and then incubated at 37°C for 30 minutes. Spread Escherichia coli SS320 on a 2YT-Car + -Tet + plate and culture overnight in a 37°C incubator. Calculate the phage input, output, etc., prepare the input phage by scraping and perform 3 rounds of screening.
  • the 2000 clones obtained in the third round of screening were selected and cultured overnight in 96-well deep-well plates. Centrifuge to remove the supernatant. Dilute anti-human IgG (STAR161, Bio-rad) to 2 ⁇ g/mL with PBS, coat the ELISA plate, add 30 ⁇ L to each well, and overnight at 4°C. The plate was washed 3 times with PBST (PBS, pH 7.4+0.1% Tween 20). 1% BSA was blocked at room temperature for 1 hour, and the plate was washed 3 times with PBST. Add 30 ⁇ L of supernatant after centrifugation, room temperature for 2 hours, and wash the plate 3 times with PBST.
  • PBST PBS, pH 7.4+0.1% Tween 20
  • the above 364 clones were inoculated into 50 mL of 2YT-Car + -Tet + medium at a ratio of 1:1000, and cultured overnight at 37°C. Transfer the bacterial solution to a 50mL centrifuge tube. Centrifuge at room temperature, discard the supernatant, add 0.5 mL of 4°C pre-cooled protein lysate (10 mM Tris-HCl pH 9.0, 1 mM EDTA, 5 mM MgCl 2 , 25 U/mL Bezonase nuclease (Merck)), Incubate on ice for 1 hour. Centrifuge at 4°C to collect the supernatant for later use.
  • IL-2R ⁇ -Fc (CJ82, Novoprotein) (GenBank Accession No: NP_000869.1) was diluted to 8 ⁇ g/mL with PBS, coated on an ELISA plate, 30 ⁇ L was added to each well, and left overnight at 4°C. Wash the plate 3 times with PBST. 1% BSA was blocked at room temperature for 1 hour, and the plate was washed 3 times with PBST. After incubating the obtained Fab lysate with biotinylated human Fc-tagged IL-2 (2.5 ⁇ g/mL) at room temperature for 1 hour, add 30 ⁇ L to each well, room temperature for 1 hour, and wash the plate 3 times with PBST.
  • the heavy chain and light chain variable regions of the six antibodies in Example 1 were connected to the human IgG1 heavy chain constant region and the kappa or lamda chain constant region to form a full-length fully human anti-IL-2 antibody.
  • the heavy chain constant region carries L234A and L235A mutations (LALA mutations) to remove the possible ADCC function of the antibody.
  • LALA mutations L234A and L235A mutations
  • HC is the constant region of human IgG1 heavy chain
  • LC is the constant region of kappa chain
  • the above sequence was synthesized, digested with BamHI and XhoI, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) through the BamHI/XhoI restriction site.
  • the expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator for 4- 5 days. After the expressed antibody is recovered by centrifugation, the antibody is purified according to a conventional method, and after identification, the full-length fully human antibody of the present disclosure is obtained.
  • Embodiment 3 anti-IL-2 antibody can block or reduce the combination of IL-2 and IL-2R ⁇ , can block the ELISA experiment of IL-2 and IL-2R ⁇ combination
  • Dilute IL-2R ⁇ -Fc (CJ78, Novoprotein) (Accession#NP_000408) to 0.5 ⁇ g/mL with PBS, coat the ELISA plate, add 30 ⁇ L to each well, and overnight at 4°C. Wash the plate 3 times with PBST. 1% BSA was blocked at room temperature for 1 hour, and the plate was washed 3 times with PBST.
  • Antibody number IC50 ( ⁇ g/mL) A2-22 N.A. A3-21 1.456 B2-15 N.A. C2-53 0.6591 D2-60 N.A. D3-68 1.196
  • Embodiment 4 anti-IL-2 antibody blocks or reduces the combination of IL-2 and IL-2R ⁇ , blocks the Octet experiment of IL-2 and IL-2R ⁇ combination
  • Streptavidin biosensors (Fortebio, #18-5020) were soaked in 200 ⁇ L of KB buffer (PBS, pH 7.4, 0.05% tween-20, 0.1% BSA) for 60 seconds for wet treatment. Then, biotinylated IL-2R ⁇ -Fc (CJ78, Novoprotein) was diluted to 10 ⁇ g/mL with KB buffer, and the sensor was placed in 200 ⁇ L of this solution for 150 seconds. Soak the sensor in KB buffer for 60 seconds to elute excess IL-2R ⁇ .
  • KB buffer PBS, pH 7.4, 0.05% tween-20, 0.1% BSA
  • IL-2-His (CX66, Novoprotein) was mixed with anti-IL-2 antibody, diluted with KB buffer to final concentrations of 100 nM and 500 nM, respectively, and incubated at room temperature for 30 minutes. The sensor was placed in this mixed solution for 300 seconds to bind. The greater the ability of the anti-IL-2 antibody to block or reduce the binding of IL-2 to IL-2R ⁇ , the lower the reading. Read the value at 230 seconds from the start of binding, as shown in Table 6.
  • Anti-IL-2 antibodies block or reduce the binding of IL-2 to IL-2R ⁇ in the Octet assay
  • Streptavidin biosensors (Fortebio, #18-5020) were soaked in 200 ⁇ L of KB buffer (PBS, pH 7.4, 0.05% tween-20, 0.1% BSA) for 60 seconds for wet treatment. Then, biotinylated IL-2-Fc (Sanyou Biopharmaceutical) was diluted to 10 ⁇ g/mL with KB buffer, and the sensor was placed in 200 ⁇ L of this solution for 300 seconds. Soak the sensor in KB buffer for 60 seconds to elute excess IL-2. The anti-IL-2 antibody was diluted with KB buffer to a final concentration of 500nM, and the sensor was placed in the antibody solution to bind for 300 seconds.
  • KB buffer PBS, pH 7.4, 0.05% tween-20, 0.1% BSA
  • Antibody number The value of Octet level on IL-2R ⁇ blocking A3-21 0.003 C2-53 0.017 D3-68 0.027 A2-22 0.042 B2-15 0.056 D2-60 0.086
  • Embodiment 5 the binding ELISA experiment of anti-IL-2 antibody and IL-2
  • Dilute anti-human IgG (STAR161, Bio-rad) to 2 ⁇ g/mL with PBS, coat the ELISA plate, add 30 ⁇ L to each well, and leave overnight at 4°C. Wash the plate 3 times with PBST. 1% BSA was blocked at room temperature for 1 hour, and the plate was washed 3 times with PBST.
  • Example 6 Affinity and kinetics Biacore experiment of anti-IL-2 antibody and IL-2
  • Antibodies were captured with a Protein A sensor chip (GE, Cat#29127556) from Biacore Instruments (Biacore T200, GE), in which anti-IL-2 antibody was diluted to 1 ⁇ g/mL with 1 ⁇ HBS-EP at a flow rate of 10 ⁇ L/min for continuous 30 seconds. Then, a series of concentration-gradient IL-2 (C013, Novoprotein) was passed through the surface of the chip at a flow rate of 30 ⁇ L/min, and the binding lasted for 120 seconds. The dissociation flow rate was 30 ⁇ L/min for 300 seconds, the reaction signal was detected in real time, and the association and dissociation curves were obtained.
  • GE Protein A sensor chip
  • Biacore T200 Biacore Instruments
  • Example 7 Determination of the phosphorylation activity of STAT5 in human peripheral blood PBMC by the non-covalent complex of anti-IL-2 antibody and IL-2
  • Basal medium RPMI 1640+10% fetal bovine serum.
  • CD3 APC-Cy7 (BD 557832), CD4 BB515 (BD 564419), CD8 BB700 (BD 566452), CD25 BV421 (BD 564033), pSTAT5 AF647 (BD 562076).
  • Human PBMC STAT5 phosphorylation experiment freshly isolated human PBMC cells were adjusted to a density of 6.5 ⁇ 10 6 cells/mL with basal medium, and 80 ⁇ L was placed in a 96-well plate. Different concentrations of anti-IL-2 antibodies were premixed with different concentrations of IL-2 at room temperature for 30 minutes, 20 ⁇ L was added to 90 ⁇ L PBMC, and stimulated at 37°C for 20 minutes. Immediately thereafter, the cells were fixed with pre-warmed BD Cytofix buffer (BD, Cat No. 554655) at 37° C. for 15 minutes, and then continued to fix on ice for 15 minutes. Centrifuge at 400g for 7 minutes at 4°C.
  • BD Cytofix buffer BD, Cat No. 554655
  • CD8 + T cells are defined as CD3 + CD4 - CD8 + cells
  • Treg are defined as CD3 + CD4 + CD8 + CD25 + cells.
  • IgG1 isotype did not have any effect on IL-2 activation of pSTAT5 in Treg and CD8 + T cells with increasing antibody concentration.
  • A3-21 and C2-53 basically did not affect the activity of IL-2 on Treg in a large concentration range, but reduced the activity of IL-2 on CD8 + T cells to a large extent. Only at high concentrations, such as 100nM A3-21 and 33.3nM C2-53, the activity of IL-2 on Treg decreased to a certain extent, while under this condition, IL-2 had no activity on CD8 + T cells at all.
  • A3-21 and C2-53 can inhibit or more reduce the activity of IL-2 on immune effector cells such as CD8 + T cells while not affecting or less reducing the activity of IL-2 on Treg.
  • D3-68 can also reduce the activity of IL-2 on CD8 + T cells more than Treg, although this ability may not be as significant as A3-21 and C2-53.
  • Embodiment 8 Preparation of fusion protein
  • the non-covalent complex of anti-IL-2 antibody and IL-2 may be easily dissociated in vivo to generate free IL-2, causing toxicity.
  • G 4 S 5 linker i.e., GGGGSGGGGSGGGGSGGGGSGGGGS, SEQ ID No: 190
  • the fusion protein For antibody numbering, if IL-2 is at the N-terminal of the heavy chain of the antibody, the fusion protein will have the suffix a; if IL-2 is at the N-terminal of the light chain of the antibody, the fusion protein will have the suffix b.
  • IL-2 For the fused IL-2, it has a mutation of T3A (ie, the third amino acid is mutated from Thr to Ala) to remove possible glycosylation.
  • Human IL-2 mature protein does not contain amino acid M at position 1, so the numbering starts from amino acid A at position 2.
  • the amino acid sequences of the 12 fusion proteins are as follows:
  • Example 2 According to the method in Example 2, the above 12 fusion proteins were expressed and purified, and the expression amount and purity in SEC-HPLC are shown in Table 11.
  • IL-2 was coupled to the N-terminus of the heavy chain or light chain, which would greatly affect the expression and purity of the fusion protein.
  • the low expression level or purity indicates that IL-2 and the antibody in the fusion protein may not form a normal intramolecular combination, but form an intermolecular combination, thereby causing protein aggregation.
  • Fusion proteins with higher expression (at least 20 mg/L) and purity (at least 90%) were selected for subsequent identification.
  • Embodiment 10 Determination of the phosphorylation activity of fusion protein on human peripheral blood (PBMC) STAT5
  • the STAT5 phosphorylation activity of the fusion protein on Treg and CD8 + T cells in human peripheral blood (PBMC) was determined.
  • PBMC peripheral blood
  • the experimental results showed that compared with IL-2, all the fusion proteins had reduced activity on Treg and CD8 + T cells.
  • the activity of these fusion proteins decreased more on CD8+ T cells than on Treg Many, suggesting that IL-2 in these fusion proteins can be more biased to activate Treg.
  • NA the value is still low at the highest tested concentration, and cannot be fitted to obtain an accurate EC 50 ).
  • Example 11 Determination of the influence of fusion protein on peripheral blood immune cells of Balb/c mice
  • BALB/c mice purchased from Beijing Charles River Experimental Animal Technology Co., Ltd.
  • Female, 6-8 weeks old, weighing 18-20 g were adaptively fed for 5 days before the formal experiment.
  • All BALB/c mice were kept in an IVC constant temperature and pressure system in an SPF grade animal room, where the temperature was 20 to 26°C, the humidity was 40 to 70%, and the light cycle was 12 hours light/12 hours dark.
  • No more than 6 BALB/c mice were housed in each cage. The mice were grouped according to their body weight, and the administration was started after grouping. See Table 16 for the type, dosage and route of administration.
  • the day of model grouping is Day 0.
  • the anticoagulated blood collected freshly at each time point was lysed with erythrocyte lysate and washed once with PBS.
  • the mixed staining solution includes CD3 APC-Cy7 (Biolegend 100329), CD8 PE (Biolegend 100708), CD4 PE-Cy7 (eBioscience 25-0042-82), CD25 PerCP-Cy5.5 (BD 561112).
  • the membrane was fixed and permeated with True-Nuclear TM Transcription Factor Buffer Set (Biolegend 424401) for 60 minutes, and 100 ⁇ L of anti-mouse Foxp3 antibody (Biolegend 126405) and anti-mouse Ki67 antibody (eBioscience, 25-5698-82) were incubated at room temperature for 60 minutes. Wash twice with PBS (pH 7.4), and finally resuspend with 500 ⁇ L PBS, pH 7.4 washing solution, and analyze on the machine.
  • CD8 + T cells are defined as CD3 + CD4 - CD8 + cells
  • Treg are defined as CD3 + CD4 + CD25 + Foxp3 + cells.
  • Example 12 Determination of the influence of the fusion protein on the spleen immune cells of Balb/c mice immunized with chicken ovalbumin OVA
  • OVA Chicken ovalbumin
  • CFA Freund's complete adjuvant
  • Male C57B16/J mice purchased from Shanghai Experimental Animal Center), 6-8 weeks old, weighing 18-20 g.
  • the mice were grouped according to body weight.
  • 8 mL of OVA solution was taken, mixed with 8 mL of CFA, and emulsified into a water-in-oil state.
  • Each animal was immunized by intraperitoneal injection of 200 ⁇ L.
  • mice in each group were injected subcutaneously (sc) with 10 mL/kg of the fusion protein to be tested and PBS control. See Table 17 for the type of administration, dosage and route of administration.
  • the method comprises: soaking in 75% ethanol for 5 minutes, taking out the mouse spleen under aseptic conditions, rinsing with PBS for 1-2 times, and cutting the mouse spleen into pieces. Put the strainer in a 50mL centrifuge tube, transfer the spleen tissue fragments to the strainer, grind, and add fresh PBS continuously during the process. Centrifuge at 1800 rpm for 3 minutes and discard the supernatant.
  • the detected cells and their markers are as follows:
  • GEB Germinal Center B cells
  • Follicular T helper cells (Tfh, follicular T helper cells) (PD1 high, CXCR5 high, FOXP3-CD4+);
  • Follicular regulatory T cells (Tfr, follicular regulatory T cells) (PD1 high, CXCR5 high, FOXP3+CD4+);
  • A2-22-b, B2-15-b, D2-60-b can stimulate the proliferation of Treg and Tfr in the spleen, and inhibit the number of Tfh and GCB in the spleen, indicating that A2-22-b, B2-15-b, D2-60-b has the effect of suppressing the immune system by activating Treg.
  • Example 13 Pharmacodynamic experiment of fusion protein in mouse delayed hypersensitivity reaction model
  • Male ICR mice purchased from Shanghai Experimental Animal Center), 6 weeks old, weighing 18-20 g. Mice were grouped according to body weight. On the 0th day of the experiment, 50 ⁇ L of 1% DNFB solution was smeared on the abdomen of the mice for immunization.
  • mice Male DBA/1 pure-line mice were purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • the arthritis model was induced by immunization with bovine type II collagen (Biolead, 20022). On the 0th day of the experiment, take 2.5mL bovine II collagen/glacial acetic acid (2mg/mL), and then fully suspend it with 2.5mL complete Freund's adjuvant to form a water-in-oil emulsion. Intradermal injection at the base of the tail of each mouse, followed by a booster injection 3 weeks later.
  • the model animals were randomly divided into 6 groups: normal control group, model group, IL-2 (C013, novoprotein) group, A2-22-b group, B2-15-b group, with 8 animals in each group. At the same time, normal control animals were selected as the normal control group, and the groups are shown in Table 19.
  • mice were weighed and semi-quantitatively scored (clinical scores) for the severity of arthritis every 3 days from the second immunization:
  • the maximum score is 4 points per paw and 16 points per mouse. Arthritis condition observation continued until the end of the experiment.
  • Detection of serum anti-bovine type II collagen antibody concentration At the end of the experiment, blood was collected, serum was separated, and stored at -80°C. ELISA detection kit (Chondrex, 20322T) was used to detect the level of anti-bovine type II collagen antibody in serum. The specific steps are carried out according to the instructions.
  • Example 15 Effects of different tether lengths on the expression and purity of fusion proteins
  • the (G 4 S) 5 linking arm between IL-2 in B2-15-b and the light chain of the anti-IL-2 antibody B2-15 was shortened to 4 repetitions of G4S, while the heavy chain remained unchanged to obtain B2
  • the light chain sequence of -15-b variant B2-15-b-(G 4 S) 4 is shown in Table 20.
  • B2-15-b was expressed and purified according to the method in Example 2, and the expression level was still 47 mg/L, and the SEC-HPLC purity was 99%. It is proved that different tether lengths can realize the expression of fusion protein.
  • Example 16 Determination of the phosphorylation activity of human peripheral blood (PBMC) STAT5 by fusion proteins between anti-IL-2 antibodies and different IL-2 variants
  • Mutate IL-2 introduce T3E, or T3Q, or remove the first 3 amino acids at the N-terminus of IL-2 (B2-15-b-Del3), or remove the first 7 amino acids at the N-terminus of IL-2 (B2-15-b-Del3).
  • Del7 or directly using wild-type IL-2, were conjugated with anti-IL-2 antibody.
  • the following fusion proteins were generated (the heavy chain sequence of each antibody remained unchanged), see Table 21.
  • the fusion protein of the above-mentioned anti-IL-2 antibody and the IL-2 variant was tested for its STAT5 phosphorylation activity on Treg and CD8 + T cells in human peripheral blood (PBMC).
  • PBMC peripheral blood
  • the experimental results show that these IL-2 variants basically do not affect the activity of the antibody fusion protein on Treg and CD8+ T cells.
  • These fusion proteins still favor activation of Tregs rather than CD8 + T cells.
  • Example 17 Drug efficacy experiment of fusion protein in MRL/lpr spontaneous lupus erythematosus mouse model
  • Female BALB/c mice and female MRL/lpr mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Taking female BALB/c mice as normal controls, female MRL/lpr mice were randomly divided into 3 groups according to proteinuria level and body weight, respectively: MRL/lpr control group (model control group), 1 and 3 mg/kg administration Group. D2-60-b-T3E was administered by subcutaneous injection, administered once every 3 days, at a dose of 1.3 mg/kg; administered at the age of 8 weeks, and administered continuously for 8 weeks.
  • the urine protein content of the mice in each group was measured every two weeks; after the administration for 8 consecutive weeks, the following detections were carried out: 1 Determination of the contents of IgM and IgG against double-stranded DNA in the serum of mice in each group; 2 Determination of The contents of blood creatinine (CRE) and blood urea nitrogen (BUN) in the serum of mice; 3Determination of the contents of IL-6, IL-10 and IFN- ⁇ in the serum of mice in each group; 4The renal tissues of mice in each group were Pathological analysis and scoring.
  • CRE blood creatinine
  • BUN blood urea nitrogen
  • the urine of mice was collected by the tail-lifting reflex method, and the urine protein concentration of the mice was detected by the urine protein detection kit (CBB method, Nanjing Jiancheng Institute of Bioengineering, #C035-2-1).
  • CBB method Nanjing Jiancheng Institute of Bioengineering, #C035-2-1.
  • the kidneys will be damaged due to the deposition of dsDNA antibodies, which will lead to the decline of glomerular filtration function and reabsorption capacity, and the increase of urinary protein concentration.
  • the level of urinary protein in mice will gradually increase.
  • Anti-double-stranded DNA (anti-dsDNA) IgG and IgM levels in serum were detected by ELISA.
  • ELISA Anti-double-stranded DNA
  • 3mg/kg D2-60-b-T3E administration group had significantly lower serum anti-dsDNA IgG and IgM levels than the model control group (P ⁇ 0.01), although the 1mg/kg group had IgG and IgM levels
  • the trend was lower than that of the model control group, but the difference with the model control group was not statistically significant.
  • the experimental results are shown in Fig. 11A and Fig. 11B.
  • This experiment uses creatinine (CRE) assay kit (Nanjing Jiancheng Bioengineering Institute, #C011-2-1) and urea nitrogen (BUN) test kit (Nanjing Jiancheng Bioengineering Institute, #C013-2-1) detection experiment The contents of serum creatinine and urea nitrogen in mouse serum at the end.
  • CRE creatinine
  • BUN urea nitrogen
  • Blood urea nitrogen and creatinine are the metabolites of human protein and muscle respectively, and the kidney is the final excretion organ. After renal function is damaged, blood urea nitrogen and creatinine cannot be effectively excreted, and blood urea nitrogen (BUN) and serum creatinine (CRE) The concentration gradually increases due to retention. Therefore, the levels of BUN and CRE in serum are the main indexes reflecting renal function clinically. Compared with normal mice, the contents of creatinine (CRE) and blood urea nitrogen (BUN) in the serum of MRL/lpr control group (model control group) mice were significantly increased (P ⁇ 0.0001), suggesting that MRL/lpr model group mice Functional renal impairment occurs.
  • the content of creatinine in the serum of mice in the 1 and 3mg/kg administration groups was significantly lower than that of the model control group (P ⁇ 0.01, P ⁇ 0.0001), which can improve the renal function of MRL/lpr mice to a certain extent, and 3mg/kg administration
  • the content of creatinine in the serum of the mice in the drug group was significantly lower than that in the 1mg/kg administration group.
  • the content of blood urea nitrogen in the serum of mice in the 3mg/kg administration group was also significantly lower than that in the model control group (P ⁇ 0.05), indicating that 3mg/kg of D2-60-b-T3E can reverse the spontaneous renal failure of MRL/lpr mice. damage.
  • the results are shown in Figures 12A and 12B.
  • the levels of cytokines IFN- ⁇ , IL-6 and IL-10 in serum were detected by ELISA method.
  • the left kidney of the mouse was fixed in 4% paraformaldehyde, embedded in paraffin and stained with H&E.
  • the sections were observed under a microscope and scored for pathology. Grading and scoring are carried out, and the scoring criteria are shown in Table 24.
  • MRL/lpr control group In the MRL/lpr control group (model control group), severe glomerulus, renal interstitium, and vascular pathological damage occurred in the kidneys of the mice, with glomerulosclerosis, partial inward depression, and a large number of cells infiltrated in the perivascular and interstitial areas. Severe swelling and hyperplasia of the glomerulus. Compared with normal mice, the renal pathological score of mice in the MRL/lpr control group (model control group) was significantly increased (P ⁇ 0.001), suggesting that the kidneys of the mice in the model control group had obvious lesions.
  • the glomerulus, renal interstitium and vascular pathological damage of mice in the administration group were alleviated to a certain extent.
  • the renal pathological scores of the 1 and 3 mg/kg administration groups were significantly lower than those of the model control group (P ⁇ 0.05, P ⁇ 0.0001), indicating that D2-60-b-T3E can significantly reduce MRL/lpr spontaneous renal lesions.
  • the results are shown in Figure 15.
  • a method for the analysis of the separation of solutes based on the relative relationship between the pore size of the gel pores and the coil size of the polymer sample molecules.
  • SEC% SEC monomer content percentage
  • a monomer is the peak area of the main peak monomer in the sample, and A total is the sum of all peak areas).
  • ⁇ SEC% SEC% of the formulation before the forced degradation test - SEC% of the formulation after the forced degradation test.
  • NR-CE% A main peak/A total ⁇ 100% (A main peak is the peak area of the light chain main peak+heavy chain main peak in the sample, and A is always the sum of all peak areas.
  • ⁇ NR-CE% Preparation before forced degradation test NR-CE % - NR-CE % of the formulation after the forced degradation test.
  • CE determination instrument Beckman model plus800
  • iCIEF% neutral peak area/total area ⁇ 100% (the total area is the sum of the areas of acidic peak, neutral peak and basic peak).
  • ⁇ iCIEF% iCIEF% of the formulation before the forced degradation test - iCIEF% of the formulation after the forced degradation test.
  • the freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point depression value and the molar concentration of the solution, a high-sensitivity temperature-sensing element is used to measure the freezing point of the solution, which is converted into osmotic pressure by electricity. Instrument manufacturer Luo Ze Loser, model OM815.
  • the fusion protein used in the following examples is D2-60-b-T3E.
  • the concentration of the fusion protein was measured with a protein concentration meter.
  • Protein concentration determination instrument UV-Vis spectrophotometer, model: Nano Drop oneC, optical path 1mm.
  • Preparations containing the buffer system shown in Table 25, 30 mg/mL fusion protein, 80 mg/mL sucrose, and 0.2 mg/mL polysorbate 80 (PS80) were prepared.
  • the sample was subjected to a forced degradation study (placed at 40°C for 1 month, 25°C, and shaken at 300rpm for 6 days).
  • SEC and iCIEF as evaluation indicators, the effects of different buffer systems on the stability of the fusion protein were investigated.
  • the buffer system is preferably AA and His-HCl, and the pH range is preferably 5.5-6.5.
  • AA acetic acid-sodium acetate
  • SA succinic acid-sodium succinate
  • His-HCl stands for histidine-histidine hydrochloride
  • Shake D6 Shake at 300rpm at 25°C for 6 days; 40°C M1: Store at 40°C for 1 month, the same below.
  • Preparations containing 10mM AA buffer (pH 5.5), the fusion protein of the protein concentration shown in Table 26, 80mg/mL sucrose, and 0.2mg/mL PS80 were prepared.
  • the sample was subjected to a forced degradation study (placed at 40°C for 1 month), and the effects of different protein concentrations on the stability of the fusion protein were investigated using SEC, NR-CE and iCIEF as evaluation indicators.
  • FT5C 5 cycles of freezing and thawing, and so on.
  • the anti-IL-2 protein preparation containing 36mg/mL sucrose, 12mg/mL glycine, 0.8mg/mL PS80 protein concentration and 10mg/mL.
  • the specific ionic strength settings are as follows:
  • the preparation is filtered, filled, stoppered and capped.
  • the preparation sample was freeze-dried, and the freeze-drying program was pre-freezing, primary drying and secondary drying (see Table 33 for parameters). After the freeze-drying procedure, stoppering with vacuum. Then, the lyophilized preparation was reconstituted with water for injection to obtain an anti-IL-2 antibody reconstitution solution with a protein concentration of 10 mg/mL, and the appearance, NR-CE and SEC purity of the preparation before lyophilization and the reconstitution solution after lyophilization were investigated. The change. The experimental results are shown in Table 34. The results show that the anti-IL-2 antibody preparations maintain good stability before and after lyophilization.
  • anti-IL-2 containing 45mg/mL sucrose, 10mg/mL glycine, 0.8mg/mL PS80 protein concentration and 10mg/mL protein preparations.
  • the preparation is filtered, filled, stoppered and capped.
  • the preparation sample was freeze-dried, and the freeze-drying program was pre-freezing, primary drying and secondary drying (see Table 35 for parameters). After the freeze-drying procedure, stoppering with vacuum. Then, the lyophilized preparation was reconstituted with water for injection to obtain an anti-IL-2 antibody reconstitution solution with a protein concentration of 10 mg/mL, and the changes in the appearance and SEC purity of the preparation before lyophilization and the reconstitution solution after lyophilization were investigated. The experimental results are shown in Table 36. The results show that the anti-IL-2 antibody preparations maintain good stability before and after lyophilization.

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Abstract

涉及一种含融合蛋白的药物组合物；具体地，涉及包含含有共价结合的IL-2与抗IL-2抗体的融合蛋白的药物组合物及其用途。

Description

一种含融合蛋白的药物组合物 技术领域
本披露属于药物制剂领域，具体地，本披露涉及一种包含含有共价结合的IL-2与抗IL-2抗体的融合蛋白的药物组合物，以及其作为药物的用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
人白细胞介素-2(Interleukin-2，IL-2)，也称为T细胞生长因子(TCGF)，基因位于4号染色体(4q27)，包括共7kb的序列，由133个氨基酸组成，分子量约15kD。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通过细胞表面的IL-2R发生作用。IL-2R包括三个亚基，IL-2Rα(即CD25)、IL-2Rβ(即CD122)和IL-2Rγ(即CD132)。三个亚基可以形成三种受体形式：高结合力受体包含所有三个亚基IL-2Rα/β/γ，中结合力受体包含IL-2Rβ/γ，低结合力受体为IL-2Rα。其中，IL-2Rβ和IL-2Rγ是IL-2激活下游信号通路所必需的，当IL-2同时结合IL-2Rβ和IL-2Rγ时，两个受体亚基形成异源二聚体，磷酸化细胞内的STAT5，进入细胞核导致相应的基因转录和表达；IL-2Rα并非信号传导所必需，但可以增强IL-2与IL-2Rβ和IL-2Rγ的结合。Treg持续表达高结和力受体IL-2Rα/β/γ，而未被激活的CD8+ T细胞和NK细胞等只表达中结合力受体IL-2Rβ/γ。因此，低剂量IL-2优先结合并激活Treg，而高剂量IL-2则也可以激活CD8+ T细胞和NK细胞等免疫效应细胞。
由于Treg具有免疫抑制作用，而低剂量IL-2具有优先激活Treg的性质，因此低剂量IL-2被报道可以用于一系列免疫相关，如自身免疫疾病和炎性疾病的治疗，、例如系统性红斑狼疮(systemic lupus erythematosus，SLE)、I型糖尿病(type I diabetes，T1D)、慢性移植物对抗宿主疾病(chronic Graft versus Host Disease，cGvHD)、骨髓移植(hematopoietic stem cell transplantation，HSCT)、斑秃(alopecia areata)、丙型肝炎病毒诱导的血管炎(hepatitis C virus-induced vasculitis)、慢性GvHD、哮喘、皮炎、斑秃、急性肺损伤、慢性铍尘病、败血症等。在很多接受低剂量IL-2处理的患者中，Treg与效应T细胞的比例均有上升，症状也有所缓解。然而一方面，IL-2半衰期较短，需要频繁给药；另一方面，IL-2的给药窗口较小，如果剂量略高，则可能激活免疫效应细胞，加重自身免疫疾病。这些IL-2本身的性质或缺点使得IL-2难以在患者体内保持持续的低剂量，限制了其进一步应用。
发明内容
本披露提供一种含融合蛋白的药物组合物。该组合物具有治疗活性。此外，该组合物还可具有稳定性好，适于冻干等优势。
在一些实施方案中，本披露提供一种药物组合物，包含融合蛋白和缓冲剂，其中所述融合蛋白包含共价结合的IL-2与抗IL-2抗体，所述缓冲剂为组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂。在一些实施方案中，所述缓冲剂为组氨酸缓冲剂或醋酸盐缓冲剂。在一些实施方案中，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂或醋酸-醋酸钠缓冲剂。在一些实施方案中，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂。
在一些实施方案中，如上任一项所述的药物组合物，其中所述缓冲剂的pH为5.0-6.5。在一些实施方案中，所述缓冲剂的pH为5.0-6.0。在一些实施方案中，所述缓冲剂的pH为5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4或6.5。在一些实施方案中，所述缓冲剂的pH为5.5-6.0。在一些实施方案中，所述缓冲剂的pH为6.0。
在一些实施方案中，所述药物组合物的pH相比其含有的缓冲剂的pH具有不超过±0.3的差异。在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物的pH为5.0-6.5。在一些实施方案中，所述药物组合物的pH为6.0-6.5。在一些实施方案中，所述药物组合物的pH为6.0-6.3。在一些实施方案中，所述药物组合物的pH为5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4或6.5。
在一些实施方案中，如上任一项所述的药物组合物，其中所述融合蛋白的浓度为5mg/mL至50mg/mL。在一些实施方案中，所述融合蛋白的浓度为5mg/mL至30mg/mL。在一些实施方案中，所述融合蛋白的浓度为10mg/mL至30mg/mL。在一些实施方案中，所述融合蛋白的浓度为5mg/mL、10mg/mL、15mg/mL、20mg/mL、25mg/mL、30mg/mL。在一些实施方案中，所述融合蛋白的浓度为30mg/mL。在一些实施方案中，所述融合蛋白的浓度为10mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物包含表面活性剂。在一些实施方案中，所述表面活性剂是非离子表面活性剂。在一些实施方案中，所述表面活性剂选自聚山梨酯、聚山梨酯20、聚山梨酯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-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、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。在一些实施方案中，所述表面活性剂为聚山梨酯或泊洛沙姆。在一些实施方案中，所述表面活性剂为聚山梨酯80(PS80)、聚山梨酯20(PS20)或泊洛沙姆188(PF68)。在一些实施方案中，所述表面活性剂为聚山梨酯80或泊洛沙姆188。在一些实施方案中，所述表面活性剂为泊洛沙姆188。在一些实施方案中，所述表面活性剂为聚山梨酯80。
在一些实施方案中，如上任一项所述的药物组合物，其中所述表面活性剂浓度为0.05mg/mL至2mg/mL。在一些实施方案中，所述表面活性剂浓度为0.05mg/mL至1.0mg/mL。在一些实施方案中，所述表面活性剂浓度为0.1mg/mL至0.8mg/mL。在一些实施方案中，所述表面活性剂浓度为0.1mg/mL至0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为0.2mg/mL至0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为0.05mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1.0mg/mL、1.1mg/mL、1.2mg/mL、1.3mg/mL、1.4mg/mL、1.5mg/mL、1.6mg/mL、1.7mg/mL、1.8mg/mL、1.9mg/mL或2.0mg/mL。在一些实施方案中，所述表面活性剂浓度为1.6mg/mL。在一些实施方案中，所述表面活性剂浓度为1.5mg/mL。在一些实施方案中，所述表面活性剂浓度为0.8mg/mL。在一些实施方案中，所述表面活性剂浓度为0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为0.4mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物包含糖。在一些实施方案中，所述糖选自常规组合物(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。所述的糖可选自葡萄糖，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。在一些实施方案中，所述糖选自蔗糖、甘露醇和海藻糖中的一种或多种。在一些实施方案中，所述糖为蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，在一些实施方案中，所述糖的浓度为20mg/mL至100mg/mL。在一些实施方案中，所述糖的浓度为30mg/mL至80mg/mL。在一些实施方案中，所述糖的浓度为40mg/mL至80mg/mL。在一些实施方案中，所述糖的浓度为30mg/mL至50mg/mL。在一些实施方案中，所述糖的浓度为20mg/mL、30mg/mL、36mg/mL、40mg/mL、45mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、87mg/mL、90mg/mL或100mg/mL。在一些实施方案中，所述糖的浓度为87mg/mL。在一些实施方案中，所述糖的浓度为80mg/mL。在一些实施方案中，所述糖的浓度为50mg/mL。在一些实施方案中，所述糖的浓度为45mg/mL。在一些实施方案中，所述糖的浓度为40mg/mL。在一些实施方案中，所述糖的浓度为36mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物还 包含辅料。在一些实施方案中，所述辅料为甘氨酸、甲硫氨酸、脯氨酸、组氨酸、苯丙氨酸、盐酸精氨酸、谷氨酸、天冬氨酸或乙二胺四乙酸二钠。在一些实施方案中，所述辅料为甘氨酸。
在一些实施方案中，如上任一项所述的药物组合物，其中所述辅料的浓度为1mg/mL至30mg/mL；优选地，辅料的浓度为5mg/mL至15mg/mL。在一些实施方案中，如上任一项所述的药物组合物，其中所述辅料的浓度为12mg/mL。在一些实施方案中，如上任一项所述的药物组合物，其中所述辅料的浓度为10mg/mL。在一些实施方案中，如上任一项所述的药物组合物，其中所述辅料的浓度为9mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，所述缓冲剂的浓度为5mM至100mM。在一些实施方案中，所述缓冲剂的浓度为5mM、10mM、20mM、30mM、40mM、50mM、60mM、70mM、80mM、90mM或100mM。在一些实施方案中，所述缓冲剂的浓度为5mM至30mM。在一些实施方案中，所述缓冲剂的浓度为10mM至30mM。在一些实施方案中，所述缓冲剂的浓度为5mM至10mM。在一些实施方案中，所述缓冲剂的浓度为10mM。在一些实施方案中，所述缓冲剂的浓度为5mM。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体与IL-2的亲合力的KD值≤5nM。在另一些实施方案中，所述抗IL-2抗体与IL-2的亲合力的KD值＞5nM或≥10nM，且≤200nM或≤150nM或≤100nM或≤80nM或≤60nM。在一些实施方案中，所述KD值＞5nM且≤60nM，＞5nM且≤50nM，≥10nM且≤50nM，≥10nM且≤60nM，＞5nM且≤80nM，≥10nM且≤80nM，＞5nM且≤100nM，≥10nM且≤100nM，＞5nM且≤150nM，或≥10nM且≤150nM。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
SEQ ID NO：13、19、25、31、37或43所示的HCDR1，和/或
SEQ ID NO：14、20、26、32、38或44所示的HCDR2，和/或
SEQ ID NO：15、21、27、33、39或45所示的HCDR3，和/或
SEQ ID NO：16、22、28、34、40或46所示的LCDR1，和/或
SEQ ID NO：17、23、29、35、41或47所示的LCDR2，和/或
SEQ ID NO：18、24、30、36、42或48所示的LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
SEQ ID NO：13、25或37所示的HCDR1，和/或
SEQ ID NO：14、26或38所示的HCDR2，和/或
SEQ ID NO：15、27或39所示的HCDR3，和/或
SEQ ID NO：16、28或40所示的LCDR1，和/或
SEQ ID NO：17、29或41所示的LCDR2，和/或
SEQ ID NO：18、30或42所示的LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
SEQ ID NO：19、31或43所示的HCDR1，和/或
SEQ ID NO：20、32或44所示的HCDR2，和/或
SEQ ID NO：21、33或45所示的HCDR3，和/或
SEQ ID NO：22、34或46所示的LCDR1，和/或
SEQ ID NO：23、35或47所示的LCDR2，和/或
SEQ ID NO：24、36或48所示的LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
SEQ ID NO：13-15所示的HCDR1-3，和/或SEQ ID NO：16-18所示的LCDR1-3；
SEQ ID NO：19-21所示的HCDR1-3，和/或SEQ ID NO：22-24所示的LCDR1-3；
SEQ ID NO：25-27所示的HCDR1-3，和/或SEQ ID NO：28-30所示的LCDR1-3；
SEQ ID NO：31-33所示的HCDR1-3，和/或SEQ ID NO：34-36所示的LCDR1-3；
SEQ ID NO：37-39所示的HCDR1-3，和/或SEQ ID NO：40-42所示的LCDR1-3；
SEQ ID NO：43-45所示的HCDR1-3，和/或SEQ ID NO：46-48所示的LCDR1-3。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
如SEQ ID NO：1、3、5、7、9、11之一所示或与之具有至少90％、95％、98％、99％同一性的VH；和/或
如SEQ ID NO：2、4、6、8、10、12之一所示或与之具有至少90％、95％、98％、99％同一性的VH。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
如SEQ ID NO：1所示或与之具有至少90％同一性的VH，如SEQ ID NO：2所示或与之具有至少90％同一性的VL；
如SEQ ID NO：3所示或与之具有至少90％同一性的VH，如SEQ ID NO：4所示或与之具有至少90％同一性的VL；
如SEQ ID NO：5所示或与之具有至少90％同一性的VH，如SEQ ID NO：6所示或与之具有至少90％同一性的VL；
如SEQ ID NO：7所示或与之具有至少90％同一性的VH，如SEQ ID NO：8所示或与之具有至少90％同一性的VL；
如SEQ ID NO：9所示或与之具有至少90％同一性的VH，如SEQ ID NO：10所示或与之具有至少90％同一性的VL；或
如SEQ ID NO：11所示或与之具有至少90％同一性的VH，如SEQ ID NO：12所示或与之具有至少90％同一性的VL。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体的可变区与CDR采用Kabat编号系统。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体的重链可变区(VH)与人IgG1重链恒定区连接，轻链可变区(VL)与kappa或lamda 链恒定区连接。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
如SEQ ID NO：157、159、161、163、165、167之一所示或与之具有至少80％、90％、95％、98％、99％同一性的HC；和/或
如SEQ ID NO：158、160、162、164、166、168之一所示或与之具有至少80％、90％、95％、98％、99％同一性的LC。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含
如SEQ ID NO：157所示或与之具有至少80％、90％、95％、98％、99％同一性的全长重链(HC)，如SEQ ID NO：158所示或与之具有至少80％、90％、95％、98％、99％同一性的全长轻链(LC)；
如SEQ ID NO：159所示或与之具有至少80％、90％、95％、98％、99％同一性的HC，如SEQ ID NO：160所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；
如SEQ ID NO：161所示或与之具有至少80％、90％、95％、98％、99％同一性的HC，如SEQ ID NO：162所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；
如SEQ ID NO：163所示或与之具有至少90％同一性的HC，如SEQ ID NO：164所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；
如SEQ ID NO：165所示或与之具有至少90％同一性的HC，如SEQ ID NO：166所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；或
如SEQ ID NO：167所示或与之具有至少80％、90％、95％、98％、99％同一性的HC，如SEQ ID NO：168所示或与之具有至少80％、90％、95％、98％、99％同一性的LC。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体为Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如具体为scFv、Fv、Fab或Fab’片段。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白中的IL-2与抗IL-2抗体的轻链可变区或重链可变区连接，例如，IL-2与抗IL-2抗体的轻链可变区或重链可变区的N端连接。在一些实施方案中，IL-2与抗IL-2抗体的轻链可变区的N端连接。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白中的IL-2与抗IL-2抗体通过连接子连接。在一些实施方案中，所述连接子是柔性肽连接子。在一些实施方案中，所述连接子的氨基酸长度为5-50、10-40、20-30或25。在一些实施方案中，所述连接子选自：如(G  mS  n) x或(GGNGT) x或(YGNGT) x 所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，x独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些具体实施方案中，所述连接子具有如(G 4S)x所示的氨基酸序列，x独立地选自1-6的整数(例如，x为4或5)。所述连接子还可以为与上述(G mS n) x或(GGNGT) x或(YGNGT) x所示的氨基酸序列具有类似柔性和长度的其他连接子。在一些实施方案中，所述连接子的氨基酸序列如SEQ ID No：190所示。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白选择性增强高表达IL-2Ra的细胞的活性或促进其增殖，所述高表达IL-2Ra的细胞例如为调节性T(Treg)细胞。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白中的IL-2存在第三位氨基酸(作为一个示例，T3)的氨基酸取代或N端缺失。一些具体实施方案中，IL-2的第三位氨基酸取代为Ala、Gln或Glu(作为一个示例，T3A、T3Q、T3E)，N端缺失为N端前3个或前7个氨基酸缺失。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白包含选自以下组的HC和LC：
SEQ ID No：169所示的HC，SEQ ID No：158所示的LC；
SEQ ID No：171所示的HC，SEQ ID No：160所示的LC；
SEQ ID No：159所示的HC，SEQ ID No：172所示的LC；
SEQ ID No：173所示的HC，SEQ ID No：162所示的LC；
SEQ ID No：175所示的HC，SEQ ID No：164所示的LC；
SEQ ID No：163所示的HC，SEQ ID No：176所示的LC；
SEQ ID No：177所示的HC，SEQ ID No：166所示的LC；
SEQ ID No：179所示的HC，SEQ ID No：168所示的LC；
SEQ ID No：167所示的HC，SEQ ID No：180所示的LC；
SEQ ID No：161所示的HC，SEQ ID No：174、181-186任一所示的LC；
SEQ ID No：157所示的HC，SEQ ID No：170、187所示的LC；
SEQ ID No：165所示的HC，SEQ ID No：178、188、189任一所示的LC；
或与上述HC、LC具有至少80％、90％、95％、98％、99％同一性的序列。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白选择性的增强了IL-2对Treg活性的作用(例如促增殖活性、促STAT5磷酸化活性)，和/或选择性地提高IL-2促Treg细胞中FOXP3、CD25和Icos中的一种或多种表达的能力。
在一些实施方案中，如上任一项所述的药物组合物，所述融合蛋白还具有(i)-(iii)中的一项或多项：
(i)对Treg的活性激活程度高于对CD8+T细胞的活性激活程度；
(ii)相较于在CD8+T细胞中，在Treg细胞中对STAT5磷酸化的促进程度更 高；
(iii)相较于CD8+T细胞、CD4+T细胞或NK细胞的增殖，对Treg细胞的增殖促进程度更高。
在一些实施方案中，如上任一项所述的药物组合物，所述抗IL-2抗体包含重链可变区和轻链可变区，所述重链可变区具有：包含SEQ ID NO：37的氨基酸序列的HCDR1、包含SEQ ID NO：38的氨基酸序列的HCDR2和包含SEQ ID NO：39的氨基酸序列的HCDR3，所述轻链可变区具有：包含SEQ ID NO：40的氨基酸序列的LCDR1、包含SEQ ID NO：41的氨基酸序列的LCDR2和包含SEQ ID NO：42的氨基酸序列的LCDR3。在一些实施方案中，所述重链可变区包含SEQ ID NO：9的氨基酸序列，所述轻链可变区包含SEQ ID NO：10的氨基酸序列。在一些实施方案中，所述抗IL-2抗体包含SEQ ID NO：165的氨基酸序列的重链和SEQ ID NO：166的氨基酸序列的轻链。
在一些实施方案中，如上任一项所述的药物组合物，所述IL-2直接或通过连接子融合至抗IL-2抗体的轻链的N端。在一些实施方案中，所述IL-2的第3位氨基酸残基为E。在一些实施方案中，所述融合蛋白包含SEQ ID NO：165的氨基酸序列的重链和SEQ ID NO：189的氨基酸序列的轻链。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.05mg/mL至2mg/mL的聚山梨酯或泊洛沙姆，(c)20mg/mL至100mg/mL的糖，和(d)10mM至30mM的组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂，所述缓冲剂的pH为5.5至6.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.8mg/mL的聚山梨酯或泊洛沙姆，(c)30mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂，所述缓冲剂的pH为5.5至6.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.8mg/mL的聚山梨酯或泊洛沙姆，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂，所述缓冲剂的pH为5.5至6.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.6mg/mL的聚山梨酯或泊洛沙姆，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂，所述缓冲剂的pH为5.5至6.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.8mg/mL的聚山梨酯或泊洛沙姆，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的 组氨酸缓冲剂，所述组氨酸缓冲剂的pH为5.5至6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.6mg/mL的聚山梨酯或泊洛沙姆，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸缓冲剂，所述组氨酸缓冲剂的pH为5.5至6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的醋酸盐缓冲剂，所述醋酸盐缓冲剂的pH为5.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂，所述缓冲剂的pH为5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸缓冲剂，所述组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸缓冲剂，所述组氨酸缓冲剂的pH为6.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的醋酸盐缓冲剂，所述醋酸盐缓冲剂的pH为5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.2mg/mL的聚山梨酯80，(c)80mg/mL的海藻糖，和(d)10mM的醋酸盐缓冲剂，所述醋酸盐缓冲剂的pH为5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.6mg/mL的泊洛沙姆188，(c)80mg/mL的蔗糖，和(d)10mM的醋酸盐缓冲剂，所述醋酸盐缓冲剂的pH为5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.6mg/mL的泊洛沙姆188，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸缓冲剂，所述组氨酸缓冲剂的pH为5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.6mg/mL的泊洛沙姆188，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为5.5至6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.6mg/mL的泊洛沙姆188，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.6mg/mL的泊洛沙姆188，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.6mg/mL的聚山梨酯80，(c)80mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.4mg/mL的聚山梨酯80，(c)87mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)87mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)30mg/mL的所述融合蛋白，(b)1.5mg/mL的聚山梨酯80，(c)87mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.8mg/mL的聚山梨酯或泊洛沙姆，(c)30mg/mL至80mg/mL的蔗糖，(d)1mg/mL至30mg/mL的甘氨酸，和(e)5mM至10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL至30mg/mL的所述融合蛋白，(b)0.1mg/mL至0.8mg/mL的聚山梨酯80，(c)30mg/mL至50mg/mL的蔗糖，(d)5mg/mL至15mg/mL的甘氨酸，和(e)5mM至10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)1.6mg/mL的聚山梨酯80，(c)36mg/mL 的蔗糖，(d)12mg/mL甘氨酸，和(e)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)36mg/mL的蔗糖，和(d)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)36mg/mL的蔗糖，(d)12mg/mL甘氨酸，和(e)5mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)36mg/mL的蔗糖，(d)12mg/mL甘氨酸，和(e)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)1.6mg/mL的聚山梨酯80，(c)36mg/mL的蔗糖，(d)12mg/mL甘氨酸，和(e)5mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)50mg/mL的蔗糖，(d)9mg/mL甘氨酸，和(e)5mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)50mg/mL的蔗糖，(d)9mg/mL甘氨酸，和(e)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)45mg/mL的蔗糖，(d)10mg/mL甘氨酸，和(e)5mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)45mg/mL的蔗糖，(d)10mg/mL甘氨酸，和(e)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)40mg/mL 的蔗糖，(d)10mg/mL甘氨酸，和(e)5mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)10mg/mL的所述融合蛋白，(b)0.8mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，(d)10mg/mL甘氨酸，和(e)10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
本披露的数值范围旨在包括该范围内的每个数字和数字子集，无论是否被明确地公开。
在一些实施方案中，如上任一项所述的药物组合物，所述药物组合物是液体制剂。在一些实施方案中，所述液体制剂的溶剂是水。在一些实施方案中，所述组合物的施用途径为皮下注射、皮内注射、静脉注射或肌内注射。在一些实施方案中，所述组合物的施用途径为皮下注射。
本披露还提供一种的冻干制剂，其特征在于所述冻干制剂复溶后可形成如上任一项所述的药物组合物。本披露还提供一种的冻干制剂，其为如上任一项所述的药物组合物的冻干形式。
本披露还提供一种制备冻干制剂的方法，其中包括将如上任一项所述的药物组合物进行冷冻干燥的步骤。
本披露还提供一种冻干制剂，所述制剂通过将如上任一项所述的药物组合物经冷冻干燥获得。
本披露还提供一种复溶溶液，其特征在于所述复溶溶液是通过将如上任一项所述的冻干制剂复溶制备的。本披露还提供一种的冻干制剂，其为如上任一项所述的冻干制剂的复溶形式。
本披露还提供一种制品，其包括容器，该容器中装有如上任一项所述的药物组合物、如上任一项所述的冻干制剂或如上任一项所述的复溶溶液。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品在制备治疗自身免疫疾病或延缓自身免疫疾病进展的药物中的用途。
本披露还提供一种治疗自身免疫疾病或延缓自身免疫疾病进展的方法，包括给予患者有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品，其用于治疗自身免疫疾病或延缓自身免疫疾病进展。
在一些实施方案中，所述自身免疫疾病选自以下的任一项或组合：狼疮、移植物抗宿主病、丙型肝炎诱导的脉管炎、I型糖尿病、多发性硬化、炎性肠病、慢性GvHD、哮喘、皮炎、斑秃、急性肺损伤、败血症、反应性关节炎、类风湿性关节 炎。
附图说明
图1A和图1B分别为抗IL-2抗体阻断IL-2与IL-2Rα和IL-2Rβ结合的ELISA实验结果；其中图1A为抗IL-2抗体阻断IL-2与IL-2Rα结合的ELISA结果；图1B为抗IL-2抗体阻断IL-2与IL-2Rβ结合的ELISA结果。
图2为通过ELISA实验检测抗IL-2抗体与IL-2的结合能力的结果。
图3A至图3H为抗IL-2抗体与IL-2的非共价复合体刺激人外周血Treg和CD8 +T细胞STAT5磷酸化的实验结果。
图4A至图4D为融合蛋白刺激人外周血调节性T细胞Treg和CD8 +T细胞中STAT5磷酸化活性的实验结果。
图5A至图5F为融合蛋白对Balb/c小鼠体内外周血中淋巴细胞增殖影响的实验结果。
图6为融合蛋白对经过OVA免疫的小鼠脾脏中淋巴细胞增殖影响的实验结果。
图7为融合蛋白在小鼠延迟性过敏反应模型中的药效结果。
图8A至图8B为融合蛋白在小鼠关节炎模型中的药效结果。
图9A至图9F为抗IL-2抗体与不同IL-2变体形成的融合蛋白刺激人外周血调节性T细胞Treg和CD8 +T细胞中STAT5磷酸化活性的实验结果。
图10为D2-60-b-T3E对MRL/lpr小鼠尿中蛋白含量影响的实验结果(n＝7至10)。
图11A和图11B分别为D2-60-b-T3E对MRL/lpr小鼠血清中dsDNA IgG、IgM含量影响的实验结果(**P<0.01 vs.模型对照组)。
图12A和图12B分别为D2-60-b-T3E对MRL/lpr小鼠肾功能影响的实验结果。*P<0.05，**P<0.01，****P<0.0001 vs.模型对照组，n＝9至10。
图13A和图13B分别为D2-60-b-T3E对MRL/lpr小鼠血清IL-6、INF-γ含量影响的实验结果。*P<0.05，**P<0.01，****P<0.0001 vs.模型对照组，n＝9至10。
图14为D2-60-b-T3E对MRL/lpr小鼠血清IL-10含量影响的实验结果。*P<0.05，**P<0.01，****P<0.0001 vs.模型对照组，n＝9至10。
图15为D2-60-b-T3E对MRL/lpr小鼠病理评分的影响。*P<0.05，**P<0.01，****P<0.0001 vs.模型对照组，n＝9至10。
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的 一般技术人员通常理解的含义。
本披露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”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，活性成份例如抗体)、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。该术语涵盖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。该术语还涵盖未加工的IL-2以及源自细胞中的加工的任何形式的IL-2，还涵盖天然存在的IL-2变体，例如剪接变体或等位变体，还涵盖IL-2的保守修饰变体。
“IL-2Rα”、“CD25”或“IL-2受体的α亚基”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CD25，包括“全长”的未加工的CD25以及源自细胞中的加工的任何形式的CD25，还包括天然存在的CD25变体，例如剪接变体或等位变体。在某些实施方案中，CD25是人CD25，例示性序列如SEQ ID NO.37所示。
“高亲合力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、p70或IL-2Rβ)和受体α亚基(也称为CD25、p55、IL-2Rα)组成。比较而言，“中等亲合力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))。
“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)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。
“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、提高的活性。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。优选的氨基酸突变是氨基酸取代。为了改变例如抗IL-2抗体的结合特性，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。优选的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。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原-特异性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘，并鉴定参与抗原结合位点的残基。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。
Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。
Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。
AbM编号系统使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号系统使用知识数据库和de-novo方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab  Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。
接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。
构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。
其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本文使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。本文使用的方法可利用根据这些方法中的任一种定义的CDR。对于包含超过一个CDR的任何给定实施例，可根据Kabat、Chothia、延伸的、AbM、IMGT、接触和/或构象定义中的任一个来定义CDR。
“单克隆抗体”或“单抗”指从基本上同质的抗体群体获得的抗体，即除了可能以少量存在的可能天然存在的突变之外，群体包含的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。此外，与多克隆抗体不同，每种单克隆抗体针对抗原上的单个决定簇。修饰语“单克隆”指示如从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本披露使用的单克隆抗体可通过首先由Kohler和Milstein，1975，Nature256：495描述的杂交瘤方法来制备，或者可通过例如美国专利号4,816,567中所述的重组DNA方法来制备。例如，单克隆抗体也可从使用McCafferty等，1990，Nature 348：552-554中描述的技术，从所生成的噬菌体文库中分离。
“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本披露的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
“人抗体”或“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突 变。
“抗原(Ag)”指用于免疫接种免疫活性的脊椎动物的分子，以产生识别Ag的抗体(Ab)；或在表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)中进行筛选所使用的分子或模拟物。在本文中，Ag被更广义地定义，并且一般预期包括被Ab识别的靶分子。因此，Ag包括用于产生Ab的免疫接种过程或用于选择Ab的文库筛选中使用的分子、或其部分或模拟物。因此，对于本披露的与IL-2结合的抗体，来自哺乳动物物种的全长IL-2(例如人、猴、小鼠和大鼠IL-2)，包括其单体和多聚体，例如二聚体、三聚体等，以及IL-2的截短变体和其它变体均被称为抗原。
“表位”是指抗原上与免疫球蛋白(或抗体)特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括例如3至15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
“抗体片段”包括单链抗体；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段。产生和制备这些抗体片段的方法在本领域是公知的(参见例如Verma等人,1998,Journal ofImmunological Methods,216,165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本披露的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异性例如双特异性或可以是单特异性的(参见例如WO92/22583和WO05/113605)，后者的一个实例是描述于WO 92/22583中的Tri-Fab(或TFM)。
“与IL-2结合”，指能与IL-2或其表位相互作用，所述IL-2或其表位可以是人源的。
“抗原结合位点”指抗原上连续或不连续的，由本披露抗体或抗原结合片段识别的三维空间位点。
“调节性T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。
“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列变体 Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。“ADCC”是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A、P329G；IgG2/4chimera，IgG4的F234A/L235A突变。“CDC”是指通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的ADCC、CDC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价CDC。
“Fc受体”或“FcR”描述结合抗体Fc区的受体。优选的FcR是人FcR。此外，优选的FcR是结合IgG抗体的FcR(γ受体)，且包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和选择性剪接形式。FcγRII受体包括FcγRIIA(“激活受体”)和FcγRIIB(“抑制受体”)，它们具有主要在其胞质结构域中不同的相似氨基酸序列。激活受体FcγRIIA在其胞质结构域中包含基于免疫受体酪氨酸的激活基序(ITAM)。抑制受体FcγRIIB在其胞质结构域中包含基于免疫受体酪氨酸的抑制基序(ITIM)(参见综述M.Daeron,Annu.Rev.Immunol.15:203-234(1997))。FcR综述于Ravetch和Kinet,Annu.Rev.Immunol.9:457-92(1991)；Capel等,Immunomethods 4:25-34(1994)；及de Haas等,J.Lab.Clin.Med.126:330-41(1995)中。本文的术语“FcR”涵盖了其他FcR，包括有待在将来鉴定的那些。该术语还包括负责将母体IgG转移至胎儿的新生儿受体FcRn(Guyer等，J.Immunol.117:587(1976)和Kim等,J.Immunol.24:249(1994))。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人IL-2或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲合力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲合力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
“亲合力”在本文中用作两个分子(例如抗体或其抗原结合片段与抗原)之间的非共价相互作用的强度量度。术语“亲合力”用于描述单价相互作用(固有活性)。两个分子之间的结合亲合力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。K D通过方程K D＝kd/ka与ka和kd有 关。解离常数的值可通过众所周知的方法直接确定，并且可通过方法例如Caceci等人(1984，Byte 9：340-362)中所述的那些甚至对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定K D。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本文其它地方例举的其它测定。抗体的结合动力学和结合亲合力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用Biacore TM系统或KinExA来评价。可通过比较各个抗体/融合蛋白的K D值来比较与不同分子相互作用相关的结合亲合力，例如，不同抗体对于给定抗原的结合亲合力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的K D值与非目的相互作用(例如已知不结合IL-2的对照抗体)的K D值进行评价。在一些实施方案中，本披露的抗IL-2抗体能够与其靶结合的亲合力比它与另一种非IL-2分子结合的亲合力大至少2倍、10倍、50倍、100倍、200倍、500倍、1,000倍或10,000倍，这里不属于限制性定义。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。抑制和阻断也旨在包括与抗IL-2抗体接触时，与未与抗IL-2抗体接触的IL-2相比，任何可测量的IL-2结合亲合力、促细胞增殖(例如T细胞)活性降低。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“药物组合物”表示含有一种或多种本文所述融合蛋白，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本披露中，“药物组合物”和“制剂”并不互相排斥。
“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。
“药学可接受的载体”或“药学可接受的赋形剂”包括当与活性成分组合时，允许该成分保留生物学活性并且不与受试者的免疫系统反应的任何材料。例子包 括但不限于任何标准药物载体，例如磷酸盐缓冲盐水溶液、水、乳剂如油/水乳剂、和各种类型的润湿剂。在一些实施例中，用于气雾剂或肠胃外施用的稀释剂是磷酸盐缓冲盐水(PBS)或生理(0.9％)盐水。包含此类载体的组合物通过众所周知的常规方法配制。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、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剂。
“组氨酸缓冲剂”是包含组氨酸的缓冲剂。组氨酸缓冲剂的实例包括组氨酸-盐酸组氨酸，组氨酸-醋酸组氨酸，组氨酸-磷酸组氨酸，组氨酸-硫酸组氨酸等缓冲剂，优选组氨酸-盐酸组氨酸缓冲剂。组氨酸-盐酸组氨酸缓冲剂可由组氨酸与盐酸配制而成，或者由组氨酸与盐酸组氨酸配制而成。
“枸橼酸盐缓冲剂”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-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。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠盐、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠盐。示例性的，所述的琥珀酸-琥珀酸钠可由琥铂酸与氢氧化钠配制而成，或由琥铂酸与琥珀酸钠盐配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂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-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、组氨酸-醋酸组氨酸、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠。
“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。
本披露所述的药物组合物能够达到一种稳定的效果：其中的融合蛋白在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。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，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月或6个月在内的时段后表现出期望的特征。稳定性的典型的例子：通过 SEC-HPLC测得，通常不超过约10％、优选不超过约5％的融合蛋白发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色澄明液体，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，优选不超过±5％的变化。所述制剂通常形成不超过约10％、优选不超过约5％的聚集。
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，融合蛋白没有显示出显著的聚集增加、沉淀和/或变性，那么所述融合蛋白在药物制剂中“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。
如果融合蛋白没有显示出显著的化学改变，那么所述融合蛋白在药物制剂中“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CE-SDS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)。
如果融合蛋白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，那么所述融合蛋白在药物制剂中“保留它的生物活性”。
“施用”、“给予”和“处理”，当其应用于动物、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。“处理”当其应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种的药物组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领 域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
在以上说明书中提出了本披露一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方式。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权利要求书限定。
实施例-融合蛋白
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。PCT/CN2021/076806(优先权：CN202010107662.4)通过援引完整收入本披露。
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。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗IL-2抗体的筛选
A)人源天然噬菌体Fab库的筛选
将保存的天然库噬菌体悬液用BSA稀释封闭，与磁珠Dynabeads(M-280，invitrogen)共孵育，收集负筛孵育后的噬菌体。按照Kingfisher磁珠筛选系统方法(Thermo Scientific)对磁珠进行结合和清洗，将5％BSA与磁珠孵育。用生物素标记的带有人Fc标签的IL-2(Sanyou Biopharmaceutical，GenBank Accession No：P60568-1)包被封闭Dynabeads，将负筛后收集的噬菌体悬液与所述Dynabeads孵育，并按照Kingfisher磁珠筛选系统方法进行结合和清洗。用胰酶洗脱噬菌体。洗脱后噬菌体溶液与生长对数期的大肠杆菌SS320细胞(Sanyou Biopharmaceutical)充分混合后，37℃静置孵育30min。将大肠杆菌SS320涂布在2YT-Car +-Tet +平板上，37℃培养箱过夜培养。计算噬菌体输入、输出等，刮板制备输入的噬菌体并进行3轮筛选。
B)ELISA初筛能结合IL-2的表达了抗IL-2的Fab的单克隆噬菌体
挑选第3轮筛选获得的2000个克隆在96孔深孔板中培养过夜。离心取上清。将抗人IgG(STAR161，Bio-rad)用PBS稀释到2μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST(PBS，pH 7.4+0.1％Tween 20)洗板3次。1％BSA 室温封闭1小时，PBST洗板3次。加入离心后的上清30μL，室温2小时，PBST洗板3次。将用生物素标记的带有人Fc标签的IL-2用PBS稀释到2μg/mL每孔加入30μL，室温1小时，PBST洗板3次。加入1:8000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。最终获得364株具有特异序列的克隆。
C)ELISA初筛能够阻断IL-2与IL-2Rβ结合的抗IL-2的Fab
将上述364株克隆按照1:1000的比例接种到50mL体积的2YT-Car +-Tet +培养基中，37℃培养过夜。将菌液转移至50mL离心管。常温条件下离心处理，弃掉上清，向其中加入4℃预冷的蛋白裂解液(10mM Tris-HCl pH 9.0，1mM EDTA，5mM MgCl 2，25U/mL Bezonase核酸酶(Merck))0.5mL，冰上静置孵育1小时。4℃条件下离心处理收集上清备用。
将IL-2Rβ-Fc(CJ82，Novoprotein)(GenBank登录No：NP_000869.1)用PBS稀释到8μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将获得的Fab裂解液与用生物素标记的带有人Fc标签的IL-2(2.5μg/mL)室温温育1小时后，每孔加入30μL，室温1小时，PBST洗板3次。加入1:8000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。
获得具有较强的阻断IL-2与IL-2Rβ结合能力的6个Fab。这6个全人源抗体的重链和轻链可变区序列如表1所示，不同编号系统的互补决定区(CDR)如表2所示。
表1.全人源抗IL-2抗体重链(HCVR)和轻链(LCVR)可变区序列
Figure PCTCN2022114858-appb-000001
Figure PCTCN2022114858-appb-000002
表2.全人源抗IL-2抗体的重链和轻链互补决定区(CDR)序列
Figure PCTCN2022114858-appb-000003
Figure PCTCN2022114858-appb-000004
Figure PCTCN2022114858-appb-000005
实施例2:全长全人源抗IL-2抗体的制备
将实施例1中6个抗体的重链和轻链可变区与人IgG1重链恒定区和kappa或lamda链恒定区连接，构造形成全长全人源抗IL-2抗体。其中重链恒定区带有L234A和L235A突变(LALA突变)，用以去除抗体可能的ADCC功能。6个抗体的全长序列如表3所示。
表3.全人源抗IL-2抗体的重轻链全长序列
Figure PCTCN2022114858-appb-000006
Figure PCTCN2022114858-appb-000007
Figure PCTCN2022114858-appb-000008
(注：HC的下划线部分为人IgG1重链恒定区，LC的下划线部分为kappa链恒定区)
合成上述序列，用BamHI和XhoI消化后，通过BamHI/XhoI酶切位点插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。表达的抗体通过离心回收后，按常规方法进行抗体纯化，经鉴定，得到本披露的全长全人源抗体。
实施例3:抗IL-2抗体能阻断或降低IL-2与IL-2Rα结合，能阻断IL-2和IL-2Rβ结合的ELISA实验
A)抗体阻断或降低IL-2与IL-2Rα结合的ELISA实验
将IL-2Rα-Fc(CJ78，Novoprotein)(Accession#NP_000408)用PBS稀释到0.5μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将抗IL-2抗体用PBS，pH7.4做浓度梯度稀释至10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.63μg/mL，0.31μg/mL，0.16μg/mL，0.08μg/mL，分别与用生物素标记的带有人Fc标签的IL-2(终浓度1μg/mL)室温温育15分钟后，每孔加入30μL，室温1小时，PBST洗板3次。加入1:6000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次。加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。
结果如图1A所示。结果显示，C2-53、D3-68和A3-21可以完全阻断IL-2与IL-2Rα的结合，其余克隆在10μg/mL高浓度下可能仅有微弱的阻断(即降低)IL-2与IL-2Rα结合的能力。各个抗体的IC 50如表4。
表4.抗IL-2抗体阻断IL-2与IL-2Rα的结合的IC 50
抗体编号 IC 50(μg/mL)
A2-22 N.A.
A3-21 1.456
B2-15 N.A.
C2-53 0.6591
D2-60 N.A.
D3-68 1.196
(注：N.A.：由于阻断效果较弱，无法拟合曲线获得有意义的IC50。)
B)抗体阻断IL-2与IL-2Rβ结合的ELISA实验
将IL-2Rβ-Fc(CJ82，Novoprotein)用PBS稀释到8μg/mL,包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将抗IL-2抗体用PBS，pH7.4做浓度梯度稀释至10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.63μg/mL，0.31μg/mL，0.16μg/mL，0.08μg/mL，分别与用生物素标记的带有人Fc标签的IL-2(终浓度5μg/mL)室温温育15分钟后，每孔加入30μL，室温1小时，PBST洗板3次。加入1:6000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次。加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。
结果如图1B所示。其中，所有抗体都可以完全阻断IL-2与IL-2Rβ的结合。各个抗体的IC 50如表5。
表5.抗IL-2抗体阻断IL-2与IL-2Rβ的结合的IC 50
抗体编号 IC 50(μg/mL)
A2-22 2.728
A3-21 3.475
B2-15 3.045
C2-53 1.441
D2-60 8.272
D3-68 2.134
实施例4:抗IL-2抗体阻断或降低IL-2与IL-2Rα的结合，阻断IL-2和IL-2Rβ结合的Octet实验
A)抗体阻断或降低IL-2与IL-2Rα结合的Octet实验
将Streptavidin生物传感器(Fortebio，#18-5020)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.05％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将生物素化的IL-2Rα-Fc(CJ78，Novoprotein)稀释到10μg/mL，将传感器置于200μL该溶液中150秒。将传感器浸泡于KB缓冲液中60秒，以洗脱多余的IL-2Rα。将IL-2-His(CX66，Novoprotein)与抗IL-2抗体混合，并以KB缓冲液稀释至终浓度分别为100nM和500nM，室温温育30分钟。将传感器置于该混合溶液中结合300秒。抗IL-2抗体阻断或降低IL-2与IL-2Rα结合的能力越强，则读数越低。读取开始结合230秒时的数值，如表6所示。
表6.抗IL-2抗体在Octet实验中阻断或降低IL-2与IL-2Rα的结合
抗体编号 Octet水平对IL-2Rα阻断数值
C2-53 0.410
A3-21 0.428
D3-68 0.473
B2-15 0.700
D2-60 0.754
A2-22 0.848
B)抗体阻断IL-2与IL-2Rβ结合的Octet实验
将Streptavidin生物传感器(Fortebio，#18-5020)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.05％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将生物素化的IL-2-Fc(Sanyou Biopharmaceutical)稀释到10μg/mL，将传感器置于200μL该溶液中300秒。将传感器浸泡于KB缓冲液中60秒，以洗脱多余的IL-2。将抗IL-2抗体以KB缓冲液稀释至终浓度500nM，将传感器置于抗体溶液中结合300秒。将传感器浸泡于KB缓冲液中60秒，以洗脱多余的抗IL-2抗体。将IL-2Rβ-Fc(CJ82，Novoprotein)以KB缓冲液稀释至终浓度8000nM，将传感器置于抗体溶液中结合300秒。抗IL-2抗体阻断IL-2与IL-2Rβ结合的能力越强，则读数越低。读取在IL-2Rβ-Fc开始结合230秒时的数值，如表7所示。
表7.抗IL-2抗体在Octet实验中阻断IL-2与IL-2Rβ的结合
抗体编号 Octet水平对IL-2Rβ阻断数值
A3-21 0.003
C2-53 0.017
D3-68 0.027
A2-22 0.042
B2-15 0.056
D2-60 0.086
实施例5:抗IL-2抗体与IL-2的结合ELISA实验
将抗人-IgG(STAR161，Bio-rad)用PBS稀释到2μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将抗IL-2抗体用PBS梯度稀释到20μg/mL，10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.625μg/mL，0.3125μg/mL，0.15625μg/mL，加30μL抗体到ELISA板中，室温1小时，PBST洗板3次。将用生物素标记的带有人Fc标签的IL-2(Sanyou Biopharmaceutical)用PBS稀释到2μg/mL每孔加入30μL，室温1小时，PBST洗板3次。加入1:6000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。结果如图2和表8所示。结果显示，在该实验方法下，所有抗体均与IL-2有结合，其中C2-53结合较强。
表8.抗IL-2抗体与IL-2结合的EC 50
抗体编号 EC 50(μg/mL)
A2-22 1.289
A3-21 1.350
B2-15 2.117
C2-53 0.7301
D2-60 1.428
D3-68 1.205
实施例6：抗IL-2抗体与IL-2的亲合力和动力学Biacore实验
用Biacore仪器(Biacore T200，GE)的Protein A传感芯片(GE，Cat#29127556)捕获抗体，其中抗IL-2抗体用1×HBS-EP稀释至1μg/mL，以10μL/min的流速持续30秒。然后，在芯片表面以30μL/min的流速流经一系列浓度梯度的IL-2(C013，Novoprotein)，结合持续120秒。解离流速30μL/min，持续300秒，实时检测反应信号，获得结合和解离曲线。在每个循环解离完成后，用10mM Gly-HCl pH 2.0将芯片洗净再生。实验得到的数据以1:1结合模型进行拟合，得出抗IL-2抗体与IL-2的结合力数值，见表9。结果显示，所有抗体均与IL-2有结合，亲合力跨度从C2-53的0.267nM到A2-22的39.8nM不等。
表9.抗IL-2抗体与IL-2的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) KD(M)
C2-53 1.64E+07 4.37E-03 2.67E-10
D3-68 1.16E+07 5.46E-02 4.72E-09
D2-60 5.09E+05 6.51E-03 1.28E-08
B2-15 1.46E+06 4.48E-02 3.06E-08
A2-22 1.33E+06 5.27E-02 3.98E-08
实施例7：抗IL-2抗体与IL-2的非共价复合体对人外周血PBMC中STAT5磷酸化活性的测定
依据在不同浓度的抗IL-2抗体与IL-2非共价复合体处理下，人外周血中各细胞群体(包括Treg，CD4 +T细胞，CD8 +T细胞)中STAT5磷酸化水平，检测抗体对IL-2活性的调节能力。
基础培养基：RPMI 1640+10％胎牛血清。
抗体混合物：CD3 APC-Cy7(BD 557832)，CD4 BB515(BD 564419)，CD8 BB700(BD 566452)，CD25 BV421(BD 564033)，pSTAT5 AF647(BD 562076)。
人PBMC STAT5磷酸化实验：将新鲜分离的人PBMC细胞用基础培养基调整至6.5×10 6个细胞/mL的密度，取80μL置于96孔板中。将不同浓度的抗IL-2抗体与不同浓度的IL-2室温预混30分钟，取20μL加入90μL PBMC中，37℃刺激20分钟。之后立即用预热的BD Cytofix缓冲液(BD，Cat No.554655)在37℃固定细胞15分钟，然后在冰上继续固定15分钟。400g 4℃离心7分钟。去除上清液，用150μL PBS洗涤一次。加入150μL在-20℃预冷的BD Phosflow Perm Buffer III(BD，Cat No.558050)-20℃破膜过夜。500g 4℃离心7分钟。去除上清液， 加入150μL PBS，pH 7.4洗涤两次。加入100μL按1:200稀释的FcR封闭剂，在4℃下温育20分钟。500g 4℃离心7分钟。去除上清液，加入50μL抗体混合物，对细胞在4℃染色1小时。500g 4℃离心7分钟。去除上清液，加入200μL PBS，pH 7.4洗涤一次，用150μL PBS，pH 7.4重悬细胞，用流式细胞仪(BD FACSCelesta)检测。CD8 +T细胞定义为CD3 +CD4 -CD8 +的细胞，Treg定义为CD3 +CD4 +CD8 +CD25 +的细胞。
对上述两个细胞群的pSTAT5荧光数值(MFI)进行统计，计算每个浓度下的数值与完全激活状态下的最大pSTAT5 MFI数值的百分比，采用计算机程序或四参数回归计算法进行拟合。结果如图3A至图3H所示。
结果表明，随着抗体浓度增加，IgG1同种型并不会对IL-2激活Treg和CD8 +T细胞的pSTAT5产生任何影响。相反，A3-21，C2-53在很大的浓度范围内，基本不影响IL-2对Treg的活性，却在很大程度上降低了IL-2对CD8 +T细胞的活性。只有在高浓度下，比如100nM A3-21和33.3nM C2-53，IL-2对Treg的活性有一定下降，而在该条件下，IL-2对CD8 +T细胞完全没有活性。也即，A3-21和C2-53可以在不影响或较少的降低IL-2对Treg的活性的同时，抑制或更多的降低IL-2对CD8 +T细胞等免疫效应细胞的活性。类似的，D3-68也可以相对于Treg更多的降低IL-2对CD8 +T细胞的活性，虽然这种能力可能不如A3-21和C2-53显著。
实施例8：融合蛋白的制备
抗IL-2抗体与IL-2的非共价复合体体内可能容易解离，生成游离的IL-2，造成毒性。我们通过(G 4S) 5连接臂(即，GGGGSGGGGSGGGGSGGGGSGGGGS，SEQ ID No：190)将人IL-2偶联至抗IL-2抗体重链或轻链的N端，得到12个抗IL-2抗体与IL-2的融合蛋白。
对于抗体编号，若IL-2在抗体重链的N端，则该融合蛋白带有后缀a；若IL-2在抗体轻链的N端，则该融合蛋白带有后缀b。
对于所融合的IL-2，其带有T3A(即第3位氨基酸由Thr突变为Ala)的突变，以去除可能的糖基化。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。12个融合蛋白氨基酸序列如下：
表10.融合蛋白的全长序列
Figure PCTCN2022114858-appb-000009
Figure PCTCN2022114858-appb-000010
Figure PCTCN2022114858-appb-000011
Figure PCTCN2022114858-appb-000012
依照实施例2中的方法对上述12个融合蛋白进行表达和纯化，表达量和在 SEC-HPLC中的纯度如下表11。
表11.融合蛋白的表达量和纯度
抗体编号 表达量(mg/L) 纯度(％)
A2-22-a 39 90.780
A2-22-b 104 96.308
A3-21-a 82 89.358
A3-21-b 2 82.095
B2-15-a 89 53.821
B2-15-b 70 99.839
C2-53-a N.A. 54.940
C2-53-b 6 26.598
D2-60-a 2.4 40.248
D2-60-b 129 97.540
D3-68-a 0.3 N.A.
D3-68-b 106 100
结果显示，对于不同抗IL-2抗体，IL-2偶联在重链或轻链的N端，会极大的影响融合蛋白的表达量和纯度。表达量或纯度低，提示在融合蛋白中IL-2与抗体可能无法形成正常的分子内结合，而是形成分子间结合，从而引起蛋白质的聚集。挑选表达量(至少20mg/L)和纯度(至少90％)较高的融合蛋白进行后续鉴定。
实施例9：融合蛋白与IL-2Rα和IL-2Rβ结合的Octet实验
将Protein A生物传感器(Fortebio，#18-5010)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.02％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将融合蛋白稀释到10μg/mL，将传感器置于200μL该溶液中，待读数为1.2nm时停止。将传感器浸泡于KB缓冲液中100秒，以洗脱多余的抗体-IL-2融合蛋白。将IL-2Rα(ILA-H52H9，Acrobiosystem)用KB缓冲液以2倍梯度稀释至100nM-3.125nM之间。将传感器置于该溶液中结合60秒。将传感器置于KB缓冲液中解离60秒。采用动态1：1结合方式拟合，则融合蛋白与IL-2Rα的亲合力如表12所示。结果显示，与IL-2相比，偶联了抗IL-2抗体的IL-2与IL-2Rα的结合均有减弱，其中A3-21-a，D3-68-b完全不结合IL-2Rα。
表12.融合蛋白与IL-2Rα的亲合力
待测物 Kon(1/Ms) Koff(1/s) KD(M)
IL-2 1.40E+06 2.51E-02 1.80E-08
A2-22-a 9.22E+05 3.75E-02 4.06E-08
A2-22-b 1.63E+06 4.13E-02 2.53E-08
A3-21-a N.A. N.A. 不结合
B2-15-b 1.23E+06 3.30E-02 2.68E-08
D2-60-b 1.24E+06 5.61E-02 4.51E-08
D3-68-b N.A. N.A. 不结合
(注：N.A.：不结合，所以无法拟合得到数值。其中IL-2与IL-2Rα的亲合力实验，IL-2Rα作为固定相，IL-2作为流动相)。
与上述方法类似，用Octet采用稳态模式拟合数据，测得抗体-IL-2融合蛋白与IL-2Rβ(CD2-H5221，Acrobiosystem)的亲合力如表13所示。其中，抗体融合蛋白作为固定相，IL-2Rβ作为流动相。结果显示，与IL-2相比，偶联了抗IL-2抗体的IL-2绝大多数完全不结合，个别抗体-IL2融合蛋白，如D3-68-b与IL-2Rβ的结合减弱，但仍然有一定程度的结合。其中IL-2与IL-2Rβ的亲合力实验，IL-2Rβ作为固定相，IL-2作为流动相。
表13.抗IL-2抗体与IL-2融合蛋白与IL-2Rβ的亲合力
待测物 KD(nM)
IL-2 4.6E-07
A2-22-a 不结合
A2-22-b 不结合
A3-21-a 不结合
B2-15-b 不结合
D2-60-b 不结合
D3-68-b 1.1E-06
实施例10：融合蛋白对人外周血(PBMC)STAT5磷酸化活性的测定
依照实施例7中的方法，测定融合蛋白对人外周血(PBMC)中Treg和CD8 +T细胞中的STAT5磷酸化活性。如图4A至图4D所示，实验结果显示，相比IL-2，所有融合蛋白对Treg和CD8 +T细胞的活性均有所降低。但是对于A2-22-a，A2-22-b，B2-15-b，D2-60-b，A3-21-b，这些融合蛋白对CD8+ T细胞的活性降低相比对Treg的活性降低更多，提示了这些融合蛋白中的IL-2可以更偏向于激活Treg。
而对于D3-68-b，其对Treg的活性降低相比对CD8 +T细胞的活性降低更多(图4C和图4D所示)。融合蛋白激活Treg和CD8 +T细胞中pSTAT5的EC50如表14和表15所示。
表14.融合蛋白激活Treg细胞中pSTAT5的EC 50
待测物 EC 50(nM)
IL-2 0.0003714
A2-22-a 0.04439
A2-22-b 0.05677
B2-15-b 0.01072
D2-60-b 0.1940
D3-68-b 0.1731
A3-21-a 0.1923
表15.融合蛋白激活CD8 +T细胞中pSTAT5的EC 50
待测物 EC 50(nM)
IL-2 0.4343
A2-22-a N.A.
A2-22-b N.A.
B2-15-b N.A.
D2-60-b N.A.
D3-68-b 31.06
A3-21-a N.A.
(注：N.A.：在最高测试浓度下数值依然低，无法拟合获得准确EC 50)。
实施例11：融合蛋白对Balb/c小鼠外周血免疫细胞影响的测定
BALB/c小鼠(购自北京Charles River实验动物技术有限公司)，雌性，6-8周龄，体重18-20g，在正式实验前，适应性饲养5天。所有的BALB/c小鼠饲养于SPF级动物房IVC恒温恒压系统中，其中温度20至26℃，湿度40至70％，光照周期12小时明/12小时暗。每个笼盒内饲养不多于6只BALB/c小鼠。小鼠按照体重进行分组，分组后开始给药，给药种类、给药剂量和给药途径见表16。模型分组当天为第0天。
表16.小鼠给药方案
Figure PCTCN2022114858-appb-000013
每个时间点新鲜采集的抗凝血用红细胞裂解液裂解红细胞，PBS洗一次。用含有1％FBS的PBS配制混合染色液。混合染色液包括CD3 APC-Cy7(Biolegend 100329)、CD8 PE(Biolegend 100708)、CD4 PE-Cy7(eBioscience 25-0042-82)、CD25 PerCP-Cy5.5(BD 561112)。每个样本加入100μL混合染色液，4度温育30分钟。用含有1％FBS的PBS洗两次。用True-Nuclear TM Transcription Factor Buffer Set(Biolegend 424401)进行固定破膜60分钟，100μL抗小鼠Foxp3抗体(Biolegend 126405)和抗小鼠Ki67抗体(eBioscience，25-5698-82)室温孵育60分钟。用PBS(pH 7.4)洗两次，最后用500μLPBS，pH 7.4洗液重悬，上机分析。其中，CD8 +T细胞定义为CD3 +CD4 -CD8 +的细胞，Treg定义为CD3 +CD4 +CD25 +Foxp3 +的细胞。
实验结果如图5A至图5F所示。对于A2-22-b，B2-15-b，D2-60-b，与第0天相比，小鼠全血中Treg占CD4 +T细胞的百分比从第2天开始上升，Treg中Ki67+ 的百分比也从第2天开始显著上升，表明A2-22-b，B2-15-b，D2-60-b可以强烈刺激Treg的增殖。相反，对于CD4 +CD25 -T细胞和CD8+ T细胞，Ki67+的百分比则几乎没有变化或变化较小，CD8 +T细胞占CD3 +T细胞的百分比也几乎未变，说明A2-22-b，B2-15-b，D2-60-b几乎不活化CD8 +T细胞。
实施例12：融合蛋白对接受鸡卵清蛋白OVA免疫的Balb/c小鼠的脾脏免疫细胞的影响的测定
鸡卵清蛋白(OVA)，Sigma A5503，使用PBS溶解，配制成0.5mg/mL。弗氏完全佐剂(CFA，含灭活结核分枝杆菌1mg/mL)，Sigma，F5881。雄性C57B16/J小鼠(购自上海实验动物中心)，6-8周龄，体重18-20g。小鼠按照体重进行分组，实验第1天，取OVA溶液8mL，与8mL CFA混匀、乳化呈油包水状态。每只动物腹腔注射200μL进行免疫。分别于第3天和第8天，各组小鼠皮下注射(sc)待测融合蛋白和PBS对照10mL/kg。给药种类、给药剂量和给药途径见表17。
表17.小鼠免疫方案
Figure PCTCN2022114858-appb-000014
第10天时，小鼠安乐死，取脾脏处理后流式细胞仪检测相关免疫细胞。方法包括：75％乙醇浸泡5分钟后在无菌条件下取出小鼠脾脏，PBS漂洗1-2次后，将小鼠脾脏剪碎。将过滤网置于50mL离心管中，将脾脏组织碎片转移到筛网上，研磨，过程中不断加入新鲜的PBS。1800转/分钟离心3分钟，弃上清。加入10mL红细胞裂解液冰上静置5分钟后加入PBS终止裂解，1800转/分钟离心3分钟两次。根据BioLegend流式抗体染色步骤进行染色后，将细胞用PBS重悬离心两次，过筛，进行流式细胞检测。
检测细胞及其标记物如下：
脾脏生殖中心B细胞(GCB，Germinal Center B cells)(Fas+GL-7+B220+)；
卵泡辅助性T细胞(Tfh，follicular T helper cells)(PD1高，CXCR5高，FOXP3-CD4+)；
卵泡调节性T细胞(Tfr，follicular regulatory T cells)(PD1高，CXCR5高，FOXP3+CD4+)；
调节性T细胞Treg细胞(FOXP3+CD25+CD4+CXCR5低)；
实验结果如图6所示。A2-22-b，B2-15-b，D2-60-b均可以刺激脾脏中的Treg和Tfr增殖，抑制脾脏中的Tfh和GCB数量，表明A2-22-b，B2-15-b，D2-60-b具有通过激活Treg抑制免疫系统的作用。
实施例13：融合蛋白在小鼠延迟性过敏反应模型中的药效实验
实验方法：取2，4二硝基氟苯(DNFB，Sigma，42085-50G)，用丙酮：橄榄油＝4：1溶解，配制成1％(1g/100mL)溶液备用，另稀释成0.5％(0.5g/100mL)溶液备用。雄性ICR小鼠(购自上海实验动物中心)，6周周龄，体重18-20g。小鼠按照体重进行分组。实验第0天，小鼠腹部涂抹1％的DNFB溶液50μL免疫，实验第5天时，小鼠右耳内外侧分别涂抹0.5％的DNFB溶液10μL(共20μL)进行激发，分别于实验第6天使用8mm直径打孔器取下左侧和右侧耳朵组织称重，并计算左、右两侧耳朵组织重量的差值。待测药从实验开始前2天(第-2天)开始，每5天皮下给药1次。
给药种类、给药剂量和给药途径见表18。
表18.小鼠免疫方案
组号 数量 待测物 剂量(mpk) 给药方式 给药时间
1 5 PBS NA 皮下 第-2和3天
2 5 A2-22-b 1 皮下 第-2和3天
3 5 A2-22-b 0.3 皮下 第-2和3天
4 5 A2-22-b 0.1 皮下 第-2和3天
5 5 B2-15-b 1 皮下 第-2和3天
6 5 B2-15-b 0.3 皮下 第-2和3天
7 5 B2-15-b 0.1 皮下 第-2和3天
8 5 D2-60-b 1 皮下 第-2和3天
9 5 D2-60-b 0.3 皮下 第-2和3天
10 5 D2-60-b 0.1 皮下 第-2和3天
实验结果如图7所示。A2-22-b，B2-15-b，D2-60-b均可以降低耳朵肿胀程度，并呈现剂量效应，表明A2-22-b，B2-15-b，D2-60-b具有抑制免疫系统的作用。
实施例14：融合蛋白在小鼠关节炎模型中的药效实验
雄性DBA/1纯系小鼠购自上海斯莱克实验动物有限公司。关节炎模型由牛II型胶原(Biolead，20022)免疫诱发。实验第0天，取2.5mL牛II胶原/冰乙酸(2mg/mL)中，再与2.5mL完全弗氏佐剂充分混悬，成油包水乳状，取0.1毫升在乙醚轻麻醉下于每个小鼠的尾根部皮内注射，3周后加强注射一次。造模动物随机分为6组，分别为：正常对照组，造模型组，IL-2(C013，novoprotein)组、A2-22-b组、B2-15-b组，每组各8只。同时选用正常对照动物作为正常对照组， 分组见表19。
表19.小鼠免疫方案
Figure PCTCN2022114858-appb-000015
(注：Q5D，每五天一次；Q2D，每两天一次；i.p.，腹腔给药；s.c.，皮下给药。)
关节炎症评价：小鼠从二次免疫开始，每3天进行称重和关节炎病情严重程度半定量计分(clinical scores)：
0分：无红肿；
1分：在爪上有一处红肿炎症；
2分：整爪上有轻微炎症或者有两处及以上红肿；
3分：整爪上有中度红肿；
4分：整个爪上有严重红肿甚至关节僵硬、畸形及活动障碍。
每只爪最高得分4分，每只鼠最高得分16分。关节炎病情观察一直持续到实验结束。
血清抗牛II型胶原抗体浓度检测：实验结束时，收集血，分离血清，-80℃保存。采用ELISA检测试剂盒(Chondrex，20322T)检测血清中抗牛II型胶原抗体的水平。具体步骤按说明书进行。
实验结果如图8A和图8B所示。和正常对照组相比，模型对照组小鼠体重明显下降，关节炎评分显著升高(p<0.001)；和模型对照组相比，B2-15-b可显著抑制关节炎造模小鼠体重减轻，可显著降低关节炎评分(p<0.05)。和正常对照组相比，模型对照组血清抗牛II型胶原IgG2a抗体的浓度显著增加(p<0.001)；和模型对照组相比，B2-15-b给药可显著下调关节炎小鼠血清抗牛II型胶原IgG2a抗体的分泌(p<0.01)。
实施例15：不同连接臂长度对融合蛋白表达量和纯度的影响
将B2-15-b中IL-2与抗IL-2抗体B2-15轻链之间的(G 4S) 5连接臂缩短G4S的重复次数至4次，而重链保持不变，获得B2-15-b的变体B2-15-b-(G 4S) 4，其 轻链序列如表20。
表20.融合蛋白中抗体的全长轻链序列
Figure PCTCN2022114858-appb-000016
按照实施例2中的方法对上述B2-15-b的变体进行表达和纯化，仍可以达到表达量47mg/L，SEC-HPLC纯度为99％。证明不同连接臂长度能实现融合蛋白的表达。
实施例16：抗IL-2抗体与不同IL-2变体之间的融合蛋白对人外周血(PBMC)STAT5磷酸化活性的测定
将IL-2进行突变，引入T3E，或T3Q，或去除IL-2N端前3个氨基酸(B2-15-b-Del3)，或去除IL-2N端前7个氨基酸(B2-15-b-Del7)，或直接使用野生型IL-2，分别与抗IL-2抗体进行偶联。产生如下融合蛋白(每一个抗体的重链序列保持不变)，见表21。
表21.融合蛋白中抗体的轻链全长序列
Figure PCTCN2022114858-appb-000017
Figure PCTCN2022114858-appb-000018
依照实施例7中的方法，测定上述抗IL-2抗体与IL-2变体的融合蛋白对人外周血(PBMC)中Treg和CD8 +T细胞中的STAT5磷酸化活性。如图9A至图9F、表22所示，实验结果显示，这些IL-2变体基本不影响抗体融合蛋白对Treg和CD8+ T细胞的活性。这些融合蛋白依然更偏向于激活Treg，而不是CD8 +T细胞。
表22.抗IL-2抗体与不同IL-2变体的融合蛋白激活Treg细胞中pSTAT5的EC 50
融合蛋白编号 EC 50(nM)
A2-22-b 0.168
A2-22-b-Del3 0.144
B2-15-b 0.016
B2-15-b-T3E 0.031
B2-15-b-WT 0.041
B2-15-b-T3Q 0.029
B2-15-b-Del3 0.038
B2-15-b-Del7 0.055
D2-60-b 0.114
D2-60-b-Del3 0.229
D2-60-b-T3E 0.168
实施例17：融合蛋白在MRL/lpr自发性红斑狼疮小鼠模型中的药效实验
雌性BALB/c小鼠和雌性MRL/lpr小鼠购自上海斯莱克实验动物有限公司(Shanghai SLAC Laboratory Animal Co.,Ltd)。以雌性BALB/c小鼠为正常对照，雌性MRL/lpr小鼠按蛋白尿水平及体重随机分为3组，分别为：MRL/lpr对照组(模型对照组)，1和3mg/kg给药组。D2-60-b-T3E经皮下注射给药，每3天给药一次，剂量为1、3mg/kg；8周龄时开始给药，连续给药8周。
表23.实验设计与分组
Figure PCTCN2022114858-appb-000019
给药期间，每两周测定各组小鼠尿蛋白含量；连续8周给药结束后进行如下检测：①测定各组小鼠血清中抗双链DNA的IgM和IgG的含量；②测定各组小鼠血清中血肌酐(CRE)和血尿素氮(BUN)含量；③测定各组小鼠血清中IL-6、IL-10和IFN-γ的含量；④对各组小鼠的肾脏组织进行病理分析和评分。
1)小鼠尿蛋白含量的检测
本实验使用提尾反射法收集小鼠尿液，采用尿蛋白检测试剂盒(CBB法，南京建成生物工程研究所，#C035-2-1)检测小鼠尿蛋白浓度。MRL/lpr小鼠红斑狼疮发病的过程中，肾脏会因dsDNA抗体沉积而发生损伤，进而导致肾小球滤过功能和重吸收能力下降，尿蛋白浓度升高。随着MRL/lpr小鼠狼疮肾炎的发展，小鼠尿蛋白水平会逐渐升高。每两周进行的小鼠尿蛋白水平检测显示，最初每组小鼠尿蛋白含量无明显差异，随着实验的进行，MRL/lpr对照组(模型对照组)小 鼠尿蛋白水平逐渐升高并明显高于正常组，说明由于自身抗体的沉积，MRL/lpr小鼠出现严重的自发性肾损伤。1和3mg/kg D2-60-b-T3E给药组小鼠平均尿蛋白含量低于模型对照组并呈现剂量依赖性，但由于数据个体差异较大，和模型对照组相比无统计学差异。数据见图10。
2)血清中抗双链DNA的IgM和IgG的含量检测
采用ELISA法检测血清中抗双链DNA(抗-dsDNA)IgG、IgM的含量。具体方法参见ELISA试剂盒(Alpha Diagnostic，#5120和#5130)说明书。3mg/kg的D2-60-b-T3E给药组小鼠血清中抗-dsDNA IgG和IgM含量的平均值显著低于模型对照组(P<0.01)，1mg/kg组虽然有IgG和IgM含量低于模型对照组的趋势，但和模型对照组的差异无统计学意义。实验结果见图11A和图11B。
3)D2-60-b-T3E对MRL/lpr小鼠肾功能的影响
本实验使用肌酐(CRE)测定试剂盒(南京建成生物工程研究所，#C011-2-1)和尿素氮(BUN)测试盒(南京建成生物工程研究所，#C013-2-1)检测实验结束时小鼠血清中血肌酐和尿素氮的含量。
尿素氮和肌酐分别是人体蛋白质和肌肉的代谢产物，肾脏是其终排泄器官，肾功能受损后，血中尿素氮和肌酐无法有效排出，血尿素氮(BUN)和血肌酐(CRE)的浓度因潴留而逐渐升高。因此血清中BUN和CRE水平是临床上反映肾功能的主要指标。与正常小鼠相比，MRL/lpr对照组(模型对照组)小鼠血清中肌酐(CRE)和尿素氮(BUN)含量均显著升高(P<0.0001)，提示MRL/lpr模型组小鼠出现功能性肾受损。
1和3mg/kg给药组小鼠血清中肌酐的含量显著低于模型对照组(P<0.01，P<0.0001)，可以一定程度上改善MRL/lpr小鼠的肾功能，并且3mg/kg给药组小鼠血清中肌酐的含量显著低于1mg/kg给药组。3mg/kg给药组小鼠血清中尿素氮的含量同样显著低于模型对照组(P<0.05)，说明3mg/kg的D2-60-b-T3E可以逆转MRL/lpr小鼠的自发性肾损伤。结果见图12A和图12B。
4)D2-60-b-T3E对MRL/lpr小鼠血清IL-6，IL10和INF-γ含量的影响
采用ELISA方法检测血清中细胞因子IFN-γ、IL-6和IL-10的含量。
与正常小鼠相比，MRL/lpr对照组(模型组)小鼠血清中IL-6和IFN-γ的含量显著升高(P<0.0001)。3mg/kg给药组小鼠血清中IL-6的含量显著低于模型对照组与1mg/kg给药组(P<0.0001)，呈现出剂量依赖关系。3mg/kg给药组小鼠血清中IFN-γ的含量显著低于模型对照组(P<0.01)。结果见图13A和图13B。
与正常小鼠相比，MRL/lpr对照组(模型对照组)小鼠血清中IL-10的含量显著下降(P<0.0001)。3mg/kg D2-60-b-T3E给药组小鼠血清中IL-10的含量显著高于模型对照组(P<0.001)。结果见图14。
5)D2-60-b-T3E对MRL/lpr小鼠肾脏病变的影响
实验结束时，取小鼠左肾用4％多聚甲醛中固定，石蜡包埋切片后H&E染色， 在显微镜下观察切片，并进行病理评分，对肾小球、肾间质以及血管等病理损伤进行分级评分，评分标准见表24。
表24.肾脏病理评分标准
Figure PCTCN2022114858-appb-000020
MRL/lpr对照组(模型对照组)小鼠肾脏出现严重的肾小球、肾间质及血管病理损伤，肾小球硬化，部分向内凹陷，大量细胞浸润在血管周及间质区域，肾小球出现严重的肿胀与增生。与正常小鼠相比，MRL/lpr对照组(模型对照组)小鼠肾脏病理评分显著升高(P<0.001)，提示模型对照组小鼠的肾脏出现明显的病变。
给药组小鼠的肾小球、肾间质及血管病理损伤有一定程度的减轻。1和3mg/kg给药组小鼠肾脏病理评分显著低于模型对照组(P<0.05，P<0.0001)，表明D2-60-b-T3E能显著减轻MRL/lpr自发性肾脏病变。结果见图15。
实施例-制剂
SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。
SEC％(SEC单体含量百分比)＝A单体/A总×100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和)。ΔSEC％＝强制降解实验前制剂的SEC％-强制降解实验后制剂的SEC％。
SEC测定用仪器：安捷伦1260；柱子：waters，XBrige
Figure PCTCN2022114858-appb-000021
SEC(300×7.8mm 3.5μm)
NR-CE毛细管凝胶电泳：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
NR-CE％＝A主峰/A总×100％(A主峰为样品中轻链主峰+重链主峰的峰面积，A总为所有峰面积之和。ΔNR-CE％＝强制降解实验前制剂的NR-CE％-强制降解实验后制剂的NR-CE％。
CE测定用仪器：Beckman型号plus800
iCIEF成像毛细管等点聚焦电泳：
根据蛋白质等电点pI不同进行分离的技术。
iCIEF％＝中性峰面积/总面积×100％(总面积为酸性峰、中性峰和碱性峰面积之和)。ΔiCIEF％＝强制降解实验前制剂的iCIEF％-强制降解实验后制剂的iCIEF％。
iCIEF测定所用仪器厂家simple protein，型号muarice。
渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM815。
融合蛋白
以下实施例所采用的融合蛋白为D2-60-b-T3E。融合蛋白的浓度采用蛋白浓度计。蛋白浓度测定仪器：紫外可见分光光度计，型号：Nano Drop oneC，光程为1mm。
制剂实施例1.pH和缓冲体系筛选
制备含表25所示的缓冲体系、30mg/mL融合蛋白、80mg/mL蔗糖、0.2mg/mL聚山梨酯80(PS80)的制剂。对样品进行强制降解研究(40℃放置1个月、25℃，300rpm振摇6天)，以SEC和iCIEF为评价指标，考察不同缓冲体系对融合蛋白稳定性的影响。
结果见表25。SEC数据显示，振摇6天后含His-HCl的制剂的单体纯度优于含AA或SA的制剂。iCIEF数据显示，振摇后组间无显著差异。40℃放置一个月后，含AA(pH 5.5)的制剂与含His-HCl(pH 6.0-6.5)的制剂优于其他组。综上所述，缓冲体系优选AA和His-HCl，pH范围优选5.5-6.5。
表25.pH和缓冲体系筛选结果
Figure PCTCN2022114858-appb-000022
Figure PCTCN2022114858-appb-000023
AA代表醋酸-醋酸钠；SA代表琥珀酸-琥珀酸钠；His-HCl代表组氨酸-盐酸组氨酸；
振摇D6：25℃ 300rpm振摇6天；40℃ M1：40℃放置1个月，下同。
制剂实施例2.蛋白浓度筛选
制备含10mM AA缓冲液(pH 5.5)、表26所示的蛋白浓度的融合蛋白、80mg/mL蔗糖、0.2mg/mL PS80的制剂。对样品进行强制降解研究(40℃放置1个月)，以SEC、NR-CE和iCIEF为评价指标，考察不同蛋白浓度对融合蛋白稳定性的影响。
结果见表26。蛋白浓度10-30mg/mL时，高温放置过程中外观及纯度项无明显差异，纯度项变化值均在可接受范围内，说明在该浓度范围内，融合蛋白样品具有良好的稳定性。
表26.蛋白浓度筛选结果
组别 浓度(mg/mL) 条件 ΔSEC％ ΔNR-CE％ ΔiCIEF％
1 30 40℃ M1 7.3 3.2 15.4
2 10 40℃ M1 6.7 3.5 14.9
制剂实施例3.含不同糖的制剂
在10mM AA pH5.5缓冲液中，制备蛋白浓度为10mg/mL，含0.2mg/mL PS80，含不同糖种类的抗IL-2蛋白制剂。具体糖种类和浓度如下：
1)80mg/mL蔗糖
2)80mg/mL海藻糖
将每种制剂过滤，灌装，加塞，轧盖。将样品进行25℃300rpm振摇稳定性考察，以SEC为评价指标，结果见表27，蔗糖稳定性优于海藻糖。
表27.糖的筛选结果
Figure PCTCN2022114858-appb-000024
制剂实施例4.表面活性剂浓度筛选
制备含30mg/mL的融合蛋白、80mg/mL蔗糖、表28所示的表面活性剂、和10mM AA缓冲液(pH 5.5)的制剂。对样品进行强制降解实验(40℃恒温条件下 放置1个月)，以SEC和iCIEF为评价指标，考察样品稳定性。结果见表28。结果显示，含泊洛沙姆188(PF68)的制剂的蛋白单体纯度未有明显变化。
表28.融合蛋白表面活性剂种类及浓度筛选结果
组别 表面活性剂 条件 ΔSEC％ ΔiCIEF％
1 0.1mg/mL PF68 40℃ M1 7.0 17.1
2 0.2mg/mL PF68 40℃ M1 7.4 20.9
3 0.4mg/mL PF68 40℃ M1 6.9 19.9
4 0.6mg/mL PF68 40℃ M1 7.0 18.9
制剂实施例5.表面活性剂筛选
以10mM pH6.0 His-HCl为缓冲体系，制备含80mg/mL蔗糖，含有不同种类的表面活性剂，蛋白浓度为30mg/mL的抗IL-2蛋白制剂。将每种制剂过滤，灌装，加塞，轧盖。取样品进行4℃长期研究，以外观和SEC为评价指标，考察样品稳定性，试验结果见表29。具体表面活性剂设定如下：
1)0.6mg/mL PS80
2)0.6mg/mL PF68
实验结果显示，4℃M13后相同表面活性剂浓度下PF68组SEC单体纯度与PS80组无明显差异。外观上，相同表面活性剂浓度下PS80组外观较优于PF68组。综合以上数据优选PS80作为抗IL-2蛋白制剂的表面活性剂。
表29.抗IL-2蛋白表面活性剂种类筛选结果
Figure PCTCN2022114858-appb-000025
制剂实施例6.表面活性剂浓度筛选
以10mM pH6.0 His为缓冲体系，制备含87mg/mL蔗糖，含有不同浓度PS80，蛋白浓度为30mg/mL的抗IL-2蛋白制剂。具体PS80浓度设定如下：
1)0.4mg/mL PS80
2)0.8mg/mL PS80
3)1.5mg/mL PS80
将每种制剂过滤，灌装，加塞，轧盖。进行冻融考察，以外观、SEC为评价指标，考察样品稳定性，试验结果见表30。冻融MFI结果显示，随着PS80浓度增大，≥2μm颗粒数减少。
表30.表面活性剂浓度筛选结果
组别 条件 外观 MFI(≥2μm)
1 FT5C 澄明 180
2 FT5C 澄明 129
3 FT5C 澄明 100
FT5C：冻融5个循环，以此类推。
制剂实施例7.辅料筛选
在10mM pH6.0 His-HCl缓冲液中，制备蛋白浓度为10mg/mL，含0.8mg/mL PS80，含不同辅料的抗IL-2蛋白制剂。具体如下：
1)36mg/mL蔗糖
2)36mg/mL蔗糖，12mg/mL甘氨酸
将每种制剂过滤，灌装，加塞，轧盖。将样品进行冻融考察，以外观、DLS和MFI为评价指标，结果见表31。外观DLS和MFI数据显示组间无显著差异。考虑到皮下制剂，减少注射刺激性，渗透压控制为等渗最佳，因此对辅料浓度进行筛选，确定等渗浓度。只有36mg/mL蔗糖渗透压偏低，36mg/mL蔗糖加12mg/mL甘氨酸或40mg/mL蔗糖加10mg/mL甘氨酸或45mg/mL蔗糖加10mg/mL甘氨酸或50mg/mL蔗糖加9mg/mL甘氨酸可控制在等渗范围内。
表31.辅料筛选结果
Figure PCTCN2022114858-appb-000026
制剂实施例8.不同离子强度筛选
以pH6.0 His-HCl为缓冲体系，制备含36mg/mL蔗糖、12mg/mL甘氨酸，含0.8mg/mLPS80蛋白浓度为10mg/mL的抗IL-2蛋白制剂。具体不同离子强度设定如下：
1)10mM
2)5mM
将每种制剂过滤，灌装，加塞，轧盖。进行冻融考察，以外观和SEC为评价指标，考察样品稳定性，试验结果见表32。结果显示组间外观、SEC无显著差异。
表32.不同离子强度筛选结果
Figure PCTCN2022114858-appb-000027
制剂实施例9.冻干制剂
以10mM pH6.0 His-HCl为缓冲体系，制备含50mg/mL蔗糖加9mg/mL甘氨酸(组别1)或40mg/mL蔗糖加10mg/mL甘氨酸(组别2)，含0.8mg/mL PS80蛋白浓度为10mg/mL的抗IL-2蛋白制剂。
将制剂过滤，灌装，加塞，轧盖。将制剂样品进行冻干，冻干程序为预冻、一次干燥和二次干燥(参数见表33)。冻干程序结束后，真空加塞。然后，用注射用水复溶冻干制剂，获得蛋白浓度10mg/mL的抗IL-2抗体复溶溶液，考察冻干前制剂及冻干后复溶溶液的外观、NR-CE和SEC纯度等指标的变化。实验结果见表34，结果表明，抗IL-2抗体制剂冻干前后均保持良好的稳定性。
表33.抗IL-2抗体制剂冻干程序
Figure PCTCN2022114858-appb-000028
表34.抗IL-2抗体制剂冻干前后制剂对比实验结果
Figure PCTCN2022114858-appb-000029
制剂实施例10.冻干制剂
分别以10mM pH6.0 His-HCl或5mM pH6.0 His-HCl为缓冲体系，制备含45mg/mL蔗糖、10mg/mL甘氨酸，含0.8mg/mL PS80蛋白浓度为10mg/mL的抗IL-2蛋白制剂。
将制剂过滤，灌装，加塞，轧盖。将制剂样品进行冻干，冻干程序为预冻、一次干燥和二次干燥(参数见表35)。冻干程序结束后，真空加塞。然后，用注射用水复溶冻干制剂，获得蛋白浓度10mg/mL的抗IL-2抗体复溶溶液，考察冻干 前制剂及冻干后复溶溶液的外观和SEC纯度等指标的变化。实验结果见表36，结果表明，抗IL-2抗体制剂冻干前后均保持良好的稳定性。
表35.抗IL-2抗体制剂冻干程序
Figure PCTCN2022114858-appb-000030
表36.抗IL-2抗体制剂冻干前后制剂对比实验结果
Figure PCTCN2022114858-appb-000031

Claims (20)

  1. 一种药物组合物，包含融合蛋白和缓冲剂，其中所述融合蛋白包含共价结合的IL-2与抗IL-2抗体，所述缓冲剂为组氨酸缓冲剂、醋酸盐缓冲剂或琥珀酸盐缓冲剂；
    优选地，所述缓冲剂为组氨酸缓冲剂或醋酸盐缓冲剂；
    更优选地，所述缓冲剂为组氨酸-盐酸组氨酸缓冲剂或醋酸-醋酸钠缓冲剂。
  2. 根据权利要求1所述的药物组合物，其中所述融合蛋白的浓度为5mg/mL至50mg/mL；
    优选地，所述融合蛋白的浓度为10mg/mL至30mg/mL。
  3. 根据权利要求1或2所述的药物组合物，其中所述药物组合物包含表面活性剂；
    优选地，所述表面活性剂为聚山梨酯或泊洛沙姆；
    更优选地，所述表面活性剂为聚山梨酯80、聚山梨酯20或泊洛沙姆188。
  4. 根据权利要求3所述的药物组合物，其中所述表面活性剂的浓度为0.05mg/mL至2mg/mL；
    优选地，所述表面活性剂的浓度为0.1mg/mL至0.8mg/mL。
  5. 根据权利要求1至4任一项所述的药物组合物，其中所述药物组合物包含糖；
    优选地，所述糖为蔗糖、甘露醇和海藻糖中的一种或多种；
    更优选地，所述糖为蔗糖。
  6. 根据权利要求5所述的药物组合物，其中所述糖的浓度为20mg/mL至100mg/mL；
    优选地，所述糖的浓度为30mg/mL至80mg/mL；
    更优选地，所述糖的浓度为30mg/mL至50mg/mL。
  7. 根据权利要求6所述的药物组合物，其中所述药物组合物还包含辅料；优选地，所述辅料为甘氨酸、甲硫氨酸、脯氨酸、组氨酸、苯丙氨酸、盐酸精氨酸、谷氨酸、天冬氨酸或乙二胺四乙酸二钠；更优选地，所述辅料为甘氨酸。
  8. 根据权利要求7所述的药物组合物，其中所述辅料的浓度为1mg/mL至30 mg/mL；优选地，辅料的浓度为5mg/mL至15mg/mL。
  9. 根据权利要求1至8任一项所述的药物组合物，其中所述缓冲剂的浓度为5mM至100mM；
    优选地，所述缓冲剂的浓度为5mM至30mM；
    更优选地，所述缓冲剂的浓度为5mM至10mM。
  10. 根据权利要求1至9任一项所述的药物组合物，其中所述缓冲剂的pH为5.0至6.5；
    优选地，所述缓冲剂的pH为5.5至6.0；
    更优选地，所述缓冲剂的pH为6.0。
  11. 根据权利要求1至10任一项所述的药物组合物，所述抗IL-2抗体包含重链可变区和轻链可变区，所述重链可变区具有：包含SEQ ID NO：37的氨基酸序列的HCDR1、包含SEQ ID NO：38的氨基酸序列的HCDR2和包含SEQ ID NO：39的氨基酸序列的HCDR3，所述轻链可变区具有：包含SEQ ID NO：40的氨基酸序列的LCDR1、包含SEQ ID NO：41的氨基酸序列的LCDR2和包含SEQ ID NO：42的氨基酸序列的LCDR3；
    优选地，所述重链可变区包含SEQ ID NO：9的氨基酸序列，所述轻链可变区包含SEQ ID NO：10的氨基酸序列；
    更优选地，所述抗IL-2抗体包含SEQ ID NO：165的氨基酸序列的重链和SEQ ID NO：166的氨基酸序列的轻链。
  12. 根据权利要求1至11任一项所述的药物组合物，所述IL-2直接或通过连接子融合至抗IL-2抗体的轻链的N端；
    优选地，所述IL-2的第3位氨基酸残基为E；
    更优选地，所述融合蛋白包含SEQ ID NO：165的氨基酸序列的重链和SEQ ID NO：189的氨基酸序列的轻链。
  13. 根据权利要求1至12任一项所述的药物组合物，其包含如下组分：
    a)10mg/mL至30mg/mL的所述融合蛋白，
    b)0.1mg/mL至0.8mg/mL的聚山梨酯或泊洛沙姆，
    c)30mg/mL至80mg/mL的蔗糖，
    d)1mg/mL至30mg/mL的甘氨酸，和
    e)5mM至10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0；
    优选地，所述药物组合物包含如下组分：
    a)10mg/mL至30mg/mL的所述融合蛋白，
    b)0.1mg/mL至0.8mg/mL的聚山梨酯80，
    c)30mg/mL至50mg/mL的蔗糖，
    d)5mg/mL至15mg/mL的甘氨酸，和
    e)5mM至10mM的组氨酸-盐酸组氨酸缓冲剂，所述组氨酸-盐酸组氨酸缓冲剂的pH为6.0。
  14. 根据权利要求1至13任一项所述的药物组合物，其中所述组合物是液体制剂；
    优选地，所述组合物的施用途径为皮下注射、皮内注射、静脉注射或肌内注射；
    更优选地，所述组合物的施用途径为皮下注射。
  15. 一种冻干制剂，所述冻干制剂复溶后可形成权利要求1至14任一项所述的药物组合物。
  16. 一种制备冻干制剂的方法，其中包括将权利要求1至14中任一项所述的药物组合物进行冷冻干燥的步骤。
  17. 一种冻干制剂，所述制剂通过将权利要求1至14中任一项所述的药物组合物经冷冻干燥获得。
  18. 一种复溶溶液，所述复溶溶液是通过将权利要求15或17所述的冻干制剂复溶制备的。
  19. 一种制品，其包括容器，该容器中装有如权利要求1至14任一项所述的药物组合物、权利要求15或17所述的冻干制剂或权利要求18所述的复溶溶液。
  20. 一种治疗自身免疫疾病或延缓自身免疫疾病进展的方法，所述方法包括向有需要的受试者施用治疗有效量的权利要求1至14任一项所述的药物组合物、权利要求15或17所述的冻干制剂、权利要求18所述的复溶溶液或权利要求19所述制品；
    优选地，所述自身免疫疾病选自以下的任一项或组合：狼疮、移植物抗宿主病、丙型肝炎诱导的脉管炎、I型糖尿病、多发性硬化、炎性肠病、慢性GvHD、哮喘、皮炎、斑秃、急性肺损伤、败血症、反应性关节炎和类风湿性关节炎。
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US12129302B2 (en) * 2021-08-25 2024-10-29 Ibio, Inc. Anti-CD-25 antibody

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