WO2020200099A1 - 包含抗il-5抗体的药物组合物及其用途 - Google Patents

包含抗il-5抗体的药物组合物及其用途 Download PDF

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Publication number
WO2020200099A1
WO2020200099A1 PCT/CN2020/081701 CN2020081701W WO2020200099A1 WO 2020200099 A1 WO2020200099 A1 WO 2020200099A1 CN 2020081701 W CN2020081701 W CN 2020081701W WO 2020200099 A1 WO2020200099 A1 WO 2020200099A1
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Prior art keywords
seq
variable region
chain variable
antibody
heavy chain
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PCT/CN2020/081701
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English (en)
French (fr)
Inventor
吴婷婷
李晧
刘洵
陶维康
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
Original Assignee
Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Priority to CA3134401A priority Critical patent/CA3134401A1/en
Priority to MX2021011530A priority patent/MX2021011530A/es
Priority to AU2020251627A priority patent/AU2020251627B2/en
Priority to JP2021557128A priority patent/JP7641908B2/ja
Priority to EP20782113.3A priority patent/EP3939611A4/en
Priority to CN202410290438.1A priority patent/CN118340881A/zh
Priority to KR1020217034029A priority patent/KR102962504B1/ko
Priority to CN202080018182.9A priority patent/CN113507938B/zh
Application filed by Jiangsu Hengrui Medicine Co Ltd, Shanghai Hengrui Pharmaceutical Co Ltd filed Critical Jiangsu Hengrui Medicine Co Ltd
Priority to BR112021019272A priority patent/BR112021019272A2/pt
Priority to US17/598,610 priority patent/US20220144937A1/en
Publication of WO2020200099A1 publication Critical patent/WO2020200099A1/zh
Priority to ZA2021/06721A priority patent/ZA202106721B/en
Anticipated expiration legal-status Critical
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing an anti-IL-5 antibody or an antigen-binding fragment thereof, and its use as a diagnostic agent and a therapeutic drug for IL-5 related diseases.
  • Interleukin-5 is one of the important members of the interleukin family, also known as T cell replacement factor (T cell replacing factor, TRF), B cell growth factor-II (B cell growth factor-II) , BCGF-II), IgA-enhancing factor (IgA-enhancing factor, IgA-EF), acidic granulocyte differentiation factor (eosinophil differentiation factor, EDF), are a kind of the same two secreted by helper T cells 2 (Th2) Aggregate glycoprotein.
  • T cell replacement factor T cell replacing factor
  • B cell growth factor-II B cell growth factor-II
  • BCGF-II B cell growth factor-II
  • IgA-enhancing factor IgA-enhancing factor
  • IgA-EF acidic granulocyte differentiation factor
  • EDF acidic granulocyte differentiation factor
  • Human IL-5 consists of 134 amino acid residues, including a 22 amino acid signal peptide and 2 glycosylation sites. Active IL-5 takes the form of an oligodi
  • EOS Eosinophil
  • Asthma is a chronic inflammatory disease of the respiratory tract.
  • IL-5 and its receptor IL-5R play an important role in the pathogenesis of asthma.
  • the most effective method for the treatment of asthma is to administer steroid drugs through the nose or mouth to inhibit the expression of some key mediators (including IL-5) in asthma to reduce lung inflammation.
  • IL-5 key mediators
  • long-term use of steroids has many side effects. Therefore, it is necessary to find new drug targets for the treatment of asthma.
  • anti-IL-5 antibodies to inhibit the binding of IL-5 to its receptor can significantly reduce the accumulation of eosinophils in the lungs, reduce the level of eosinophils in blood, tissues, and sputum, and It can reduce the inflammatory response mediated by eosinophils, improve lung function, and has a good effect on severe eosinophilic asthma and recurrent asthma (Drugs.2017 May; 77(7):777-784).
  • Antibody drugs have large molecular weights and complex structures, and problems of denaturation, aggregation, contamination and particle formation often occur during production, transportation and storage. To keep the antibody effective, the antibody must maintain its biological activity during production, purification, transportation, and storage. At present, new production and purification technologies have been developed for the production of large quantities of highly purified monoclonal antibodies. However, how to stabilize these antibodies for transportation and storage, and provide antibodies in a dosage form suitable for administration, has always been a problem.
  • the only anti-IL-5 antibodies currently on the market are mepolizumab from GSK and reslizumab from Teva Pharma.
  • Related patents include WO2018119016, WO2017033121, WO2014141149, WO2016040007, WO2015095539, WO2012138958, WO9535375 and so on.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an IL-5 antibody or an antigen-binding fragment thereof, a buffer and a surfactant, the buffer being selected from the group consisting of acetic acid-sodium acetate, succinic acid-sodium succinate, histidine- Any one of hydrochloride and citrate-sodium citrate buffer, preferably acetic acid-sodium acetate or succinate-sodium succinate buffer; wherein said anti-IL-5 antibody or antigen-binding fragment thereof Contains heavy chain variable region and light chain variable region, where:
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 16, 17, 18, respectively, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 19, 20, 21, respectively LCDR1, LCDR2 and LCDR3 shown;
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 22, 23, 24, respectively, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 25, 26, 27, respectively LCDR1, LCDR2 and LCDR3 shown;
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 28, 29, 30, respectively, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 31, 32, 33, respectively LCDR1, LCDR2 and LCDR3 shown;
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 34, 35, 36, respectively, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 37, 38, 39, respectively LCDR1, LCDR2 and LCDR3 shown;
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 40, 41, and 42 amino acid sequences, respectively, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 43, 44, and 45, respectively.
  • LCDR1, LCDR2 and LCDR3 as shown; or
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 34, 82, 36, respectively, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 37, 38, 39, respectively LCDR1, LCDR2 and LCDR3 shown.
  • the pH of the buffer in the pharmaceutical composition is from about 5.0 to about 6.5, preferably from about 5.5 to about 6.5, preferably from about 6.0 to about 6.5, preferably from about 5.0 to about 6.0, It is preferably about 5.5 to about 6.0, preferably about 5.0 to about 5.5, preferably about 5.0 to about 5.8, preferably about 5.2 to about 5.8; non-limiting examples of the pH value of the buffer include about 5.0, about 5.1 , About 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, and most preferably about 5.5.
  • the concentration of the buffer in the pharmaceutical composition is about 10 mM to about 40 mM, about 10 mM to about 30 mM, preferably about 15 mM to about 30 mM, preferably about 20 mM to about 30 mM, preferably about 25 mM To about 30mM, preferably about 10mM to about 25mM, preferably about 15mM to about 25mM, preferably about 20mM to about 25mM, preferably about 10mM to about 15mM; non-limiting examples of the buffer concentration include about 10mM, about 12mM, about 14mM, about 16mM, about 18mM, about 20mM, about 22mM, about 24mM, about 26mM, about 28mM, about 30mM, about 32mM, about 34mM, most preferably about 30mM.
  • the concentration of the anti-IL-5 antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1 mg/ml to about 120 mg/ml, preferably about 1 mg/ml to about 100 mg/ml, preferably It is about 10mg/ml to about 120mg/ml, preferably about 20mg/ml to about 120mg/ml, preferably about 30mg/ml to about 120mg/ml, preferably about 40mg/ml to about 120mg/ml, preferably about 50mg/ml to about 120mg/ml, preferably about 60mg/ml to about 120mg/ml, preferably about 70mg/ml to about 120mg/ml, preferably about 80mg/ml to about 120mg/ml, preferably about 90mg/ml ml to about 120 mg/ml, preferably about 100 mg/ml to about 120 mg/ml, preferably about 110 mg/ml to about 120 mg/ml,
  • the surfactant contained in the pharmaceutical composition may be selected from polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium lauryl sulfonate, lauryl sulfonic acid Sodium, sodium octyl 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, cocaamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmamidopropyl-betaine, isostearamidopropyl -Betaine, coca
  • the concentration of polysorbate 80 in the pharmaceutical composition is about 0.05 mg/ml to about 0.6 mg/ml, preferably about 0.1 mg/ml to about 0.6 mg/ml, preferably about 0.2 mg /ml to about 0.6 mg/ml, preferably about 0.3 mg/ml to about 0.6 mg/ml, preferably about 0.4 mg/ml to about 0.6 mg/ml, preferably about 0.5 mg/ml to about 0.6 mg/ml , Preferably about 0.2mg/ml to about 0.5mg/ml, preferably about 0.3mg/ml to about 0.5mg/ml, preferably about 0.4mg/ml to about 0.5mg/ml, preferably about 0.3mg/ml ml to about 0.4 mg/ml, as a non-limiting example, the concentration of the surfactant in the pharmaceutical composition is about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.45
  • the pharmaceutical composition further includes excipients, wherein the excipients are selected from stabilizers.
  • the stabilizer is selected from sugars or amino acids;
  • the sugar can be selected from sucrose, trehalose, mannitol or sorbitol, preferably sucrose.
  • the amino acid is selected from glycine, methionine and proline.
  • the concentration of the sugar is about 50 mg/ml to about 80 mg/ml, preferably about 60 mg/ml to about 80 mg/ml, preferably about 70 mg/ml to about 80 mg/ml, preferably It is about 75 mg/ml to about 80 mg/ml, preferably about 70 mg/ml to about 75 mg/ml; as a non-limiting example, the concentration of the stabilizer in the pharmaceutical composition includes about 70 mg/ml, about 71 mg/ml, About 72mg/ml, about 73mg/ml, about 74mg/ml, about 75mg/ml, about 76mg/ml, about 77mg/ml, about 78mg/ml, about 79mg/ml, about 80mg/ml, more preferably about 72mg /ml.
  • the concentration of said amino acid is about 8 mg/ml.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition preferably comprises:
  • the anti-IL-5 antibody or antigen-binding fragment thereof in the pharmaceutical combination described in the present disclosure is a murine antibody, a chimeric antibody or a humanized antibody.
  • the anti-IL-5 humanized antibody in the pharmaceutical composition contains the heavy chain variable region shown in SEQ ID NO: 49, 57, 63, 69 or 75 or a variant thereof;
  • the variant has 1-10 amino acid back mutations in the heavy chain variable region sequence shown in SEQ ID NO: 49, 57, 63, 69 or 75.
  • the variant is selected from any of the following variants:
  • amino acid back mutation selected from one or more of R38K, M48I, R67K, V68A, M70L, R72V, T74K and L83F in the heavy chain variable region shown in SEQ ID NO: 63;
  • the heavy chain variable region shown in SEQ ID NO: 69 has an amino acid back mutation selected from one or more of F29I, R38K, V48I, R72A, and T97F and/or N55V mutation in the CDR; or
  • the heavy chain variable region shown in SEQ ID NO: 75 has an amino acid back mutation selected from one or more of R38K, M48I, R67K, V68A, R72A, T74K, M81L, L83F and D89E.
  • the anti-IL-5 humanized antibody in the pharmaceutical composition comprises a heavy chain variable region as shown in SEQ ID NO: 50 or 51; or
  • It comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 76, 77, 78, and 79.
  • the anti-IL-5 humanized antibody in the pharmaceutical composition contains the light chain variable region shown in SEQ ID NO: 46, 54, 60, 67 or 72 or a variant thereof;
  • the variant has 1-10 amino acid back mutations in the light chain variable region shown in SEQ ID NO: 46, 54, 60, 67 or 72.
  • the variant is selected from any of the following variants:
  • amino acid back mutation selected from one or more of A43S, L47V, G66R, T69S, F71Y and Y87F in the light chain variable region shown in SEQ ID NO: 46;
  • the light chain variable region shown in SEQ ID NO: 60 has an amino acid back mutation selected from E1D, I2T, I57V, V84T and Y86F or a combination thereof;
  • amino acid back mutation selected from one or more of M4L, A42S, L45P and L46W in the light chain variable region shown in SEQ ID NO: 67; or
  • the light chain variable region shown in SEQ ID NO: 72 has an amino acid back mutation selected from one or more of A43S, I48V and F71Y.
  • the anti-IL-5 humanized antibody in the pharmaceutical composition comprises a light chain variable region as shown in SEQ ID NO: 47 or 48; or
  • the anti-IL-5 humanized antibody in the pharmaceutical composition comprises:
  • the light chain variable region shown or having 95% sequence identity with it; preferably, the anti-IL-5 humanized antibody comprises:
  • the aforementioned amino acid sequence having at least 95% sequence identity preferably, it has at least 95%, 96%, 97%, 98%, or 99% sequence identity, more preferably, it has 97%, 98%, or 99% sequence identity. % Or more, most preferably having at least 99% or more sequence identity.
  • the above-mentioned amino acid sequence having at least 95% sequence identity includes one or more amino acid deletion, insertion or substitution mutations.
  • the anti-IL-5 antibody in the pharmaceutical composition includes a human antibody constant region, preferably comprising the human antibody heavy chain constant region shown in SEQ ID NO: 52 and SEQ ID NO: 53 The human antibody light chain constant region.
  • the anti-IL-5 antibody in the pharmaceutical composition comprises:
  • the anti-IL-5 antibody in the pharmaceutical composition is a monoclonal antibody or antigen-binding fragment thereof that competes with the aforementioned anti-IL-5 antibody or antigen-binding fragment thereof for binding to IL-5 .
  • the antigen-binding fragment in the pharmaceutical composition of the present disclosure is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region ( Diabodies) and disulfide bond stabilized V regions (dsFv).
  • the present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, which includes the step of replacing the stock solution of the anti-IL-5 antibody with a buffer.
  • the buffer is preferably an acetic acid-sodium acetate buffer.
  • the pH of the buffer is about 5.0 to about 6.5, preferably about 5.5 to about 6.5, preferably about 6.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.0, preferably about 5.0 to about 5.5, preferably From about 5.2 to about 5.8, non-limiting examples of the buffer pH value include about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, About 6.0, about 6.5, and most preferably about 5.5.
  • the concentration of the buffer in the pharmaceutical composition is about 10 mM to about 30 mM, preferably about 15 mM to about 30 mM, preferably about 20 mM to about 30 mM, preferably about 25 mM to about 30 mM, preferably About 5mM to about 25mM, preferably about 10mM to about 25mM, preferably about 15mM to about 25mM, preferably about 20mM to about 25mM, preferably about 5mM to about 20mM, preferably about 10mM to about 15mM; the buffer
  • concentrations include about 10mM, about 12mM, about 14mM, about 16mM, about 18mM, about 20mM, about 22mM, about 24mM, about 26mM, about 28mM, about 30mM, and most preferably about 30mM.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate, pH 5.5 and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate, pH 5.5 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 5.0 and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 5.5 and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 6.0 and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 5.0 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 5.5 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 6.0 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 6.5 and 0.2 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 5.5 and 0.2 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 6.0 and 0.2 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 6.5 and 0.05 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 5.5 and 0.05 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 6.0 and 0.05 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 6.5 and 0.2 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM citrate-sodium citrate, pH 5.5 and 0.2 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM histidine-hydrochloric acid, pH 6.0 and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM histidine-hydrochloric acid, pH 6.5 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM histidine-hydrochloric acid, pH 5.5 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM histidine-hydrochloric acid, pH 6.0 and 0.05 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 5.0 and 0.1 mg/mL polysorbate 20.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate, pH 5.0 and 0.1 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate pH 5.0, 50 mg/mL sucrose, and 0.1 mg/mL polysorbate Ester 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate pH 5.0, 50 mg/mL trehalose and 0.1 mg/mL polysorbate Alcohol ester 80.
  • the pharmaceutical composition comprises: 100 mg/ml anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate pH 5.0, 50 mg/mL mannitol and 0.1 mg/mL polysorbate Ester 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate pH 5.0, 50 mg/mL sorbitol and 0.1 mg/mL polysorbate Ester 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate pH 5.0, 8 mg/mL glycine and 0.1 mg/mL polysorbate Ester 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate pH 5.0, 8 mg/mL methionine and 0.1 mg/mL Polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate pH 5.0, 8 mg/mL proline and 0.1 mg/mL poly Sorbitol ester 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM succinate-sodium succinate pH 5.5, 70 mg/mL sucrose, and 0.4 mg/mL polysorbate Ester 80.
  • the pharmaceutical composition comprises: 80 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.8, and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.4, and 0.6 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 80 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.4, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 80 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.0, and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.4, and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 80 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.8 and 0.6 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 120 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.0, and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 80 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.0 and 0.6 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 120 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.8, and 0.6 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 120 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.4, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.4, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 120 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.0 and 0.6 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 120 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.8 and 0.2 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.0, and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.8 and 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate pH 5.5, 70 mg/ml sucrose and 0.4 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 20 mM acetic acid-sodium acetate pH 5.5, 70 mg/ml sucrose and 0.4 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 30 mM acetic acid-sodium acetate pH 5.5, 70 mg/ml sucrose and 0.4 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 30 mM acetic acid-sodium acetate pH 5.5, 73 mg/ml sucrose and 0.4 mg/mL polysorbate 80 .
  • the pharmaceutical composition comprises: 100 mg/ml of anti-IL-5 antibody h1705-008, 30 mM acetic acid-sodium acetate pH 5.5, 75 mg/ml sucrose and 0.4 mg/mL polysorbate 80 .
  • the pharmaceutical composition of the present disclosure is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. It can be stable at 25°C for at least 3 months, at least 6 months.
  • the present disclosure also provides a method for preparing a freeze-dried preparation containing an anti-IL-5 antibody, which includes the step of freeze-drying the aforementioned pharmaceutical composition.
  • the freeze-drying includes the steps of pre-freezing, primary drying and secondary drying in sequence. Freeze drying is performed by freezing the preparation and then sublimating water at a temperature suitable for one-time drying. Under this condition, the product temperature is lower than the eutectic point or decomposition temperature of the formulation. Under a suitable pressure usually in the range of about 50-250 millitorr, generally, the storage temperature range for the primary drying is about -30 to 25°C (assuming the product remains frozen during the primary drying process).
  • the size and type of the formulation, the container (e.g., glass vial) holding the sample, and the volume of the liquid determine the time required for drying, which can range from a few hours to several days (e.g., 40-60 hours).
  • the secondary drying stage can be carried out at about 0-40°C, depending on the type and size of the container and the type of protein used.
  • the secondary drying time is determined by the desired residual humidity level in the product, and usually requires at least about 5 hours.
  • the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%.
  • the pressure may be the same as the pressure applied in the primary drying step.
  • the pressure of the secondary drying is lower than that of the primary drying. Freeze drying conditions can vary with the formulation and vial size.
  • the present disclosure also provides a freeze-dried preparation containing an IL-5 antibody prepared by the aforementioned method for preparing a freeze-dried preparation containing an anti-IL-5 antibody.
  • the lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present disclosure also provides a method for preparing a reconstituted solution of a freeze-dried preparation containing an anti-IL-5 antibody, which includes the step of reconstituted the aforementioned freeze-dried preparation, and the solution used for the reconstitution includes, but is not limited to, water for injection, physiological saline or Glucose solution.
  • the present disclosure also provides a reconstitution solution of a lyophilized preparation containing an IL-5 antibody prepared by the aforementioned method of a reconstitution solution of a lyophilized preparation containing an anti-IL-5 antibody.
  • the present disclosure further provides an article or kit comprising a container containing any stable pharmaceutical composition described herein.
  • the container is a neutral borosilicate glass vial.
  • the present disclosure also provides a product, which includes a container containing the aforementioned pharmaceutical composition or a lyophilized preparation or a reconstituted solution of the lyophilized preparation.
  • the present disclosure also provides a method for treating IL-5 mediated diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized preparation or reconstitution solution Or products, wherein the IL-5 mediated disease is preferably asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, Ceratocystis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, worm infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilia Hypergranulocytosis bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or product of the lyophilized preparation in the preparation of drugs for treating IL-5 mediated diseases, wherein the IL-5 mediated diseases Preferred asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocercia dermatitis, intermittent angioedema, eosinophils Myalgia syndrome, eosinophilic gastroenteritis, worm infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic bronchitis, nodular arteritis, Sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, Onchocerciasis dermatitis
  • the present disclosure also provides the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or product of the lyophilized preparation for use as a therapeutic drug, wherein the drug is used to treat IL-5 mediated diseases, wherein the IL- 5 mediated diseases are preferably asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocercia dermatitis, intermittent angioedema , Eosinophilic myalgia syndrome, eosinophilic gastroenteritis, worm infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic bronchitis, nodules Arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivit
  • Figure 1 shows the results of FACS experiment in which anti-IL-5 antibody blocks IL-5 binding to IL-5 receptor
  • Figure 2 shows the detection results of the binding specificity of anti-IL-5 antibody and Th2 cytokine
  • FIG. 3 shows that anti-IL-5 antibodies enhance intermittent respiration (Penh) levels.
  • G1 normal control group (PBS);
  • G2 model group (IgG);
  • G3 h1705-008 antibody 10mpk group;
  • G4 h1705-008 antibody 2mpk group;
  • G5 h1706-009 antibody 10mpk group;
  • G6 h1706-009 antibody 2mpk group;
  • G7 Hu39D10 10mpk group; among them, *p ⁇ 0.05, ** ⁇ 0.01 (compared with G2 group by ANOVA/Bonferroni);
  • Figure 4A shows the BALF eosinophil level in the lungs of asthmatic mice
  • Figure 4B shows the airway mucosal thickness score of asthmatic mice.
  • G1 normal control group
  • G2 model group
  • G3 h1705-008 antibody 10mpk group
  • G4 h1705-008 antibody 2mpk group
  • G5 h1706-009 antibody 10mpk group
  • G6 h1706-009 antibody 2mpk group
  • G7 Hu39D10 10mpk group
  • Figure 4C shows the percentage of BALF eosinophils in the lungs of asthmatic mice
  • Figures 5A and 5B show the ability of IL5 monoclonal antibody to reduce the level of eosinophils in BALF.
  • Buffering agent refers to a buffering agent that withstands pH changes through the action of its acid-base conjugated component.
  • buffers that control the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, and fumarate. Salt, glycylglycine and other organic acid buffers.
  • Hetidine salt buffer is a buffer containing histidine radical ions.
  • the histidine salt buffer include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and the like buffers, preferably histidine-acetate Buffer or histidine-hydrochloride buffer, histidine-acetate buffer is prepared from histidine and acetic acid, and histidine buffer is prepared from histidine and hydrochloric acid.
  • citrate buffer is a buffer that includes citrate ions.
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • the preferred citrate buffer is citrate-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-succinate calcium salt and the like.
  • the preferred succinate buffer is succinate-sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • the preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • Acetate buffer is a buffer including acetate ions.
  • acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, promote the administration to the organism, and facilitate the absorption of the active ingredient to exert its biological activity.
  • the "sugar” in the present disclosure includes conventional (CH 2 O) n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like.
  • the preferred sugar is a non-reducing disaccharide, more preferably sucrose.
  • the solvent therein is all water.
  • “Lyophilized preparation” means a pharmaceutical composition in liquid or solution form or a preparation or pharmaceutical composition obtained after a liquid or solution preparation is subjected to a vacuum freeze-drying step.
  • the freeze-drying in the present disclosure includes pre-freezing, primary drying, and secondary drying.
  • the purpose of pre-freezing is to freeze the product and obtain a crystalline solid.
  • Pre-freezing temperature and pre-freezing speed are two important process parameters.
  • the pre-freezing temperature is set to -45°C and the pre-freezing speed is set to 1°C/min.
  • Primary drying is also called main drying and is the main stage of freeze drying of samples. The purpose is to remove ice from the product while maintaining the shape of the product, minimizing damage to the product. If the temperature and vacuum degree of the first drying are not selected properly, the product will collapse. Higher temperature and vacuum will accelerate freeze-drying efficiency, but at the same time will also increase the risk of product collapse.
  • the temperature of the primary drying in the present invention can be a conventional temperature in the field, for example, -30°C to 0°C.
  • Secondary drying is also called analytical drying, which is the main step of removing bound water in the product by pumping an ultimate vacuum (0.01mbar) and increasing the temperature (20-40°C). Since most biological products are sensitive to temperature, the secondary drying temperature is selected at the low point of the temperature range, namely 25°C.
  • the freeze-drying time is related to the freezer, the dose of the freeze-dried preparation, and the container of the freeze-dried drug. This time adjustment is well known to those skilled in the art.
  • the terms “about” and “approximately” mean that the index value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value partly depends on how to measure or determine (ie the limit of the measurement system). For example, in every practice in the art, “about” can mean within one or more than one standard deviation. Alternatively, “about” or “substantially comprising” may mean a range of at most ⁇ 20%, for example, a pH of about 5.5 means a pH of 5.5 ⁇ 1.1. Furthermore, especially for biological systems or processes, the term can mean at most an order of magnitude or at most 5 times the value. Unless otherwise specified, when a specific value appears in the application and claims, the meaning of "about” or “substantially includes” should be within the acceptable error range of the specific value.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: the antibody therein substantially retains its physical stability and/or chemical stability and/or biological activity after storage.
  • the drug The composition basically retains its physical and chemical stability and its biological activity after storage.
  • the shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are many analytical techniques for measuring protein stability, which can measure the stability after storage at a selected temperature for a selected period of time.
  • a stable pharmaceutical antibody preparation is a preparation in which no significant changes in physical, and/or chemical, and/or biological activity are observed under the following conditions: storage at a refrigerated temperature (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 preparations include liquid preparations that exhibit desired characteristics after being stored at a temperature of 25°C for a period of time including 1 month, 3 months, and 6 months. Stability of a typical formulation: measured by SEC-HPLC, usually no more than about 10%, preferably no more than about 5% of the antibody monomer is degraded. Through visual analysis, the drug antibody preparation is pale yellow, almost colorless, clear liquid or colorless, or clear to slightly milky white.
  • the concentration, pH and osmolality of the preparation have no more than ⁇ 10% change. A reduction of not more than about 10%, preferably not more than about 5% is generally observed. Generally no more than about 10%, preferably no more than about 5% of aggregates are formed.
  • the antibody does not show a significant increase in aggregation, precipitation and/or denaturation, Then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).
  • the antibody does not show a significant chemical change, then the antibody "retains its chemical stability" in the pharmaceutical formulation.
  • chemical stability can be assessed.
  • Degradation processes that often change the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS, etc.) Method to evaluate), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.) and isomerization (by measuring isoaspartic acid content, Peptide mapping etc. to evaluate).
  • the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical preparation is prepared, then the antibody "retains its biological activity" in the pharmaceutical preparation.
  • the biological activity of an antibody can be determined, for example, by an antigen binding assay.
  • antibody refers to an immunoglobulin.
  • a complete antibody is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the antibody light chain described in the present disclosure may further comprise a light chain constant region, and the light chain constant region comprises human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain described in the present disclosure may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (LCVR, VL) and heavy chain variable region (HCVR, VH) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known Kabat numbering rules (LCDR1-3, HCDR1-3).
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • the "antigen-binding fragment of an antibody” or “functional fragment” as used in the present disclosure refers to Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to antibodies.
  • the Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies into a polypeptide chain, which is called a single chain antibody or single chain Fv (sFv).
  • antigen-binding site in the present disclosure refers to a continuous or discontinuous three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • murine antibody in the present disclosure is a monoclonal antibody to human IL-5 prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with IL-5 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-derived specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed, and convert the mouse variable region
  • the gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the IL-5 chimeric antibody further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the IL-5 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising the human IgG2 or IgG4 heavy chain constant region, or using amino acid mutations without ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germlines The antibody produced in the antibody framework sequence. It can overcome the strong heterologous reaction induced by the chimeric antibody due to the large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase ), and in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation for CDR by phage display.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • antibody-dependent cell-mediated cytotoxicity means that cells expressing Fc receptors directly kill the antibody-encased cells by recognizing the Fc segment of the antibody.
  • the target cells are.
  • the ADCC effect function of antibodies can be reduced or eliminated by modifying the Fc section of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2/4 chimera, and F234A/L235A mutation of IgG4.
  • the “mutation” in the mutant sequence described in the present disclosure includes but is not limited to "back mutation", “conservative modification” or “conservative substitution or substitution”.
  • the “conservative modification” or “conservative substitution or substitution” mentioned in the present disclosure refers to the substitution of other amino acids in proteins with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.)
  • the amino acids that make it possible to make frequent changes without changing the biological activity of the protein are those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity.
  • the "mutated sequence” mentioned in the present disclosure refers to the nucleotide sequence and/or amino acid sequence of the present disclosure after appropriate substitutions, insertions or deletions and other mutation modifications are made.
  • the amino acid sequence has nucleotide sequences and/or amino acid sequences with different percent sequence identity.
  • the sequence identity described in this disclosure may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparison and determination of the percent identity between the two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • binding to IL-5" refers to the ability to interact with IL-5, preferably human IL-5.
  • anti-IL-5 antibody and "IL-5 antibody” are used interchangeably, and both are antibodies that bind to IL-5.
  • the fusion protein described in the present disclosure is a protein product obtained by co-expressing two genes through DNA recombination.
  • Recombinant IL-5 extracellular region Fc fusion protein is a fusion protein that expresses IL-5 extracellular region and human antibody Fc fragment through DNA recombination.
  • the IL-5 extracellular region refers to the part of IL-5 protein expressed outside the cell membrane, and the sequence is shown in SEQ ID NO:1.
  • the methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Chapters 5-8 and 15 of the "Antibody Experimental Technology Guide” published by Cold Spring Harbor Laboratory.
  • Mice can be immunized with human IL-5 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments described in the present disclosure are genetically engineered to add one or more human FR regions to non-human CDR regions.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human IL-5. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions that interact with animals, humans, and recipients. Contact with subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition containing any one of the combination compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered to the patient or population to be treated in an amount effective to relieve one or more symptoms of the disease, in order to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can assess whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • the disease related to IL-5 there is no restriction on the disease related to IL-5, as long as it is a disease related to IL-5.
  • the therapeutic response induced by the molecule of the present disclosure can be combined with human IL-5 and then inhibit or inhibit eosinophil stimulation. Effect.
  • the pharmaceutical composition of the present disclosure is very useful for people who suffer from allergies and/or atopic reactions, or are related to eosinophils
  • eosinophils For example, but not limited to, asthma, exacerbation of asthma, malignant asthma attack, chronic pneumonia, allergic rhinitis, perennial allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, special Atopic Dermatitis, Onchocerciasis Dermatitis, Intermittent Angioedema, Eosinophilic Myalgia Syndrome, Eosinophilic Gastroenteritis, Worm Infection, Hodgkin's Disease, Nasal Polyps, Loeffler's Syndrome, Urticaria, eosinophilic bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis
  • this treatment inhibits or reduces the lung tissue infiltration of eosinophils.
  • the number of administration of the pharmaceutical composition can range from three times a day to once every 6 months, and the route of administration can be intravenous, subcutaneous, intramuscular, parenteral or topical.
  • the formulations of the present disclosure can be used to treat IL-5 mediated diseases.
  • IL-5 mediated diseases For example, but not limited to inhibiting or reducing IL-5 mediated inflammatory response and maturation, activation, degranulation or tissue infiltration of eosinophils in vivo and in vitro; inhibiting excessive stress of smooth muscle caused by IL-5; reducing IL-5 Levels in the lungs, airways, or blood.
  • the formulations of the present disclosure can be used to treat IL-5 mediated diseases.
  • the IL-5 mediated diseases are selected from asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophils Cytocytosis, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, worm infection, He Jie King's disease, nasal polyps, Loeffler's syndrome, urticaria, eosinophilic bronchitis, nodular arteritis, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis , Allergic conjunctivitis, Onchocerciasis dermatitis, endometriosis or steroid-dependent eosinophilic bronchitis.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Replacement refers to the replacement of the solvent system that dissolves the antibody protein.
  • the high salt or hypertonic solvent system containing the antibody protein is replaced by physical manipulation using a buffer system of a stable formulation, so that the antibody protein exists in the stable formulation.
  • the physical operation methods include but are not limited to ultrafiltration, dialysis or centrifugation and reconstitution.
  • the sequence of the fusion protein encoding the extracellular domain of human IL-5 with His tag, rhesus IL-5, mouse IL-5, rat IL-5, human IL-5R ⁇ receptor containing human IgG1-Fc fragment was cloned to On the phr vector, construct an expression plasmid, and then transfect HEK293. On the 6th day of transfection, take a sample and centrifuge at 4500 rpm for 10 min to collect the cell supernatant. The supernatant containing recombinant IL-5 and IL-5 ⁇ receptor proteins is purified by nickel column, and the recombinant human IL-5-Fc fusion protein is used Protein A affinity chromatography column for purification. The purified protein can be used in subsequent experiments.
  • the protein sequence of the specific antigen is as follows:
  • the italicized part is His6-tag.
  • the italicized part is His6-tag.
  • the italicized part is His6-tag.
  • the italicized part is the human IgG1-Fc tag.
  • Example 2 Construction and identification of recombinant IL-5 ⁇ receptor and IL-5 ⁇ / ⁇ receptor cell lines
  • the present disclosure constructs a CHO-S/IL-5 ⁇ cell line expressing IL-5 ⁇ and a CHO-S/IL-5 ⁇ /IL-5 ⁇ cell line expressing both IL-5 ⁇ and IL-5 ⁇
  • the full-length human IL-5 ⁇ gene (Q01344) was cloned into the mammalian cell expression vector pTargeT, the linearized plasmid was electrotransfected into CHO-S cells, screened by G418 for 2 weeks, and then subjected to two limiting dilutions.
  • the IL-5 ⁇ gene on the cell surface was detected by FACS, and the CHO-S/IL-5 ⁇ cell line with high IL-5 ⁇ expression was selected.
  • the linearized pcDNA3.1-IL-5 ⁇ was electrotransfected, and G418 Screened with zeocin for 2 weeks, and then performed two limiting dilutions.
  • the IL-5 ⁇ and IL-5 ⁇ genes on the cell surface were detected by FACS, and CHO-S/IL-5 ⁇ /IL with high expression of IL-5 ⁇ and IL-5 ⁇ was selected.
  • -5 ⁇ cell line The IL-5 ⁇ and IL-5 ⁇ genes on the cell surface were detected by FACS, and CHO-S/
  • mice/group mice/group mice Use recombinant protein rhIL-5-his and Freund’s adjuvant CFA (Sigma, Lot#SLBQ1109V), IFA (Sigma, Lot#SLBJ2845V) two doses of 100g/50g/50g and 25g/12.5g/12.5g to immunize two doses respectively.
  • the specific immune response of IL-5 was determined by ELISA to detect serum titer, ligand receptor blocking test and TF-1 proliferation inhibition test. Select mice with better specific immune response, and after execution, take spleen cells and fuse with myeloma cells.
  • the initial screening was performed with an ELISA binding experiment against human IL-5. After transferring the hybridoma cells to a 24-well plate, use ELISA binding experiments for human, cynomolgus monkey, and mouse IL-5, ELISA-based receptor blocking experiments for IL-5 and TF-1 proliferation inhibition experiments , And re-screen the supernatant. After the selected positive clones undergo two rounds of subcloning, hybridoma clones are obtained, used for antibody production, and purified by affinity chromatography.
  • the purified antibodies were subjected to SEC-HPLC, endotoxin content detection, biacore determination of affinity to various IL-5, FACS-based receptor blocking experiment for IL-5, and TF-1 proliferation inhibition experiment.
  • the adhesion test of acidic granulocytes, as well as the efficacy evaluation on the mouse asthma model and the guinea pig neutralization model, the monoclonal hybridoma cell lines mAb1705, mAb1706, mAb1780, mAb1773 and mAb1779 with good activity in vivo and in vitro were screened.
  • the process of cloning sequences from positive hybridomas is as follows: collect logarithmic growth phase hybridoma cells, extract RNA with Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions, and use PrimeScript TM Reverse Transcriptase kit to reverse transcription ( Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced.
  • amino acid sequences corresponding to the DNA sequences of the heavy chain and light chain variable regions of mAb1705, mAb1706, mAb1780, mAb1773, and mAb1779 were obtained (VH/VL CDR amino acid residues were determined and annotated by the Kabat numbering system).
  • Example 4 Purification of IL-5 related recombinant protein, and purification of hybridoma antibodies and recombinant antibodies
  • the collected eluate was further purified by ion exchange (Hiload 16/600 superdex 200 column). Equilibrate about 2 column volumes with PBS to ensure pH 7.4. Concentrate the elution buffer containing the target protein and load the sample, collect the sample, and identify it as correct by SDS-PAGE and LC-MS.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, the hybridoma expression supernatant was purified by a Protein G column, and the Fc fusion protein expression supernatant was purified by a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the target protein was eluted with 100mM acetic acid pH3.0, and neutralized with 1M Tris-HCl, pH8.0.
  • the eluted sample is appropriately concentrated and further purified by PBS-equilibrated gel chromatography Superdex200 (GE), and the depolymerized peak is collected and divided into equipment for use.
  • the humanization of the murine anti-human IL-5 monoclonal antibody is carried out according to the methods published in many documents in the field.
  • the constant region of the murine antibody is replaced with a human constant region, and the CDR of the murine antibody is grafted onto the human template with the highest FR homology, and the amino acids in the FR region are backmutated.
  • the heavy chain and light chain variable region germlines with high amino acid sequence identity with mAb-1705, mAb-1706, mAb1780, mAb1773 and mAb1779 antibodies were selected respectively
  • the CDRs of the murine antibody were transplanted into the corresponding human templates to form the variable regions in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acid residues are identified and annotated by the Kabat numbering system.
  • VL light chain variable region
  • VH heavy chain variable region
  • the VH of h1705 selects IGHV3-23*04 as the template, and the VL selects IGKV1-12*01 as the template.
  • the CDR of mAb1705 is transplanted to the human template, and the embedded residues are found through the software to directly interact with the CDR region. And back mutate the residues of, to obtain different light chain and heavy chain variable regions of the derivatized antibody, as shown in Table 3.
  • Grafted represents the sequence of mouse antibody CDR implanted into human germline FR region.
  • A43S means that the amino acid sequence is numbered according to the natural sequence, and the grafted position 43 A is mutated back to S.
  • This table shows the sequence of various mutation combinations.
  • the humanized murine antibody h1705-007 contains two mutants of the light chain h1705_VL.1A and the heavy chain h1705_VH.1A, and others can be deduced by analogy.
  • variable region of humanized antibody No. h1705 is as follows:
  • the light chain variable region and the light chain constant region sequence are combined to form a light chain sequence, and each heavy chain variable region and the heavy chain constant region are combined to form a heavy chain sequence.
  • An exemplary humanized antibody constant region sequence is shown below:
  • the VH of h1706 selected IGHV3-23*04 as the template, and the VL selected IGKV1-12*01 as the template.
  • the CDR region of the murine antibody mab1706 was transplanted to the selected humanized template, and FR amino acids were backmutated to obtain The humanized antibody light chain and heavy chain variable regions are shown in Table 5.
  • Grafted represents mouse antibody CDR implanted into human germline FR region.
  • A43S means that the amino acid sequence is numbered according to the natural sequence, and the 43th A grafted is mutated back to S.
  • the designed humanized molecules were combined into different antibodies in the following table, as shown in Table 6.
  • variable region of the humanized antibody No. h1706 is as follows:
  • the light chain variable region and the light chain constant region sequence are combined to form a light chain sequence.
  • Each heavy chain variable region and heavy chain constant region are combined to form a heavy chain sequence.
  • the VH of h1780 selected IGHV1-2*02 as the template, and the VL selected IGKV3-11*01 as the template.
  • the CDR region of the mouse antibody mAb1780 was transplanted to the selected humanized template, and the FR region amino acids were multiple-mutated ,
  • the humanized antibody light chain and heavy chain variable regions are obtained, as shown in Table 7.
  • Grafted represents mouse antibody CDR implanted into human germline FR region.
  • E1D represents the numbering according to the natural sequence of the amino acid sequence, and the grafted position 1 E is mutated back to D.
  • variable region of the humanized antibody h1780 is as follows:
  • the light chain variable region and the light chain constant region sequence are combined to form a light chain sequence.
  • Each heavy chain variable region and heavy chain constant region are combined to form a heavy chain sequence.
  • the CDR region of the murine antibody mab1773 was transplanted to the selected humanized template, and back mutation was performed to obtain the human source.
  • the light chain and heavy chain variable regions of the modified antibody are shown in Table 9.
  • mutation of N in HCDR2 (RIDPANGDTKHGPKFQG) of h1773 to V forms the heavy chain variable region of the HCDR2 mutation (the sequence of the mutated HCDR2 is SEQ ID NO: 82: RIDPAVGDTKHGPKFQG).
  • Grafted represents mouse antibody CDR implanted into human germline FR region.
  • M4L means that the amino acid sequence is numbered according to the natural sequence, and the M of the grafted position 4 is mutated back to L.
  • variable region of humanized antibody No. h1773 is as follows:
  • the light chain variable region and the light chain constant region sequence shown in SEQ ID NO:53 are combined to form the final complete heavy and light chain sequence.
  • Each heavy chain variable region is combined with the heavy chain constant region shown in SEQ ID NO: 52 to form the final heavy chain sequence.
  • the VH of h1779 selected IGHV1-2*02 as the template, and the VL selected IGKV1-33*01 as the template.
  • the CDR region of the mouse antibody h1779 was transplanted to the selected humanized template, and back mutation was performed to obtain the human source.
  • the light chain and heavy chain variable regions of the modified antibody are shown in Table 11.
  • Grafted represents mouse antibody CDR implanted into human germline FR region.
  • A43S means that the amino acid sequence is numbered according to the natural sequence, and the 43th A grafted is mutated back to S.
  • variable region of the humanized antibody No. h1779 is as follows:
  • the light chain variable region is combined with the light chain constant region sequence shown in SEQ ID NO:53 to form a light chain sequence.
  • Each heavy chain variable region is combined with the heavy chain constant region shown in SEQ ID NO: 52 to form a heavy chain sequence.
  • This disclosure uses the antibody Hu39D10 against IL5 in WO2012083370A1 as a positive control, and its heavy chain and light chain sequences are shown in SEQ ID NO: 80 and SEQ ID NO: 81, respectively.
  • the positive antibody molecules obtained from hybridoma screening are sequenced to obtain the variable region coding gene sequence.
  • Design the head and tail primers based on the sequence obtained by sequencing, use the sequencing gene as a template, construct each antibody VH/VK gene fragment by PCR, and then combine the expression vector pHr (with signal peptide and hIgG1/hkappa constant region gene (CH1-Fc/CL) fragment)
  • pHr with signal peptide and hIgG1/hkappa constant region gene (CH1-Fc/CL) fragment
  • the recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr was constructed to obtain five chimeric antibodies of Ch1705, Ch1706, Ch1780, Ch1773 and Ch1779.
  • Humanized antibody sequence after codon optimization, the coding gene sequence of human codon preference is obtained, primer PCR is designed to construct each antibody VH/VK gene fragment, and then the expression vector pHr (with signal peptide and hIgG1/hkappa constant region gene ( CH1-Fc/CL) fragment) undergo homologous recombination to construct a humanized antibody full-length expression plasmid VH-CH1-Fc-pHr/VL-CL-pHr.
  • Plasmids expressing antibody light and heavy chains were transfected into HEK293E cells. After 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with a protein A column. Rinse the column with PBS until the A280 reading drops to baseline. Elute the target protein with pH3.0-pH3.5 acidic eluent, and neutralize with 1M Tris-HCl, pH8.0-9.0. After the eluted sample is properly concentrated, it is further purified by PBS-balanced gel chromatography Superdex200 (GE) to remove aggregates, collect monomer peaks, and divide them for use.
  • PBS-balanced gel chromatography Superdex200 GE
  • test methods are used to verify the performance and beneficial effects of the antibodies of the present disclosure.
  • Biacore measures the binding of murine IL-5 antibody to IL-5 of different species
  • a Biacore T200 (GE) instrument was used to determine the affinity between the mouse IL-5 antibody to be tested and human IL-5.
  • Biacore measures the affinity of IL-5 humanized antibody to IL-5 of different species
  • a Biacore T200 (GE) instrument was used to determine the affinity between the humanized IL-5 antibody to be tested and human IL-5.
  • the protein A biosensing chip was used to affinity capture the molecule to be tested, and then the antigen (prepared in Example 1) was flowed on the surface of the chip, and the reaction signal was detected in real time with the Biacore T200 instrument to obtain the binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with glycine-hydrochloric acid regeneration solution (pH 1.5). Using BIAevaluation version 4.1, GE software fits the data with a (1:1) Langmuir model to obtain the affinity value, as shown in Table 14.
  • Test Example 3 ELISA-based murine IL-5 antibody blocking IL-5 binding to IL-5 ⁇ receptor experiment
  • IL-5 5 ⁇ g/ml in PBS was coated on an ELISA plate and incubated at 37°C for 1 hour. After discarding the liquid, add 200 ⁇ l/well of 5% skim milk blocking solution diluted with PBS, and incubate for 2.5 hours in a 37° C. incubator for blocking.
  • PBST buffer pH 7.4 PBS containing 0.05% Tween-20
  • 25 ⁇ l of 10 ⁇ g/labeled with a biotin labeling kit Dongren Chemical, LK03
  • ml of IL-5R ⁇ in 1% BSA
  • 25 ⁇ l of serially diluted antibody The antibody and IL-5R ⁇ compete to bind to IL-5, and incubate at 37°C for 1 hour.
  • Test Example 4 FACS-based IL-5 antibody blocking IL-5 binding to IL-5 receptor experiment
  • IL-5 antibody screened can block the IL-5 receptor on the cell surface
  • a CHOS recombinant cell line that highly expresses the two receptors of IL-5R ⁇ / ⁇ was constructed. This experiment identified that IL-5 antibodies can block the binding of IL-5 to the recombinant IL-5 ⁇ / ⁇ receptors on the surface of the CHOS cell line.
  • CD-CHO containing 100ng/ml G418, 25ng/ml zeozin to culture CHO-S-IL-5R ⁇ and ⁇ .
  • concentration should not exceed 3 ⁇ 10 6 cells/ml.
  • Centrifuge the IL-5R ⁇ / ⁇ -CHOS cells in good condition 1000rpm, 5min
  • wash them with 10% FBS in PBS and count them.
  • IL-5 can induce the proliferation of TF-1 cells, and IL-5 antibody can prevent IL-5 from stimulating proliferation of TF-1 cells.
  • TF-1 cells ATCC, CRL-2003
  • 10% FBS 10% FBS
  • 2ng/mL rhGM-CSF LinkBio, Catalog No. 96-AF-300-03-20
  • RPMI1640 RPMI1640
  • the cells in the logarithmic growth phase were washed three times with PBS and centrifuged at 800 rpm for 5 min, and the cell density was adjusted to 6000 cells/well/90 ⁇ l with RPMI1640 (FBS: 2%, recombinant human IL-5: 10ng/ml), and added After 10 ⁇ l of the antibody to be tested is cultured in 96 wells for 3 days, 30 ⁇ l of cell titers are added to the test, and the IC50 is calculated based on the reading.
  • the test results are shown in Table 17 below.
  • IL-5 humanized antibody inhibits IL-5 induced TF1 cell proliferation test results
  • antibody IC50(nM) antibody IC50(nM) Hu39D10 0.30 h1706-003 0.31 h1705-004 0.30 h1706-004 0.30 h1705-005 0.30 h1706-006 0.34 h1705-007 0.25 h1706-007 0.28 h1705-008 0.20 h1706-009 0.25
  • IL5 can induce the differentiation, maturation, migration and activation of eosinophils, causing inflammation of the respiratory tract and leading to asthma.
  • This experiment uses the principle that IL-5 cytokines can promote the adhesion and activation of eosinophils, collect and purify eosinophils from human peripheral whole blood to test the effect of IL-5 specific antibodies on IL-5.
  • the blocking effect of the pathway is to detect the blocking effect of IL-5 antibody on IL5-mediated eosinophil number adhesion in vitro.
  • human peripheral whole blood is diluted 5 times with PBS containing 2mM EDTA, and PercollTM (density gradient 1.088) is used to separate monocytes and granulocytes, carefully aspirate the red blood cell layer containing granulocytes, and use red blood cell lysis solution to remove red blood cells; To count the remaining cells, add the separation magnetic beads (Miltenyi Biotec, Catalog No. 130-045-701) with CD16 antibody in proportion, and pass through the magnetic bead column after incubating for 30 min. Negative selection collects the direct outflow cell subgroup, which is the main For eosinophils.
  • PercollTM density gradient 1.088
  • the isolated eosinophil count is added to a 96-well cell culture plate pre-coated with IgG antibody, about 1 ⁇ 10 4 cells per well, and human IL-5 (20ng/ml) and different concentrations of IL- 5 Antibody molecules (starting at 10 ⁇ g/ml, 3 times dilution, 10 concentration points); the cell culture plate is placed in a 37°C, 5% CO2 incubator and incubated for 1 hour, the culture plate is taken out, 0.3% CTAB is added to lyse the cells, and finally added The oxidase reaction substrate TMB develops the color reaction, and the microplate reader reads the OD450 absorption value.
  • Table 18 The results are as follows in Table 18:
  • IL-5 is one of Th2 cytokines.
  • Th2 and related cytokines including IL2 (R&D) ,202-IL-010/CF), IL4(R&D,204-IL-050/CF), IL-5(R&D,205-IL-025/CF), IFNgamma, IL6(R&D,7270-IL-025/ CF), IL9(R&D,209-IL-010/CF), IL10(R&D,217-IL-025/CF), IL13(R&D,213-ILB-025/CF), IL25(R&D,8134-IL- 025/CF), IL31 (R&D, 2824-IL-010/CF), and IL3 (203-IL-050/CF) and GMCSF (R&D, 215-GM-010/CF), and GMCSF (R&D, 215-GM-010/CF), IL3 (203-IL-050/CF) and
  • Test Example 8 Evaluation of the efficacy of IL-5 antibody in OVA-induced mouse asthma model
  • This test is based on airway inflammation and airway remodeling to evaluate the efficacy of IL-5 antibody in ovalbumin (OVA) aerosol-induced BALB/c mouse asthma model.
  • OVA ovalbumin
  • mice were randomly divided into 7 groups according to their body weight, each with 10 mice: normal control group (G1); model group (G2); 2 test antibodies h1705-008 (G3 and G4) and h1706-009 (G5 and G6) , Each test antibody 2 doses (10mpk and 2mpk) treatment group; and positive antibody Hu39D10 control group (G7, 10mpk).
  • G1 normal control group
  • G2 model group
  • G3 and G4 2 test antibodies h1705-008
  • h1706-009 G5 and G6
  • Hu39D10 control group G7, 10mpk
  • mice were sensitized by intraperitoneal injection of sensitizing solution.
  • the six groups of mice outside the first group were challenged by aerosol with OVA challenge solution for 30 minutes.
  • mice were intraperitoneally injected with different doses of different antibodies, once a day for three consecutive days. Prepare fresh test antibody before each injection, and complete the preparation within half an hour of administration.
  • the mice in the first group were treated with PBS for aerosol attack for 30 minutes as a normal control group.
  • Phosphate buffer was injected intraperitoneally 2 hours before the attack, once a day for three consecutive days.
  • the tracheal tube with a diameter of 1.2 mm is inserted into the trachea and fixed, and the lungs will be lavaged twice, each time 0.8 ml of phosphate buffer containing 1% BSA and 0.6mM EDTA. Record the recovered volume of lavage fluid.
  • the BALF was centrifuged at 300g at 4 degrees Celsius for 5 minutes, and the supernatant was retained for cytokine analysis. After centrifugation, the cells were resuspended in 1.5 ml of PBS (containing 1% BSA and 0.6 mM EDTA) for cell counting. The total number of cells in BALF was counted by hemocytometer and trypan blue staining experiment. After the cell smear, Wright staining solution was stained for one minute, followed by Giemsa staining for 7 minutes to distinguish eosinophils, neutrophils, macrophages and lymphocytes. Count under an optical microscope.
  • the antibody molecules h1705-008 and h1706-009 in the present disclosure can significantly improve lung function, and the mode of action is in a dose-dependent mode, while high doses (10mpk) of positive compounds cannot improve lung function (see Figure 3).
  • the two antibodies significantly reduced the level of eosinophils and the thickness of the mucosa, and showed a stronger ability to reduce eosinophils than the positive antibody at the same dose (10 mpk) (see Figures 4A and 4B).
  • Test Example 9 Guinea pig acute asthma model induced by exogenous human IL-5 to evaluate the in vivo efficacy of IL-5 antibody
  • Guinea pigs are divided into 9 groups, each with 8-10 animals: normal control group, model group, hu39D10 (1mg/kg) group, h1705-008 (1mg/kg) group, h1706-009 (1mg/kg) group, h1780 -017 (1mg/kg) group, h1773-007 (1mg/kg) group and h1779-014 (1mg/kg) group.
  • the guinea pigs of the model group and the administration group were injected with 100 ⁇ l of human IL5 (containing 5 ⁇ g of IL5 antigen) on the first day, respectively, and the normal control group was injected with PBS in the trachea.
  • the administration group was given 1 mg/kg of the above-mentioned IL5 monoclonal antibody by intraperitoneal injection 2 hours before challenge, the administration volume was 5ml/kg, the model group was given the corresponding IgG antibody, and the normal control group was given PBS solvent.
  • the cell concentration is adjusted to 5 ⁇ 10 6 /ml, drop 15 ⁇ l on a glass slide, dry and fix, perform HE staining, and count the total number of cells and cytokines under a 400X microscope. Number of eosinophils and calculate the percentage of eosinophils.
  • Fig. 5A and Fig. 5B indicating that all the 5 humanized antibodies of the present disclosure can significantly reduce the level of eosinophils in BALF.
  • the first step Take a certain amount of purified anti-IL-5 antibody solution, use a buffer without antibody (such as 30mM, pH5.5 acetic acid-sodium acetate buffer) for solvent replacement (preferably ultrafiltration), and ultrafiltration
  • a buffer without antibody such as 30mM, pH5.5 acetic acid-sodium acetate buffer
  • the membrane is replaced by at least 6 times the volume, and the protein is concentrated to about 120 mg/mL.
  • the product is filtered and tested for sterility by central control sampling. Pass the stock solution through a 0.22 ⁇ m PVDF filter element and collect the filtrate.
  • the second step adjust the filling volume to 1.2ml, fill the filtrate into a 2ml vial, stopper, sample and control the difference in filling volume at the beginning, middle, and end of filling.
  • the third step Turn on the capping machine, add aluminum caps, and perform capping.
  • Step 4 Visual inspection to confirm that the product has no defects such as inaccurate loading.
  • Printing and pasting labels for vials printing paper box labels, folding paper boxes, boxing, sticker box labels.
  • the buffer systems were 10mM acetic acid-sodium acetate (AA) pH 5.0, 5.5; 10mM succinic acid-sodium succinate (SA) pH 5.0, 5.5, 6.0; 10mM citrate-sodium citrate (CA) pH 5. 5. 6.0, 6.5; 10mM histidine-hydrochloride (His) pH 5.5, 6.0, 6.5; 10mM phosphate (PB) pH 6.0, 6.5. Each preparation is filtered, filled, stoppered, and capped. The samples were subjected to a forced degradation experiment, with appearance, SEC, and iCIEF as evaluation indicators.
  • the results are shown in Table 19.
  • the appearance data shows that the samples with shaking (300rpm, 25°C) and 40°C have different degrees of particles.
  • the overall appearance is better when the pH is lower.
  • the buffer system acetic acid-sodium acetate and succinic acid-sodium succinate Better;
  • SEC data shows that AA pH 5.5 group is slightly better at 40°C;
  • iCIEF data shows that AA pH 5.5, His pH 5.5, CA pH 6.5 groups are slightly better at 40°C; considering physical and chemical stability, AA is preferred pH 5.5.
  • N/A means no detection
  • D means day
  • T0 means day 0.
  • h1705-008 preparation with protein concentration of 100mg/ml, 10mM SA pH5.5, 70mg/ml sucrose, 0.4mg/ml PS20 or PS80.
  • Test case 13 DOE prescription design and screening
  • the DOE (Design of valent) experiment was designed with 10mM acetate (AA) buffer pH, protein concentration and Tween concentration as variables.
  • the factors and levels were pH5.0 ⁇ 5.8, PS80 concentration 0.2 ⁇ 0.6mg/mL, antibody
  • the h1705-008 concentration is 80 ⁇ 120mg/mL
  • a series of prescriptions are designed, and the prescriptions are shown in Table 22.
  • the samples are subjected to forced degradation at 40°C.
  • the appearance, SEC, non-reducing CE, and iCIEF are used as evaluation indicators. The results are shown in Table 23.
  • the formulation contained a protein concentration of 80-120mg/ml , 0.2-0.6mg/ml PS80, pH5.0-5.8.
  • the optimal prescription is protein concentration 100mg/ml, 0.4mg/ml PS80, pH5.5.
  • the h1705-008 formulation with protein concentration of 100mg/ml, 10mM AA pH5.5, 70mg/ml sucrose, 0.4mg/ml PS80 was prepared, and the samples were tested for stability at 4°C and 25°C. The results are shown in Table 24.
  • the results show that when the sugar concentration is 70-75mg/ml, the osmotic pressure is in the best isotonic range (290-310mosm); according to the osmotic pressure data of the 70mg/ml and 73mg/ml groups, when the sugar concentration is 72mg/ml, The osmotic pressure can reach the best value, about 300 mosm.
  • M means month
  • T means time
  • N/A means not determined.
  • anti-IL-5 antibody pharmaceutical formulations with other formulations, including but not limited to:
  • the experimental results show that the IL-5 antibody preparations of the above formulations have good stability and can be applied to the preparation of IL-5 antibody drugs.
  • the h1705-008 antibody preparation containing 72 mg/ml sucrose, 0.4 mg/ml polysorbate 80 and an anti-IL-5 antibody concentration of 100 mg/ml was prepared with 30 mM acetic acid-sodium acetate buffer at pH 5.5.
  • the antibody was filled into a 6 mL vial with 2.15 mL/bottle, put into a lyophilization box, and lyophilized.
  • the freeze-drying procedures are pre-freezing, primary drying and secondary drying. After the freeze-drying process, vacuum stoppering.
  • the reconstituted samples were compared before and after freeze-drying. The results show that the reconstituted solution can maintain the good performance of the liquid formulation.

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Abstract

本申请公开了包含抗IL-5抗体的药物组合物及其用途。具体而言，本申请公开了一种药物组合物，其包含在缓冲液中的IL-5抗体或其抗原结合片段。除此之外，该药物组合物还包含糖和非离子型表面活性剂。

Description

包含抗IL-5抗体的药物组合物及其用途 技术领域
本公开属于药物制剂领域，具体涉及一种包含抗IL-5抗体或其抗原结合片段的药物组合物，以及其作为IL-5相关疾病诊断剂和治疗药物的用途。
背景技术
这里的陈述仅是提供与本公开有关的背景信息，而不必然地构成现有技术。
白细胞介素-5(IL-5)是白细胞介素家族的重要成员之一，又称为T细胞替代因子(T cell replacing factor，TRF)、B细胞生长因子-II(B cell growth factor-II，BCGF-II)、IgA增强因子(IgA-enhancing factor，IgA-EF)、酸性粒细胞分化因子(eosinophil differentiation factor，EDF)，是一种主要由辅助性T细胞2(Th2)分泌的同二聚体糖蛋白。人的IL-5由134个氨基酸残基组成，包括由22个氨基酸组成的信号肽和2个糖基化位点。具有活性的IL-5呈现寡二聚体形式，两条肽链以二硫键连接，以反向平行的构型存在，而IL-5的单体则无生物学活性(Adv Im-munol.1994；57:145-90)。
嗜酸性粒细胞(eosinophil，EOS)与肺部多种炎性疾病包括与过敏性反应有关的变应性病症有关。哮喘是一种慢性呼吸道炎症疾病，全球约有3亿患者，发病率为10％。其发病机制与多种细胞因子有关，IL-5及其受体IL-5R在哮喘的发病过程中扮演着重要的角色。目前对哮喘治疗最有效的方法是通过鼻腔或口腔给予固醇类药物来抑制哮喘中一些关键介质(包括IL-5)的表达，以减轻肺部炎症。然而长期使用固醇类药物具有很多副作用。因此，有必要寻找新的治疗哮喘的药物靶点。已有研究证明，使用抗IL-5的抗体抑制IL-5与其受体结合，可显著降低嗜酸性粒细胞在肺内的聚集，降低血液、组织、痰液中的嗜酸性粒细胞水平，并可减少嗜酸性粒细胞所介导的炎症反应，提高肺功能，对严重的嗜酸性粒细胞哮喘和复发性哮喘均具有很好的疗效(Drugs.2017May；77(7):777-784)。
抗体药物其分子量大，结构复杂，在生产、运输和储存的过程中经常会出现变性、聚集、污染和粒子形成的问题。为了保持抗体有效，抗体必须在生产、纯化、运输和储存期间维持其生物活性。目前，已开发出新的生产和纯化技术用于生产大量的高度纯化的单克隆抗体。然而，如何使这些抗体稳定以便运输和储存，并且提供适合于给药的剂型的抗体，一直是一个难题。
现有上市的抗IL-5抗体只有GSK公司的美泊利单抗(mepolizumab)和Teva Pharma公司的瑞利珠单抗(reslizumab)，相关专利有WO2018119016、WO2017033121、WO2014141149、WO2016040007、WO2015095539、WO2012138958、WO9535375等。
发明内容
本公开提供一种药物组合物，其包含IL-5抗体或其抗原结合片段、缓冲剂以及表面活性剂，所述缓冲剂选自醋酸-醋酸钠、琥珀酸-琥珀酸钠、组氨酸-盐酸盐和枸橼酸-枸橼酸钠缓冲剂中的任一种，优选为醋酸-醋酸钠或琥珀酸-琥珀酸钠缓冲剂；其中所述的抗IL-5抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
(i)重链可变区包含分别如SEQ ID NO：16、17、18氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：19、20、21氨基酸序列所示的LCDR1、LCDR2和LCDR3；
(ii)重链可变区包含分别如SEQ ID NO：22、23、24氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：25、26、27氨基酸序列所示的LCDR1、LCDR2和LCDR3；
(iii)重链可变区包含分别如SEQ ID NO：28、29、30氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：31、32、33氨基酸序列所示的LCDR1、LCDR2和LCDR3；
(iv)重链可变区包含分别如SEQ ID NO：34、35、36氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：37、38、39氨基酸序列所示的LCDR1、LCDR2和LCDR3；
(v)重链可变区包含分别如SEQ ID NO：40、41、42氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：43、44、45氨基酸序列所示的LCDR1、LCDR2和LCDR3；或
(vi)重链可变区包含分别如SEQ ID NO：34、82、36氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：37、38、39氨基酸序列所示的LCDR1、LCDR2和LCDR3。
在可选的实施方案中，药物组合物中所述缓冲剂的pH值为约5.0至约6.5，优选为约5.5至约6.5，优选为约6.0到约6.5，优选为约5.0至约6.0，优选为约5.5至约6.0，优选为约5.0至约5.5，优选为约5.0至约5.8，优选为大约5.2至约5.8；所述缓冲剂pH值的非限制的实施例包括约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0、约6.5，最优选为约5.5。
在可选的实施方案中，药物组合物中所述缓冲剂浓度为大约10mM至约40mM，大约10mM至约30mM，优选为约15mM至约30mM，优选为约20mM至约30mM，优选为约25mM至约30mM，优选为约10mM至约25mM，优选为约15mM至约25mM，优选为约20mM至约25mM，优选为约10mM至约15mM；所述缓冲剂浓度的非限制性实施例包括为约10mM、约12mM、约14mM、约16mM、约18mM、约20mM、约22mM、约24mM、约26mM、约28mM、约30mM、约32mM、约 34mM，最优选为约30mM。
在可选的实施方案中，药物组合物中所述抗IL-5抗体或其抗原结合片段的浓度为大约1mg/ml至约120mg/ml，优选为约1mg/ml至约100mg/ml，优选为约10mg/ml至约120mg/ml，优选为约20mg/ml至约120mg/ml，优选为约30mg/ml至约120mg/ml，优选为约40mg/ml至约120mg/ml，优选为约50mg/ml至约120mg/ml，优选为约60mg/ml至约120mg/ml，优选为约70mg/ml至约120mg/ml，优选为约80mg/ml至约120mg/ml，优选为约90mg/ml至约120mg/ml，优选为约100mg/ml至约120mg/ml，优选为约110mg/ml至约120mg/ml，优选为约20mg/ml至约100mg/ml，优选为约30mg/ml至约100mg/ml，优选为约40mg/ml至约100mg/ml，优选为约50mg/ml至约100mg/ml，优选为约60mg/ml至约100mg/ml，优选为约70mg/ml至约100mg/ml，优选为约80mg/ml至约100mg/ml，优选为约90mg/ml至约100mg/ml；作为非限制性实施例，所述抗IL-5抗体或其抗原结合片段浓度为约80mg/ml、约85mg/ml、约90mg/ml、约91mg/ml、约92mg/ml、约93mg/ml、约94mg/ml、约95mg/ml、约96mg/ml、约97mg/ml、约98mg/ml、约99mg/ml、约100mg/ml、约101mg/ml、约102mg/ml、约103mg/ml、约104mg/ml、约105mg/ml、约106mg/ml、约107mg/ml、约108mg/ml、约109mg/ml、约110mg/m、约115mg/m、约120mg/m，最优选为约100mg/ml。
在可选实施方案中，药物组合物中包含的表面活性剂可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-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等。优选的表面活性剂是聚山梨醇醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯80。
在可选的实施方案中，药物组合物中聚山梨酯80的浓度为约0.05mg/ml至约0.6mg/ml，优选为约0.1mg/ml至约0.6mg/ml，优选为约0.2mg/ml至约0.6mg/ml，优选为约0.3mg/ml至约0.6mg/ml，优选为约0.4mg/ml至约0.6mg/ml，优选为约0.5mg/ml至约0.6mg/ml，优选为约为0.2mg/ml至约0.5mg/ml，优选为约0.3mg/ml至约0.5mg/ml，优选为约0.4mg/ml至约0.5mg/ml，优选为约0.3mg/ml至约0.4mg/ml，作为非限制性实施例，所述药物组合物中表面活性剂的浓度约为0.2mg/ml、约0.3mg/ml、约0.4mg/ml、约0.45mg/ml、约0.5mg/ml、约0.55mg/ml、约0.6mg/ml，最优选为约0.4mg/ml。
进一步地，在可选的实施方案中，药物组合物还包括辅料，其中所述的辅料 选自稳定剂。
在可选的实施方案中，其中所述的稳定剂选自糖或氨基酸；其中所述的糖可选自蔗糖、海藻糖、甘露醇或山梨醇，优选为蔗糖。其中所述的氨基酸选自甘氨酸、甲硫氨酸和脯氨酸。
在可选的实施方案中其中所述的糖的浓度为约50mg/ml至约80mg/ml，优选为约60mg/ml至约80mg/ml，优选为约70mg/ml至约80mg/ml，优选为约75mg/ml至约80mg/ml，优选为约70mg/ml-约75mg/ml；作为非限制性实施例，药物组合物中稳定剂的浓度为包括约70mg/ml、约71mg/ml、约72mg/ml、约73mg/ml、约74mg/ml、约75mg/ml、约76mg/ml、约77mg/ml、约78mg/ml、约79mg/ml、约80mg/ml，更优选为约72mg/ml。
在可选实施方案中，其中所述的氨基酸的浓度为约8mg/ml。
在可选的实施方案中，所述药物组合物包含：
(a)约1mg/ml-约120mg/ml的抗IL-5抗体或其抗原结合片段；(b)约10mM-约30mM的醋酸-醋酸钠缓冲剂，pH为约5.0-6.5；和(c)约0.1mg/ml-约0.6mg/ml聚山梨醇酯80。
在可选实施方案中，所述药物组合物包含：
(a)约80mg/ml-约100mg/ml的抗IL-5抗体或其抗原结合片段；(b)约10mM-约30mM的醋酸-醋酸钠缓冲剂，pH为约5.0-约6.0和(c)约0.1mg/ml-约0.4mg/ml聚山梨醇酯80。
在可选的实施方案中，所述药物组合物包含：
(d)约80mg/ml-约120mg/ml的IL-5抗体或其抗原结合片段；(e)约10mM-约30mM的醋酸-醋酸钠缓冲剂，pH为约5.0-约5.8；(f)约0.2mg/ml-约0.6mg/ml聚山梨醇酯80；和(g)约70mg/ml-约75mg/ml蔗糖；优选地，所述药物组合物优选包含：
(h)约100mg/ml的IL-5抗体或其抗原结合片段，(i)约30mM的醋酸-醋酸钠缓冲剂，pH为约5.5，(j)约0.4mg/ml的聚山梨醇酯80和(k)约72mg/ml的蔗糖。
在一些优选的实施方式中，本公开所述药物组合中的抗IL-5抗体或其抗原结合片段为鼠源抗体、嵌合抗体或人源化抗体。
在可选的实施方案中，所述药物组合物中的抗IL-5人源化抗体含有SEQ ID NO：49、57、63、69或75所示的重链可变区或其变体；所述变体是在SEQ ID NO：49、57、63、69或75所示的重链可变区序列上具有1-10个氨基酸回复突变。
在可选的实施方案中，所述的变体选自如下任一所示的变体：
(i)在SEQ ID NO：49所示的重链可变区上具有选自S49T，V93T和K98S中的一个或多个的氨基酸回复突变；
(ii)在SEQ ID NO：57所示的重链可变区上具有选自S49T,V93T和K98T中的一个或多个的氨基酸回复突变；
(iii)在SEQ ID NO：63所示的重链可变区上具有选自R38K，M48I，R67K，V68A，M70L，R72V，T74K和L83F中的一个或多个的氨基酸回复突变；
(iv)在SEQ ID NO：69所示的重链可变区上具有选自F29I，R38K，V48I，R72A，和T97F中的一个或多个的氨基酸回复突变和/或CDR中N55V突变；或
(v)在SEQ ID NO：75所示的重链可变区上具有选自R38K，M48I，R67K，V68A，R72A，T74K，M81L，L83F和D89E中的一个或多个的氨基酸回复突变。
在可选的实施方案中，所述药物组合物中的抗IL-5人源化抗体包含如SEQ ID NO：50或51所示的重链可变区；或
包含如SEQ ID NO：58或59所示的重链可变区；或
包含选自SEQ ID NO：64、65和66中任一个所示的重链可变区；或
包含如SEQ ID NO：70或71所示的重链可变区；或
包含选自SEQ ID NO：76、77、78和79中任一个所示的重链可变区。
在可选的实施方案中，所述药物组合物中的抗IL-5人源化抗体含有SEQ ID NO：46、54、60、67或72所示的轻链可变区或其变体；所述变体是在SEQ ID NO：46、54、60、67或72所示的轻链可变区上具有1-10个氨基酸回复突变。
在可选的实施方案中，其中所述的变体选自如下任一所示的变体：
(i)在SEQ ID NO：46所示的轻链可变区上具有选自A43S，L47V，G66R，T69S，F71Y和Y87F中的一个或多个的氨基酸回复突变；
(ii)在SEQ ID NO：54所示的轻链可变区上具有选自A43S，L47M，F71Y和Y87F中的一个或多个的氨基酸回复突变；
(iii)在SEQ ID NO：60所示的轻链可变区上具有选自E1D，I2T，I57V，V84T和Y86F或其组合的氨基酸回复突变；
(iv)在SEQ ID NO：67所示的轻链可变区上具有选自M4L，A42S，L45P和L46W中的一个或多个的氨基酸回复突变；或
(v)在SEQ ID NO：72所示的轻链可变区上具有选自A43S，I48V和F71Y中的一个或多个的氨基酸回复突变。
在可选的实施方案中，所述药物组合物中的抗IL-5人源化抗体包含如SEQ ID NO：47或48所示的轻链可变区；或
包含如SEQ ID NO：55或56所示的轻链可变区；或
包含如SEQ ID NO：61或62中所示的轻链可变区；或
包含SEQ ID NO：68所示的轻链可变区；或
包含如SEQ ID NO：73或74所示的轻链可变区。
在可选的实施方案中，所述药物组合物中的抗IL-5人源化抗体包含：
(i)如SEQ ID NO：49、50、51中任一所示或与其具有95％序列同一性的重链可变区或和如SEQ ID NO：46、47、48中任一所示或与其具有95％序列同一性的轻链可变区；
(ii)如SEQ ID NO：57、58、59中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：54、55、56中任一所示或与其具有95％序列同一性的轻链可变区；
(iii)如SEQ ID NO：63、64、65、66中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：60、61、62中任一所示或与其具有95％序列同一性的轻链可变区；
(iv)如SEQ ID NO：69、70、71中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：67、68任一所示或与其具有95％序列同一性的轻链可变区；或
(v)如SEQ ID NO：75、76、77、78、79中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：72、73、74中任一所示或与其具有95％序列同一性的轻链可变区；优选地，所述的抗IL-5人源化抗体包含：
(a)SEQ ID NO：51所示的重链可变区和SEQ ID NO：47所示的轻链可变区；
(b)SEQ ID NO：65所示的重链可变区和SEQ ID NO：62所示的轻链可变区；
(c)SEQ ID NO：58所示的重链可变区和SEQ ID NO：56所示的轻链可变区；
(d)SEQ ID NO：71所示的重链可变区和SEQ ID NO：68所示的轻链可变区；或
(e)SEQ ID NO：79所示的重链可变区和SEQ ID NO：73所示的轻链可变区。
上述具有至少95％序列同一性的氨基酸序列，优选地，其具有至少95％、96％、97％、98％、或99％序列一致性，更优选地，其具有97％、98％或99％以上，最优选的具有至少99％以上序列一致性，上述具有至少95％序列同一性的氨基酸序列包括通过一个或者多个氨基酸缺失、插入或替换突变获得。
在可选的实施方案中，所述药物组合物中的抗IL-5抗体包括人抗体恒定区，优选包含SEQ ID NO：52所示的人抗体重链恒定区和SEQ ID NO：53所示的人抗体轻链恒定区。
在可选的实施方案中，所述药物组合物中的抗IL-5抗体包含：
(i)SEQ ID NO：83所示的重链和SEQ ID NO：84所示的轻链；
(ii)SEQ ID NO：85所示的重链和SEQ ID NO：86所示的轻链；
(iii)SEQ ID NO：87所示的重链和SEQ ID NO：88所示的轻链；
(iv)SEQ ID NO：89所示的重链和SEQ ID NO：90所示的轻链；或
(v)SEQ ID NO：91所示的重链和SEQ ID NO：92所示的轻链。
在可选的实施方案中，所述药物组合物中的抗IL-5抗体是一种与上述的抗IL-5 抗体或其抗原结合片段竞争结合IL-5的单克隆抗体或其抗原结合片段。
在一种优选的实施方式中，本公开药物组合物中所述的抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
本公开还提供制备前述药物组合物的方法，其中包括将抗IL-5抗体原液经缓冲液置换的步骤。在可选的实施方案中，所述缓冲液优选醋酸-醋酸钠缓冲剂。所述缓冲剂的pH值为约5.0到约6.5，优选约5.5至约6.5，优选约6.0至约6.5，优选约5.0至约6.0，优选约5.5至约6.0，优选约5.0至约5.5，优选为约5.2至约5.8，所述缓冲剂pH值的非限制的实施例包括约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0、约6.5，最优选约5.5。在可选的实施方案中，药物组合物中所述缓冲剂浓度为约10mM至约30mM，优选为约15mM至约30mM，优选为约20mM至约30mM，优选为约25mM至约30mM，优选为约5mM至约25mM，优选为约10mM至约25mM，优选为约15mM至约25mM，优选为约20mM至约25mM，优选为约5mM至约20mM，优选为约10mM至约15mM；所述缓冲剂浓度的非限制性实施例包括约10mM、约12mM、约14mM、约16mM、约18mM、约20mM、约22mM、约24mM、约26mM、约28mM、约30mM，最优选为约30mM。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠，pH5.5和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠，pH5.5和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH5.0和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH5.5和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH6.0和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH5.0和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH5.5和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH6.0和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH6.5和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体 h1705-008，10mM枸橼酸-枸橼酸钠，pH5.5和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH6.0和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH6.5和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH5.5和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH6.0和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH6.5和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM枸橼酸-枸橼酸钠，pH5.5和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM组氨酸-盐酸，pH6.0和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM组氨酸-盐酸，pH6.5和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM组氨酸-盐酸，pH5.5和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM组氨酸-盐酸，pH6.0和0.05mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH5.0和0.1mg/mL聚山梨醇酯20。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠，pH5.0和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，50mg/mL蔗糖和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，50mg/mL海藻糖和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，50mg/mL甘露醇和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，50mg/mL山梨醇和0.1mg/mL聚山梨 醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，8mg/mL甘氨酸和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，8mg/mL甲硫氨酸和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.0，8mg/mL脯氨酸和0.1mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM琥珀酸-琥珀酸钠pH5.5，70mg/mL蔗糖和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：80mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.8和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.4和0.6mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：80mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.4和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：80mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.0和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.4和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：80mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.8和0.6mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：120mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.0和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：80mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.0和0.6mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：120mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.8和0.6mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：120mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.4和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.4和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：120mg/ml的抗IL-5抗体 h1705-008，10mM醋酸-醋酸钠pH5.0和0.6mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：120mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.8和0.2mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.0和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.8和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，10mM醋酸-醋酸钠pH5.5,70mg/ml蔗糖和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，20mM醋酸-醋酸钠pH5.5,70mg/ml蔗糖和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，30mM醋酸-醋酸钠pH5.5,70mg/ml蔗糖和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，30mM醋酸-醋酸钠pH5.5,73mg/ml蔗糖和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，所述的药物组合物包含：100mg/ml的抗IL-5抗体h1705-008，30mM醋酸-醋酸钠pH5.5，75mg/ml蔗糖和0.4mg/mL聚山梨醇酯80。
在一些实施方案中，本公开中的药物组合物于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在25℃可稳定存在至少3个月，至少6个月。
本公开还提供一种制备包含抗IL-5抗体的冻干制剂的方法，其中包括将前述药物组合物经冷冻干燥的步骤。
在可选的实施方案中，制备包含抗IL-5抗体的冻干制剂的方法中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。通过冷冻制剂和随后在适于一次干燥的温度使水升华，进行冷冻干燥。在此条件下，产物温度低于制剂的低共熔点或分解温度。在通常约50-250毫托范围的合适压力下，通常，一次干燥的存放温度范围为约-30至25℃(假设产物在一次干燥过程中保持冷冻)。制剂、容纳样品的容器(例如，玻璃小瓶)的大小和类型以及液体的体积决定了干燥所需的时间，所述时间的范围可为几小时至几天(例如40-60小时)。二次干燥阶段可在约0-40℃进行，这主要取决于容器的类型和大小以及采用的蛋白的类型。二次干燥时间由产物中的期望残余湿度水平决定，通常需要至少约5小时。通常，低压冻干的制剂的含水量小于约5％，优选小于约3％。压力可与在一次干燥步骤中应用的压力相同，优选的，二次干燥的压力低于一次干燥。冷冻干燥条件可以随制剂和小瓶大小而变化。
本公开还提供一种经前述制备包含抗IL-5抗体的冻干制剂的方法制备的含IL-5抗体的冻干制剂。
在一些实施方案中，该冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本公开还提供制备含有抗IL-5抗体的冻干制剂的复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液包括但不限于注射用水、生理盐水或葡萄糖溶液。
本公开还提供一种经前述含有抗IL-5抗体的冻干制剂的复溶溶液的方法制备的含有IL-5抗体的冻干制剂的复溶溶液。
本公开进一步提供一种制品或试剂盒，其包含装有本文所述任何稳定的药物组合物的容器。在一些实施方案中，该容器为中性硼硅玻璃管制注射剂瓶。
本公开还提供一种制品，其包括容器，该容器中装有前述的药物组合物或冻干制剂或冻干制剂的复溶溶液。
本公开还提供一种治疗IL-5介导的疾病的方法，所述方法包括向此有需要的受试者施用治疗有效量的如前所述的药物组合物或冻干制剂或复溶溶液或制品，其中所述的IL-5介导的疾病优选哮喘、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位或类固醇依赖性嗜酸细胞性支气管炎。
本公开还提供前述的药物组合物或冻干制剂或冻干制剂的复溶溶液或制品在制备治疗IL-5介导的疾病的药物中的用途，其中所述的IL-5介导的疾病优选哮喘、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位或类固醇依赖性嗜酸细胞性支气管炎。
本公开还提供用作治疗药物的前述药物组合物或冻干制剂或冻干制剂的复溶溶液或制品，其中所述的药物用于治疗IL-5介导的疾病，其中所述的IL-5介导的疾病优选哮喘、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾 丝虫皮炎、子宫内膜异位或类固醇依赖性嗜酸细胞性支气管炎。
如本领域技术人员所熟知的，本公开中所述各个实施方案的一项、一些或所有特性可以进一步组合以形成本公开的其它实施方案。本公开的以上实施方案和通过组合得到的其他实施方案通过下面的详述进一步说明。
附图说明
图1显示抗IL-5抗体阻断IL-5结合IL-5受体的FACS实验结果；
图2显示抗IL-5抗体与Th2细胞因子的结合特异性检测结果；
图3显示抗IL-5抗体增强呼吸间歇值(Penh)水平。G1：正常对照组(PBS)；G2：模型组(IgG)；G3：h1705-008抗体10mpk组；G4：h1705-008抗体2mpk组；G5：h1706-009抗体10mpk组；G6：h1706-009抗体2mpk组；G7：Hu39D10 10mpk组；其中，*p<0.05，**<0.01(通过ANOVA/Bonferroni与G2组进行比较)；
图4A显示哮喘小鼠肺部BALF嗜酸性粒细胞水平；图4B显示哮喘小鼠气管粘膜厚度评分。G1：正常对照组；G2：模型组；G3：h1705-008抗体10mpk组；G4：h1705-008抗体2mpk组；G5：h1706-009抗体10mpk组；G6：h1706-009抗体2mpk组；G7：Hu39D10 10mpk组；图4C显示哮喘小鼠肺部BALF嗜酸性粒细胞百分比；
图5A和图5B显示IL5单抗降低BALF中嗜酸性粒细胞水平的能力。
具体实施方式
术语
为了更容易理解本公开，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公开所属领域的一般技术人员通常理解的含义。
“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、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸盐缓冲剂”是包含组氨酸根离子的缓冲剂。组氨酸盐缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂，优选组氨酸-醋酸盐缓冲剂或组氨酸-盐酸盐缓冲剂，组氨酸-醋酸盐缓冲剂是组氨酸与醋酸配制而成，组氨酸盐缓冲剂是组氨酸与盐酸配制而成。
“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、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。
“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、醋酸组氨酸盐、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。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持抗体活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本文中，“药物组合物”和“制剂”在本文中可互换使用。
本公开的“糖”包含常规(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。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，木糖醇l，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。优选的糖为非还原性二糖，更优选为蔗糖。
本公开中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“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。
本公开的冷冻干燥包括预冻，一次干燥，二次干燥。预冻目的是冷冻产品，获得结晶固体。预冻温度和预冻速度是两个重要工艺参数，本公开中将预冻温度设定为-45℃，预冻速度设定为1℃/min。一次干燥也称为主干燥，是样品冷冻干燥的主要阶段。目的是移除产品里冰的同时，保持产品形状，最小程度的减少对产品的破坏。若一次干燥的温度和真空度选择不当，会导致制品的塌陷。较高的温度和真空度均会加快冻干效率，但同时也会增加制品塌陷的风险。本发明所述一次干燥的温度可为本领域常规的温度，例如-30℃-0℃。二次干燥也称为解析干燥，是通过抽极限真空(0.01mbar)和升高温度(20-40℃)除去制品中结合水的主要步骤。由于大多数生物制品对温度比较敏感，所以二次干燥温度选择在温度范围的低点，即25℃。冷冻干燥的时间与冷冻机，冻干制剂剂量，冻干药剂的容器有关。这种时间的调整是在本领域技术人员所熟知的。
本文所用术语“约”、“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或 者，“约”或“基本上包含”可意味着至多±20％的范围，例如，约5.5的pH意指pH5.5±1.1。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该为在该具体值的可接受误差范围内。
本公开所述的药物组合物能够达到一种稳定的效果：其中的抗体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。
稳定的药物抗体制剂是在下述情况下没有观察到物理、和/或化学、和/或生物学活性显著变化的制剂：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、6个月在内的时段后表现出期望的特征。稳定性的典型的制剂：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生降解。通过视觉分析，药物抗体制剂是淡黄色近无色澄明液体或者无色，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的减少。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)测定，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“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。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体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，那么所述抗体在药物制剂中“保留它的生物活性”。抗体的生物活性可以例如通过抗原结合测定来确定。
本公开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。
本公开所述的“抗体”指免疫球蛋白，完整抗体是由两条相同的重链和两条 相同的轻链通过链间二硫键连接而成的四肽链结构。
在本公开中，本公开所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本公开中，本公开所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
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(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)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(LCVR，VL)和重链可变区(HCVR，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。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
本公开中所述的“抗体的抗原结合片段”或“功能片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与抗体结合的Fv片段scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。
本公开的术语“抗原结合位点”指抗原上连续或不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人IL-5的单克隆抗体。制备时用IL-5抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的IL-5嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的IL-5嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC (antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
本公开中所述的“ADCC”，即抗体依赖的细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity)，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；IgG2/4嵌合体，IgG4的F234A/L235A突变。
本公开中所述的突变序列中的“突变”包括但不限于“回复突变”、“保守修饰”或“保守置换或取代”。本公开中所述的“保守修饰”或“保守置换或取代”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))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
本公开所述的“突变序列”是指对本公开的核苷酸序列和/或氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本公开的核苷酸序列和/或氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和/或氨基酸序列。本公开中所述的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
术语“与IL-5结合”，指能与IL-5，优选人IL-5相互作用。
术语“抗IL-5抗体”与“IL-5抗体”可互换使用，均为与IL-5结合的抗体。
本公开中所述的融合蛋白是一种通过DNA重组，将得到的两个基因共表达的蛋白产物。重组IL-5胞外区Fc融合蛋白是通过DNA重组，把IL-5胞外区和人抗体Fc片段共表达的融合蛋白。所述的IL-5胞外区，是指IL-5蛋白表达在细胞膜以外的部分，序列见SEQ ID NO:1。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港实验室出版的《抗体实验技术指南》第5-8章和15章。小鼠可以用人IL-5或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人IL-5特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲剂的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链CDR区”意味着特定序列的抗体重链CDR区可以但不必须存在。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“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。“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，例如包含本公开的任一种结合化合物的组合物，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可测量的 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。尽管本公开的实施方案(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。
对与IL-5相关的疾病没有限制，只要它是与IL-5相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类IL-5然后阻遏或抑制嗜酸性粒细胞刺激作用而产生。因此，当处于适于治疗应用的制备物和制剂中时，本公开的药物组合物对这样一些人是非常有用的，他们患有变态和/或特应性反应，或与嗜酸性粒细胞有关的反应，例如但不限于，哮喘、哮喘恶化、哮喘恶性发作、慢性肺炎、过敏性鼻炎、常年过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位、类固醇依赖性嗜酸细胞性支气管炎等。在优选的实施方案中，这种治疗能抑制或减轻嗜酸性细胞的肺组织浸润。药物组合物给予的次数可以从每日三次到每6个月一次，给予的途径可以是静脉、皮下、肌肉、胃肠外或局部途径。
本公开制剂可用于治疗IL-5介导的疾病。例如，但不限于抑制或减轻IL-5介导的炎症反应和嗜酸性细胞在体内外的成熟、活化、脱颗粒或组织浸润；抑制IL-5引起的平滑肌的过度应激；降低IL-5在肺、气道或血液中的水平。优选地，本公开制剂可用于治疗IL-5介导的疾病，优选地，所述IL-5介导的疾病选自哮喘、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位或类固醇依赖性嗜酸细胞性支气管炎。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、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。
“置换”是指溶解抗体蛋白的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体蛋白的高盐或高渗溶剂体系置换，从而使抗体蛋白存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
实施例与测试例
以下结合实施例进一步描述本公开，但这些实施例并非是对本公开范围的限制。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如参照冷泉港实验室出版的《抗体技术实验手册》，《分子克隆手册》；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.IL-5抗原及检测用蛋白的制备
1.1 IL-5抗原设计及表达
编码带His标签的人IL-5，恒河猴IL-5，小鼠IL-5，大鼠IL-5，含人IgG1-Fc片段的人IL-5Rα受体胞外区融合蛋白序列克隆至phr载体上，构建成表达质粒，然后转染HEK293。待转染第6天，取样，4500rpm离心10min收集细胞上清，将含重组的IL-5和IL-5α受体蛋白上清利用镍柱进行纯化，重组的人IL-5-Fc融合蛋白利用Protein A亲和层析柱进行纯化。纯化的蛋白可用于后续实验中。具体抗原的蛋白序列如下：
带his标签的人IL-5氨基酸序列(rhIL-5-his)
Figure PCTCN2020081701-appb-000001
注释：斜体部分为His6-tag标记
                            SEQ ID NO：1
带his标签的食蟹猴IL-5氨基酸序列
Figure PCTCN2020081701-appb-000002
注释：斜体部分为His6-tag标记。
带his标签的小鼠IL-5氨基酸序列
Figure PCTCN2020081701-appb-000003
注释：斜体部分为His6-tag标记。
带his标签的大鼠IL-5氨基酸序列
Figure PCTCN2020081701-appb-000004
注释：斜体部分为His6-tag标记。
融合人Fc片段的人IL-5α受体氨基酸序列
Figure PCTCN2020081701-appb-000005
注释：斜体部分为人IgG1-Fc标签。
实施例2.重组IL-5α受体和IL-5α/β受体细胞系的构建和鉴定
为筛选有功能的抗体，本公开构建了表达IL-5α的CHO-S/IL-5α细胞株，以及同时表达IL-5α和IL-5β的CHO-S/IL-5α/IL-5β细胞株
具体为将人IL-5α全长基因(Q01344)克隆到哺乳动物细胞表达载体pTargeT上，将线性化质粒电转染至CHO-S细胞中，经G418筛选2周，再进行2次有限稀释。通过FACS检测细胞表面的IL-5α基因，挑选出IL-5α表达量高的CHO-S/IL-5α细胞株，在此基础上电转染线性化的pcDNA3.1-IL-5β，经G418和zeocin筛选2周，再进行2次有限稀释，通过FACS检测细胞表面的IL-5α和IL-5β基因，挑选出IL-5α和IL-5β表达量高的CHO-S/IL-5α/IL-5β细胞株。
实施例3.抗人IL-5鼠源单克隆抗体的制备
用重组蛋白rhIL-5-his和弗氏佐剂CFA(Sigma，Lot#SLBQ1109V)、IFA(Sigma，Lot#SLBJ2845V)高低两个剂量100g/50g/50g和25g/12.5g/12.5g分别免疫两组Balb/c(5只/组)小鼠，四组SJL(5只/组)小鼠。IL-5的特异性免疫反应由检测 血清效价的ELISA，配体受体阻断实验和TF-1增殖抑制实验来测定。选取有较好特异性免疫反应的小鼠，处死后，取脾细胞，与骨髓瘤细胞融合。
初次筛选用针对人IL-5的ELISA结合实验进行。当将杂交瘤细胞转移到24孔板后，用针对人、食蟹猴、小鼠IL-5的ELISA结合实验，基于ELISA的针对IL-5的受体阻断实验和TF-1增殖抑制实验，对其上清进行复筛。筛选出的阳性克隆经过两轮亚克隆后，得到杂交瘤克隆，用于抗体生产，并通过亲和层析方法纯化。
纯化后的抗体分别进行SEC-HPLC，内毒素含量检测，biacore测定对各种属IL-5的亲和力，基于FACS的针对IL-5的受体阻断实验，和TF-1增殖抑制实验，嗜酸性粒细胞的粘附实验，以及小鼠哮喘模型和豚鼠体内中和模型上的药效评价，筛选出体内外活性好的单克隆杂交瘤细胞株mAb1705、mAb1706、mAb1780、mAb1773和mAb1779。
从阳性杂交瘤中克隆序列过程如下：收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TM Reverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后测序。得到mAb1705和mAb1706，mAb1780，mAb1773，mAb1779的重链和轻链可变区DNA序列对应的氨基酸序列(VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释)。
mAb1705鼠源重链可变区序列
Figure PCTCN2020081701-appb-000006
mAb1705鼠源轻链可变区序列
Figure PCTCN2020081701-appb-000007
mAb1706鼠源重链可变区序列
Figure PCTCN2020081701-appb-000008
mAb1706鼠源轻链可变区序列
Figure PCTCN2020081701-appb-000009
Figure PCTCN2020081701-appb-000010
mAb1780鼠源重链可变区序列
Figure PCTCN2020081701-appb-000011
mAb1780鼠源轻链可变区序列
Figure PCTCN2020081701-appb-000012
mAb1773鼠源重链可变区序列
Figure PCTCN2020081701-appb-000013
mAb1773鼠源轻链可变区序列
Figure PCTCN2020081701-appb-000014
mAb1779鼠源重链可变区序列
Figure PCTCN2020081701-appb-000015
mAb1779鼠源轻链可变区序列
Figure PCTCN2020081701-appb-000016
其中各抗体轻重链中CDR序列如表1所示。
表1.各抗体重链及轻链CDR区序列
Figure PCTCN2020081701-appb-000017
Figure PCTCN2020081701-appb-000018
其Biacore活性检测结果见表2。
表2.IL-5鼠源抗体的体外活性
Figure PCTCN2020081701-appb-000019
结果显示，本公开中的鼠源抗体与抗原亲和力高。
实施例4.IL-5相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
4.1 IL-5-Flag-His重组蛋白的纯化步骤：
将样品高速离心去除杂质，并浓缩至适当体积。利用PBS平衡NI-NTA亲和柱(QIAGEN，Cat No.30721)，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用PBS冲洗柱子，至A280读数降至基线。用PBS冲洗柱子，冲洗杂蛋白，并收集。依次用洗涤缓冲液(咪唑20mM)和洗脱缓冲液(咪唑300mM)洗脱目的蛋白，并收集洗脱峰。
收集的洗脱液用离子交换(Hiload 16/600 superdex 200柱)进一步纯化。用PBS平衡2个柱体积左右，保证pH7.4。将鉴定含目的蛋白的洗脱缓冲液浓缩后上样，收集样品，SDS-PAGE和LC-MS鉴定为正确后分装备用。
4.2杂交瘤表达抗体，Fc融合蛋白的纯化
将细胞表达上清样品高速离心去除杂质，杂交瘤表达上清用Protein G柱，Fc融合蛋白表达上清用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.0洗脱目的蛋白，用1M Tris-HCl,pH8.0中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，去聚体的峰收集好后分装备用。
实施例5.抗人IL-5单克隆抗体的人源化设计
鼠源抗人IL-5单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，鼠源抗体的恒定区替换为人恒定区，且将鼠源抗体的CDR移植到FR同源性最高人源模板上，并对FR区的氨基酸进行回复突变。
通过比对IMGT人类抗体重轻链可变区种系基因数据库，分别挑选与mAb-1705，mAb-1706，mAb1780，mAb1773和mAb1779抗体氨基酸序列同一性高的重链和轻链可变区种系作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区。其中氨基酸残基由Kabat编号系统确定并注释。
人源FR区的选择和氨基酸的回复突变
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，从人源种系数据库中搜索轻链可变区(VL)和重链可变区(VH)的同源序列，按FR的同源性由高到低排序，选取FR同源性最高的种系作为主要模板，将鼠源抗体的CDR区移植到人源模板上，然后对FR的残基进行突变，并对氨基酸残基优化，得到最终的人源化分子。
5.1杂交瘤克隆mAb1705号人源化构架选择
h1705的VH选取IGHV3-23*04作为模板，VL选取IGKV1-12*01作为模板，将mAb1705的CDR移植到人源模板上，并通过软件找出包埋残基、与CDR区有直接相互作用的残基并回复突变，得到源化抗体不同的轻链和重链可变区，如表3所示。
表3.h1705的模板选择和回复突变设计
Figure PCTCN2020081701-appb-000020
注：Grafted代表鼠抗体CDR植入人种系FR区序列。如A43S表示依照氨基酸序列自然顺序编号，将grafted的第43位A突变回S。
表4.h1705人源化抗体重轻链可变区相互组合表
  h1705_VH.1 h1705_VH.1A h1705_VH.1B
h1705_VL.1 h1705-003 h1705-004 h1705-005
h1705_VL.1A h1705-006 h1705-007 h1705-008
h1705_VL.1B h1705-009 h1705-010 h1705-011
注：该表表示各种突变组合所得的序列。如h1705-007表示，人源化的鼠抗体h1705-007含轻链h1705_VL.1A、重链h1705_VH.1A两种突变体，其它类推。
h1705号人源化抗体可变区具体序列如下：
>h1705_VL.1(SEQ ID NO：46)
Figure PCTCN2020081701-appb-000021
>h1705_VL.1A(SEQ ID NO：47)
Figure PCTCN2020081701-appb-000022
>h1705_VL.1B(SEQ ID NO：48)
Figure PCTCN2020081701-appb-000023
>h1705_VH.1(SEQ ID NO：49)
Figure PCTCN2020081701-appb-000024
>h1705_VH.1A(SEQ ID NO：50)
Figure PCTCN2020081701-appb-000025
>h1705_VH.1B(SEQ ID NO：51)
Figure PCTCN2020081701-appb-000026
Figure PCTCN2020081701-appb-000027
将上述轻链可变区与轻链恒定区序列组合形成轻链序列，将各重链可变区与重链恒定区组合形成重链序列。示例性的人源化抗体恒定区序列如下所示：
>重链IgG1恒定区：
Figure PCTCN2020081701-appb-000028
注：下划线为M252Y、S254T、T256E突变
>轻链kappa恒定区：
Figure PCTCN2020081701-appb-000029
5.2杂交瘤克隆mAb1706号人源化构架选择
h1706的VH选取了IGHV3-23*04作为模板，VL选取了IGKV1-12*01作为模板，将鼠源抗体mab1706的CDR区移植到选择的人源化模板上，并FR氨基酸进行回复突变，得到人源化抗体轻链和重链可变区，如表5所示。
表5.h1706的模板选择和回复突变设计
Figure PCTCN2020081701-appb-000030
注：Grafted代表鼠抗体CDR植入人种系FR区。如A43S表示依照氨基酸序列的自然顺序编号，将grafted的第43位A突变回S。
将设计的人源化分子按下表组合成不同的抗体，如表6所示。
表6.h1706人源化抗体重轻链可变区相互组合表
  h1706_VH.1 h1706_VH.1A h1706_VH.1B
h1706_VL.1 h1706-002 h1706-003 h1706-004
h1706_VL.1A h1706-005 h1706-006 h1706-007
h1706_VL.1B h1706-008 h1706-009 h1706-010
h1706号人源化抗体可变区具体序列如下：
>h1706_VL.1(SEQ ID NO：54)
Figure PCTCN2020081701-appb-000031
>h1706_VL.1A(SEQ ID NO：55)
Figure PCTCN2020081701-appb-000032
>h1706_VL.1B(SEQ ID NO：56)
Figure PCTCN2020081701-appb-000033
>h1706_VH.1(SEQ ID NO：57)
Figure PCTCN2020081701-appb-000034
>h1706_VH.1A(SEQ ID NO：58)
Figure PCTCN2020081701-appb-000035
>h1706_VH.1B(SEQ ID NO：59)
Figure PCTCN2020081701-appb-000036
将上述轻链可变区与轻链恒定区序列组合形成轻链序列。将各重链可变区与重链恒定区组合形成重链序列。
5.3杂交瘤克隆mAb 1780号人源化构架选择
h1780的VH选取了IGHV1-2*02作为模板，VL选取了IGKV3-11*01作为模板，将鼠源抗体mAb1780的CDR区移植到选择的人源化模板上，并对FR区氨基酸进行复突变，得到人源化抗体轻链和重链可变区，如表7所示。
表7.h1780的模板选择和回复突变设计
Figure PCTCN2020081701-appb-000037
Figure PCTCN2020081701-appb-000038
注：Grafted代表鼠抗体CDR植入人种系FR区。如E1D表示依照氨基酸序列的自然顺序编号，将grafted的第1位E突变回D。
将设计的人源化分子按下表组合成不同的分子，如表8所示。
表8 h1780人源化抗体重轻链可变区相互组合表
  h1780_VH.1 h1780_VH.1A h1780_VH.1B h1780_VH.1C
h1780_VL.1 h1780-007 h1780-008 h1780-009 h1780-010
h1780_VL.1A h1780-011 h1780-012 h1780-013 h1780-014
h1780_VL.1B h1780-015 h1780-016 h1780-017 h1780-018
h1780号人源化抗体可变区具体序列如下：
>h1780_VL.1(SEQ ID NO：60)
Figure PCTCN2020081701-appb-000039
>h1780_VL.1A(SEQ ID NO：61)
Figure PCTCN2020081701-appb-000040
>h1780_VL.1B(SEQ ID NO：62)
Figure PCTCN2020081701-appb-000041
>h1780_VH.1(SEQ ID NO：63)
Figure PCTCN2020081701-appb-000042
>h1780_VH.1A(SEQ ID NO：64)
Figure PCTCN2020081701-appb-000043
>h1780_VH.1B(SEQ ID NO：65)
Figure PCTCN2020081701-appb-000044
>h1780_VH.1C(SEQ ID NO：66)
Figure PCTCN2020081701-appb-000045
将上述轻链可变区与轻链恒定区序列组合形成轻链序列。将各重链可变区与重链恒定区组合形成重链序列。
5.4杂交瘤克隆mAb1773号人源化构架选择
h1773的VH选取了IGHV3-73*01作为模板，VL选取了IGKV1-39*01作为模板，将鼠源抗体mab1773的CDR区移植到选择的人源化模板上，并进行回复突变，得到人源化抗体的轻链和重链可变区，如表9所示。同时，将h1773的HCDR2(RIDPANGDTK HGPKFQG)中N突变为V(即N55V)，形成HCDR2突变(突变后的HCDR2序列为SEQ ID NO:82：RIDPAVGDTKHGPKFQG)的重链可变区。
表9.h1773的模板选择和回复突变设计
Figure PCTCN2020081701-appb-000046
注：Grafted代表鼠抗体CDR植入人种系FR区。如M4L表示依照氨基酸序列的自然顺序编号，将grafted的第4位M突变回L。
将设计的人源化分子按下表组合成不同的分子，如表10所示。
表10.h1773人源化抗体重轻链可变区相互组合表
  h1773_VH.1 h1773_VH.1A h1773_VH.1B
h1773_VL.1 h1773-002 h1773-003 h1773-004
h1773_VL.1A h1773-005 h1773-006 h1773-007
h1773号人源化抗体可变区具体序列如下：
>h1773_VL.1(SEQ ID NO：67)
Figure PCTCN2020081701-appb-000047
>h1773_VL.1A(SEQ ID NO：68)
Figure PCTCN2020081701-appb-000048
>h1773_VH.1(SEQ ID NO：69)
Figure PCTCN2020081701-appb-000049
>h1773_VH.1A(SEQ ID NO：70)
Figure PCTCN2020081701-appb-000050
>h1773_VH.1B(SEQ ID NO：71)
Figure PCTCN2020081701-appb-000051
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成最终的完整重轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成最终的各重链序列。
5.5杂交瘤克隆mAb1779号人源化构架选择
h1779的VH选取了IGHV1-2*02作为模板，VL选取了IGKV1-33*01作为模板，将鼠源抗体h1779的CDR区移植到选择的人源化模板上，并进行回复突变，得到人源化抗体的轻链和重链可变区，如表11所示。
表11.h1779的模板选择和回复突变设计
Figure PCTCN2020081701-appb-000052
注：Grafted代表鼠抗体CDR植入人种系FR区。如A43S表示依照氨基酸序列的自然顺序编号，将grafted的第43位A突变回S。
将设计的人源化分子按下表组合成不同的分子，如表12所示。
表12.h1779人源化抗体重轻链可变区相互组合表
  h1779_VH.1 h1779_VH.1A h1779_VH.1B h1779_VH.1C h1779_VH.1D
h1779_VL.1 h1779-005 h1779-006 h1779-007 h1779-008 h1779-009
h1779_VL.1A h1779-010 h1779-011 h1779-012 h1779-013 h1779-014
h1779_VL.1B h1779-015 h1779-016 h1779-017 h1779-018 h1779-019
h1779号人源化抗体可变区具体序列如下：
>h1779_VL.1(SEQ ID NO：72)
Figure PCTCN2020081701-appb-000053
>h1779_VL.1A(SEQ ID NO：73)
Figure PCTCN2020081701-appb-000054
>h1779_VL.1B(SEQ ID NO：74)
Figure PCTCN2020081701-appb-000055
>h1779_VH.1(SEQ ID NO：75)
Figure PCTCN2020081701-appb-000056
>h1779_VH.1A(SEQ ID NO：76)
Figure PCTCN2020081701-appb-000057
>h1779_VH.1B(SEQ ID NO：77)
Figure PCTCN2020081701-appb-000058
>h1779_VH.1C(SEQ ID NO：78)
Figure PCTCN2020081701-appb-000059
>h1779_VH.1D(SEQ ID NO：79)
Figure PCTCN2020081701-appb-000060
将上述轻链可变区与如SEQ ID NO:53所示的轻链恒定区序列组合形成轻链序列。将各重链可变区与如SEQ ID NO:52所示的重链恒定区组合形成重链序列。
示例性的人源化抗体全长序列如下：
h1705-008重链：
Figure PCTCN2020081701-appb-000061
Figure PCTCN2020081701-appb-000062
h1705-008轻链：
Figure PCTCN2020081701-appb-000063
h1706-009重链：
Figure PCTCN2020081701-appb-000064
h1706-009轻链：
Figure PCTCN2020081701-appb-000065
h1780-017重链：
Figure PCTCN2020081701-appb-000066
Figure PCTCN2020081701-appb-000067
h1780-017轻链：
Figure PCTCN2020081701-appb-000068
h1773-007重链：
Figure PCTCN2020081701-appb-000069
h1773-007轻链：
Figure PCTCN2020081701-appb-000070
h1779-014重链：
Figure PCTCN2020081701-appb-000071
Figure PCTCN2020081701-appb-000072
h1779-014轻链：
Figure PCTCN2020081701-appb-000073
本公开将WO2012083370A1中针对IL5的抗体Hu39D10作为阳性对照，其重链和轻链序列分别如SEQ ID NO:80和SEQ ID NO:81所示。
Hu39D10的重链序列
Figure PCTCN2020081701-appb-000074
Hu39D10的轻链序列
Figure PCTCN2020081701-appb-000075
实施例6.重组嵌合抗体以及人源化抗体的制备
1.重组嵌合抗体的分子克隆
杂交瘤筛选所获得的阳性抗体分子经过测序后，得到可变区编码基因序列。以测序所得序列设计首尾引物，以测序基因为模板，经过PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG1/hkappa恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建重组嵌合抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr，得到Ch1705、Ch1706、Ch1780、Ch1773和Ch1779五个嵌合抗体。
2.人源化抗体的分子克隆
人源化抗体序列，经过密码子优化后得到人密码子偏好的编码基因序列，设计引物PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG1/hkappa恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建人源化抗体全长表达质粒VH-CH1-Fc-pHr/VL-CL-pHr。
3.重组嵌合抗体以及人源化抗体的表达与纯化
分别表达抗体轻重链的质粒，转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl,pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
以下用测试方法验证本公开抗体性能及有益效果。
体外活性生物学评价
测试例1.Biacore测定鼠源IL-5抗体与不同种属IL-5的结合
用Biacore T200(GE)仪器测定待测鼠源IL-5抗体和人IL-5的亲和力。
用Protein A生物传感芯片亲和捕获待测分子，然后于芯片表面流经抗原(实施例1制备所得的重组人，猴IL-5)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)将生物传感芯片洗净再生。用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，如表13所示。
表13.IL-5鼠源抗体与不同种属IL-5亲和力的BIAcore检测结果
Figure PCTCN2020081701-appb-000076
本实施例证明，本公开的抗体mAb1705、mAb1706、mAb1780、mAb1773和mAb1779与不同种属(人、猴)的IL-5均有较高的亲和力。
测试例2.Biacore测定IL-5人源化抗体与不同种属IL-5的亲和力
用Biacore T200(GE)仪器测定待测人源化IL-5抗体和人IL-5的亲和力。
用Protein A生物传感芯片亲和捕获待测分子，然后于芯片表面流经抗原(实施例1制备所得)，用Biacore T200仪器实时检测反应信号获得结合和解离曲线。在每个实验循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)将生物传感芯片洗净再生。用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得 出亲和力数值，如表14所示。
表14.IL-5人源化抗体与人IL-5亲和力的BIAcore检测结果
抗体 KD(M) 抗体 KD(M)
h1705-003 3.35E-09 h1706-003 1.89E-11
h1705-006 4.11E-09 h1706-006 1.73E-11
h1705-009 4.55E-09 h1706-009 5.45E-11
h1705-004 2.14E-11 h1780-017 7.78E-11
h1705-007 2.21E-11 h1773-007 2.07E-10
h1705-010 2.05E-11 h1779-014 4.12E-10
h1705-005 2.16E-11    
h1705-008 3.42E-11    
h1705-011 2.30E-11    
结果显示，人源化IL-5抗体与人IL-5均具有较高的亲和力。
测试例3.基于ELISA的鼠源IL-5抗体阻断IL-5结合IL-5α受体实验
为鉴定IL-5抗体阻断IL-5结合到重组表达的IL-5α受体蛋白胞外区的能力，将IL-5(5μg/ml in PBS)包被ELISA板，37℃孵育1小时，弃去液体后，加入用PBS稀释的5％脱脂牛奶封闭液200μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％Tween-20)洗板5次后，先加入25μl用生物素标记试剂盒(东仁化学，LK03)标记的10μg/ml的IL-5Rα(in 1％BSA)，再加入25μl梯度稀释的抗体，抗体和IL-5Rα竞争结合IL-5，37℃孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入50μl/孔用样品稀释液以1:600浓度稀释的Streptavidin-Peroxidase Polymer(Sigma，S2438-250UG)于37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-10min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算IL-5抗体阻断IL-5与IL-5Rα结合的IC50值，结果如表15所示，本公开抗体均可有效抑制IL-5与其受体的结合。
表15.鼠源IL-5抗体阻断IL-5结合IL-5α受体的ELISA检测结果
  mAb1705 mAb1706
IC50(μg/ml) 0.42 0.40
测试例4.基于FACS的IL-5抗体阻断IL-5结合IL-5受体实验
为了鉴定筛选到的IL-5抗体可以阻断细胞表面的IL-5受体，构建了高表达 IL-5Rα/β的两种受体的CHOS重组细胞系。本实验鉴定IL-5抗体可以分别阻断IL-5结合到CHOS细胞系表面的重组IL-5α/β受体。
具体方法：用含100ng/ml G418,25ng/ml zeozin的CD-CHO来培养CHO-S-IL-5Rα和β，细胞培养过程中，浓度不要超过3×10 6细胞/ml。将状态良好的IL-5Rα/β-CHOS细胞离心(1000rpm，5min)，用10％FBS的PBS洗一遍，并且计数，将细胞浓度调整浓度至4×10 6细胞/ml,取25μl加入到圆底的96孔板中。用含10％FBS的PBS溶液稀释待测抗体，初始浓度为200μg/ml，按1:10倍比稀释8个梯度。将25μl 100ng/ml的用生物素标记试剂盒(东仁化学，LK03)标记的IL-5和50μl稀释的各浓度抗体混合均匀，加到已加入细胞的96孔板中，在4℃孵育1小时。孵育结束后，4℃离心(400g，5min)，弃掉上清，用200μl的预冷PBS离心洗涤，重复两次后加入1:1333稀释的PE-Avidin二抗，4℃避光孵育40min后，4℃离心(400g，5min，弃掉上清，加入200μl的预冷PBS，吹起细胞，4℃离心洗涤，重复三次，加入100μl PBS，上机读板，根据荧光信号值计算IL-5抗体阻断IL-5与IL-5Rα/β结合的IC50值。结果如表16和图1所示。
表16.IL-5抗体阻断IL-5与IL-5Rα/β结合的检测结果
抗体 IgG hu39D10 h1705-008 h1706-009 h1780-017 h1779-014
IC50(ng/ml) 8777 25.07 14.51 24.74 16 49.64
结果显示，抗体h1705-008、h1706-009、h1780-017、h1779-014显示出较强的阻断IL-5结合到细胞表面IL-5受体的能力。
测试例5.IL-5抗体抑制IL-5诱导的TF1细胞增殖实验
IL-5可以诱导TF-1细胞增殖，IL-5抗体可以阻止IL-5对TF-1细胞的刺激增殖作用。
具体为：TF-1细胞(ATCC,CRL-2003)培养于10％FBS以及2ng/mL rhGM-CSF(联科生物,Catalog No.96-AF-300-03-20)的RPMI1640，放置与37℃，5％CO 2培养箱中，细胞密度不超过1×10 6个/ml。检测抗体时，取对数生长期的细胞用PBS洗三遍800rpm离心5min，用RPMI1640(FBS：2％，重组人IL-5:10ng/ml)调整细胞密度6000个/孔/90μl，并加入10μl梯度稀释的待测抗体到96孔培养3d后，加入30μl细胞滴度混匀后进行检测，根据读值计算IC50。检测结果如下表17所示。
表17.IL-5人源化抗体抑制IL-5诱导的TF1细胞增殖试验结果
抗体 IC50(nM) 抗体 IC50(nM)
Hu39D10 0.30 h1706-003 0.31
h1705-004 0.30 h1706-004 0.30
h1705-005 0.30 h1706-006 0.34
h1705-007 0.25 h1706-007 0.28
h1705-008 0.20 h1706-009 0.25
h1705-010 0.30 h1773-007 0.38
h1705-011 0.28 h1780-017 0.16
    h1779-014 0.20
测试例6.IL5抗体抑制IL5诱导的嗜酸性粒细胞粘附实验
IL5可以诱导嗜酸性粒细胞的分化，成熟，迁移和活化，引起呼吸道炎症而导致哮喘。本实验利用IL-5细胞因子能够促进嗜酸性粒细胞粘附并使之激活的原理，收集和纯化来源于人外周全血的嗜酸性粒细胞来测试IL-5特异性抗体对IL-5该通路的阻断作用，在体外检测IL-5抗体对IL5介导的嗜酸性粒细胞数粘附效果的阻断。
具体为：人外周全血用含2mM EDTA的PBS稀释5倍，采用PercollTM(密度梯度为1.088)分离单核细胞和粒细胞，小心吸取含有粒细胞的红细胞层，利用红细胞裂解液去除红细胞；将剩余的细胞计数，按比例加人带有CD16抗体的分离磁珠(美天旎，Catalog No.130-045-701)，孵育30min后过磁珠柱，负选收集直接流出细胞亚群即主要为嗜酸性粒细胞。分离得到的嗜酸性粒细胞计数，加入预先以IgG抗体包被的96孔细胞培养板内，每孔约1×10 4个，加人IL-5(20ng/ml)，以及不同浓度的IL-5抗体分子(10μg/ml起始，3倍稀释，10个浓度点)；细胞培养板置于37℃，5％CO2培养箱孵育1h，取出培养板，加入0.3％CTAB裂解细胞，最后加入过氧化物酶反应底物TMB显色反应，酶标仪读取OD450吸收值。最大吸附值为只加IL-5孔读值，不加IL-5及抗体药物孔为背景对照，计算各浓度下抗体药物相对于最大吸附孔的抑制值＝(最大吸附值-[抗体药物])/(最大吸附值-背景对照值)×100％，计算IC50。结果如下表18：
表18.IL-5抗体阻断IL-5诱导的嗜酸性粒细胞粘附
  Hu39D10 h1705-008 h1706-009 h1780-017
IC50(ng/ml) 11.79 4.85 4.3 21.19
结果表明，本公开的人源化抗体显示较强的抑制IL5介导的嗜酸性粒细胞粘附的能力。
测试例7.人源化IL-5抗体Th2细胞因子的特异性评价
IL-5是Th2细胞因子中的一种，为了验证IL-5抗体只特异性针对IL-5而与其他细胞因子没有交叉反应，用Fortebio检测了12种Th2以及相关细胞因子，包括IL2(R&D,202-IL-010/CF)，IL4(R&D,204-IL-050/CF)，IL-5(R&D,205-IL-025/CF)，IFNgamma，IL6(R&D,7270-IL-025/CF)，IL9(R&D,209-IL-010/CF)，IL10(R&D,217-IL-025/CF)，IL13(R&D,213-ILB-025/CF)，IL25(R&D,8134-IL-025/CF)，IL31(R&D,2824-IL-010/CF)，以及与IL-5共用α受体的IL3(203-IL-050/CF)和GMCSF(R&D,215-GM-010/CF)。
具体为：用Protein A Biosensor(PALL Fortebio,18-5010)来捕获抗体，记录捕获信号，然后再进样40nM各细胞因子，记录新的结合信号。最后将与IL-5的结合信号定义为100％，看其他细胞因子与抗体的的结合信号，结果如图2所示。
结果显示，12种相关细胞因子中，IL-5人源化抗体h1705-008和h1706-009只特异性结合IL-5，与其他Th2细胞因子无交叉反应。
体内活性生物学评价
测试例8.IL-5抗体在OVA诱导的小鼠哮喘模型中的药效评价
本测试是基于气道炎性反应以及气道重塑来评估IL-5抗体在卵清蛋白(OVA)气雾诱导的BALB/c小鼠哮喘模型中的药效。
小鼠根据体重随机分成7组，每组10只小鼠：正常对照组(G1)；模型组(G2)；2个测试抗体h1705-008(G3和G4)和h1706-009(G5和G6)，每个测试抗体2个剂量(10mpk和2mpk)的治疗组；以及阳性抗体Hu39D10对照组(G7，10mpk)。在第1天和第14天，对所有小鼠腹腔注射致敏溶液进行致敏。在第28、29、30天，用OVA攻击溶液对第一组外的其他六组小鼠进行气雾攻击30分钟。攻击前2小时，第二组(G2)腹腔注射磷酸缓冲液，第三组至第七组(G3-G7)小鼠腹腔注射不同剂量的不同抗体，一天一次，连续三天。每次注射前配制新鲜的测试抗体，配制完成至给药半小时内完成。第一组的小鼠作为正常对照组用PBS进行气雾攻击30分钟，攻击前2小时腹腔注射磷酸缓冲液，一天一次，连续三天。
在第31天，使用WBP系统对动物进行气道高反应性的检测。所有动物均用雾化吸入1.5625、3.125、6.25、12.5、25、50mg/mL倍增浓度的乙酰甲胆碱，测定相应浓度下的增强呼气间歇值。
在第31天，WBP系统检测气道反应1小时后，直径1.2毫米的气管插管插入气管内并且固定，肺将被灌洗两次，每次0.8毫升的磷酸缓冲液含1％的BSA和0.6mM的EDTA。记录灌洗液的回收体积。
将BALF在4摄氏度300g下离心5分钟，上清液保留用于细胞因子分析。离心后，细胞重悬在1.5毫升的PBS里(含1％的BSA和0.6mM的EDTA)进行细胞计数。通过血球计数器和台盼蓝染色实验对BALF中的总细胞数进行计数。细胞涂片后，莱特染色液染色一分钟，接着用吉姆沙染色7分钟，从而区分出嗜酸细胞、中性白细胞、巨噬细胞和淋巴细胞。在光学显微镜下计数。
灌洗后，收集肺组织并用10％的中性甲醛溶液浸染，然后固定在10％的中性甲醛溶液中。固定的组织进行石蜡包埋，切边，H&E染色，评分。测试结果如图3和图4A、图4B所示。
结果显示，本公开中抗体分子h1705-008和h1706-009可明显提高肺功能，且作用方式呈剂量依赖模式，而高剂量(10mpk)的阳性化合物却不能改善肺功能(参见图3)。同时，两个抗体显著降低了嗜酸性粒细胞水平和粘膜厚度，在同样剂量 下(10mpk)显示出比阳性抗体更强的降低嗜酸性粒细胞的能力(参见图4A和4B)。而在同样类型小鼠哮喘模型中，重复实验也同样验证了1mg/ml的h1705-008，h1706-009和h1780-017均比阳性抗体更显著地降低BalF中的嗜酸性粒细胞水平(参见图4C)。
测试例9.外源性人IL-5诱导的豚鼠急性哮喘模型评价IL-5抗体的体内药效
本实验选用雄性豚鼠建立人IL-5诱导的急性哮喘模型，以hu39D10为阳性抗体，评估本公开5个IL-5人源化单抗对人IL-5诱导的豚鼠肺支气管灌流液(BALF)中的嗜酸性粒细胞增加是否有抑制作用。豚鼠共分为9组，每组8-10只：正常对照组、模型组、hu39D10(1mg/kg)组、h1705-008(1mg/kg)组、h1706-009(1mg/kg)组、h1780-017(1mg/kg)组、h1773-007(1mg/kg)组和h1779-014(1mg/kg)组。其中模型组和给药组豚鼠分别于第1天气管注射100μl人IL5(含IL5抗原5μg)激发，正常对照组气管注射PBS。给药组激发前2小时腹腔注射分别给予1mg/kg上述IL5单抗，给药体积为5ml/kg，模型组给予相应的IgG抗体，正常对照组腹腔给予PBS溶剂。气管注射后24h后麻醉豚鼠抽取肺支气管灌流液，待细胞浓度调整至5^10 6/ml时取15μl滴在载玻片上，晾干固定后进行HE染色，400倍镜下计数细胞总数和嗜酸性粒细胞数并计算嗜酸性粒细胞百分比。结果如图5A和图5B所示，表明本公开5个人源化抗体均可以显著降低BALF中嗜酸性粒细胞水平。
制剂成分的筛选及稳定性评价
示例性抗体药物组合物(制剂)制备工艺：
第一步：取一定量的纯化的抗IL-5抗体溶液，用不含抗体的缓冲剂(如30mM，pH5.5醋酸-醋酸钠缓冲剂)进行溶剂置换(优选超滤)，经超滤膜至少6倍体积置换，蛋白浓缩到约120mg/mL。加入一定体积的蔗糖母液，混匀，使最终蔗糖浓度为72mg/mL。加入一定体积的聚山梨醇酯80母液，混匀，使最终聚山梨醇酯80浓度为0.4mg/mL。加10mM pH 5.5醋酸-醋酸钠缓冲剂定容，使蛋白浓度为100mg/mL(其他待测试制剂或稳定性制剂参照相似步骤进行配制)。
产品经过滤后中控取样检测无菌。将原液过0.22μm PVDF滤芯，收集滤液。
第二步：调节装量至1.2ml，将滤液灌装于2ml西林瓶中，加塞，分别于灌装开始、灌装中间、灌装结束时取样中控检测装量差异。
第三步：开启轧盖机，加铝盖，进行轧盖。
第四步：目检，确认产品无装量不准等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
测试例10.缓冲体系的筛选
在pH5.0-6.5一系列缓冲液中，配制蛋白浓度为100mg/mL的h1705-008制剂， 其中振摇样品含0.2mg/ml聚山梨醇酯80(PS80)，其他样品含0.05mg/mL PS80。缓冲体系分别为10mM醋酸-醋酸钠(AA)pH5.0、5.5；10mM琥珀酸-琥珀酸钠(SA)pH5.0、5.5、6.0；10mM枸橼酸-枸橼酸钠(CA)pH5.5、6.0、6.5；10mM组氨酸-盐酸盐(His)pH5.5、6.0、6.5；10mM磷酸盐(PB)pH6.0、6.5。将每种制剂过滤，灌装，加塞，轧盖。将样品进行强制降解实验，以外观、SEC、iCIEF为评价指标。
结果见表19，外观数据显示，振摇(300rpm，25℃)及40℃样品出现不同程度颗粒，整体来看pH较低时外观较好，缓冲体系醋酸-醋酸钠及琥珀酸-琥珀酸钠较优；SEC数据显示，40℃条件下AA pH 5.5组略优；iCIEF数据显示，40℃条件下AA pH 5.5、His pH 5.5、CA pH 6.5组略优；综合考虑物理化学稳定性，优选AA pH 5.5。
表19.pH和缓冲体系筛选结果
Figure PCTCN2020081701-appb-000077
Figure PCTCN2020081701-appb-000078
注：N/A表示未检测，D表示天，T0表示第0天。
测试例11.制剂的辅料筛选
在含下列不同种类辅料的10mM SA(pH5.0)缓冲液中，制备蛋白浓度为100mg/mL的h1705-008制剂。具体辅料如下：
1)0.1mg/mL聚山梨醇酯20(PS20)
2)0.1mg/mL聚山梨醇酯80(PS80)
3)50mg/mL蔗糖+0.1mg/mL PS80
4)50mg/mL海藻糖+0.1mg/mL PS80
5)50mg/mL甘露醇+0.1mg/mL PS80
6)50mg/mL山梨醇+0.1mg/mL PS80
7)8mg/ml精氨酸(Arg)+0.1mg/mL PS80
8)8mg/ml赖氨酸(Lys)+0.1mg/mL PS80
9)8mg/ml甘氨酸(Gly)+0.1mg/mL PS80
10)8mg/ml甲硫氨酸(Met)+0.1mg/mL PS80
11)8mg/ml脯氨酸(Pro)+0.1mg/mL PS80
12)8mg/ml氯化钠(NaCl)+0.1mg/mL PS80
每种制剂经过滤灌装，加塞，轧盖后备用。将样品进行40℃高温强制降解实验，结果表明(表20)：各组样品SEC、CE、iCIEF的检测结果无显著差异，Arg/Lys/NaCl组外观较差，其他组间无显著差异。蔗糖、海藻糖、甘露醇、山梨醇、甘氨酸、脯氨酸、甲硫氨酸对蛋白稳定性均较好。
表20.辅料筛选结果
Figure PCTCN2020081701-appb-000079
注：D表示天。
测试例12.表面活性剂的筛选
制备蛋白浓度100mg/ml，10mM SA pH5.5，70mg/ml蔗糖，0.4mg/ml PS20或PS80的h1705-008制剂。
将样品置于4℃条件考察稳定性，结果见表21，结果显示：4℃M4后PS80组外观澄明，SEC、CE、iCIEF无显著变化，稳定性良好，而PS20组出现大量颗粒，因此PS80优于PS20，另外可以看出处方中同时加入PS80对h1705-008有较好的稳定作用，该处方稳定性较好。
表21.h1705-008 4℃稳定性结果
Figure PCTCN2020081701-appb-000080
注：M表示月；N/A表示未检测。
测试例13.DOE处方设计和筛选
以10mM醋酸盐(AA)缓冲液pH、蛋白浓度和吐温浓度为变量进行DOE(Design of expriment)实验设计，因子及水平为pH5.0～5.8、PS80浓度0.2～0.6mg/mL、抗体h1705-008浓度80～120mg/mL，设计系列处方，处方见表22，将样品进行40℃高温强制降解，以外观、SEC、非还原CE、iCIEF为评价指标，结果见表23。
表22.DOE处方筛选实验处方设计
Figure PCTCN2020081701-appb-000081
表23.DOE处方筛选实验结果
Figure PCTCN2020081701-appb-000082
Figure PCTCN2020081701-appb-000083
注：D表示天。
结果显示，各处方外观均澄明，SEC、CE、iCIEF分别下降<2％、<2％、约14％，在可接受范围内，处方稳定性较好，因此制剂包含蛋白浓度80-120mg/ml， 0.2-0.6mg/ml PS80，pH5.0-5.8。最优处方为蛋白浓度100mg/ml，0.4mg/ml PS80，pH5.5。
测试例14.稳定性检测
制备蛋白浓度100mg/ml，10mM AA pH5.5，70mg/ml蔗糖，0.4mg/ml PS80的h1705-008制剂，将样品进行4℃、25℃稳定性考察，结果见表24。
结果显示，高温条件下h1705-008制剂SEC、CE、iCIEF略有下降，但下降幅度均在可接受范围，其他条件下各项指标均无显著变化，该处方稳定性较好，可保证h1705-008在4℃6个月内的稳定性。
表24.h1705-008 25℃及4℃稳定性结果
Figure PCTCN2020081701-appb-000084
注：M表示月。
测试例15.离子强度的筛选
含下列不同离子强度的醋酸(钠)缓冲液中，制备蛋白浓度100mg/mL，70mg/mL蔗糖，0.4mg/mL PS80的h1705-008制剂，测定置换用缓冲液及最终制剂pH，结果见表25。结果显示离子强度越高，pH漂移越小，当离子强度为30mM时，pH漂移小于0.1。
表25.不同离子强度制剂pH结果
离子强度 缓冲液-pH 制剂原液-pH △pH
10mM 5.50 5.71 0.21
20mM 5.50 5.66 0.16
30mM 5.50 5.59 0.09
测试例16.糖浓度的筛选
在含下列不同蔗糖浓度的缓冲液中，制备蛋白浓度为100mg/mL，含30mM AA pH5.5，0.4mg/mlPS80的h1705-008制剂，测定渗透压，结果见表26。
结果显示，当糖浓度为70-75mg/ml时，渗透压处于最佳等渗范围(290-310mosm)；根据70mg/ml和73mg/ml组渗透压数据推算，糖浓度为72mg/ml时，渗透压可达最佳值，约300mosm。
表26.h1705-008不同糖浓度处方渗透压比较
糖浓度(mg/ml) 70 73 75
渗透压(mosm) 290 306 310
测试例17.制剂稳定性测试
制备蛋白浓度为100mg/mL，含30mM AA pH5.5，72mg/ml蔗糖，0.4mg/mlPS80的h1705-008制剂，考察其4℃及25℃稳定性，结果见表27。结果显示，高温条件下h1705-008制剂SEC、CE、IEC略有下降，但下降幅度均在可接受范围，4℃条件下各项指标均无显著变化，该处方稳定性较好。
表27.h1705-008制剂稳定性
条件 外观 SEC(％) IEC中性峰(％) 非还原CE-SDS(％)
T0 澄明 98.4 63.5 97.6
25℃-M3 N/A 96.8 55.0 96.5
4℃–M3 澄明 98.4 61.9 97.3
注：M表示月，T表示时间，N/A表示未测定。
测试例18.其它可选择制剂配方
此外，本公开还提供其它制剂配方的抗IL-5抗体药物制剂，包含但不限于：
(1)1mg/ml抗IL-5抗体(h1705-008)，72mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH 5.5的10mM醋酸-醋酸钠缓冲剂；
(2)100mg/ml抗IL-5抗体(h1705-008)，72mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH为5.5的20mM醋酸-醋酸钠缓冲剂；
(3)120mg/ml抗IL-5抗体(h1705-008)，72mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH为5.5的40mM醋酸-醋酸钠缓冲剂；
(4)100mg/ml抗IL-5抗体(h1705-008)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和pH为5.0的30mM醋酸-醋酸钠缓冲剂；
(5)80mg/ml抗IL-5抗体(h1705-008)，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和pH为5.4的20mM醋酸-醋酸钠缓冲剂；
(6)100mg/ml抗IL-5抗体(h1705-008)，80mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH为5.5的30mM醋酸-醋酸钠缓冲剂；
(7)90mg/ml抗IL-5抗体(h1705-008)，74mg/ml蔗糖，0.5mg/ml的聚山梨醇酯80，和pH为5.6的25mM醋酸-醋酸钠缓冲剂；
(8)90mg/ml抗IL-5抗体(h1705-008)，76mg/ml蔗糖，0.3mg/ml的聚山梨醇酯80，和pH为5.4的35mM醋酸-醋酸钠缓冲剂；
(9)80mg/ml抗IL-5抗体(h1705-008)，72mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH为5.6的30mM醋酸-醋酸钠缓冲剂；
(10)100mg/ml抗IL-5抗体(h1705-008)，72mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH为5.5的40mM醋酸-醋酸钠缓冲剂；
(10)100mg/ml抗IL-5抗体(h1705-008)，80mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和pH为5.5的40mM醋酸-醋酸钠缓冲剂。
实验结果表明，上述制剂配方的IL-5抗体制剂均具有良好的稳定性，可应用于IL-5抗体药物的配制。
测试例19.抗IL-5抗体制剂的冻干
用pH5.5的30mM醋酸-醋酸钠缓冲液，制备含72mg/ml蔗糖，0.4mg/ml聚山梨醇酯80的抗IL-5抗体浓度为100mg/ml的h1705-008抗体制剂。将抗体以2.15mL/瓶填充入6mL西林瓶中，装入冻干箱中，冻干。冻干程序为预冻、一次干燥和二次干燥。冻干程序结束后，真空加塞。复溶样品进行冻干前后对比。结果表明，复溶溶液可保持液体制剂良好的性能。
表28.制剂的冻干步骤
Figure PCTCN2020081701-appb-000085
注：N/A(Not applicable)表示本表对其不适用

Claims (27)

  1. 一种药物组合物，其包含抗IL-5抗体或其抗原结合片段、缓冲剂和表面活性剂，其中所述缓冲剂选自醋酸-醋酸钠、琥珀酸-琥珀酸钠、组氨酸-盐酸盐和枸橼酸-枸橼酸钠缓冲剂中的任一种，优选为醋酸-醋酸钠或琥珀酸-琥珀酸钠缓冲剂；其中所述的抗IL-5抗体或其抗原结合片段包含选自i)至vi)中任一项所示的重链可变区和轻链可变区：
    i)重链可变区包含分别如SEQ ID NO：16、17、18氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：19、20、21氨基酸序列所示的LCDR1、LCDR2和LCDR3；
    ii)重链可变区包含分别如SEQ ID NO：22、23、24氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：25、26、27氨基酸序列所示的LCDR1、LCDR2和LCDR3；
    iii)重链可变区包含分别如SEQ ID NO：28、29、30氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：31、32、33氨基酸序列所示的LCDR1、LCDR2和LCDR3；
    iv)重链可变区包含分别如SEQ ID NO：34、35、36氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：37、38、39氨基酸序列所示的LCDR1、LCDR2和LCDR3；
    v)重链可变区包含分别如SEQ ID NO：40、41、42氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：43、44、45氨基酸序列所示的LCDR1、LCDR2和LCDR3；和
    vi)重链可变区包含分别如SEQ ID NO：34、82、36氨基酸序列所示的HCDR1、HCDR2和HCDR3，轻链可变区包含分别如SEQ ID NO：37、38、39氨基酸序列所示的LCDR1、LCDR2和LCDR3。
  2. 根据权利要求1所述的药物组合物，其中所述的缓冲剂的pH为约5.0至约6.5，优选为约5.0至约5.8，最优选为约5.5。
  3. 根据权利要求1或2所述的药物组合物，其中所述缓冲剂浓度为约10mM至约40mM，优选为约20mM至约30mM。
  4. 根据权利要求1至3中任一项所述的药物组合物，其中所述抗IL-5抗体或其抗原结合片段浓度为约1mg/ml至约120mg/ml，优选为约80mg/ml至约120mg，最优选为约100mg/ml。
  5. 根据权利要求1至4中任一项所述的药物组合物，其中所述的表面活性剂 为聚山梨醇酯80。
  6. 根据权利要求5所述的药物组合物，其中所述的聚山梨酯80的浓度为约0.1mg/ml至约0.6mg/ml，优选为约0.2mg/ml至约0.6mg/ml，更优选为约0.4mg/ml。
  7. 根据权利要求1至6中任一项所述的药物组合物，其还包括稳定剂，其中所述的稳定剂为糖或氨基酸，其中所述的糖选自蔗糖、海藻糖、甘露醇和山梨醇，优选为蔗糖；其中所述的氨基酸选自甘氨酸、甲硫氨酸和脯氨酸。
  8. 根据权利要求7所述的药物组合物，其中所述糖的浓度为约50mg/ml至约80mg/ml，优选为约70mg/ml至约75mg/ml，最优选为约72mg/ml。
  9. 根据权利要求7所述的药物组合物，其中所述氨基酸的浓度为约8mg/ml。
  10. 根据权利要求1至9中任一项所述的药物组合物，其包含如下成分：
    约1mg/ml至约120mg/ml的抗IL-5抗体或其抗原结合片段；约10mM至约40mM的醋酸-醋酸钠缓冲剂，pH为约5.0至约6.5；和约0.1mg/ml至约0.6mg/ml聚山梨酯80。
  11. 根据权利要求1至9中任一项所述的药物组合物，其包含：
    约80mg/ml至约120mg/ml的抗IL-5抗体或其抗原结合片段；约10mM至约30mM的醋酸-醋酸钠缓冲剂，pH为约5.0至约5.8；约0.2mg/ml至约0.6mg/ml聚山梨酯80和约70mg/ml至约80mg/ml的蔗糖；
    优选地，所述药物组合物包含：
    约100mg/ml的抗IL-5抗体或其抗原结合片段，约30mM的醋酸-醋酸钠缓冲剂，pH为约5.5，约0.4mg/ml的聚山梨醇酯80和约72mg/ml的蔗糖。
  12. 根据权利要求1至11中任一项所述的药物组合物，其中所述的抗IL-5抗体或其抗原结合片段为鼠源抗体、嵌合抗体或人源化抗体。
  13. 根据权利要求12所述的药物组合物，其中所述的抗IL-5人源化抗体包含SEQ ID NO：49、57、63、69或75所示的重链可变区或其变体；所述变体是在SEQ ID NO：49、57、63、69或75所示的重链可变区序列上分别具有1-10个氨基酸回复突变。
  14. 根据权利要求13所述的药物组合物，其中所述的变体选自如下任一项所示的变体：
    i)在SEQ ID NO：49所示的重链可变区上具有选自S49T，V93T和K98S中的一个或多个的氨基酸回复突变；
    ii)在SEQ ID NO：57所示的重链可变区上具有选自S49T，V93T和K98T中的一个或多个的氨基酸回复突变；
    iii)在SEQ ID NO：63所示的重链可变区上具有选自R38K，M48I，R67K，V68A，M70L，R72V，T74K和L83F中的一个或多个的氨基酸回复突变；
    iv)在SEQ ID NO：69所示的重链可变区上具有选自F29I，R38K，V48I，R72A和T97F中的一个或多个的氨基酸回复突变和/或HCDR2中N55V突变；或
    v)在SEQ ID NO：75所示的重链可变区上具有选自R38K，M48I，R67K，V68A，R72A，T74K，M81L，L83F和D89E中的一个或多个的氨基酸回复突变。
  15. 根据权利要求14所述的药物组合物，其中所述的抗IL-5人源化抗体包含如SEQ ID NO：50或51所示的重链可变区；或
    包含如SEQ ID NO：58或59所示的重链可变区；或
    包含选自SEQ ID NO：64、65和66中任一个所示的重链可变区；或
    包含如SEQ ID NO：70或71所示的重链可变区；或
    包含选自SEQ ID NO：76、77、78和79中任一个所示的重链可变区。
  16. 根据权利要求12所述的药物组合物，其中所述的抗IL-5人源化抗体含有如SEQ ID NO：46、54、60、67或72所示的轻链可变区或其变体；所述变体是在SEQ ID NO：46、54、60、67或72所示的轻链可变区上具有1-10个氨基酸回复突变。
  17. 根据权利要求16所述的药物组合物，其中所述的变体选自如下任一所示的变体：
    i)在SEQ ID NO：46所示的轻链可变区上具有选自A43S，L47V，G66R，T69S，F71Y和Y87F中的一个或多个的氨基酸回复突变；
    ii)在SEQ ID NO：54所示的轻链可变区上具有选自A43S，L47M，F71Y和Y87F中的一个或多个的氨基酸回复突变；
    iii)在SEQ ID NO：60所示的轻链可变区上具有选自E1D，I2T，I57V，V84T和Y86F中的一个或多个的氨基酸回复突变；
    iv)在SEQ ID NO：67所示的轻链可变区上具有选自M4L，A42S，L45P和L46W中的一个或多个的氨基酸回复突变；或
    v)在SEQ ID NO：72所示的轻链可变区上具有选自A43S，I48V和F71Y中的一个或多个的氨基酸回复突变。
  18. 根据权利要求17所述的药物组合物，其中所述抗IL-5人源化抗体包含如 SEQ ID NO：47或48所示的轻链可变区；或
    包含如SEQ ID NO：55或56所示的轻链可变区；或
    包含如SEQ ID NO：61或62所示的轻链可变区；或
    包含如SEQ ID NO：68所示的轻链可变区；或
    包含如SEQ ID NO：73或74所示的轻链可变区。
  19. 根据权利要求12所述的药物组合物，其中所述的抗IL-5人源化抗体包含：
    i)如SEQ ID NO：49、50、51中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：46、47、48中任一所示或与其具有95％序列同一性的轻链可变区；
    ii)如SEQ ID NO：57、58、59中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：54、55、56中任一所示或与其具有95％序列同一性的轻链可变区；
    iii)如SEQ ID NO：63、64、65、66中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：60、61、62中任一所示或与其具有95％序列同一性的轻链可变区；
    iv)如SEQ ID NO：69、70、71中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：67、68任一所示或与其具有95％序列同一性的轻链可变区；或
    v)如SEQ ID NO：75、76、77、78、79中任一所示或与其具有95％序列同一性的重链可变区和如SEQ ID NO：72、73、74中任一所示或与其具有95％序列同一性的轻链可变区；优选地，所述的抗IL-5人源化抗体包含：
    a)SEQ ID NO：51所示的重链可变区和SEQ ID NO：47所示的轻链可变区；
    b)SEQ ID NO：65所示的重链可变区和SEQ ID NO：62所示的轻链可变区；
    c)SEQ ID NO：58所示的重链可变区和SEQ ID NO：56所示的轻链可变区；
    d)SEQ ID NO：71所示的重链可变区和SEQ ID NO：68所示的轻链可变区；或
    e)SEQ ID NO：79所示的重链可变区和SEQ ID NO：73所示的轻链可变区。
  20. 根据权利要求1至19中任一项所述的药物组合物，其中所述抗IL-5抗体包括人抗体恒定区；优选包含SEQ ID NO：52所示的人抗体重链恒定区和SEQ ID NO：53所示的人抗体轻链恒定区。
  21. 根据权利要求20所述的药物组合物，所述抗IL-5抗体包含：
    i)SEQ ID NO：83所示的重链和SEQ ID NO：84所示的轻链；
    ii)SEQ ID NO：85所示的重链和SEQ ID NO：86所示的轻链；
    iii)SEQ ID NO：87所示的重链和SEQ ID NO：88所示的轻链；
    iv)SEQ ID NO：89所示的重链和SEQ ID NO：90所示的轻链；或
    v)SEQ ID NO：91所示的重链和SEQ ID NO：92所示的轻链。
  22. 根据权利要求1至21中任一项所述的药物组合物，其中所述抗IL-5抗体是一种与权利要求1至21中任一项所述的抗IL-5抗体或其抗原结合片段竞争结合IL-5的单克隆抗体或其抗原结合片段。
  23. 制备权利要求1至22中任一项所述的药物组合物的方法，包括将抗IL-5抗体或其抗原结合片段原液经缓冲液置换的步骤，其中所述的缓冲液优选为醋酸-醋酸钠缓冲液。
  24. 一种含抗IL-5抗体或其抗原结合片段的冻干制剂，所述冻干制剂通过将权利要求1至22中任一项所述的药物组合物经冷冻干燥获得；优选地，所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
  25. 一种含有抗IL-5抗体或其抗原结合片段的复溶溶液，其中所述的复溶溶液是通过将权利要求24所述的冻干制剂经复溶获得。
  26. 一种制品，其包括容器，该容器中装有如权利要求1至22中任一项所述的药物组合物，或权利要求24所述的冻干制剂，或权利要求25所述的复溶溶液。
  27. 一种治疗IL-5介导的疾病的方法，包括向有此需要的受试者施用治疗有效量的根据权利要求1至22中任一项所述的药物组合物，或权利要求24所述的冻干制剂，或权利要求25所述的复溶溶液，或权利要求26所述制品，其中所述的IL-5介导的疾病优选为哮喘、慢性肺炎、过敏性鼻炎、过敏性支气管肺曲霉病、嗜酸粒细胞增多症、Churg-Strauss综合征、特应性皮炎、盘尾丝虫皮炎、间歇性血管水肿、嗜酸粒细胞性肌痛综合征、嗜酸粒细胞性胃肠炎、蠕虫感染、何杰金氏病、鼻息肉、Loeffler’s综合征、荨麻疹、嗜酸粒细胞过度增多性支气管炎、结节性动脉炎、鼻窦炎、嗜酸粒细胞性食管炎、过敏性嗜酸粒细胞性食管炎、变应性结膜炎、盘尾丝虫皮炎、子宫内膜异位或类固醇依赖性嗜酸细胞性支气管炎。
PCT/CN2020/081701 2019-03-29 2020-03-27 包含抗il-5抗体的药物组合物及其用途 Ceased WO2020200099A1 (zh)

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AU2020251627A AU2020251627B2 (en) 2019-03-29 2020-03-27 Pharmaceutical composition containing antibody against IL-5 and use thereof
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EP20782113.3A EP3939611A4 (en) 2019-03-29 2020-03-27 PHARMACEUTICAL COMPOSITION CONTAINING ANTI-IL-5 ANTIBODY AND USE THEREOF
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BR112021019272A BR112021019272A2 (pt) 2019-03-29 2020-03-27 Composição farmacêutica contendo anticorpo contra il-5 e uso da mesma
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