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WO2022116858A1 - 一种抗tslp抗体药物组合物及其用途 - Google Patents

一种抗tslp抗体药物组合物及其用途 Download PDF

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Publication number
WO2022116858A1
WO2022116858A1 PCT/CN2021/132037 CN2021132037W WO2022116858A1 WO 2022116858 A1 WO2022116858 A1 WO 2022116858A1 CN 2021132037 W CN2021132037 W CN 2021132037W WO 2022116858 A1 WO2022116858 A1 WO 2022116858A1
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Prior art keywords
seq
pharmaceutical composition
antibody
tslp
heavy chain
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PCT/CN2021/132037
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English (en)
French (fr)
Inventor
王志万
吴婷婷
刘洵
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CA3200767A priority Critical patent/CA3200767A1/en
Priority to EP21899892.0A priority patent/EP4257604A4/en
Priority to JP2023534070A priority patent/JP2024500308A/ja
Priority to MX2023006596A priority patent/MX2023006596A/es
Priority to KR1020237021784A priority patent/KR20230116857A/ko
Priority to US18/039,604 priority patent/US20240016931A1/en
Priority to CN202180081285.4A priority patent/CN116601295A/zh
Publication of WO2022116858A1 publication Critical patent/WO2022116858A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • 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
    • C07K2317/92—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

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition comprising an anti-TSLP antibody and its use as a medicine for treating diseases or conditions.
  • Asthma is a serious chronic inflammatory disease of the airways, and there are approximately 334 million asthma patients worldwide. As the environment deteriorates and air pollution increases, more people may suffer from this disease, which seriously endangers human life and health.
  • Thymic stromal lymphopoietin is a cytokine produced by epithelial cells in response to pro-inflammatory stimuli, and promotes allergic inflammatory responses mainly through its activity on dendritic cells and mast cells.
  • TSLP is an interleukin-7 (IL-7)-like cytokine first discovered in the conditioned medium of thymic stromal cells in mice.
  • IL-7 interleukin-7
  • TSLP is mainly expressed in lung, skin and intestinal epithelial cells.
  • TSLP is composed of four ⁇ -helices and two loops of AB and CD. There are three pairs of disulfide bonds composed of six cysteines in the molecule, two N glycosylation sites, and the molecular weight is about 15-20kD.
  • the receptor of TSLP is a complex, including two parts, one part is TSLPR and the other part is IL-7R ⁇ .
  • TSLP first binds to TSLPR with relatively low affinity, then recruits IL-7R ⁇ with high affinity, and finally activates signaling pathways such as stat5, leading to the maturation of DCs and the differentiation of T cells.
  • Bone marrow-derived dendritic cells are the main effector cells of TSLP.
  • TSLP acts on immature mDCs, and mDCs secrete cytokines IL-8, eotaxin-2, TARC and MDC, and at the same time highly express OX40L. .
  • OX40L binds to native CD4+ T cells to differentiate into Th2 cells, which in turn secrete Th2 cytokines such as IL-5, IL-4, IL-9, IL-13 and TNF , induce the body's Th2 inflammatory response.
  • TSLP can also induce DC cells to produce the cytokine IL-8, which in turn recruits neutrophils, leading to neutrophil innate immune inflammation.
  • TSLP can also induce DC to produce eotaxin-2, eotaxin-2 recruits eosinophils, and works together with IL-5 to rapidly enter the inflammatory state of eosinophil infiltration.
  • TSLP also acts on mast cells, natural killer cells, and mediates natural inflammation by inducing the production of IL-4, IL-6, IgE, etc.
  • TSLP can cause both natural inflammation and Th2 inflammation, which in turn increases tissue mucus, airway remodeling leads to tracheal stenosis, and severe cellular fibrosis, which gradually evolves into three major allergic diseases, including asthma, allergic dermatitis, and allergic rhinitis. disease. Therefore, blocking TSLP is a potentially effective strategy for the treatment of diseases such as asthma and atopic dermatitis.
  • the present disclosure provides a pharmaceutical composition of an anti-TSLP antibody and uses thereof.
  • the aforementioned pharmaceutical composition comprises an anti-TSLP antibody and a buffer, wherein the buffer is a histidine buffer or a succinate buffer.
  • the aforementioned pharmaceutical composition comprises an anti-TSLP antibody and a buffer, wherein the buffer is a histidine buffer.
  • the aforementioned pharmaceutical composition wherein the buffer is a histidine-acetate buffer or a histidine-hydrochloride buffer.
  • the aforementioned pharmaceutical composition wherein the buffer is a succinic acid-sodium succinate buffer.
  • the aforementioned pharmaceutical composition wherein the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 26, SEQ ID NO: 94 and SEQ ID NO: 28, respectively;
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:29, SEQ ID NO:113 and SEQ ID NO:31, respectively;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, respectively;
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:70, SEQ ID NO:24 and SEQ ID NO:25, respectively;
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 45, respectively;
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 54, respectively; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, respectively;
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, respectively.
  • the aforementioned pharmaceutical composition wherein the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region as shown in any one of the following:
  • the aforementioned pharmaceutical composition, wherein the anti-TSLP antibody comprises:
  • the aforementioned pharmaceutical composition wherein the concentration of the buffer is 5mM-50mM, including but not limited to 5mM-10mM, 10mM-15mM, 15mM-25mM, 25mM-35mM or 35mM-45mM.
  • the aforementioned pharmaceutical composition wherein the concentration of the buffer is 10 mM-30 mM.
  • the aforementioned pharmaceutical composition wherein the concentration of the buffer is 5mM-50mM, including but not limited to 5mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, and these points Any range between values.
  • the aforementioned pharmaceutical composition wherein the concentration of the buffer is 10 mM-20 mM.
  • the aforementioned pharmaceutical composition wherein the concentration of the buffer is about 20 mM.
  • the aforementioned pharmaceutical composition wherein the pH of the buffer is 5.0-6.5, including but not limited to 5.0-5.5, 5.5-6.0, or 6.0-6.5.
  • the aforementioned pharmaceutical composition wherein the pH of the buffer is 5.0-6.5, including but not limited to 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 , 6.1, 6.2, 6.3, 6.4, 6.5, and any range between these point values.
  • the aforementioned pharmaceutical composition wherein the pH of the buffer is 5.5-6.5.
  • the aforementioned pharmaceutical composition wherein the pH of the buffer is about 6.0.
  • the aforementioned pharmaceutical composition wherein the pH of the buffer is about 6.2.
  • the pH of the aforementioned pharmaceutical compositions may drift by about ⁇ 0.3.
  • the aforementioned pharmaceutical composition wherein the pH of the pharmaceutical combination is 5.0-6.5.
  • the aforementioned pharmaceutical composition wherein the pH of the pharmaceutical combination is 5.5-6.5.
  • the aforementioned pharmaceutical composition wherein the pH of the pharmaceutical combination is about 5.8-6.3.
  • the aforementioned pharmaceutical composition wherein the pH of the pharmaceutical combination is about 6.0-6.3.
  • the aforementioned pharmaceutical composition wherein the pH of the pharmaceutical combination is about 6.3.
  • the aforementioned pharmaceutical composition wherein the concentration of the anti-TSLP antibody is 1 mg/mL-150 mg/mL, including but not limited to 80 mg/mL-150 mg/mL, 80 mg/mL-120 mg/mL, 80mg/mL-100mg/mL, 100mg/mL-150mg/mL or 100mg/mL-120mg/mL.
  • the aforementioned pharmaceutical composition wherein the concentration of the anti-TSLP antibody is 1 mg/mL-150 mg/mL, including but not limited to 1 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, and these Any range between point values.
  • the aforementioned pharmaceutical composition wherein the concentration of the anti-TSLP antibody is 100 mg/mL-150 mg/mL.
  • the aforementioned pharmaceutical composition wherein the concentration of the anti-TSLP antibody is about 100 mg/mL.
  • the aforementioned pharmaceutical composition wherein the pharmaceutical composition further comprises a surfactant.
  • the aforementioned pharmaceutical composition wherein the surfactant is a polysorbate.
  • the aforementioned pharmaceutical composition wherein the surfactant is polysorbate 80 or polysorbate 20.
  • the aforementioned pharmaceutical composition wherein the surfactant is polysorbate 80.
  • the aforementioned pharmaceutical composition wherein the surfactant concentration is 0.01 mg/mL-1.0 mg/mL, including but not limited to 0.05 mg/mL-1.0 mg/mL, 0.05 mg/mL-0.8 mg/mL, 0.1mg/mL-1.0mg/mL, 0.1mg/mL-0.8mg/mL, 0.1mg/mL-0.6mg/mL, 0.1mg/mL-0.4mg/mL, 0.2mg/mL-1.0 mg/mL, 0.2 mg/mL-0.8 mg/mL, 0.2 mg/mL-0.6 mg/mL, or 0.2 mg/mL-0.4 mg/mL.
  • the aforementioned pharmaceutical composition wherein the surfactant concentration is from 0.1 mg/mL to 0.8 mg/mL.
  • the aforementioned pharmaceutical composition wherein the surfactant concentration is 0.01 mg/mL-1.0 mg/mL, including but not limited to 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.15 mg/mL mg/mL, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, 0.35mg/mL, 0.4mg/mL, 0.45mg/mL, 0.5mg/mL, 0.55mg/mL, 0.6mg/mL, 0.65 mg/mL, 0.7 mg/mL, 0.75 mg/mL, 0.8 mg/mL, 0.85 mg/mL, 0.9 mg/mL, 0.95 mg/mL, 1.0 mg/mL, and any range between these point values.
  • the aforementioned pharmaceutical composition wherein the surfactant concentration is about 0.8 mg/mL.
  • the aforementioned pharmaceutical composition wherein the composition further comprises a stabilizer, wherein the stabilizer is selected from one or more of sugars, amino acids, and EDTA.
  • the aforementioned pharmaceutical composition wherein the sugar is selected from one or more of trehalose, sucrose, sorbitol, and mannitol.
  • the aforementioned pharmaceutical composition wherein the amino acid is selected from one or more of histidine, tryptophan, and methionine.
  • the aforementioned pharmaceutical composition wherein the stabilizer is sucrose.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a saccharide at a concentration of 20 mg/mL-100 mg/mL, including but not limited to 30 mg/mL-90 mg/mL, 30 mg/mL-70 mg/mL , 50mg/mL-90mg/mL or 50mg/mL-70mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a sugar, and its concentration is 30 mg/mL-100 mg/mL, including but not limited to 40 mg/mL-90 mg/mL, 40 mg/mL-80 mg/mL , 50mg/mL-80mg/mL, 60mg/mL-90mg/mL or 60mg/mL-80mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a saccharide at a concentration of 20 mg/mL-100 mg/mL, including but not limited to 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL , 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, and any range between these point values.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a sugar at a concentration of 50 mg/mL to 70 mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a sugar at a concentration of 50 mg/mL to 60 mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a sugar at a concentration of about 70 mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a sugar, and its concentration is 30 mg/mL-70 mg/mL, wherein the sugar is trehalose, sucrose, sorbitol or mannitol.
  • the aforementioned pharmaceutical composition wherein the stabilizer is a sugar, wherein the sugar is trehalose at a concentration of about 60 mg/mL to 70 mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is sucrose at a concentration of 70 mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer is sorbitol or mannitol at a concentration of 50 mg/mL.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 5mM-50mM, including but not limited to 10mM-50mM, 10mM-40mM, 10mM-30mM, or 10mM-20mM.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 5mM-50mM, including but not limited to 5mM-40mM, 5mM-30mM, 5mM-20mM, 20mM-30mM, 20mM- 40mM or 30mM-40mM.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 10 mM-30 mM.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of about 30 mM.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 5mM-50mM, including but not limited to 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, and any range between these point values.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 5 mM to 50 mM, wherein the amino acid is selected from the group consisting of histidine, tryptophan and methionine.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 10 mM to 30 mM, wherein the amino acid is selected from the group consisting of histidine, tryptophan and methionine.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises an amino acid at a concentration of 30 mM, wherein the amino acid is selected from the group consisting of histidine, tryptophan and methionine.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises EDTA at a concentration of 0.1 mM-10 mM, including but not limited to 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 1 mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, and any range between these point values.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises EDTA at a concentration of 0.1 mM-10 mM, including but not limited to 0.1 mM-8 mM, 0.1 mM-6 mM, 0.1 mM-4 mM or 0.1 mM -2mM.
  • the aforementioned pharmaceutical composition wherein the stabilizer further comprises EDTA at a concentration of 0.37mM-10mM, including but not limited to 0.37mM-9mM, 0.37mM-7mM, 0.37mM-5mM, 0.37mM -3mM.
  • the aforementioned pharmaceutical composition wherein the stabilizer is 30mg/mL-70mg/mL sugar and 10mM-30mM amino acid; wherein the sugar is selected from trehalose, sucrose, sorbitol and mannose One or more of alcohols, wherein the amino acid is selected from one or more of histidine, tryptophan and methionine.
  • the aforementioned pharmaceutical composition wherein the stabilizer is 30mg/mL-70mg/mL sugar and 0.37mM-10mM EDTA; wherein the sugar is selected from trehalose, sucrose, sorbitol and One or more of mannitol.
  • the aforementioned pharmaceutical composition wherein the stabilizer is 30mg/mL-70mg/mL sugar, 10mM-30mM amino acid and 0.37mM-10mM EDTA; wherein the sugar is selected from trehalose, One or more of sucrose, sorbitol and mannitol, wherein the amino acid is selected from one or more of histidine, tryptophan and methionine.
  • the aforementioned pharmaceutical composition, wherein the stabilizer is:
  • sugar selected from trehalose, sucrose, sorbitol and mannitol, wherein said amino acid is selected from histidine, tryptophan and methionine.
  • the aforementioned pharmaceutical composition, wherein the stabilizer is:
  • the aforementioned pharmaceutical composition, wherein the stabilizer is:
  • the aforementioned pharmaceutical composition wherein the pH of the pharmaceutical composition is 5.0-6.5. In some embodiments, the aforementioned pharmaceutical composition, wherein the pH of the pharmaceutical composition is 5.5-6.5. In some embodiments, the aforementioned pharmaceutical composition, wherein the pH of the pharmaceutical composition is about 6.0-6.3.
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition wherein the sugar is selected from trehalose, sucrose, sorbitol and mannitol; and/or the polysorbate is selected from polysorbate 20 and polysorbate 80; And/or the amino acid is selected from histidine, tryptophan and methionine; and/or the histidine buffer is selected from histidine-hydrochloride buffer and histidine- Acetate buffer.
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • anti-TSLP antibody 100mg/mL-150mg/mL of anti-TSLP antibody, wherein said anti-TSLP antibody comprises a heavy chain as shown in SEQ ID NO:139 and a light chain as shown in SEQ ID NO:140;
  • the pharmaceutical composition comprises:
  • anti-TSLP antibody 100mg/mL-150mg/mL of anti-TSLP antibody, wherein said anti-TSLP antibody comprises a heavy chain as shown in SEQ ID NO:139 and a light chain as shown in SEQ ID NO:140;
  • the pharmaceutical composition comprises:
  • anti-TSLP antibody 100mg/mL-150mg/mL of anti-TSLP antibody, wherein said anti-TSLP antibody comprises a heavy chain as shown in SEQ ID NO:139 and a light chain as shown in SEQ ID NO:140;
  • the sugar is selected from trehalose, sucrose, sorbitol and mannitol,
  • the amino acid is selected from histidine, tryptophan and methionine,
  • the polysorbate is selected from polysorbate 80 and polysorbate 20; and
  • the pH of the pharmaceutical composition is 5.5-6.5; preferably, the pH is about 6.0-6.3.
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • an anti-TSLP antibody 100 mg/mL of an anti-TSLP antibody, wherein the anti-TSLP antibody comprises a heavy chain as shown in SEQ ID NO: 139 and a light chain as shown in SEQ ID NO: 140;
  • (b) 0.8 mg/mL 20 mM histidine-hydrochloride buffer;
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the aforementioned pharmaceutical composition comprising the following components:
  • the present disclosure also provides a lyophilized formulation comprising an anti-TSLP antibody, wherein the lyophilized formulation is obtained by lyophilizing the pharmaceutical composition as described in any of the foregoing.
  • the present disclosure also provides a method of preparing the pharmaceutical composition of any preceding one, the method comprising the step of buffer-exchanging a stock solution of an anti-TSLP antibody.
  • the present disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition as hereinbefore described or a lyophilized formulation as hereinbefore described.
  • the present disclosure also provides a method of treating a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the foregoing, or a lyophilized formulation as described above.
  • the present disclosure also provides the use of the pharmaceutical composition as described in any of the foregoing, or the use of the lyophilized preparation as described above in the preparation of a medicament for treating a disease or condition.
  • a pharmaceutical composition as described in any of the foregoing, or a lyophilized formulation as described above, can be used as a medicament for the treatment of a disease or disorder.
  • the disease or disorder is selected from allergic diseases, cancer, and immune diseases;
  • the allergic diseases are selected from the group consisting of: asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, Allergic rhinitis, allergic sinusitis, urticaria, Netherton Syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchitis Pulmonary aspergillosis, allergic fungal sinusitis and chronic pruritus;
  • the cancer is selected from: breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer;
  • the immune disease is selected from: rheumatoid arthritis, chronic obstructive Lung disease, systemic sclerosis, multiple sclerosis, keloids, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Chur
  • the aforementioned disease or disorder is associated with TSLP.
  • Figure 1 Results of antibodies blocking the binding activity of TSLP to the TSLP receptor.
  • Figure 2 Results of antibodies blocking the binding activity of TSLP to cell surface TSLP receptors.
  • Figure 3 Antibodies inhibit TSLP-induced proliferative activity of BaF3 cells.
  • Figure 4A Antibody activity to inhibit TSLP-induced chemokine TARC production.
  • Figure 4B Antibody activity to inhibit TSLP-induced chemokine OPG production.
  • Figure 5A Antibody activity to inhibit the production of the Th2 cytokine IL-13.
  • Figure 5B Antibody activity to inhibit the production of the Th2 cytokine IL-4.
  • Figure 5C Antibody activity to inhibit the production of the Th2 cytokine TNF-[alpha].
  • Figure 5D Antibody activity to inhibit the production of the Th2 cytokine IL-5.
  • TSLP thymic stromal lymphopoietin
  • IL-7 interleukin-7
  • DC dendritic cells
  • TSLP includes variants, isoforms, homologs, orthologs and paralogs of TSLP.
  • Buffer refers to a buffer that resists changes in pH through the action of its acid-base conjugated component.
  • buffers that control pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumaric acid Salt, glycylglycine and other organic acid buffers.
  • a “histidine buffer” is a buffer containing histidine ions.
  • histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and the like, preferably histidine-acetate Buffer or histidine-hydrochloride buffer; histidine-acetate buffer is prepared from histidine and acetic acid; histidine-hydrochloride buffer is prepared from histidine and hydrochloric acid .
  • citrate buffer is a buffer that includes citrate ions (ie, citrate ions).
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • a preferred citrate buffer is citrate-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, and the like.
  • a preferred succinate buffer is succinate-sodium succinate.
  • the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or can be prepared from succinic acid and sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • phosphate buffers 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 hydrogen phosphate.
  • acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, acetate-histidine, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate.
  • “Surfactant” refers to a surface-active agent, preferably a nonionic surfactant.
  • the use of surfactants can reduce protein aggregation and/or particle formation in the formulation.
  • the amount of surfactant added is such that it reduces aggregation of proteins in the formulation and minimizes particle formation.
  • the surfactants of the present disclosure may be selected from polysorbates (including but not limited to polysorbate 20, polysorbate 80), polyhydroxyalkenes, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, octyl Sodium Glycoside, Lauryl-Sulfobetaine, Myristyl-Sulfobetaine, Linoleyl-Sulfobetaine, Stearyl-Sulfobetaine, Lauryl-Saccharine, Myristyl - sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine , Coca-Amidopropyl-Betaine, Linoleamidopropyl-Betaine, Myristamidopropyl-Betaine, Palmamidopropyl-
  • Stabilizer refers to a component that helps maintain the structural integrity of a biopharmaceutical drug, especially during freezing and/or lyophilization and/or storage (especially when exposed to stress). This stabilizing effect can occur for a variety of reasons, usually the stabilizing agent acts as a penetrant that reduces protein denaturation.
  • stabilizers include sugars, amino acids, and EDTA; amino acids in stabilizers described herein are amino acids added in addition to those in the buffer.
  • the "sugars” of the present disclosure include conventional compositions (CH2O ) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like.
  • Sugars in the present disclosure may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol , Milibiose, melanotriose, raffinose, mannose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.
  • Preferred sugars are sucrose, trehalose, sorbitol and mannitol, more preferably trehalose or sucrose, most preferably sucrose.
  • Displacement refers to the displacement of the solvent system that dissolves the antibody protein, eg, the presence of the antibody protein in the stable formulation by physically manipulating a high-salt or hypertonic solvent system containing the antibody protein using the buffer system of the stable formulation.
  • the so-called physical manipulations include, but are not limited to, ultrafiltration, dialysis or reconstitution after centrifugation.
  • the terms “about”, “approximately” refer to an index value within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, “about” or “substantially comprising” may mean a range of ⁇ 10% of the specific numerical value indicated thereafter. Furthermore, particularly with respect to biological systems or processes, the term can mean up to one order of magnitude or up to five times the value. Unless stated otherwise, when a specific value appears in this application and in the claims, the meaning of "about” or “substantially comprising” should be assumed to be within an acceptable error range for the specific value.
  • “Pharmaceutical composition” means a mixture comprising one or more of the antibody drug conjugates described herein, or a physiological/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiological/pharmaceutical Pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, promote the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • the solvent therein is all water.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably a drug
  • the composition substantially retains its physical and chemical stability and its biological activity after storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • analytical techniques for measuring protein stability which can measure stability after storage at a selected temperature for a selected period of time.
  • a stable formulation is one in which no significant change is observed when stored at refrigerated temperature (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, even more preferably up to 2 years.
  • stable liquid formulations include liquid formulations that exhibit desirable characteristics after storage at temperatures including 25°C for periods of time including 1 month, 3 months, and 6 months.
  • Typical example of stability Typically no more than about 10%, preferably no more than about 5% of the antibody monomers aggregate or degrade as measured by SEC-HPLC. By visual analysis, the formulation was a colorless to pale yellow, clear to slightly opalescent liquid. The formulations had no more than ⁇ 10% variation in concentration, pH and osmolality. A reduction of no more than about 10%, preferably no more than about 5%, is generally observed. Typically no more than about 10% aggregates are formed, preferably no more than about 5% aggregates.
  • An Antibody Drug Conjugate that does not exhibit a significant increase in aggregation, precipitation and/or denaturation then "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An antibody "retains its chemical stability” in a pharmaceutical formulation if it does not exhibit significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that frequently change the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS (capillary electrophoresis sodiu dodecyl sulfate)), oxidation (by methods such as mass spectrometry or MALDI/TOF/ MS combined peptide mapping, etc.), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.), and isomerization (measured by isoaspartic acid content, peptide mapping, etc.).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation.
  • “Lyophilized formulation” means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.
  • antibody in this disclosure is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies or antigen binding thereof Fragments (also referred to as "antigen-binding portions”) so long as they exhibit the desired antigen-binding activity.
  • Full-length antibodies are immunoglobulins (Ig) comprising at least two heavy chains and two light chains interconnected by disulfide bonds.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE.
  • the heavy chains are ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ chains, respectively.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
  • VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • FRs conserved framework regions
  • CDRs complementarity determining regions
  • VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs).
  • complementarity determining region refer to the region within the variable domain that mainly contributes to antigen binding
  • framework or "FR” refers to the variable domain residues other than CDR residues.
  • VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3
  • VL contains 3 CDR regions: LCDR1, LCDR2, and LCDR3.
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a single VH or VL may be sufficient to confer antigen-binding specificity.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, for example: the "Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia” numbering scheme, “ABM” numbering scheme, "contact” numbering scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278) et al.
  • the relationship between the various numbering systems is well known to those skilled in the art and is shown in Table 1 below.
  • antigen-binding fragment or “functional fragment” or “antigen-binding portion” refers to one or more fragments of an intact antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment” include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) a VH consisting of a single arm of the antibody Fv fragments composed of and VL domains; (v) dsFv, a stable antigen-binding fragment formed by VH and VL through interchain disulfide bonds; (vi) scFv; Antibodies, Bispecific Antibodies and Multispecific Antibodies.
  • single-chain antibody single-chain Fv or “scFv” is meant to comprise an antibody heavy chain variable domain (or region, VH) and an antibody light chain variable domain (or region, VL) joined by a linker molecule.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Numerous linkers suitable for linking antibody VH and VL have been disclosed in the prior art, for example consisting of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl . Acad. Sci.
  • Diabodies are antibody fragments in which scFv or Fab are dimerized, and are antibody fragments having bivalent antigen-binding activity. In bivalent antigen binding activity, the two antigens can be the same or different.
  • Bispecific and multispecific antibodies refer to antibodies that bind two or more antigens or antigenic determinants simultaneously, including scFv or Fab fragments that bind TSLP.
  • inventions of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region
  • exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed reengineering and amino acid substitutions of the heavy chain constant region, specifically replacing YTE as known in the art Mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations that acquire a knob-into-hole structure (giving the antibody heavy chain a combination of knob-Fc and hole-Fc) that have been shown to confer antibodies with novel performance, but does not alter the function of the variable region of the antibody.
  • full-length antibody intact antibody
  • complete antibody completely antibody
  • whole antibody whole antibody
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody binds with an affinity (KD) of about less than 10-8 M, eg, about less than 10-9 M, 10-10 M , 10-11 M, 10-12 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • an antibody of the present disclosure binds TSLP with a dissociation equilibrium constant (KD) of less than about 10-7 M, eg, less than about 10-8 M or 10-9 M, eg, the affinity of the antibody for a cell surface antigen in the present disclosure KD values were determined by FACS or Biacore method.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Amino acid sequence identity refers to the alignment of amino acid sequences and, where necessary, the introduction of gaps to achieve a maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity, in the first sequence to the second sequence.
  • the percentage of amino acid residues that are identical For purposes of determining percent amino acid sequence identity, alignment can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • mice can be immunized with human TSLP or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the present disclosure are genetically engineered to add one or more human FR regions to non-human CDR regions.
  • Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind human TSLP. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -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 the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, eg, a composition comprising any of the binding compounds of the present disclosure, to a patient having one or more disease symptoms for which the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a patient or population to be treated, to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • Exemplary conservative substitutions are set forth in "Exemplary amino acid conservative substitutions" below in Table 2.
  • an “effective amount” or “effective dose” refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of disorders, including biochemical, tissue academic and/or behavioral symptoms.
  • beneficial or desired outcomes include clinical outcomes, such as reducing the incidence or amelioration of one or more symptoms of the various target antigen-related disorders of the present disclosure, reducing the amount of other agents required to treat the disorder dose, enhances the efficacy of another agent, and/or delays the progression of a disorder associated with the target antigen of the present disclosure in a patient.
  • Exogenous refers to a substance produced outside an organism, cell, or human body, as the case may be.
  • Endogenous refers to a substance produced in a cell, organism, or human body as the case may be.
  • Homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position . The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100.
  • sequences when sequences are optimally aligned, two sequences are 60% homologous if 6 matches or homology at 10 positions in the two sequences; if 95 matches at 100 positions in the two sequences or homologous, then the two sequences are 95% homologous.
  • comparisons are made when aligning two sequences to give the greatest percent homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are chosen to give the maximum match between the respective sequences over the entire length of the respective reference sequences.
  • the following references refer to BLAST algorithms frequently used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.
  • the disease associated with TSLP in the present disclosure is not limited as long as it is a disease associated with TSLP, for example, a therapeutic response induced using the antibodies of the present disclosure can be achieved by binding to human TSLP, then repressing TSLP binding to its receptor, or killing overexpression TSLP cells; or inhibit the growth of cells overexpressing TSLP.
  • Example 1-1 Expression of TSLP and TSLP receptors
  • HEK293E The sequences encoding human TSLP with His tag, cynomolgus monkey TSLP, human TSLP with human IgG1-Fc tag, cynomolgus monkey TSLP, and human TSLP receptor extracellular region sequences were cloned into the phr vector, constructed into an expression plasmid, and then Transfection of HEK293E.
  • the specific transfection steps were as follows: HEK293E cells were inoculated into Freestyle expression medium (containing 1% FBS) at 0.8 ⁇ 10 6 /mL the day before, and placed in a constant temperature shaker (120 rpm) at 37° C. for 24 hours.
  • the transfection plasmid and transfection reagent PEI were sterilized with a 0.22 ⁇ m filter, and then the transfection plasmid was adjusted to 100 ⁇ g/100 mL cells, and the mass ratio of PEI (1 mg/mL) and plasmid was 3:1.
  • the transfection of 200mL HEK293E cells as an example, mix 10mL Opti-MEM and 200 ⁇ g plasmid, and let stand for 5 minutes (min); take another 10mL Opti-MEM and 600 ⁇ g PEI, mix well, and let stand for 5 minutes.
  • the plasmid and PEI were mixed and allowed to stand for 15 min.
  • the plasmid and PEI mixture was slowly added to 200 mL of HEK293E cells, and cultured in a shaker at 8% CO 2 , 120 rpm, and 37°C. On day 3 of transfection, feed medium was supplemented with 10% volume. On the 6th day of transfection, sample and centrifuge at 4500rpm for 10min to collect the cell supernatant, filter, and purify the recombinant TSLP and TSLP receptor protein supernatant according to Examples 1-2. The purified protein can be used in the experiments of the following examples. . The correlation sequence is shown below.
  • the underline is the signal peptide sequence; the italicized part is the Flag-His6-tag tag.
  • the underline is the signal peptide sequence; the italicized part is the linker-human Fc-tag tag.
  • the underline is the signal peptide sequence; the italicized part is the flag-His6-tag mark.
  • the underline is the signal peptide sequence; the italicized part is the linker-human Fc-tag tag.
  • Fc-tagged human TSLP receptor extracellular domain (human TSLPR-Fc-ECD) amino acid sequence
  • the underlined part is the extracellular region of human TSLPR, and the italicized part is the linker-human Fc-tag.
  • the cell expression supernatant was centrifuged at high speed to remove impurities, filtered, the nickel column was equilibrated with PBS solution, and washed 10 times the column volume. The filtered supernatant was applied to the column. The column was rinsed with 30 mM imidazole in PBS until the A280 reading dropped to baseline. The target protein was eluted with a PBS solution containing 300 mM imidazole, and the elution peaks were collected. PBS was concentrated and exchanged, and LC-MS was identified as correct and then used. His-tagged human TSLP and cynomolgus TSLP were obtained.
  • the cell expression supernatant was centrifuged at high speed to remove impurities, and the recombinant antibody expression supernatant was purified with Protein A column.
  • the column was rinsed with PBS until the A280 reading dropped to baseline.
  • the target protein was eluted with 100 mM acetic acid (pH 3.5) and neutralized with 1 M Tris-HCl, pH 8.0. Concentrate and change the medium, and the obtained protein was identified as correct by electrophoresis and LC-MS, and then separated for use.
  • CHO-K1 and BaF3 cell lines expressing both human TSLP receptor and human IL-7R ⁇ were constructed.
  • the target gene TSLPR/IL-7R ⁇ was packaged by lentivirus and cloned into the target cell line to form a stable high-expression cell line.
  • human TSLPR and human IL-7R ⁇ genes were cloned into pCDH-CMV-MCS-EF1-puro and pCDH-CMV-MCS-EF1-Neo (SBI, CD500B-1) plasmids, respectively, and then by lentivirus infection.
  • Human TSLPR was cloned into CHO-K1 and BaF3 cell lines, and was selected and cultured for three weeks under the selection pressure of 10 ⁇ g/mL puromycin (Gibco, US). On this basis, a second round of infection was carried out, and the human IL-7R ⁇ gene was cloned in and screened with 1 mg/mL G418 (Gibco, US) and 10 ⁇ g/mL puromycin for two to three weeks. Finally, by flow sorting, the CHO-K1 and BaF3 monoclonal cell lines with high expression of TSLPR and IL-7R ⁇ were screened.
  • Anti-human TSLP monoclonal antibody was produced by immunizing mice, SJL white mice, female, 6-8 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012- 0001). Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, the temperature was 20-25°C, and the humidity was 40-60%.
  • mice were immunized with recombinant proteins huTSLP-Fc (25 ⁇ g), huTSLP-his (12.5 ⁇ g) and cyno TSLP-his (12.5 ⁇ g) with TiterMax, Alum or CpG adjuvant.
  • mice with high antibody titer in serum and the titer tending to a plateau were selected, sacrificed, and spleen cells were harvested and fused with myeloma cells.
  • splenic lymphocytes were combined with myeloma cells Sp2/0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells.
  • the monoclonal hybridoma cell lines 3, 119, 179 and 199 with good activity were obtained by screening, and the hybridoma cells in logarithmic growth phase were collected respectively, RNA was extracted with NucleoZol (MN), and reverse transcription (PrimeScript TM Reverse) was performed. Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sent to a sequencing company for sequencing.
  • the murine anti-TSLP antibodies were obtained by sequencing: mab3, mab119, mab179 and mab199, and the amino acid sequences of their variable regions are as follows (the underlined part is the complementarity determining region sequence):
  • mab3 murine heavy chain variable region sequence (SEQ ID NO: 6):
  • mab3 murine light chain variable region sequence (SEQ ID NO: 7):
  • mab119 murine heavy chain variable region sequence (SEQ ID NO: 8):
  • mab119 murine light chain variable region sequence (SEQ ID NO: 9):
  • mab179 murine heavy chain variable region sequence (SEQ ID NO: 10):
  • mab179 murine light chain variable region sequence (SEQ ID NO: 11):
  • mab199 murine heavy chain variable region sequence (SEQ ID NO: 12):
  • mab199 murine light chain variable region sequence (SEQ ID NO: 13):
  • amino acid sequence of the CDR region obtained according to the Kabat numbering rule is shown in Table 3 below:
  • the light and heavy chain variable regions of the above-mentioned murine antibodies are combined with the light and heavy chain constant regions of the human antibody (the kappa constant region shown in SEQ ID NO: 134 and the IgG1-YTE constant region shown in SEQ ID NO: 133).
  • the chimeric antibody corresponding to the mab3 clone is named Ch3
  • the chimeric antibody corresponding to the mab119 clone is named Ch119
  • the chimeric antibody corresponding to the mab179 clone is named Ch179
  • the chimeric antibody corresponding to the mab199 clone is named Ch179.
  • the conjugated antibody was named Ch199.
  • the selected mab3, mab119, mab179 and mab199 antibodies with excellent in vitro and in vivo activities were humanized.
  • Humanization of murine monoclonal antibodies is carried out according to methods disclosed in many literatures in the art. Briefly, CDR grafting was performed using human antibody constant domains in place of the parental (murine antibody) constant domains, and human germline antibody sequences selected based on the homology of the murine and human antibodies. Then, based on the three-dimensional structure of the murine antibody, by backmutating the amino acid residues of VL and VH, the constant region of the murine antibody is replaced with a human constant region to obtain the final humanized molecule.
  • the humanized VH template of mab3 is IGHV1-3*01+IGHJ6*01, and the humanized VL template is IGKV3-20+IGKJ4*01.
  • the CDR of mab3 is transplanted to the human template, and the variable obtained after transplantation is The sequence of regions is as follows:
  • Grafted represents the murine antibody CDR implanted into the human germline FR region sequence
  • L46P represents the mutation of position 46 L back to P according to the Kabat numbering system.
  • variable region sequence of the mab3 humanized antibody obtained after transplantation is as follows:
  • hu3VL1 (hu3 VL-CDR grafted) (SEQ ID NO:38):
  • hu3VH1 (hu3 VH-CDR Grafted) (SEQ ID NO: 42):
  • the single underlined part is the CDR region, and the double underlined part is the reverse mutation site.
  • the heavy chain constant region of the mab3 humanized antibody in the present disclosure is the IgG1-YTE constant region shown in SEQ ID NO: 133
  • the light chain constant region is the kappa chain constant region shown in SEQ ID NO: 134 , but can also be replaced with other constant regions known in the art.
  • the binding activity of the mab3 humanized antibody to human TSLP was detected by ELISA. The results showed that the mab3 humanized antibody had a good binding ability to human TSLP.
  • X 1 is selected from H or Y
  • X 2 is selected from N or D.
  • amino acid sequence of the light chain variable region (hu3VL4-N93D) of hu3-11 is as follows:
  • amino acid sequence of the heavy chain variable region (hu3VH2-H110Y) of hu3-11 is as follows:
  • the light and heavy chain variable regions after hot spot mutation are recombined with human germline light chain and heavy constant region sequences to form complete light and heavy chain sequences, and then antibodies with full-length sequences are obtained.
  • the molecule of hu3-11 was affinity matured.
  • the process of affinity maturation is as follows:
  • yeast library Degenerate primers were designed, and the designed mutant amino acids were introduced into the scFv mutant library of antibody hu3-11 by PCR. The size of each library was about 109. The constructed yeast library was sequenced method to verify the diversity of the library.
  • TSLP-Fc Cells with high affinity antibody library to TSLP-Fc were collected and induced for 18h at 250rpm and 20°C. The resulting enriched library was subjected to a second round of screening against biotinylated recombinant TSLP-Fc protein.
  • library cells from the previous round were mixed with biotinylated recombinant TSLP-Fc protein (0.1-1 ⁇ g/mL) and 10 ⁇ g/mL murine anti-cMyc (9E10, sigma) antibody After incubation in 0.1% PBSA for 1 hour (h) at room temperature, the mixture was washed with 0.1% PBSA to remove unbound antibody fragments. Goat anti-mouse-Alexa488 (A-11001, Life technologies) and Strepavidin-PE (S-866, Life technologies) were added and incubated at 4°C for 1 h, and the mixture was washed with 0.1% PBSA to remove unbound antibody fragments. Finally, antibodies with high affinity were screened by FACS.
  • the hu3-11-scFv mutant library was subjected to 2 rounds of MACS screening and 2 rounds of FACS screening using biotinylated TSLP-Fc antigen. Then select about 400 yeast monoclones to culture and induce expression, use FACS to detect the binding of yeast monoclones to TSLP-Fc antigen, select yeast monoclones with high affinity for sequencing verification, and compare and analyze the sequenced clones to remove redundancy. After the remaining sequences, the non-redundant sequences are converted into full-length antibodies for mammalian cell expression.
  • the light chain variable region sequence obtained by affinity maturation is as follows:
  • the single underlined part is the CDR region, and the double underlined part is the reverse mutation site.
  • the obtained light chain variable region is recombined with the heavy chain variable region of the mab3 humanized antibody to obtain a new mab3 humanized antibody.
  • huVL5 and huVL6 are combined with hu3VH2-H110Y, respectively, to obtain a new antibody Molecules hu3-12 and hu3-13, as shown in Table 9 below:
  • the obtained new mab3 humanized antibody was tested for its binding activity to human TSLP by ELISA.
  • the results showed that hu3-12 and hu3-13 still had high binding ability to human TSLP. It shows that the changes of some amino acids in LCDR3 will not affect the activity of hu3 series antibodies.
  • the CDRs of the mab3 humanized antibody have the sequences shown in Table 11 below:
  • X 1 is H or Y
  • X 3 is N or S
  • X 4 is V or G
  • X 5 is G or E.
  • the VH of mab119 selects IGHV1-69*02 and HJ6*01 as templates, VL selects IGKV4-1*01 and IGKJ2*01 and IGKV3-11*01 and IGKJ2*01 as templates, and the CDR region of the mouse antibody is transplanted to the selection On the humanized template of CDR, the FR region is backmutated to obtain different light chain and heavy chain variable regions.
  • the variable region sequences obtained by CDR transplantation are as follows:
  • hu119VL4 (Grafted, IGKV3-11*01) (SEQ ID NO: 59):
  • variable region of the mab119 humanized antibody is as follows:
  • hu119VL4 (Grafted, IGKV3-11*01) (SEQ ID NO: 59):
  • the single underlined part is the variable region, and the double underlined part is the reverse mutation.
  • the heavy chain constant region of the mab119 humanized antibody in the present disclosure is the IgG1-YTE constant region shown in SEQ ID NO: 133
  • the light chain constant region is the kappa chain constant shown in SEQ ID NO: 134. region, but can also be replaced with other constant regions known in the art.
  • the heavy and light chain variable regions of the mab119 humanized antibody are shown in Table 14.
  • the binding activity of the humanized antibody to human TSLP was detected by ELISA, and the results showed that the mab119 humanized antibody could specifically bind to human TSLP.
  • hu119VL2 and hu119VL6 mutant sequences obtained after mutation are as follows:
  • the single underlined part is the variable region, and the double underlined part is the back mutation.
  • hu119VL2, hu119VL6 mutants were combined with hu119VH to obtain new humanized hu119 antibodies, exemplarily, hu119VL2-N31S, hu119VL2-N31Q were combined with hu119VH3, respectively, resulting antibodies hu119-28 and hu119-29; hu119VL3- N31S is combined with hu119VH8 to obtain antibody hu119-30, and the variable region combination of an exemplary mutated antibody is shown in Table 16:
  • the ELISA method was used to detect the affinity of the mutated antibody with human TSLP.
  • the results showed that the hu119-28 and hu119-29 antibodies still had high affinity with human TSLP, which indicated that the N31S and N31Q mutations of LCDR2 did not affect the Anti-TSLP antibody activity.
  • the CDRs of the mab119 humanized antibody have the sequences shown in Table 17:
  • X 6 is selected from N, S and Q.
  • the VH of mab179 selects IGHV1-69*02 and IGHJ6*01 as templates, VL selects IGKV4-1*01 and IGKJ2*01 or IGKV2-29*02 and IGKJ2*01 as templates, and the CDR region of the mouse antibody is transplanted to the selected On the humanized template of , and backmutated the FR region, as shown in Table 18, light chain and heavy chain variable regions of different sequences were obtained.
  • the humanized variable region sequences and back mutations are as follows:
  • variable regions of the mab179 humanized antibody are shown below:
  • the single underlined part is the CDR, and the double underlined part is the reverse mutation site.
  • the heavy chain constant region of the mab199 humanized antibody in the present disclosure is the IgG1-YTE constant region shown in SEQ ID NO: 133
  • the light chain constant region is the kappa chain constant shown in SEQ ID NO: 134. region, but can also be replaced with other constant regions known in the art.
  • amino acid mutation is performed on GNG of HCDR2 of hu179VH1, and the mutated sequence of hu179VH1 is as follows:
  • the single underlined part is the CDR, and the double underlined part is the reverse mutation site.
  • the HCDR2 region sequence of the mab179 humanized antibody obtained after mutation is shown in Table 20:
  • the mutated CDR regions of the mab179 humanized antibody can be obtained from the above, as shown in Table 21:
  • X 7 is selected from N, Q or V, and X 8 is selected from G or V.
  • the hu179VH1 mutant obtained after mutation was combined with the humanized hu179VL to obtain a new mab179 humanized antibody.
  • An exemplary antibody combining the hu179VH1 mutant and hu179VL2 is shown in Table 22:
  • the affinity of the mutated antibody with human TSLP was detected by ELISA, and the results showed that the HCDR2 mutated antibody still maintained a high affinity with human TSLP. This indicated that the point mutations N55Q, N55V and G56V of the HCDR2 of the mab179 humanized antibody did not substantially affect the affinity activity of the antibody with TSLP.
  • N55Q, N55V and G56V point mutations (numbering in natural order) were carried out on hu179VH2, hu179VH3, hu179VH4, and hu179VH5, respectively, and the variable regions of heavy and light chains obtained by mutation were recombined to obtain a new mab179 humanized antibody .
  • the sequence of the hu179VH3 mutation is shown below:
  • the single underlined part is the CDR, and the double underlined part is the reverse mutation site.
  • the LCDR2 of the mab179 humanized antibody is subjected to amino acid mutation, and the mutated sequence of hu179VL2 is as follows:
  • the single underlined part is the CDR, and the double underlined part is the reverse mutation site.
  • the sequence of the mab179 humanized antibody LCDR2 obtained after mutation is shown in Table 23:
  • mab179 humanized antibody LCDR2 is: X 9 VX 10 X 11 X 12 X 13 T (SEQ ID NO: 118), wherein X 9 is selected from Y or E, and X 10 is selected from S, D or E, X 11 is selected from N, Q, D or E; X 12 is selected from H, Y, D or E; X 13 is selected from E or Y.
  • the CDR regions of the mab179 humanized antibody are shown in Table 24 below:
  • X 7 is selected from N, Q or V
  • X 8 is selected from G or V
  • X 9 is selected from Y or E
  • X 10 is selected from S, D or E
  • X 11 is selected from N, Q, D or E
  • X 12 is selected from H, Y, D or E
  • X 13 is selected from E or Y.
  • the hu179VL2 mutant obtained after mutation was combined with the humanized hu179 heavy chain variable region to obtain a new mab179 humanized antibody, and the exemplary hu179VL2 mutant was combined with hu179VH1 and hu179VH3 to obtain the mab179 humanized antibody
  • the CDR and heavy and light chain variable region combinations are shown in Table 25 below:
  • the affinity of the mab179 humanized antibody obtained after LCDR2 mutation with human TSLP was detected by ELISA.
  • the results showed that the antibody obtained after mutating the hot spot of LCDR2 still had a good affinity with human TSLP. It shows that the mutation of the hot spot of LCDR2 will not affect the binding activity of mab179 humanized antibody.
  • the LCDR2 of hu179VL3, hu179VL4, hu179VL5, hu179VL6, hu179VL7 and hu179VL8 were mutated with N53Q, N53D, N53S, H54Y, Y50E, S52D, S52E, N53E, H54D, H54E or Y55E.
  • the mutated light chain variable region is combined with the heavy chain variable region to form a new mab humanized antibody.
  • the mutated sequence of hu179VL8 is as follows:
  • Biacore detects the binding activity of antibodies obtained after mutation to human TSLP, and the binding activities of exemplary antibodies are shown in Table 28 below:
  • AMG157 (tezepelumab, anti-TSLP antibody) is a positive control.
  • the results showed that the hu179-33 antibody had high specific binding activity to human TSLP.
  • N55Q, N55V, G56V mutations are made on HCDR2, and N53Q, N53D, N53S, H54Y, Y50E, S52D, S52E, N53E, H54D, H54E or Y55E are made in LCDR2.
  • the mutation does not affect the binding of the antibody to human TSLP, that is, it does not affect the activity of the anti-TSLP antibody.
  • the VH of mab199 selects IGHV1-46*01 and HJ6*01 as templates, VL selects IGKV1-39*01 and IGKJ4*01 as templates, the CDR region of the mouse antibody is transplanted to the selected humanized template, and the FR The regions were backmutated to obtain light chain and heavy chain variable regions with different sequences. The backmutations are shown in Table 29.
  • I48V means according to the Kabat numbering system, mutate I back to V at position 48.
  • Grafted represents murine antibody CDR implantation of human germline FR region sequences.
  • variable regions of the mab199 humanized antibody are shown below:
  • the single underlined part is the CDR, and the double underlined part is the reverse mutation site.
  • the above-mentioned light and heavy chain variable regions are combined with the human germline light and heavy chain constant region sequences to form the final complete light and heavy chain sequences to obtain full-length antibodies.
  • the light chain constant region of the mab199 humanized antibody is the constant region set forth in SEQ ID NO: 134
  • the heavy chain constant region is the constant region set forth in SEQ ID NO: 133.
  • the obtained mab199 humanized antibody is shown in Table 30 below:
  • Table 31 The activity of mab199 humanized antibody to block the binding of TSLP to the TSLP receptor
  • the heavy chain constant regions of humanized antibodies and chimeric antibodies can be selected from the constant regions of IgG1, IgG2, IgG4 and variants thereof.
  • the IgG1-YTE constant region is used in the present disclosure, the sequence of which is as shown in SEQ ID NO: 133 shown.
  • the light chain constant region can be selected from the light chain constant regions of human kappa, lambda chains or their variants.
  • the constant region of human kappa chain is used in the present disclosure, and its sequence is shown in SEQ ID NO: 134 .
  • IgG1-YTE heavy chain constant region (SEQ ID NO: 133):
  • the humanized heavy and light chain variable regions of the present disclosure are recombined with the above-mentioned constant regions to obtain the full-length sequences of the heavy and light chains.
  • the sequences of the antibodies are as follows:
  • hu3-13 antibody heavy chain (SEQ ID NO: 135):
  • hu3-13 antibody light chain (SEQ ID NO: 136):
  • hu119-30 antibody heavy chain (SEQ ID NO: 137):
  • hu179-33 antibody heavy chain (SEQ ID NO: 139):
  • hu179-33 antibody light chain (SEQ ID NO: 140):
  • hu199-36 antibody heavy chain (SEQ ID NO: 141):
  • hu199-36 antibody light chain (SEQ ID NO: 142):
  • the underlined part is the CDR, and the italic part is the constant region.
  • the present disclosure uses AMG157 as a positive control whose sequences are shown in SEQ ID NO: 143 and SEQ ID NO: 144.
  • the present disclosure also used human TSLP receptor and human IL-7R ⁇ to construct cell lines whose sequences are shown below:
  • the underlined part is the signal peptide.
  • Human IL-7R ⁇ full-length sequence amino acid sequence (Uniprot number: P16871) (SEQ ID NO: 146):
  • the underlined part is the signal peptide.
  • the antibodies of the present disclosure can be cloned, expressed and purified by conventional gene cloning and recombinant expression methods.
  • Test Example 1 Determination of the binding of anti-TSLP antibody to human TSLP by ELISA
  • Human TSLP-his (SEQ ID NO: 1) was diluted to 1 ⁇ g/mL with pH 7.4 PBS (Shanghai Yuanpei, B320) buffer, and added to a 96-well microtiter plate (Corning, CLS3590- 100EA), incubate overnight at 4°C. After discarding the liquid, 200 ⁇ L/well of blocking solution of 5% skimmed milk (bright skim milk powder) diluted with PBS was added, and incubated in a 37° C. incubator for 2 hours for blocking.
  • PBST buffer pH 7.4 PBS containing 0.1% (v/v) tween-20
  • AMG157 positive antibody AMG157
  • the plate was washed three times with PBST, and 100 ⁇ L/well of HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, 115-035-003) diluted with sample diluent was added, and incubated at 37°C for 1 hour.
  • Test Example 2 Biacore determines the affinity of anti-TSLP humanized antibodies to different species of TSLP
  • the affinity of the humanized TSLP antibody to be tested and human and monkey TSLP was determined with a Biacore T200 (GE) instrument.
  • a protein A biosensor chip (Cat.#29127556, GE) was used to affinity capture the molecules to be tested, and then the antigens (huTSLP-his, cynoTSLP-his, prepared in Example 1-1) were flown on the surface of the chip, and Biacore T200 The instrument detects the reaction signal in real time to obtain binding and dissociation curves. After the dissociation in each experimental cycle, the biosensor chip was washed and regenerated with glycine-hydrochloric acid regeneration solution (pH 1.5 Cat.#BR-1003-54, GE). Using BIAevaluation version 4.1, GE software was used to fit the data with a (1:1) Langmuir model, and the affinity values were obtained, as shown in Table 33 below.
  • the anti-TSLP antibody of the present disclosure has a high affinity for human TSLP and can also bind to cynomolgus monkey TSLP.
  • Test Example 3 ELISA-based anti-TSLP antibody blocking TSLP binding to TSLP receptor experiment
  • the TSLP receptor has two subunits, TSLPR and IL-7R, where TSLPR is a TSLP-specific receptor and IL-7R is a shared receptor for TSLP and IL-7.
  • TSLPR is a TSLP-specific receptor
  • IL-7R is a shared receptor for TSLP and IL-7.
  • TSLP first binds to TSLPR and then to IL-7R. This test case is used to identify whether TSLP antibody can block the binding of TSLP to the extracellular domain of recombinantly expressed TSLPR receptor protein.
  • Human-TSLPR-Fc-ECD (2 ⁇ g/mL, SEQ ID NO: 5) was coated on an ELISA plate and incubated at 4°C overnight. After discarding the liquid, 200 ⁇ L/well of 5% nonfat milk blocking solution diluted with PBS was added, 37 Incubate for 2 hours in a °C incubator for blocking. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.05% (v/v) Tween 20), and the biotin-labeled huTSLP-Fc antigen was prepared to 3nM, and the antibody to be tested was prepared.
  • PBST buffer pH 7.4 PBS containing 0.05% (v/v) Tween 20
  • TMB chromogenic substrate KPL, 52-00-03
  • 1M H 2 SO 4 1M H 2 SO 4
  • NOVOStar enzyme labeling The absorber was read at 450 nm, and the IC50 value of TSLP antibody blocking the binding of TSLP to TSLPR was calculated. The results are shown in Table 34 and Figure 1.
  • Test Example 4 FACS-based TSLP antibody blocking TSLP binding to TSLP receptor experiment
  • This test case is used to identify that anti-TSLP antibodies can respectively block the binding of TSLP to the TSLPR/IL-7R receptors on the surface of CHOK1 cell line.
  • the specific method is: culture CHOK1-TSLPR/IL-7R with DME/F12 containing 10% FBS, 1mg/mL G418, 10 ⁇ g/mL puromicine, and centrifuge the CHOK1-TSLPR/IL-7R cells in good condition (1000rpm, 5min). ), washed once with 2% FBS in PBS, and counted, adjusted the cell concentration to 1 ⁇ 10 6 /mL, and added 50 ⁇ L to a round-bottomed 96-well plate.
  • the antibody to be tested was diluted with PBS solution containing 2% BSA, the initial concentration was 20nM, and 8 gradients were diluted 1:4 times.
  • Biotin-labeled TSLP-Fc antigen was prepared at 2nM, the antigen and antibody were mixed 1:1, and then placed at 37°C for 15min, 50 ⁇ L per well was added to the plated 96-well plate, and incubated at 4°C for 1 hour. After incubation, centrifuge at 4°C (800g, 5min), discard the supernatant, wash with 200 ⁇ L of pre-cooled PBS, repeat twice, add 1:1000 diluted PE-SA secondary antibody, and incubate at 4°C for 40min in the dark.
  • Test Example 5 Anti-TSLP antibody inhibits TSLP-induced chemokine production
  • TSLP can induce naive myeloid dendritic cells (mDCs) to mature and secrete the chemokines Thymus activation regulated chemokin (TARC) and osteoclast inhibitory factor (Osteoprotegerin, OPG), thereby further mediating innate and adaptive immune inflammatory response.
  • TARC Thymus activation regulated chemokin
  • OPG osteoclast inhibitory factor
  • the primary myeloid mDCs were isolated and purified from human peripheral blood mononuclear cells (PBMCs) by magnetic bead sorting (CD1c(BDCA-1) + dendritic cell isolation kit, Miltenyi Biotec).
  • PBMCs peripheral blood mononuclear cells
  • CD1c(BDCA-1) + dendritic cell isolation kit, Miltenyi Biotec The obtained mDCs were seeded in 96-well cell culture plates, and the serially diluted antibody samples and human TSLP (huTSLP-his, final concentration 50ng/mL) were pre-incubated for about 45 minutes (37°C), and then added to each cell containing mDC. In cell culture wells, mDCs were stimulated in vitro and cultured in an incubator for 48 hours.
  • TARC was detected by human CCL17/TARC Quantikine ELISA Kit of R&D Company
  • OPG content was detected by human CCL22/MDC Quantikine ELISA Kit (R&D), the results are shown in Figure 4A-4B.
  • the results show that the antibodies in the present disclosure can significantly inhibit the production of the chemokines TARC and OPG induced by TSLP, indicating that the antibodies in the present disclosure can block the occurrence of innate and adaptive inflammatory responses.
  • Anti-TSLP antibody blocks native TSLP-induced proliferation of BaF3-TLSPR/IL-7R cells
  • BaF3-hTSLPR/hIL-7R cells can proliferate under the stimulation of native TSLP, and the binding of antibody to native TSLP reduces the stimulatory effect of TSLP on BaF3-hTSLPR/hIL-7R cells.
  • Cultivate NHLF cells (BNCC340764) and HLF1 cells (BNCC337730), when the cells grow to 80%, discard the supernatant and use human 10ng/mL IL1- ⁇ (Sino Biological GMP-10139-HNAE), 20ng/mL IL13 (R&D 213-ILB-005), 20ng/mL TNF- ⁇ (PEPROTECH 300-01A) stimulated human lung fibroblasts NHLF (BNCC340764) and HLF1 (BNCC337730) for 72 hours, It is induced to produce native TSLP. After the stimulation, the cell supernatant was collected, centrifuged to remove cell debris, centrifuged at 4500 rpm for 5 minutes, and the supernatant was collected and concentrated with a concentration column, about 10 times, and filtered for use.
  • human 10ng/mL IL1- ⁇ Seo Biological GMP-10139-HNAE
  • 20ng/mL IL13 R&D 213-ILB-00
  • BaF3-hTSLPR/hIL-17R cells were cultured in RPMI1640 (10 ng/mL mIL3, R&D 213-ILB-005) in 10% FBS, adjusted to a density of 1 x 10 cells/mL, at 37°C, 5% CO 2 Incubate to logarithmic growth phase. The cells were collected, centrifuged at 800 rpm/min for 5 min, and the supernatant was discarded; the cells were washed three times with PBS to remove cytokines that stimulated their proliferation in the medium.
  • the cells were resuspended in 4% FBS RPMI1640 medium, and seeded in a 96-well plate at 4000 cells/50 ⁇ L/well, and cultured in an incubator for 2 h.
  • the antibody to be tested was diluted with native TSLP, the initial concentration of the antibody was 100nM, and the 10-fold ratio was serially diluted, and 3 gradients of 100nM, 10nM and 1nM were diluted. 50 ⁇ L/well of the diluted antibody/antigen mixture was added to the cells, the final concentrations of the antibody were 50 nM, 5 nM and 0.5 nM, and the cells were incubated at 37° C. in a 5% CO 2 incubator for 72 h.
  • the results show that the antibodies obtained in the present disclosure can significantly inhibit the activity of natural TSLP to stimulate the proliferation of BaF3, especially hu179-33, whose activity is more than 100 times that of AMG157.
  • Anti-TSLP antibody inhibits TSLP-induced proliferation of BaF3 cells overexpressing TSLPR/IL7R
  • TSLP can bind to TSLPR/IL-7R on the surface of BaF3, thereby promoting the proliferation of BaF3.
  • This test example is used to identify that the disclosed antibodies can block the BaF3 proliferation-inducing activity of TSLP.
  • BaF3 cells overexpressing TSLPR/IL-7R were cultured in RPMI1640 with 10% FBS and 2ng/mL rhIL3 (Link Bio, Catalog No. 96-AF-300-03-20) at 37°C, 5% Culture in a CO 2 incubator, and the cell density should not exceed 1 x 10 6 cells/mL.
  • Test Example 8 Humanized anti-TSLP antibody blocks TSLP-induced differentiation of native CD4 + T cells into Th2 cells
  • TSLP can induce the maturation of primary myeloid mDC cells, and mature mDC cells highly express OX40 ligands, which can bind to OX40 on the surface of natural CD4 + T cells, thereby making natural CD4 + T cells differentiate into Th2 cells and produce IL- 4/IL-5/IL-13 and other immune response-related factors make the body produce Th2 inflammatory response.
  • This test example is used to detect that the antibodies obtained in the present disclosure can block the differentiation of Th2 cells induced by TSLP.
  • the primary myeloid DCs were isolated and purified from human peripheral blood mononuclear cells (PBMCs) by magnetic bead sorting (CD1c(BDCA-1) + dendritic cell isolation kit, Miltenyi Biotec), and the obtained mDCs were seeded in In a 96-well cell culture plate, the serially diluted antibody samples and recombinantly expressed human TSLP (huTSLP-his, final concentration 50ng/mL) were pre-incubated (37°C) for about 45 minutes, and then added to each cell culture well containing mDC. Incubate at 37°C for 24 hours. Stimulated mature mDCs were collected and washed twice with PBS.
  • PBMCs peripheral blood mononuclear cells
  • CD1c(BDCA-1) + dendritic cell isolation kit Miltenyi Biotec
  • CD4 + CD45RA + naive T cells were extracted from PBMCs by magnetic bead separation (Myltenyi, Biotec). The isolated naive T cells and mature mDCs were mixed and seeded in a 96-well cell culture plate at a ratio of 5:1, and co-cultured for 6 days. Cells were collected, seeded in 96-well plates pre-coated with anti-CD3 (10 ⁇ g/mL), and anti-CD28 (1 ⁇ g/mL) was added to stimulate differentiated T cells for 24 hours. Finally, the cell culture supernatant was collected. . Th2-related cytokines secreted by cells in the supernatant were detected by ELISA.
  • IL-4 and IL-5 cytokines were detected by R&D's ELISA kit, and TNF- ⁇ and IL-13 were detected by Xinbosheng's ELISA kit.
  • the results are shown in Figures 5A-5D; wherein no antibody was added to the mDC+TSLP+T cell group and the T cell group.
  • the results show that the antibodies obtained in the present disclosure can significantly inhibit the production of Th2 cytokines IL-4, IL-5, IL-13 and TNF- ⁇ , indicating that the antibodies obtained in the present disclosure can block the differentiation of Th2 cells induced by TSLP.
  • SEC monomer content percentage SEC% A monomer / A total * 100% (A monomer is the peak area of the main peak monomer in the sample, A total is the sum of all peak areas)
  • Non-reduced CE purity percentage CE-(NR)% A main peak/A total * 100% (A main peak is the peak area of the main peak in the sample, A total is the sum of all peak areas)
  • iCIEF neutral peak content percentage iCIEF% neutral peak area/total area*100% (total area is the sum of acidic peak, neutral peak and basic peak area).
  • the instrument used for iCIEF assay is simple protein, model muarice.
  • ion exchange resin as the stationary phase, by increasing the salt ion concentration in the mobile phase, the proteins are eluted in order according to the order of the affinity between the charged protein and the immobilized ion group from weak to strong.
  • IEC neutral peak content percentage neutral peak area / total area * 100% (total area is the sum of the areas of acidic peaks, neutral peaks and basic peaks)
  • the instrument used for the IEC assay was manufactured by Agilent, model 1260Bio.
  • the freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point drop value and the molar concentration of the solution, a high-sensitivity temperature sensing element is used to measure the freezing point of the solution and convert it into osmotic pressure through electricity. Instrument manufacturer Roser Loser, model OM815.
  • Protein concentration determination instrument UV-Vis spectrophotometer, model: Nano Drop oneC.
  • Formulations containing 100 mg/mL hu179-33 antibody, 0.1 mg/mL polysorbate 80 (PS80) were prepared using the following different buffers. Antibodies were displaced into each of the following buffers using the ultrafiltration displacement method.
  • Tris(hydroxymethyl)aminomethane-hydrochloride pH 7.5.
  • Each formulation was filtered, filled, plugged, and capped separately, and samples were taken for high temperature stability (40°C), shaking (25°C, 300rpm), and freeze-thaw cycles from -35°C to 2-8°C for 5 cycles (FT5C). ) forced degradation experiments.
  • the stability of the antibody in each buffered body fluid was investigated in terms of appearance, SEC monomer, non-reduced CE monomer (CE(NR)) and iCIEF neutral peak, and the results are shown in Table 38 below.
  • D in the table means days, such as D3 means 3 days, "M” means months, such as M1 means 1 month; “0h” means the beginning of the experiment; “ ⁇ ” means the amount of change, “FT5C” means freeze-thaw 5 times, "N/A” means no detection; the same below.
  • Appearance shows that the antibody in CA pH 5.0, CA pH 5.5 buffer system, PB pH 7.0, PB pH 7.5 buffer system and TRIS pH 7.5 buffer system, at the beginning of the experiment and after repeated freezing and thawing, the appearance shows opalescence. After 7 days of shaking, the appearance did not improve or particles or turbidity appeared, indicating that the antibody was not stable in the above buffers.
  • SEC and non-reduced CE data showed that under repeated freeze-thaw and shaking conditions for 7 days, there was no significant change in SEC monomer, CE(NR) monomer and iCIEF for all formulations; but at 40 °C M1, SA pH5 0 buffer system, SEC monomer decreased by about 6.28%, CE (NR) monomer decreased by 8.98%; in SA pH5.5 buffer system, SEC monomer decreased by about 5.55%, CE (NR) monomer decreased by 8.44%; In the His-HCl and His-AA buffers, the SEC monomer decreased by about 3.78-4.72%, and the CE(NR) monomer decreased by about 4.65-7.43%, and the decrease was less than that of the SA pH5.0 and SA pH5.5 buffer systems. It shows that the stability of antibody in His-HCl and His-AA system is better than that in SA buffer system.
  • the buffer system of anti-TSLP antibody is preferably His-AA and His-HCl system, followed by SA system; pH is 5.0-6.5, preferably pH 5.5-6.5.
  • hu179-33 antibody formulations were prepared containing sucrose, trehalose, sorbitol and mannitol in 20 mM His-AA pH 6.0 buffer, 0.4 mg/mL PS80 and 100 mg/mL, respectively. Each formulation was filtered, filled, stoppered, and capped separately. The samples were subjected to high temperature stability study (40°C), shaking (25°C, 300rpm) and forced degradation experiments of 5 cycles of freeze-thaw (-35°C-2 ⁇ 8°C) to investigate the effects of different sugars on the stability of the preparation , and the results are shown in Table 39 below.
  • the content of trehalose in the table is measured by the content of ⁇ , ⁇ -dihydrate trehalose.
  • sucrose, trehalose, sorbitol and mannitol could all be used as stabilizers for TSLP antibody formulations.
  • Formulations were prepared containing 20 mM histidine-acetate buffer pH 6.0, 70 mg/mL sucrose, 100 mg/mL hu179-33 antibody and various polysorbates.
  • the specific prescription is shown in Table 40 below:
  • a formulation was prepared containing 20 mM His-AA pH 6.0, 100 mg/mL hu179-33 antibody, 70 mg/mL sucrose, and 0.8 mg/mL PS80. Sterile filtered, canned. The samples were subjected to high temperature stability studies (40°C), shaking (25°C, 300rpm) and forced degradation experiments for 5 cycles of freeze-thaw (-35°C-2 ⁇ 8°C) studies, from SEC, non-reduced CE and iCIEF In terms of the stability of the formulation, the results are shown in Table 42 below.
  • the formulation containing 20 mM His-AA pH6.0, 100 mg/mL hu179-33 antibody, 70 mg/mL sucrose, and 0.8 mg/mL PS80 was placed at 4°C, and after 3 months, the formulation was tested.
  • the results are shown in Table 43 below.
  • Formulations containing 20 mM histidine-acetate buffer, pH 6.0, 0.8 mg/mL PS80, 100 mg/mL hu179-33 antibody and various stabilizers were prepared, and the specific formulations were as follows:
  • sucrose 20 mM methionine and 10 mM EDTA.
  • the formulations of the present disclosure may also optionally incorporate the aforementioned ingredients.
  • the formulations of the present disclosure may also optionally contain EDTA components.
  • a formulation containing 20 mM His-AA pH 6.0, 150 mg/mL hu179-33 antibody, 70 mg/mL sucrose, and 0.4 mg/mL PS80 was prepared, sterile filtered, canned.
  • the samples were subjected to high temperature stability study (40°C), shaking (25°C, 300rpm) and 5 cycles of freezing and thawing (-35°C-2 ⁇ 8°C) forced degradation test to investigate the stability of the formulation.
  • the experimental results are shown in the following table 47.

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Abstract

提供了一种含抗TSLP抗体的药物组合物及其用途，所述药物组合物包含抗TSLP抗体及缓冲剂。

Description

一种抗TSLP抗体药物组合物及其用途
本申请要求申请日为2020/12/3的中国专利申请202011405207.9的优先权。本申请引用上述中国专利申请的全文。
技术领域
本披露属于药物制剂领域，具体涉及一种包含抗TSLP抗体的药物组合物，以及其作为治疗疾病或病症的药物的用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
哮喘是严重的气道慢性炎症疾病，全球约有3.34亿哮喘病人。随着环境恶化和空气污染加剧，可能会有更多人罹患此病，严重危害人类的生命健康。
胸腺基质淋巴细胞生成素(Thymic Stromal Lymphopoietin，TSLP)是上皮细胞响应促炎症刺激而产生的细胞因子，主要通过其对树突细胞和肥大细胞的活性来促进过敏性炎症反应。TSLP是一种类白细胞介素7(IL-7)细胞因子，最早从小鼠的胸腺基质细胞条件培养基中发现。TSLP主要在肺、皮肤和肠道上皮细胞中表达。TSLP由4个α-螺旋以及AB和CD两个环组成，分子内有三对由六个半胱氨酸组成的二硫键，有两个N糖基化位点，分子量约为15-20kD。TSLP的受体是个复合物，包括两部分，一部分为TSLPR，另一部分为IL-7Rα。TSLP先与TSLPR以相对较低的亲和力结合，然后以高亲和力招募IL-7Rα的结合，最终以stat5等信号通路的激活，导致DC的成熟和T细胞的分化。
骨髓源性树突状细胞(myeloid dendritic cell，mDC)是TSLP最主要的效应细胞，TSLP作用于未成熟的mDC，mDC分泌细胞因子IL-8、eotaxin-2、TARC和MDC，同时高表达OX40L。在没有IL-12的前提下，OX40L与天然CD4+T细胞结合，使其分化成Th2细胞，进而Th2细胞分泌IL-5、IL-4、IL-9、IL-13和TNF等Th2细胞因子，诱导机体Th2炎症反应。另外，TSLP还可以诱导DC细胞产生细胞因子IL-8，进而招募嗜中性粒细胞，导致嗜中性粒细胞天然免疫炎症。TSLP还可以诱导DC产生eotaxin-2，eotaxin-2招募嗜酸性粒细胞，和IL-5一起作用，使机体迅速进入嗜酸性粒细胞浸润的炎症状态。TSLP也作用于肥大细胞、自然杀伤细胞，通过诱导产生IL-4、IL-6、IgE等来介导天然炎症。综上，TSLP可同时导致天然炎症和Th2炎症，进而使组织黏液增多，气道重塑导致气管狭窄，细胞纤维化严重，进而逐步演变成哮喘、过敏性皮炎和过敏性鼻炎等三大 过敏性疾病。因此，阻断TSLP对治疗哮喘、过敏性皮炎等疾病是一个潜在的有效策略。
目前，全球范围内尚无TSLP单抗获批上市。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。然而，抗体在配制和储存过程中容易发生变性、聚集、污染等其他改变，是制备抗体制剂的重大障碍。由于抗体的多样性，没有适合于所有抗体的储存的通用配方或条件，一种抗体的最佳制剂对于该抗体通常是特定的。抗体的制剂通常是商业抗体研究和开发过程的重要部分，因此，存在开发稳定的抗体制剂的必要。
发明内容
本披露提供一种抗TSLP抗体的药物组合物及其用途。
在一些实施方案中，前述的药物组合物，其包含抗TSLP抗体和缓冲剂，其中所述的缓冲剂为组氨酸盐缓冲剂或琥珀酸盐缓冲剂。
在一些实施方案中，前述的药物组合物，其包含抗TSLP抗体和缓冲剂，其中所述的缓冲剂为组氨酸盐缓冲剂。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂为组氨酸-醋酸盐缓冲剂或组氨酸-盐酸盐缓冲剂。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂为琥珀酸-琥珀酸钠缓冲剂。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体包含重链可变区和轻链可变区，其中：
i)所述重链可变区包含分别如SEQ ID NO:26、SEQ ID NO:94和SEQ ID NO:28所示的HCDR1、HCDR2和HCDR3；和
所述轻链可变区包含分别如SEQ ID NO:29、SEQ ID NO:113和SEQ ID NO:31所示的LCDR1、LCDR2和LCDR3；
ii)所述重链可变区包含分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的HCDR1、HCDR2和HCDR3；和
所述轻链可变区包含分别如SEQ ID NO:70、SEQ ID NO:24和SEQ ID NO:25所示的LCDR1、LCDR2和LCDR3；
iii)所述重链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:45所示的HCDR1、HCDR2和HCDR3；和
所述轻链可变区包含分别如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:54所示 的LCDR1、LCDR2和LCDR3；或
iv)所述重链可变区包含分别如SEQ ID NO:32、SEQ ID NO:33和SEQ ID NO:34所示的HCDR1、HCDR2和HCDR3；和
所述轻链可变区包含分别如SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体包含如下任一项所示的重链可变区和轻链可变区：
a)如SEQ ID NO:97所示的重链可变区序列和如SEQ ID NO:119所示的轻链可变区；
b)如SEQ ID NO:69所示的重链可变区序列和如SEQ ID NO:74所示的轻链可变区；
c)如SEQ ID NO:50所示的重链可变区序列和如SEQ ID NO:52所示的轻链可变区；或
d)如SEQ ID NO:132所示的重链可变区序列和如SEQ ID NO:125所示的轻链可变区。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体包含：
a)如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
b)如SEQ ID NO:137所示的重链和如SEQ ID NO:138所示的轻链；
c)如SEQ ID NO:135所示的重链和如SEQ ID NO:136所示的轻链；或
d)如SEQ ID NO:141所示的重链和如SEQ ID NO:142所示的轻链。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂的浓度为5mM-50mM，包括但不限于5mM-10mM、10mM-15mM、15mM-25mM、25mM-35mM或35mM-45mM。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂的浓度为10mM-30mM。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂的浓度为5mM-50mM，包括但不限于5mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂的浓度为10mM-20mM。
在一些实施方案中，前述的药物组合物，其中所述的缓冲剂的浓度为约20mM。
在一些实施方案中，前述的药物组合物，其中所述的缓冲液的pH为5.0-6.5，包括但不限于5.0-5.5、5.5-6.0或6.0-6.5。
在一些实施方案中，前述的药物组合物，其中所述的缓冲液的pH为5.0-6.5，包括但 不限于5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述的缓冲液的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其中所述的缓冲液的pH为约6.0。
在一些实施方案中，前述的药物组合物，其中所述的缓冲液的pH为约6.2。
在一些实施方案中，前述的药物组合物的pH可能存在漂移，漂移程度约为±0.3。
在一些实施方案中，前述的药物组合物，其中所述的药物组合的pH为5.0-6.5。
在一些实施方案中，前述的药物组合物，其中所述的药物组合的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其中所述的药物组合的pH为约5.8-6.3。
在一些实施方案中，前述的药物组合物，其中所述的药物组合的pH为约6.0-6.3。
在一些实施方案中，前述的药物组合物，其中所述的药物组合的pH为约6.3。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体的浓度为1mg/mL-150mg/mL，包括但不限于80mg/mL-150mg/mL、80mg/mL-120mg/mL、80mg/mL-100mg/mL、100mg/mL-150mg/mL或100mg/mL-120mg/mL。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体的浓度为1mg/mL-150mg/mL，包括但不限于1mg/mL、10mg/mL、20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL、110mg/mL、120mg/mL、130mg/mL、140mg/mL、150mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体的浓度为100mg/mL-150mg/mL。
在一些实施方案中，前述的药物组合物，其中所述的抗TSLP抗体的浓度为约100mg/mL。
在一些实施方案中，前述的药物组合物，其中所述药物组合物还包含表面活性剂。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂为聚山梨酯。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂为聚山梨酯80或聚山梨酯20。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂为聚山梨酯80。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂浓度为0.01mg/mL-1.0mg/mL，包括但不限于0.05mg/mL-1.0mg/mL、0.05mg/mL-0.8mg/mL、0.1mg/mL-1.0mg/mL、0.1mg/mL-0.8mg/mL、0.1mg/mL-0.6mg/mL、0.1mg/mL-0.4mg/mL、0.2mg/mL-1.0mg/mL、0.2mg/mL-0.8mg/mL、0.2mg/mL-0.6mg/mL或0.2mg/mL-0.4mg/mL。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂浓度为0.1mg/mL-0.8mg/mL。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂浓度为0.01mg/mL-1.0mg/mL，包括但不限于0.01mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.55mg/mL、0.6mg/mL、0.65mg/mL、0.7mg/mL、0.75mg/mL、0.8mg/mL、0.85mg/mL、0.9mg/mL、0.95mg/mL、1.0mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述表面活性剂浓度为约0.8mg/mL。
在一些实施方案中，前述的药物组合物，其中所述组合物还包含稳定剂，其中所述稳定剂选自糖、氨基酸和EDTA中的一种或更多种。
在一些实施方案中，前述的药物组合物，其中所述糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种。
在一些实施方案中，前述的药物组合物，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为蔗糖。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为20mg/mL-100mg/mL，包括但不限于30mg/mL-90mg/mL、30mg/mL-70mg/mL、50mg/mL-90mg/mL或50mg/mL-70mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为30mg/mL-100mg/mL，包括但不限于40mg/mL-90mg/mL、40mg/mL-80mg/mL、50mg/mL-80mg/mL、60mg/mL-90mg/mL或60mg/mL-80mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为20mg/mL-100mg/mL，包括但不限于20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为50mg/mL-70mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为50mg/mL-60mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为约70mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其浓度为30mg/mL-70mg/mL，其中所述的糖为海藻糖、蔗糖、山梨糖醇或甘露醇。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为糖，其中所述的糖为海藻糖，其浓度为约60mg/mL-70mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为蔗糖，其浓度为70mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂为山梨糖醇或甘露醇，其浓度为50mg/mL。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为5mM-50mM，包括但不限于10mM-50mM、10mM-40mM、10mM-30mM或10mM-20mM。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为5mM-50mM，包括但不限于5mM-40mM、5mM-30mM、5mM-20mM、20mM-30mM、20mM-40mM或30mM-40mM。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为10mM-30mM。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为约30mM。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为5mM-50mM，包括但不限于5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为5mM-50mM，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为10mM-30mM，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为30mM，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包括EDTA，其浓度为0.1mM-10mM，包括但不限于0.1mM、0.2mM、0.3mM、0.4mM、0.5mM、1mM、2mM、3mM、4mM、5mM、6mM、7mM、8mM、9mM、10mM，以及这些点值之间的任意范围。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包括EDTA，其浓度为0.1mM-10mM，包括但不限于0.1mM-8mM、0.1mM-6mM、0.1mM-4mM或0.1mM-2mM。
在一些实施方案中，前述的药物组合物，其中所述稳定剂还包括EDTA，其浓度为0.37mM-10mM，包括但不限于0.37mM-9mM、0.37mM-7mM、0.37mM-5mM、0.37mM-3mM。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为30mg/mL-70mg/mL糖和10mM-30mM的氨基酸；其中所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为30mg/mL-70mg/mL糖和0.37mM-10mM的EDTA；其中所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为30mg/mL-70mg/mL糖、10mM-30mM氨基酸和0.37mM-10mM的EDTA；其中所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为：
i)30mg/mL糖和30mM的氨基酸；
ii)70mg/mL糖和0.37mM-10mM的EDTA；或
iii)70mg/mL糖、20mM氨基酸和0.37mM-10mM的EDTA；其中所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇，其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为：
i)30mg/mL蔗糖和30mM的组氨酸、色氨酸或甲硫氨酸；
ii)70mg/mL蔗糖和0.37mM-10mM的EDTA；或
iii)70mg/mL蔗糖、20mM甲硫氨酸和0.37mM-10mM的EDTA。
在一些实施方案中，前述的药物组合物，其中所述的稳定剂为：
i)30mg/mL蔗糖和30mM的组氨酸、色氨酸或甲硫氨酸；
ii)70mg/mL蔗糖和10mM的EDTA；或
iii)70mg/mL蔗糖、20mM甲硫氨酸和10mM的EDTA。
在一些实施方案中，前述的药物组合物，其中所述药物组合物的pH为5.0-6.5。在一些实施方案中，前述的药物组合物，其中所述药物组合物的pH为5.5-6.5。在一些实施方案中，前述的药物组合物，其中所述药物组合物的pH为约6.0-6.3。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山 梨酯80；和(c)10mM-30mM的组氨酸盐缓冲剂；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯80；和(c)10mM-30mM的组氨酸-盐酸盐缓冲剂；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯80；和(c)10mM-30mM的组氨酸-盐酸盐缓冲剂；其中所述药物组合物的pH为6.0-6.3。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)50mg/mL-70mg/mL的糖；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)50mg/mL-70mg/mL的糖；其中所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇，所述聚山梨酯选自聚山梨酯20和聚山梨酯80；所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)30mg/mL-70mg/mL的蔗糖和10mM-30mM的氨基酸；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)30mg/mL-70mg/mL的蔗糖和0.37mM-10mM的EDTA；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)30mg/mL-70mg/mL的蔗糖、10mM-30mM的氨基酸和0.37mM-10mM的EDTA；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其中所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇；和/或所述聚山梨酯选自聚山梨酯20和聚山梨酯80；和/或所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸；和/或所述的组氨酸盐缓冲剂选自组氨酸-盐酸盐缓冲剂和组氨酸-醋酸盐缓冲剂。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和(d)30mg/mL-100mg/mL的海藻糖、蔗糖、山梨糖醇或甘露醇；其中所述药物组合物的pH为5.5-6.5。
(a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和(d)50mg/mL-70mg/mL的海藻糖、蔗糖、山梨糖醇或甘露醇；其中所述药物组合物的pH为5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)约100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)约0.8mg/mL的聚山梨酯80；(c)约20mM组氨酸-醋酸盐缓冲剂；和(d)约70mg/mL的蔗糖；其中所述药物组合物的pH为约6.0-6.3。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
(b)0.1mg/mL-0.8mg/mL的聚山梨酯；
(c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和
(d)30mg/mL-70mg/mL的糖和10mM-30mM氨基酸；或
所述药物组合物包含：
(a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
(b)0.1mg/mL-0.8mg/mL的聚山梨酯；
(c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和
(d)30mg/mL-70mg/mL的糖和0.37mM-10mM的EDTA；或
所述药物组合物包含：
(a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
(b)0.1mg/mL-0.8mg/mL的聚山梨酯；
(c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和
(d)30mg/mL-70mg/mL的糖和0.37mM-10mM的EDTA和10mM-30mM氨基酸；
其中：
所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇，
所述氨基酸选自组氨酸、色氨酸和甲硫氨酸，
所述的聚山梨酯选自聚山梨酯80和聚山梨酯20；并且
所述药物组合物的pH为5.5-6.5；优选地，pH为约6.0-6.3。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；和(c)10mM-30mM的醋酸盐缓冲剂；其中所述药物组合物的pH为4.5-6.5；优选药物组合物的pH为4.5-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯80；和(c)10mM-30mM的醋酸盐缓冲剂；其中所述药物组合物的pH为4.5-6.5；优选药物组合物的pH为4.5-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的醋酸盐缓冲剂；和(d)30mg/mL-100mg/mL的糖；其中所述药物组合物的pH为4.5-6.5；优选药物组合物的pH为4.5-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的醋酸盐缓冲剂；和(d)50mg/mL-70mg/mL的糖；其中所述药物组合物的pH为4.5-6.5；优选药物组合物的pH为4.5-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯80；(c)10mM-30mM的醋酸盐缓冲剂；和(d)30mg/mL-70mg/mL的蔗糖；其中所述药物组合物的pH为4.5-6.5；优选药物组合物的pH为4.5-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的琥珀酸-琥珀酸钠缓冲剂，其中所述的药物组合物的pH为约5.0-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的琥珀酸-琥珀酸钠缓冲剂，其中所述的药物组合物的pH为约5.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的琥珀酸-琥珀酸钠缓冲剂，其中所述的药物组合物的pH为约5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的柠檬酸-柠檬酸钠缓冲剂，其中所述的药物组合物的pH为约5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-盐酸盐缓冲剂，其中所述的药物组合物的pH为约5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-盐酸盐缓冲剂，其中所述的药物组合物的pH为约5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-盐酸盐缓冲剂，其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20 mM的组氨酸-盐酸盐缓冲剂，其中所述的药物组合物的pH为约6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-醋酸盐缓冲剂，其中所述的药物组合物的pH为约5.5-6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-醋酸盐缓冲剂，其中所述的药物组合物的pH为约5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-醋酸盐缓冲剂，其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的组氨酸-醋酸盐缓冲剂，其中所述的药物组合物的pH为约6.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的磷酸盐缓冲剂，其中所述的药物组合物的pH为约6.0-7.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的磷酸盐缓冲剂，其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的磷酸盐缓冲剂，其中所述的药物组合物的pH为约7.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139 所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的醋酸-醋酸钠缓冲剂，其中所述的药物组合物的pH为约4.5-5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的醋酸-醋酸钠缓冲剂，其中所述的药物组合物的pH为约4.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的醋酸-醋酸钠缓冲剂，其中所述的药物组合物的pH为约5.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL的聚山梨酯80；和(c)20mM的醋酸-醋酸钠缓冲剂，其中所述的药物组合物的pH为约5.5。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.2mg/mL-0.6mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)70mg/mL的蔗糖；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.2mg/mL-0.6mg/mL的聚山梨酯20；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)70mg/mL的蔗糖；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.4mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)50mg/mL-70mg/mL的蔗糖；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139 所示的重链和如SEQ ID NO:140所示的轻链；(b)0.4mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)50mg/mL-70mg/mL的海藻糖；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.4mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)50mg/mL-70mg/mL的山梨糖醇；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.4mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)50mg/mL-70mg/mL的甘露醇；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.8mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；(d)70mg/mL的蔗糖；和(e)30mM氨基酸；其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸；所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.8mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；(d)70mg/mL的蔗糖；和(e)10mM EDTA；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.8mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；(d)70mg/mL的蔗糖；和(e)10mM EDTA和20mM氨基酸；其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸；所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.8mg/mL的聚山梨酯80；(c)20mM 的组氨酸-盐酸盐缓冲剂；(d)70mg/mL的蔗糖；和(e)10mM EDTA和20mM甲硫氨酸；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.8mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；(d)70mg/mL的蔗糖；和(e)0.37mM-10mM的EDTA；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.8mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；(d)70mg/mL的蔗糖；和(e)0.37mM-5mM的EDTA；其中所述的药物组合物的pH为约6.0。
在一些实施方案中，前述的药物组合物，其包含如下组分：
(a)150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.4mg/mL的聚山梨酯80；(c)20mM的组氨酸-盐酸盐缓冲剂；和(d)70mg/mL的蔗糖；其中所述的药物组合物的pH为约6.0。
本披露还提供一种包含抗TSLP抗体的冻干制剂，其中所述的冻干制剂通过将如前任一所述的药物组合物经冷冻干燥获得。
本披露还提供一种制备如前任一所述的药物组合物的方法，所述方法包括将抗TSLP抗体原液经缓冲液置换的步骤。
本披露还提供一种制品，其包括容器，所述容器中装有如前任一所述的药物组合物或如前所述的冻干制剂。
本披露还提供一种治疗疾病或病症的方法，所述方法包括向受试者施用治疗有效量的如前任一所述的药物组合物，或如前所述的冻干制剂。
本披露还提供一种如前任一所述的药物组合物，或如前所述的冻干制剂在制备用于治疗疾病或病症的药物中的用途。
本披露中所述的如前任一所述的药物组合物，或如前所述的冻干制剂可用作治疗疾病或病症的药物。
在一些实施方案中，所述疾病或病症选自过敏性疾病、癌症和免疫性疾病；所述过敏性疾病选自：哮喘、特发性肺纤维化、特应性皮炎、过敏性结膜炎、变应性鼻炎、过 敏性鼻窦炎、荨麻疹、内瑟顿综合征(Netherton Syndrome)、嗜酸性粒细胞性食管炎、食物过敏、过敏性腹泻、嗜酸性粒细胞性胃肠炎、过敏性支气管肺曲霉病、过敏性真菌鼻窦炎和慢性瘙痒；所述癌症选自：乳腺癌、结肠癌、肺癌、卵巢癌、前列腺癌；所述免疫性疾病选自：类风湿性关节炎、慢性阻塞性肺病、全身性硬化、多发性硬化症、瘢痕瘤、溃疡性结肠炎、鼻息肉病、慢性嗜酸性粒细胞性肺炎、嗜酸性粒细胞性支气管炎、腹腔病、Churg-Strauss综合征、嗜酸性粒细胞性肌痛综合征、高嗜酸粒细胞综合征、嗜酸性粒细胞性肉芽肿病伴随多血管炎、炎性肠病、硬皮病、间质性肺病、B型或C型慢性肝炎引发的纤维化、辐射诱发的纤维化和伤口愈合引发的纤维化。
在一些实施方案中，前述的疾病或病症与TSLP有关。
附图说明
图1：抗体阻断TSLP与TSLP受体结合活性的结果。
图2：抗体阻断TSLP与细胞表面TSLP受体结合活性的结果。
图3：抗体抑制TSLP诱导的BaF3细胞的增殖活性。
图4A：抗体抑制TSLP诱导的趋化因子TARC产生的活性。
图4B：抗体抑制TSLP诱导的趋化因子OPG产生的活性。
图5A：抗体抑制Th2细胞因子IL-13产生的活性。
图5B：抗体抑制Th2细胞因子IL-4产生的活性。
图5C：抗体抑制Th2细胞因子TNF-α产生的活性。
图5D：抗体抑制Th2细胞因子IL-5产生的活性。
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
说明书和权利要求书中所用的单数形式“一个”、“一种”和“所述”包括复数指代，除非上下文清楚表明并非如此。
除非上下文另外清楚要求，否则在整个说明书和权利要求书中，应将词语“包含”、“具有”、“包括”等理解为具有包含意义，而不是排他性或穷举性意义；也即，“包括但不仅限于”的意义。
术语“和/或”，例如“X和/或Y”应当理解为意指“X和Y”或“X或Y”并且应当被用来提供对两种含义或任一含义的明确支持。
术语“胸腺基质淋巴细胞生成素(Thymic Stromal Lymphopoietin，TSLP)”是四α-螺旋束I型细胞因子，也是响应促炎症刺激而产生的上皮细胞衍生的细胞因子，与白细胞介素-7(IL-7)密切相关，其通过刺激树突细胞(DC)起始变态反应，是调节人体免疫反应的重要因子。术语“TSLP”包括TSLP的变体、同种型、同系物、直系同源物和旁系同源物。
“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(即柠檬酸根离子)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。示例性的，所述的琥珀酸-琥珀酸钠可由琥珀酸与氢氧化钠配制而成，或由琥珀酸与琥珀酸钠配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、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。
“表面活性剂(surfactant)”是指表面活性剂(surface-active agent)，优选非离子表面活性剂。使用表面活性剂可以降低制剂中蛋白质的聚集和/或降低颗粒的形成。添加的表面活性剂的量是使得其可以在制剂中降低蛋白质的聚集，并最小化颗粒形成的量。本披露的表面活性剂可选自聚山梨酯(包括但不限于聚山梨酯20、聚山梨酯80)、聚羟亚烃、 Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨酯80或聚山梨酯20，更优选为聚山梨酯80。
“稳定剂”是指有助于维持生物制药药物的结构完整性的组分，特别是在冷冻和/或冻干和/或储存期间(特别是当暴露于应激(stress)时)。这种稳定作用可以由于多种原因而产生，通常这种稳定剂可起到减轻蛋白质变性的渗透剂的作用。如在本文中所使用的，稳定剂包括糖、氨基酸和EDTA；本文所述稳定剂中的氨基酸，是除缓冲液中的氨基酸之外，另添加的氨基酸。
本披露的“糖”包含常规组合物(CH 2O) n及其衍生物，包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。本披露中的糖可选自葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、木糖醇、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等等。优选的糖是蔗糖、海藻糖、山梨糖醇和甘露醇，更优选为海藻糖或蔗糖，最优选为蔗糖。
“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。
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”可意味着其后所示的具体数值±10％的范围。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”的含义应该假定为在该具体值的可接受误差范围内。
尽管本披露提供了含量范围或含量值，但本领域一般技术人员理解，所述含量范围或含量值涵盖了所测定具体值的可接受误差范围。
“药物组合物”表示含有一种或多种本文所述抗体药物偶联物或其生理学上/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。
本披露中，“药物组合物”和“制剂”并不互相排斥。
本披露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。
本披露所述的药物组合物能够达到一种稳定的效果：其中的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。
稳定的制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，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。稳定性的典型的例子：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生聚集或降解。通过视觉分析，制剂是无色至淡黄色、澄清透明至稍微乳白色的液体。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(ultra-violet)光散射、尺寸排阻色谱法(size-exclusion chromatography，SEC)和动态光散射(dynamic light scattering，DLS)测得，抗体药物偶联物没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体药物偶联物在药物制剂中“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CE-SDS(capillary electrophoresis sodiu dodecyl sulfate)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”。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
本披露所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。
本披露的术语“抗体”以最广义使用，其涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体或其抗原结合片段(也称“抗原结合部分”)，只要它们展现出期望的抗原结合活性。全长抗体是包含由二硫键互相连接的至少两条重链和两条轻链的免疫球蛋白(Ig)。根据免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
术语“可变区”或“可变域”指抗体重链或轻链中涉及抗体结合抗原的域。VH和VL各包含四个保守的框架区(FR)和三个互补决定区(CDR)。其中，术语“互补决定区”、“CDR”指可变结构域内主要促成抗原结合的区域；“框架”或“FR”是指除CDR残基之外的可变结构域残基。VH包含3个CDR区：HCDR1、HCDR2和HCDR3；VL包含3个CDR区：LCDR1、LCDR2、和LCDR3。每个VH和VL由从氨基末端排到羧基末端按以下顺序排列的三个CDR和四个FR构成：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。单个VH或VL可能足以赋予抗原结合特异性。
可以通过各种公知方案来确定CDR的氨基酸序列边界，例如：“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)；Front Immunol.2018 Oct 16；9:2278)等。各编号系统之间的关系是本领域技术人员熟知的，并且如下表1中所示。
表1 CDR编号系统之间的关系
Figure PCTCN2021132037-appb-000001
Figure PCTCN2021132037-appb-000002
除非另有说明，本披露实施例中的可变区和CDR序列均适用“Kabat”编号规则。
术语“抗原结合片段”或“功能片段”或“抗原结合部分”是指保持特异性结合抗原的能力的完整抗体的一个或更多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。示例性的，涵盖在术语“抗原结合片段”的结合片段的实例包括：(i)Fab片段，一种由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，一种包含通过铰链区上的二硫键桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；(vi)scFv；(vii)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域，VH)和抗体轻链可变结构域(或区域，VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。现有技术已公开众多适于连接抗体VH和VL的接头，例如由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本披露的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
双抗体是其中scFv或Fab被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
双特异性抗体和多特异性抗体是指能同时结合两个或多个抗原或抗原决定簇的抗体，其中包含能结合TSLP的scFv或Fab片段。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重 链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指轻链和重链包含恒定区的抗体。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 - 10M、10 -11M、10 -12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合TSLP，例如，在本披露中抗体与细胞表面抗原的亲和力采用FACS或Biacore法测定KD值。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人TSLP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。本披露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区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，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。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。
本披露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TSLP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，用含调整过的缓冲液的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。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“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，例如包含本披露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右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“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。示例性保守取代于下表2“示例性氨基酸保守取代”中陈述。
表2示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本披露靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本披露靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS)：Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而 不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
“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。
本披露中与TSLP相关的疾病没有限制，只要它是与TSLP相关的疾病即可，例如利用本披露的抗体诱导的治疗反应可通过结合人类TSLP，然后阻遏TSLP与其受体结合，或杀伤过表达TSLP的细胞；或抑制过表达TSLP的细胞的生长。
在以上说明书中提出了本披露一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方式。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权利要求书限定。
实施例
以下结合实施例用于进一步描述本披露，但这些实施例并非限制本披露的范围。
本披露实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，《分子克隆——实验室手册》，冷泉港实验室；《当代分子生物学方法》，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
第一部分抗体
实施例1-1 TSLP和TSLP受体的表达
分别将编码带His标签的人TSLP、食蟹猴TSLP、带人IgG1-Fc标签的人TSLP、食蟹猴TSLP、人TSLP受体胞外区序列克隆到phr载体上，构建成表达质粒，然后转染HEK293E。具体转染步骤为：前一天将HEK293E细胞以0.8×10 6/mL接种于Freestyle表达培养基(含有1％FBS)中，放置于37℃恒温摇床(120rpm)继续培养24小时。24小时后，将转染质粒和转染试剂PEI用0.22μm的滤器除菌，然后将转染质粒调整为100 μg/100mL细胞，PEI(1mg/mL)和质粒的质量比为3:1，以200mL HEK293E细胞的转染为例，取10mL的Opti-MEM和200μg质粒混匀，静置5分钟(min)；另取10mL的Opti-MEM和600μg PEI混匀，静置5min。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入200mL HEK293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养。转染第3天，补充10％体积的补料培养基。待转染第6天，取样4500rpm离心10min收集细胞上清，过滤，将重组的TSLP和TSLP受体蛋白上清按照实施例1-2进行纯化，纯化的蛋白可用于下述各实施例实验中。相关序列如下所示。
1.带his标签的人TSLP抗原(huTSLP-his)氨基酸序列
Figure PCTCN2021132037-appb-000003
注：下划线为信号肽序列；斜体部分为Flag-His6-tag标记。
2.带Fc标签的人TSLP抗原(huTSLP-Fc)氨基酸序列
Figure PCTCN2021132037-appb-000004
注：下划线为信号肽序列；斜体部分为接头-人Fc-tag标记。
3.带His标签的食蟹猴TSLP抗原(cynoTSLP-His)氨基酸序列
Figure PCTCN2021132037-appb-000005
注：下划线为信号肽序列；斜体部分为flag-His6-tag标记。
4.带Fc标签的食蟹猴TSLP抗原(cyno TSLP-Fc)氨基酸序列
Figure PCTCN2021132037-appb-000006
Figure PCTCN2021132037-appb-000007
注：下划线为信号肽序列；斜体部分为接头-人Fc-tag标记。
5.带Fc标签的人TSLP受体胞外区(人TSLPR-Fc-ECD)氨基酸序列
Figure PCTCN2021132037-appb-000008
注：下划线部分为人TSLPR的胞外区，斜体部分为接头-人Fc-tag。
实施例1-2TSLP和TSLP受体(TSLPR)重组蛋白的纯化
2.1带His标签的各种属TSLP重组蛋白的纯化
将细胞表达上清高速离心去除杂质，过滤，PBS溶液平衡镍柱，冲洗10倍柱体积。将过滤后的上清上柱。用含有30mM咪唑的PBS溶液冲洗柱子，至A 280读数降至基线。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。PBS浓缩换液，LC-MS鉴定为正确后分装备用。得到带His标签的人TSLP和食蟹猴TSLP。
2.2带人Fc标签的各种属TSLP和人TSLP受体胞外区重组蛋白的纯化
将细胞表达上清高速离心去除杂质，重组抗体表达上清用Protein A柱进行纯化。用PBS冲洗柱子，至A 280读数降至基线。用100mM乙酸(pH3.5)洗脱目的蛋白，用1M Tris-HCl，pH8.0中和。浓缩换液，所得到的蛋白经电泳、LC-MS鉴定为正确后分装备用。
实施例1-3重组TSLP受体和IL-7Rα受体细胞系的构建和鉴定
为筛选可以阻断TSLP结合TSLP受体的抗体，构建了同时表达人TSLP受体和人IL-7Rα(TSLPR/IL-7Rα)的CHO-K1和BaF3细胞株。采用慢病毒包装目的基因TSLPR/IL- 7Rα克隆至目的细胞株内形成稳定高表达细胞株。首先分别将人TSLPR和人IL-7Rα基因克隆至pCDH-CMV-MCS-EF1-puro和pCDH-CMV-MCS-EF1-Neo(SBI，CD500B-1)质粒内，然后通过慢病毒感染的方法将人TSLPR克隆至CHO-K1和BaF3细胞株内，经10μg/mL嘌呤霉素(puromycin，Gibco，US)筛选压力下选择培养三周。在此基础上进行第二轮感染，再将人IL-7Rα基因克隆进去，用1mg/mL G418(Gibco，US)和10μg/mL嘌呤霉素时筛选两至三周。最后通过流式分选的方法，筛选出同时高表达TSLPR和IL-7Rα的CHO-K1和BaF3单克隆细胞株。
实施例1-4抗人TSLP单克隆抗体的制备和筛选
抗人TSLP单克隆抗体通过免疫小鼠产生，实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠用重组蛋白huTSLP-Fc(25μg)、huTSLP-his(12.5μg)和cyno TSLP-his(12.5μg)与TiterMax、Alum或CpG佐剂免疫。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠，处死后，取脾细胞，与骨髓瘤细胞融合。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2021132037-appb-000009
CRL-8287 TM)进行融合得到杂交瘤细胞。
初次筛选用针对人和食蟹猴TSLP的ELISA结合实验、阻断人TSLP结合其受体TSLPR的实验和抑制TSLP诱导的BaF3细胞的增殖实验进行。当将杂交瘤细胞转移到24孔板后，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，并通过亲和方法纯化。
筛选得到活性好的单克隆杂交瘤细胞株3号、119号、179号和199号，分别收集对数生长期杂交瘤细胞，用NucleoZol(MN)提取RNA，并进行反转录(PrimeScript TMReverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增后送测序公司测序。经测序得到鼠源抗TSLP抗体：mab3、mab119、mab179和mab199，其可变区氨基酸序列如下(下划线部分为互补决定区序列)：
mab3鼠源重链可变区序列(SEQ ID NO:6)：
Figure PCTCN2021132037-appb-000010
mab3鼠源轻链可变区序列(SEQ ID NO:7)：
Figure PCTCN2021132037-appb-000011
mab119鼠源重链可变区序列(SEQ ID NO:8)：
Figure PCTCN2021132037-appb-000012
mab119鼠源轻链可变区序列(SEQ ID NO:9)：
Figure PCTCN2021132037-appb-000013
mab179鼠源重链可变区序列(SEQ ID NO:10)：
Figure PCTCN2021132037-appb-000014
mab179鼠源轻链可变区序列(SEQ ID NO:11)：
Figure PCTCN2021132037-appb-000015
mab199鼠源重链可变区序列(SEQ ID NO:12)：
Figure PCTCN2021132037-appb-000016
mab199鼠源轻链可变区序列(SEQ ID NO:13)：
Figure PCTCN2021132037-appb-000017
根据Kabat编号规则获得的CDR区氨基酸序列如下表3所示：
表3杂交瘤克隆来源抗体重链及轻链的CDR区序列
Figure PCTCN2021132037-appb-000018
Figure PCTCN2021132037-appb-000019
将上述鼠源抗体的轻重链可变区与人源抗体的轻、重链恒定区(如SEQ ID NO:134所示的kappa恒定区和如SEQ ID NO:133所示的IgG1-YTE恒定区)连接后形成嵌合抗体，mab3号克隆对应的嵌合抗体命名为Ch3，mab119号克隆对应的嵌合抗体命名为Ch119，mab179号克隆对应的嵌合抗体命名为Ch179，mab199号克隆对应的嵌合抗体命名为Ch199。
实施例1-5抗人TSLP单克隆抗体的人源化设计
为了降低鼠源抗体的免疫原性，将已筛选出的体内外活性优异的mab3、mab119、mab179和mab199抗体进行了人源化。鼠源单克隆抗体的人源化根据本领域许多文献公示的方法进行。简言之，使用人抗体恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。然后以鼠源抗体的三维结构为基础，通过对VL和VH的氨基酸残基进行回复突变，将鼠源抗体的恒定区替 换为人恒定区，得到最终的人源化分子。
5.1mab3的人源FR区的选择和回复突变
(1)人源FR区的选择和回复突变
mab3的人源化VH模板为IGHV1-3*01+IGHJ6*01，人源化VL的模板为IGKV3-20+IGKJ4*01，将mab3的CDR移植到人源模板上，移植后获得的可变区序列如下：
hu3VL-CDR grafted：
Figure PCTCN2021132037-appb-000020
hu3VH-CDR grafted：
Figure PCTCN2021132037-appb-000021
mab3人源化抗体的回复突变设计如下表4所示：
表4 mab3人源化抗体的回复突变
Figure PCTCN2021132037-appb-000022
注：Grafted(移植的)代表鼠抗体CDR植入人种系FR区序列，L46P表示依照Kabat编号系统，将46位L突变回P。
移植后获得的mab3人源化抗体的可变区序列如下：
hu3VL1(hu3 VL-CDR grafted)(SEQ ID NO:38)：
Figure PCTCN2021132037-appb-000023
hu3VL2(SEQ ID NO:39)：
Figure PCTCN2021132037-appb-000024
hu3VL3(SEQ ID NO:40)：
Figure PCTCN2021132037-appb-000025
hu3VL4(SEQ ID NO:41)：
Figure PCTCN2021132037-appb-000026
hu3VH1(hu3 VH-CDR Grafted)(SEQ ID NO:42)：
Figure PCTCN2021132037-appb-000027
hu3VH2(SEQ ID NO:43)：
Figure PCTCN2021132037-appb-000028
hu3VH3(SEQ ID NO:44)：
Figure PCTCN2021132037-appb-000029
注：单下划线部分为CDR区，双下划线部分为回复突变位点。
将上述轻、重链可变区与人种系轻链、重恒定区序列组合形成最终的完整轻、重链序列，进而得到全长序列的抗体。示例性的，本披露中mab3人源化抗体的重链恒定区为如SEQ ID NO:133所示IgG1-YTE恒定区，轻链恒定区为如SEQ ID NO:134所示的kappa链恒定区，但也可以更换为其他本领域内公知的恒定区。
获得的mab3人源化抗体重、轻链可变区的序列见下表5：
表5 mab3人源化抗体重、轻链可变区序列
抗体 VH(SEQ ID NO) VL(SEQ ID NO)
hu3-01 42 39
hu3-02 42 40
hu3-03 42 41
hu3-04 43 38
hu3-05 43 39
hu3-06 43 40
hu3-07 43 41
hu3-08 44 39
hu3-09 44 40
hu3-10 44 41
通过ELISA方法检测mab3人源化抗体与人TSLP的结合活性，结果显示，mab3人源化抗体与人TSLP具有很好的结合能力。
(2)hu3抗体的点突变
经检测发现，mab3人源化抗体的HCDR3的MDH序列和LCDR3的NTR序列上存在热点，因此，将相应的热点进行位点突变。突变后得到mab3人源化抗体的CDR区序列如下表6所示：
表6突变后的HCDR3、LCDR3序列
Figure PCTCN2021132037-appb-000030
注：表6中突变位点的位置采用的是根据可变区序列的自然顺序编号。
由此可得出，mab3人源化抗体CDR序列如下表7所示：
表7 mab3人源化抗体突变后的CDR
Figure PCTCN2021132037-appb-000031
其中，X 1选自H或Y，X 2选自N或D。
示例性的，突变后得到的人源化抗体hu3-11的CDR及重、轻链可变区如下表8所示：
表8 hu3-11的CDR区
Figure PCTCN2021132037-appb-000032
Figure PCTCN2021132037-appb-000033
hu3-11的轻链可变区(hu3VL4-N93D)的氨基酸序列如下：
Figure PCTCN2021132037-appb-000034
hu3-11的重链可变区(hu3VH2-H110Y)的氨基酸序列如下：
Figure PCTCN2021132037-appb-000035
将热点突变后的轻、重链可变区与人种系轻链、重恒定区序列重组形成完整轻、重链序列，进而得到全长序列的抗体。
使用ELISA方法检测突变后得到的抗体与人TSLP结合活性，结果显示，hu3-11与人TSLP的亲和活性依然较高，说明mab3人源化抗体的HCDR3和LCDR3上的热点突变，并不会影响抗体的活性。
(3)hu3-11抗体的亲和力成熟
对hu3-11的分子进行了亲和力成熟。亲和力成熟的过程如下：
酵母文库的构建：设计简并引物，通过PCR的方法将设计的突变氨基酸引入到抗体hu3-11的scFv突变体文库中，每个文库的大小约10 9左右，构建好的酵母菌文库通过测序的方法验证文库的多样性。
在第一轮筛选中，将来自hu3-11-scFv突变体文库的约5×10 10个细胞与生物素化的TSLP-Fc蛋白(1-10μg/mL)在50mL含0.1％牛血清白蛋白(BSA)-磷酸盐缓冲液(PBSA)中于室温下孵育1小时。然后，混合物用0.1％PBSA洗涤，以去除未结合的抗体片段。然后往结合生物素化TSLP-Fc的hu3-11-scFv抗体突变体文库中加入100μL链菌素微珠(Mi1envi Biotec,Auburn,CA)，并使其负载于AutoMACS系统上用于分选。收集具有对TSLP-Fc的高亲和力的抗体文库的细胞，于250rpm和20℃下诱导18h。将所得到的富集文库进行针对与生物素化的重组TSLP-Fc蛋白的第二轮筛选。
对于第三轮和第四轮的筛选，将来自前一轮的文库细胞与生物素化的重组TSLP-Fc 蛋白(0.1-1μg/mL)和10μg/mL鼠抗-cMyc(9E10,sigma)抗体在0.1％PBSA中于室温下孵育1小时(h)，混合物用0.1％PBSA洗涤，以去除未结合的抗体片段。加入羊抗鼠-Alexa488(A-11001,Life technologies)和Strepavidin-PE(S-866,Life technologies)于4℃下孵育1h，混合物用0.1％PBSA洗涤，以去除未结合的抗体片段。最后，通过FACS筛选出亲和力高的抗体。
hu3-11-scFv突变体文库利用生物素化的TSLP-Fc抗原，经过2轮MACS筛选和2轮FACS筛选。再挑选400个左右的酵母单克隆培养和诱导表达，使用FACS检测酵母单克隆对TSLP-Fc抗原的结合，挑选出亲和力高的酵母单克隆进行测序验证，对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长抗体后进行哺乳动物细胞表达。
亲和力成熟获得的轻链可变区序列如下：
hu3VL5(SEQ ID NO:51)：
Figure PCTCN2021132037-appb-000036
hu3VL6(SEQ ID NO:52)：
Figure PCTCN2021132037-appb-000037
注：单下划线部分为CDR区，双下划线部分为回复突变位点。
将获得的轻链可变区与mab3人源化抗体的重链可变区重组，得到新的mab3人源化抗体，示例性的，将huVL5和huVL6分别与hu3VH2-H110Y组合，得到新的抗体分子hu3-12和hu3-13，具体如下表9所示：
表9亲和力成熟获得的抗体
抗体 hu3VH hu3VL
hu3-12 hu3VH2-H110Y hu3VL5
hu3-13 hu3VH2-H110Y hu3VL6
亲和力成熟后，得到的mab人源化抗体的CDR序列如下表10所示：
表10亲和力成熟获得的mab3人源化抗体的CDR
Figure PCTCN2021132037-appb-000038
Figure PCTCN2021132037-appb-000039
对得到的新的mab3人源化抗体进行ELISA检测其结合人TSLP活性。结果显示，hu3-12、hu3-13与人TSLP依然具有较高的结合能力。说明，LCDR3中某些氨基酸的改变，并不会影响hu3系列抗体的活性。
综上，mab3人源化抗体的CDR具有如下表11所示的序列：
表11 mab3人源化抗体的CDR区通式序列
Figure PCTCN2021132037-appb-000040
其中，X 1为H或Y，X 3为N或S，X 4为V或G，X 5为G或E。
mab3人源化抗体热点突变亲和力成熟后的抗体重轻链可变区组合见下表12：
表12亲和力成熟抗体的序列
抗体 VH(SEQ ID NO) VL(SEQ ID NO)
hu3-11 50 49
hu3-12 50 51
hu3-13 50 52
5.2 mab119的人源FR区的选择和回复突变
(1)人源FR区的选择和回复突变
mab119的VH选取IGHV1-69*02和HJ6*01作为模板，VL选取IGKV4-1*01和IGKJ2*01以及IGKV3-11*01和IGKJ2*01作为模板，将鼠源抗体的CDR区移植到选择的人源化模板上，并对FR区进行回复突变，得到不同的轻链和重链可变区，CDR移植得到的可变区序列如下：
hu119VL(Grafted,IGKV4-1*01)(SEQ ID NO:56)：
Figure PCTCN2021132037-appb-000041
hu119VL4(Grafted,IGKV3-11*01)(SEQ ID NO:59)：
Figure PCTCN2021132037-appb-000042
hu119VH(Grafted,IGHV1-69*02)(SEQ ID NO:62)：
Figure PCTCN2021132037-appb-000043
mab119人源化抗体的回复突变见下表13：
表13 mab119回复突变
Figure PCTCN2021132037-appb-000044
Figure PCTCN2021132037-appb-000045
注：如M4L表示依照Kabat编号系统，将第4位M突变回L。Grafted(移植的)代表鼠抗体CDR植入人种系FR区序列。
mab119人源化抗体可变区具体序列如下：
hu119VL1(Grafted(IGKV4-1*01))(SEQ ID NO:56)：
Figure PCTCN2021132037-appb-000046
hu119VL2(SEQ ID NO:57)：
Figure PCTCN2021132037-appb-000047
hu119VL3(SEQ ID NO:58)：
Figure PCTCN2021132037-appb-000048
hu119VL4(Grafted,IGKV3-11*01)(SEQ ID NO:59)：
Figure PCTCN2021132037-appb-000049
hu119VL5(SEQ ID NO:60)：
Figure PCTCN2021132037-appb-000050
hu119VL6(SEQ ID NO:61)：
Figure PCTCN2021132037-appb-000051
hu119VH1(Grafted)(SEQ ID NO:62)：
Figure PCTCN2021132037-appb-000052
hu119VH2(SEQ ID NO:63)：
Figure PCTCN2021132037-appb-000053
Figure PCTCN2021132037-appb-000054
hu119VH3(SEQ ID NO:64)：
Figure PCTCN2021132037-appb-000055
hu119VH4(SEQ ID NO:65)：
Figure PCTCN2021132037-appb-000056
hu119VH5(SEQ ID NO:66)：
Figure PCTCN2021132037-appb-000057
hu119VH6(SEQ ID NO:67)：
Figure PCTCN2021132037-appb-000058
hu119VH7(SEQ ID NO:68)：
Figure PCTCN2021132037-appb-000059
hu119VH8(SEQ ID NO:69)：
Figure PCTCN2021132037-appb-000060
注：单下划线部分为可变区，双下划线部分为回复突变。
将上述轻、重链可变区与人种系轻、重链恒定区序列组合形成最终的完整轻、重链序列，进而得到全长序列的抗体。示例性的，本披露中mab119人源化抗体的重链恒定区为如SEQ ID NO:133所示的IgG1-YTE恒定区，轻链恒定区为如SEQ ID NO:134所示的kappa链恒定区，但也可以更换为其他本领域内公知的恒定区。
mab119人源化抗体的重、轻链可变区见表14。
表14 mab119人源化抗体的重、轻链可变区
抗体 VH(SEQ ID NO) VL(SEQ ID NO)
hu119-01 62 56
hu119-02 63 56
hu119-03 64 56
hu119-04 65 56
hu119-05 62 57
hu119-06 63 57
hu119-07 64 57
hu119-08 65 57
hu119-09 62 58
hu119-10 63 58
hu119-11 64 58
hu119-12 65 58
hu119-13 64 59
hu119-14 66 59
hu119-15 67 59
hu119-16 68 59
hu119-17 69 59
hu119-18 64 60
hu119-19 66 60
hu119-20 67 60
hu119-21 68 60
hu119-22 69 60
hu119-23 64 61
hu119-24 66 61
hu119-25 67 61
hu119-26 68 61
hu119-27 69 61
使用ELISA方法检测人源化抗体与人TSLP结合活性，结果显示，mab119人源化抗 体可与人TSLP特异性结合。
(2)hu119的突变
经检测发现，mab119人源化抗体的LCDR1 DNS序列存在热点，因此，将相应的位点突变为N31S或N31Q。突变后得到的LCDR1序列如表15所示：
表15 mab119人源化抗体点突变后的LCDR1
Figure PCTCN2021132037-appb-000061
注：表15中突变位点的位置采用的是自然顺序编号。
示例性的，突变后得到的hu119VL2、hu119VL6突变体序列如下：
hu119VL2-N31S(SEQ ID NO:72)
Figure PCTCN2021132037-appb-000062
hu119VL2-N31Q(SEQ ID NO:73)
Figure PCTCN2021132037-appb-000063
hu119VL6-N31S(SEQ ID NO:74)
Figure PCTCN2021132037-appb-000064
hu119VL6-N31Q(SEQ ID NO:75)
Figure PCTCN2021132037-appb-000065
注：单下划线部分为可变区，双下划线部分为回复突变。
将得到的hu119VL2、hu119VL6突变体与hu119VH组合，得到新的人源化hu119抗体，示例性的，hu119VL2-N31S、hu119VL2-N31Q分别与hu119VH3组合，得到的抗体hu119-28和hu119-29；hu119VL3-N31S与hu119VH8组合，得到抗体hu119-30，示例性的突变后抗体的可变区组合如表16所示：
表16热点突变后的人源化抗体可变区组合
  hu119VH hu119VL
hu119-28 hu119VH3 hu119VL2-N31S
hu119-29 hu119VH3 hu119VL2-N31Q
hu119-30 hu119VH8 hu119VL6-N31S
使用ELISA方法检测突变后得到的抗体与人TSLP的亲和力，结果显示，hu119-28、hu119-29抗体依然与人TSLP具有较高的亲和力，这说明，LCDR2的N31S、N31Q的突变并不会影响抗TSLP抗体活性。
综上，mab119人源化抗体的CDR具有如表17所示的序列：
表17 mab119人源化抗体的CDR
Figure PCTCN2021132037-appb-000066
其中，X 6选自N、S和Q。
5.3 mab179的人源化FR区的选择和回复突变
(1)mab179鼠源抗体人源化模板选择及回复突变
mab179的VH选取IGHV1-69*02和IGHJ6*01作为模板，VL选取IGKV4-1*01和IGKJ2*01或IGKV2-29*02和IGKJ2*01作为模板，将鼠源抗体的CDR区移植到选择的人源化模板上，并对FR区进行回复突变，如表18所示，得到不同序列的轻链和重链可变区。人源化可变区序列及回复突变如下：
hu179VL1(Grafted(IGKV4-1*01))(SEQ ID NO:77)
Figure PCTCN2021132037-appb-000067
hu179VL5(Grafted(IGKV2-29*02))(SEQ ID NO:81)
Figure PCTCN2021132037-appb-000068
hu179VH1(Grafted)(SEQ ID NO:85)
Figure PCTCN2021132037-appb-000069
表18 mab179人源化模板及回复突变
Figure PCTCN2021132037-appb-000070
注：如P43S表示依照Kabat编号系统，将43位P突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
mab179人源化抗体的可变区如下所示：
hu179VL1(Grafted(IGKV4-1*01))(SEQ ID NO:77)：
Figure PCTCN2021132037-appb-000071
hu179VL2(SEQ ID NO:78)：
Figure PCTCN2021132037-appb-000072
hu179VL3(SEQ ID NO:79)：
Figure PCTCN2021132037-appb-000073
hu179 VL4(SEQ ID NO:80)：
Figure PCTCN2021132037-appb-000074
hu179VL5(Grafted(IGKV2-29*02))(SEQ ID NO:81)：
Figure PCTCN2021132037-appb-000075
hu179VL6(SEQ ID NO:82)：
Figure PCTCN2021132037-appb-000076
hu179VL7(SEQ ID NO:83)：
Figure PCTCN2021132037-appb-000077
hu179VL8(SEQ ID NO:84)：
Figure PCTCN2021132037-appb-000078
hu179VH1(Grafted)(SEQ ID NO:85)：
Figure PCTCN2021132037-appb-000079
hu179VH2(SEQ ID NO:86)：
Figure PCTCN2021132037-appb-000080
hu179VH3(SEQ ID NO:87)：
Figure PCTCN2021132037-appb-000081
hu179VH4(SEQ ID NO:88)：
Figure PCTCN2021132037-appb-000082
Figure PCTCN2021132037-appb-000083
hu179VH5(SEQ ID NO:89)：
Figure PCTCN2021132037-appb-000084
注：单下划线部分为CDR，双下划线部分为回复突变位点。
将上述轻、重链可变区与人种系轻、重链恒定区序列组合形成最终的完整轻、重链序列，进而得到全长序列的抗体。示例性的，本披露中mab199人源化抗体的重链恒定区为如SEQ ID NO:133所示的IgG1-YTE恒定区，轻链恒定区为如SEQ ID NO:134所示的kappa链恒定区，但也可以更换为其他本领域内公知的恒定区。
获得的mab179人源化抗体重、轻链可变区的序列如下表19所示：
表19 mab179人源化抗体的重、轻链可变区组合
抗体 VH(SEQ ID NO) VL(SEQ ID NO)
hu179-01 85 77
hu179-02 85 78
hu179-03 86 77
hu179-04 86 78
hu179-05 87 77
hu179-06 87 78
hu179-07 87 79
hu179-08 87 81
hu179-09 87 82
hu179-10 87 83
hu179-11 87 84
hu179-12 88 77
hu179-13 88 78
hu179-14 89 79
hu179-15 89 80
hu179-16 89 81
hu179-17 89 82
hu179-18 89 83
hu179-19 89 84
使用ELISA方法检测mab179人源化抗体与人TSLP的亲和力，结果显示mab179人源化抗体与人TSLP具有很好的亲和力。
(2)hu179抗体的突变
经检测发现，mab179人源化抗体的HCDR2和LCDR2序列上存在热点，因此，将相应的位点突变以消除分子修饰风险。
在其中一个实施例中，对hu179VH1的HCDR2的GNG进行氨基酸突变，hu179VH1突变后的序列如下：
hu179VH1-N55Q(SEQ ID NO:90)：
Figure PCTCN2021132037-appb-000085
hu179VH1-N55V(SEQ ID NO:91)：
Figure PCTCN2021132037-appb-000086
hu179VH1-G56V(SEQ ID NO:92)：
Figure PCTCN2021132037-appb-000087
注：单下划线部分为CDR，双下划线部分为回复突变位点。
突变后得到mab179人源化抗体的HCDR2区序列如表20所示：
表20 mab179人源化抗体的HCDR2突变体
Figure PCTCN2021132037-appb-000088
注：表20中突变点的位置采用的是自然顺序编号。
由上可获得mab179人源化抗体突变后的CDR区，如表21所示：
表21 mab179人源化抗体突变后CDR
Figure PCTCN2021132037-appb-000089
其中，X 7选自N，Q或V，X 8选自G或V。
将突变后得到的hu179VH1突变体与人源化的hu179VL组合，得到新的mab179人源化抗体，示例性的hu179VH1突变体与hu179VL2组合的抗体如表22所示：
表22突变后抗体可变区组合
可变区 hu179VH1-N55Q hu179VH1-N55V hu179VH1-G56V
hu179VL2 hu179-20 hu179-21 hu179-22
使用ELISA方法检测突变后得到的抗体与人TSLP的亲和力，结果显示，HCDR2突变后的抗体与人TSLP依然保持较高的亲和力。这表明mab179人源化抗体的HCDR2的点突变N55Q、N55V和G56V基本不影响抗体与TSLP的亲和活性。
按照同样的方法，在hu179VH2、hu179VH3、hu179VH4、hu179VH5上分别进行N55Q、N55V和G56V点突变(自然顺序编号)，并将突变获得重轻链可变区重新组合，得到新的mab179人源化抗体。示例性的，hu179VH3突变的序列如下所示：
hu179VH3-N55V(SEQ ID NO:97)：
Figure PCTCN2021132037-appb-000090
注：单下划线部分为CDR，双下划线部分为回复突变位点。
在另一些实施例中，对mab179人源化抗体的LCDR2进行氨基酸突变，示例性的，hu179VL2突变后的序列如下：
hu179VL2-Y50E(SEQ ID NO:98)：
Figure PCTCN2021132037-appb-000091
hu179VL2-S52D(SEQ ID NO:99)：
Figure PCTCN2021132037-appb-000092
hu179VL2-S52E(SEQ ID NO:100)：
Figure PCTCN2021132037-appb-000093
hu179VL2-N53Q(SEQ ID NO:101)：
Figure PCTCN2021132037-appb-000094
hu179VL2-N53D(SEQ ID NO:102)：
Figure PCTCN2021132037-appb-000095
hu179VL2-N53E(SEQ ID NO:103)：
Figure PCTCN2021132037-appb-000096
hu179VL2-H54Y(SEQ ID NO:104)：
Figure PCTCN2021132037-appb-000097
hu179VL2-H54D(SEQ ID NO:105)：
Figure PCTCN2021132037-appb-000098
hu179VL2-H54E(SEQ ID NO:106)：
Figure PCTCN2021132037-appb-000099
hu179VL2-Y55E(SEQ ID NO:107)：
Figure PCTCN2021132037-appb-000100
注：单下划线部分为CDR，双下划线部分为回复突变位点。突变后获得的mab179人源化抗体LCDR2的序列如表23所示：
表23 mab179人源化抗体LCDR2突变体
Figure PCTCN2021132037-appb-000101
Figure PCTCN2021132037-appb-000102
注：双下划线部分为回复突变位点。
由上可知，mab179人源化抗体LCDR2通式为：X 9VX 10X 11X 12X 13T(SEQ ID NO:118)，其中X 9选自Y或E，X 10选自S、D或E，X 11选自N，Q，D或E；X 12选自H，Y，D或E；X 13选自E或Y。mab179人源化抗体的CDR区如下表24所示：
表24 mab179人源化抗体的CDR
Figure PCTCN2021132037-appb-000103
其中，X 7选自N，Q或V，X 8选自G或V；X 9选自Y或E；X 10选自S、D或E；X 11选自N，Q，D或E；X 12选自H，Y，D或E；X 13选自E或Y。
将突变后得到的hu179VL2突变体与人源化的hu179重链可变区组合，得到新的mab179人源化抗体，示例性的hu179VL2突变体与hu179VH1、hu179VH3组合，获得的mab179人源化抗体的CDR及重轻链可变区组合如下表25所示：
表25 LCDR2突变后mab179人源化抗体CDR区序列
Figure PCTCN2021132037-appb-000104
其中，X 5选自Y或E；X 6选自S、D或E；X 7选自N，Q，D或E；X 8选自H，Y，D或E；X 9选自E或Y。
获得的mab179人源化抗体重、轻链可变区的序列如下表26所示：
表26 LCDR2突变后mab179人源化抗体的重、轻链可变区组合
抗体 VH(SEQ ID NO) VL(SEQ ID NO)
hu179-23 85 102
hu179-24 85 104
hu179-25 87 98
hu179-26 87 99
hu179-27 87 100
hu179-28 87 101
hu179-29 87 103
hu179-30 87 105
hu179-31 87 106
hu179-32 87 107
使用ELISA的方法检测LCDR2突变后获得的mab179人源化抗体与人TSLP的亲和力，结果显示，对LCDR2的热点位点突变后，得到的抗体与人TSLP依然具有较好的亲和力。说明LCDR2的热点位点的突变也不会影响mab179人源化抗体的结合活性。
按照同样的方法，对hu179VL3、hu179VL4、hu179VL5、hu179VL6、hu179VL7和 hu179VL8的LCDR2进行N53Q、N53D、N53S、H54Y、Y50E、S52D、S52E、N53E、H54D、H54E或Y55E的突变。将突变后的轻链可变区与重链可变区组合，形成新的mab人源化抗体。在一种实施方案中，hu179VL8突变后的序列如下所示：
hu179VL8-N53E(SEQ ID NO:119)：
Figure PCTCN2021132037-appb-000105
将突变得到的hu179VL8-N53E与hu179VH3-N55V组合，得到新的抗体分子hu179-33，该分子的CDR序列如下表27所示：
表27 hu179-33抗体的CDR区
Figure PCTCN2021132037-appb-000106
Biacore检测突变后得到的抗体与人TSLP结合活性，示例性的抗体的结合活性如下表28所示：
表28 hu179-33与人TSLP的亲和力
抗体 huTSLP亲和力KD(M)
AMG157 8.12E-12
hu179-33 9.03E-13
表中，AMG157(tezepelumab，抗TSLP抗体)为阳性对照。结果显示，hu179-33抗体与人TSLP具有较高的特异性结合活性。这表明同时在HCDR2和LCDR2上进行热点的点突变，并不会影响mab179人源化抗体与人TSLP亲和力。由此可知，在mab179人源化抗体分子中，同时在HCDR2上做N55Q、N55V、G56V突变，在LCDR2中做N53Q、N53D、N53S、H54Y、Y50E、S52D、S52E、N53E、H54D、H54E或Y55E的突变，并不会影响抗体与人TSLP的结合，即不会影响抗TSLP抗体的活性。
5.4 mab199抗体人源FR区的选择和回复突变
mab199的VH选取IGHV1-46*01和HJ6*01为模板，VL选取IGKV1-39*01和IGKJ4*01为模板，将鼠源抗体的CDR区移植到选择的人源化模板上，并对FR区进行回 复突变，得到具有不同序列的轻链和重链可变区，回复突变如表29所示。
表29 mab199的回复突变设计
Figure PCTCN2021132037-appb-000107
注：如I48V表示依照Kabat编号系统，将48位I突变回V。Grafted代表鼠抗体CDR植入人种系FR区序列。
mab199人源化抗体的可变区如下所示：
hu199VL1(Grafted)(SEQ ID NO:120)：
Figure PCTCN2021132037-appb-000108
hu199VL2(SEQ ID NO:121)：
Figure PCTCN2021132037-appb-000109
hu199VL3(SEQ ID NO:122)：
Figure PCTCN2021132037-appb-000110
hu199VL4(SEQ ID NO:123)：
Figure PCTCN2021132037-appb-000111
hu199VL5(SEQ ID NO:124)：
Figure PCTCN2021132037-appb-000112
hu199VL6(SEQ ID NO:125)：
Figure PCTCN2021132037-appb-000113
hu199VH1(Grafted)(SEQ ID NO:126)：
Figure PCTCN2021132037-appb-000114
hu199VH2(SEQ ID NO:127)
Figure PCTCN2021132037-appb-000115
hu199VH3(SEQ ID NO:128)：
Figure PCTCN2021132037-appb-000116
hu199VH4(SEQ ID NO:129)：
Figure PCTCN2021132037-appb-000117
hu199VH5(SEQ ID NO:130)：
Figure PCTCN2021132037-appb-000118
hu199VH6(SEQ ID NO:131)：
Figure PCTCN2021132037-appb-000119
Figure PCTCN2021132037-appb-000120
hu199VH7(SEQ ID NO:132)：
Figure PCTCN2021132037-appb-000121
注：单下划线部分为CDR，双下划线部分为回复突变位点。
将上述轻、重链可变区与人种系轻、重链恒定区序列组合形成最终的完整轻、重链序列进而得到全长序列的抗体。在本披露中如无明确说明时，mab199人源化抗体的轻链恒定区为如SEQ ID NO:134所示的恒定区，重链恒定区为如SEQ ID NO:133所示的恒定区。
得到的mab199人源化抗体如下表30所示：
表30 mab199人源化抗体的重、轻链可变区序列
抗体 VH(SEQ ID NO) VL(SEQ ID NO)
hu199-01 127 120
hu199-02 127 121
hu199-03 127 122
hu199-04 127 123
hu199-05 127 124
hu199-06 127 125
hu199-07 128 120
hu199-08 128 121
hu199-09 128 122
hu199-10 128 123
hu199-11 128 124
hu199-12 128 125
hu199-13 129 120
hu199-14 129 121
hu199-15 129 122
hu199-16 129 123
hu199-17 129 124
hu199-18 129 125
hu199-19 130 120
hu199-20 130 121
hu199-21 130 122
hu199-22 130 123
hu199-23 130 124
hu199-24 130 125
hu199-25 131 120
hu199-26 131 121
hu199-27 131 122
hu199-28 131 123
hu199-29 131 124
hu199-30 131 125
hu199-31 132 120
hu199-32 132 121
hu199-33 132 122
hu199-34 132 123
hu199-35 132 124
hu199-36 132 125
使用ELISA方法的检测mab199人源化抗体阻断TSLP结合TSLP受体的活性，检测结果如下表31所示：
表31 mab199人源化抗体阻断TSLP结合TSLP受体的活性
Figure PCTCN2021132037-appb-000122
Figure PCTCN2021132037-appb-000123
结果显示，mab199人源化抗体依然具有较高的阻断人TSLP与TSLP受体结合的活性。
5.5抗体恒定区
人源化抗体及嵌合抗体重链恒定区可选自IgG1、IgG2、IgG4及其变体的恒定区，示例性的，本披露中使用了IgG1-YTE恒定区，其序列如SEQ ID NO:133所示。轻链恒定区可选自人源κ、λ链或其变体的轻链恒定区，示例性的，本披露中使用了人源κ链的恒定区，其序列如SEQ ID NO:134所示。
IgG1-YTE重链恒定区(SEQ ID NO:133)：
Figure PCTCN2021132037-appb-000124
注：下划线为设计的M252Y、S254T、T256E突变。
κ轻链恒定区(SEQ ID NO:134)：
Figure PCTCN2021132037-appb-000125
将本披露中的人源化的重轻链可变区与上述恒定区重组，得到重、轻链的全长序列，示例性的，抗体的序列如下所示：
hu3-13抗体重链(SEQ ID NO:135)：
Figure PCTCN2021132037-appb-000126
Figure PCTCN2021132037-appb-000127
hu3-13抗体轻链(SEQ ID NO:136)：
Figure PCTCN2021132037-appb-000128
hu119-30抗体重链(SEQ ID NO:137)：
Figure PCTCN2021132037-appb-000129
hu119-30抗体轻链(SEQ ID NO:138)：
Figure PCTCN2021132037-appb-000130
hu179-33抗体重链(SEQ ID NO:139)：
Figure PCTCN2021132037-appb-000131
hu179-33抗体轻链(SEQ ID NO:140)：
Figure PCTCN2021132037-appb-000132
hu199-36抗体重链(SEQ ID NO:141)：
Figure PCTCN2021132037-appb-000133
hu199-36抗体轻链(SEQ ID NO:142)：
Figure PCTCN2021132037-appb-000134
注：下划线部分为CDR，斜体部分为恒定区。
本披露将AMG157作为阳性对照，其序列如SEQ ID NO:143和SEQ ID NO:144所示。
AMG157的重链序列(SEQ ID NO:143)：
Figure PCTCN2021132037-appb-000135
AMG157的轻链序列(SEQ ID NO:144)：
Figure PCTCN2021132037-appb-000136
此外，在测试抗体活性时，本披露还使用了人TSLP受体和人IL-7Rα构建细胞系，其序列如下所示：
人TSLP受体全长序列氨基酸序列(SEQ ID NO:145)：
Figure PCTCN2021132037-appb-000137
注：下划线部分为信号肽。
人IL-7Rα全长序列氨基酸序列(Uniprot编号：P16871)(SEQ ID NO:146)：
Figure PCTCN2021132037-appb-000138
注：下划线部分为信号肽。
本披露的抗体可用常规基因克隆、重组表达的方法进行克隆、表达和纯化。
第二部分抗体活性检测
测试例1 ELISA测定抗TSLP抗体与人TSLP的结合
用pH7.4的PBS(上海源培，B320)缓冲液将人TSLP-his(SEQ ID NO:1)稀释至1μg/mL，以100μg/孔的体积加入96孔酶标板(Corning，CLS3590-100EA)中，于4℃ 孵育过夜。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μL/孔，37℃孵育箱孵育2小时进行封闭。封闭结束后，弃去封闭液，用PBST缓冲液(pH 7.4PBS含0.1％(v/v)tween-20)洗板3次后，加入100μL/孔用样品稀释液稀释的不同浓度待测抗体和阳性抗体AMG157，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板3次，加入100μL/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，115-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μL/孔TMB显色底物(KPL，52-00-03)，于室温孵育10-15min，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TSLP抗体对TSLP的结合EC50值，结果见下表32。
表32抗体与人TSLP结合活性结果
Figure PCTCN2021132037-appb-000139
Figure PCTCN2021132037-appb-000140
结果显示，本披露中的抗体与人的TSLP有很好的结合活性。
测试例2 Biacore测定抗TSLP人源化抗体与不同种属TSLP的亲和力
用Biacore T200(GE)仪器测定待测人源化TSLP抗体和人和猴TSLP的亲和力。
用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获待测分子，然后于芯片表面流经抗原(huTSLP-his，cynoTSLP-his，实施例1-1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 Cat.#BR-1003-54，GE)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，如下表33所示。
表33抗TSLP抗体与不同种属TSLP的亲和力
Figure PCTCN2021132037-appb-000141
结果显示，本披露中的抗TSLP抗体与人TSLP的亲和力较高，也可和食蟹猴TSLP结合。
测试例3基于ELISA的抗TSLP抗体阻断TSLP结合TSLP受体实验
TSLP受体有两个亚基，TSLPR和IL-7R，其中TSLPR为TSLP特异性受体，IL-7R为TSLP与IL-7的共用受体。TSLP先与TSLPR结合，再与IL-7R结合。本测试例用来 鉴定TSLP抗体是否可以阻断TSLP结合到重组表达的TSLPR受体蛋白胞外区。
将人-TSLPR-Fc-ECD(2μg/mL，SEQ ID NO:5)包被ELISA板，4℃孵育过夜，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％(v/v)吐温20)洗板3次，将生物素标记的huTSLP-Fc抗原配制成3nM，待测抗体从200nM开始梯度稀释，抗原和抗体1:1混匀后37℃放置15min，100μL每孔加入酶标板中，37℃放置1h，用PBST洗板3次后，加入100μL/孔用样品稀释液以1:4000浓度稀释的Streptavidin-Peroxidase Polymer，于37℃孵育1小时。用PBST洗板5次后，加入100μL/孔TMB显色底物(KPL，52-00-03)，于室温孵育3-10min，加入100μL/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在450nm处读取吸收值，计算TSLP抗体阻断TSLP与TSLPR结合的IC50值，结果如表34和图1所示。
表34抗体阻断活性结果
抗体 hu179-33 hu119-30 hu3-13 hu199-36
IC50(nM) 0.5038 0.5192 0.4975 0.5693
结果显示，本披露抗体均可较强地抑制TSLP与其受体TSLPR的结合。
测试例4基于FACS的TSLP抗体阻断TSLP结合TSLP受体实验
本测试例用来鉴定抗TSLP抗体可以分别阻断TSLP结合到CHOK1细胞系表面的TSLPR/IL-7R受体。
具体方法是：用含10％FBS，1mg/mL G418，10μg/mL puromicine的DME/F12来培养CHOK1-TSLPR/IL-7R，将状态良好的CHOK1-TSLPR/IL-7R细胞离心(1000rpm，5min)，用2％FBS的PBS洗一遍，并且计数，将细胞浓度调整浓度至1×10 6/mL，取50μL加入到圆底的96孔板中。用含2％BSA的PBS溶液稀释待测抗体，初始浓度为20nM，按1:4倍比稀释8个梯度。生物素标记的TSLP-Fc抗原配制成2nM，抗原和抗体1:1混匀后37℃放置15min，50μL每孔加到已铺细胞的96孔板中，在4℃孵育1小时。孵育结束后，4℃离心(800g，5min),弃掉上清，用200μL的预冷PBS离心洗涤，重复两次后加入1:1000稀释的PE-SA二抗，4℃避光孵育40min后，4℃离心(800g，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TSLP抗体阻断TSLP与TSLPR/IL-7R结合的IC50值。结果如表35所示。
表35抗体阻断细胞表面TSLPR结果
抗体 AMG157 hu179-33 hu119-30 hu3-13 hu199-36
IC50(nM) 0.2068 0.1867 0.1368 0.1325 0.2270
结果显示，本披露抗体均可较强地阻断TSLP与细胞表面的TSLPR/IL-7R结合。
测试例5抗TSLP抗体抑制TSLP诱导的趋化因子的产生
TSLP可以诱导初始髓样树突状细胞(mDC)成熟并分泌趋化因子胸腺激活调节趋化因子(Thymus activation regulated chemokin，TARC)和破骨细胞抑制因子(Osteoprotegerin，OPG)，从而进一步介导先天和适应性免疫炎症反应。本测试例用来验证所得抗体可以阻断TSLP诱导mDC产生趋化因子，进而阻断先天和适应性炎症反应的发生。
采用磁珠分选的方法(CD1c(BDCA-1)+树突状细胞分离试剂盒，Miltenyi Biotec)从人外周血单核细胞(peripheral blood mononuclear cell，PBMC)中分离纯化初始髓样mDC，将得到的mDC种植于96孔细胞培养板内，梯度稀释的抗体样品和人TSLP(huTSLP-his，终浓度50ng/mL)，预先孵育45分钟左右(37℃)，再分别加入到含有mDC的各细胞培养孔内，体外刺激mDC，置于培养箱中培养48小时。收集细胞培养上清，适当稀释后采用ELISA的方法检测其中趋化因子的含量。TARC采用R&D公司的人CCL17/TARC Quantikine ELISA Kit检测；OPG含量采用人CCL22/MDC Quantikine ELISA Kit(R&D)检测，结果如图4A-4B所示。
结果显示，本披露中的抗体均可显著抑制TSLP诱导的趋化因子TARC和OPG的产生，说明本披露中的抗体可以阻断先天和适应性炎症反应的发生。
测试例6抗TSLP抗体阻断天然TSLP诱导的BaF3-TLSPR/IL-7R细胞的增殖
BaF3-hTSLPR/hIL-7R细胞可在天然(native)TSLP的刺激下增殖，抗体与天然TSLP结合后降低TSLP对BaF3-hTSLPR/hIL-7R细胞的刺激作用。
培养NHLF细胞(北纳生物BNCC340764)、HLF1细胞(北纳生BNCC337730)，当细胞生长至80％时，弃掉上清，使用人10ng/mL IL1-β(Sino Biological GMP-10139-HNAE)，20ng/mL IL13(R&D 213-ILB-005)，20ng/mL TNF-α(PEPROTECH 300-01A)刺激人肺成纤维细胞NHLF(北纳生物BNCC340764)和HLF1(北纳生物，BNCC337730)72小时，诱导其产生天然TSLP。刺激结束后收集细胞上清，离心去掉细胞碎片，4500rpm，离心5分钟，收集上清后用浓缩柱进行浓缩，大约浓缩10倍，过滤后备用。
BaF3-hTSLPR/hIL-17R细胞培养于10％FBS的RPMI1640(10ng/mL mIL3，R&D 213-ILB-005)中，调整密度为1×10 4个/mL，于37℃，5％CO 2培养箱中培养至对数生长期。收集细胞，800rpm/分钟，离心5min，弃上清；用PBS洗三遍除去培养基中刺激其增殖的细胞因子。使用4％FBS RPMI1640培养基将细胞重悬，按照4000个细胞/50μL/孔接种于96孔板，培养箱中培养2h。使用天然TSLP稀释待测抗体，抗体起始浓度为100nM，10倍比进行梯度稀释，稀释3个梯度100nM、10nM和1nM。将稀释好的抗体/抗原混合物50μL/孔加入细胞中，抗体终浓度为50nM、5nM和0.5nM，37℃，5％CO 2培养箱中培养72h。然后每孔加入30μL Cell Titer-Glo(Promega)室温避光孵育10分钟，用Cytation5细胞成像仪Luminescence程序检测。结果见下表36。
表36抗TSLP抗体抑制BaF3-TLSPR/IL-7R细胞的增殖结果
抗体 AMG157 hu179-33 hu3-13 hu119-30
IC50(nM) 3.379 0.02279 0.2888 1.533
结果显示，本披露所得抗体均可以显著抑制天然TSLP刺激BaF3增殖的活性，尤其是hu179-33，其活性是AMG157的100倍以上。
测试例7抗TSLP抗体抑制TSLP诱导的过表达TSLPR/IL7R的BaF3细胞增殖实验
TSLP可以与BaF3表面的TSLPR/IL-7R结合，进而促进BaF3的增殖。本测试例用来鉴定本披露抗体可以阻断TSLP的诱导BaF3增殖的活性。
具体为：过表达TSLPR/IL-7R的BaF3细胞培养于10％FBS以及2ng/mL rhIL3(联科生物，Catalog No.96-AF-300-03-20)的RPMI1640，于37℃，5％CO 2培养箱中培养，细胞密度不要超过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5分钟，用RPMI1640(2％FBS，重组人TSLP-Fc:40ng/mL)调整细胞密度8000个/孔/90μL，并加入10μL梯度稀释的待测抗体到96孔培养2天后，加入30μL cell titer混匀后进行检测，根据读值计算IC50。结果如表37和图3所示。
表37抗体抑制BaF3细胞增殖活性
抗体 AMG157 hu179-33 hu119-30 hu3-13 hu199-36
IC50(nM) 0.5730 0.4092 0.4305 0.4436 0.4769
结果显示，本披露的抗体均具有较强的抑制TSLP介导的BaF3细胞增殖的能力。
测试例8人源化抗TSLP抗体阻断TSLP诱导的天然CD4 +T细胞往Th2细胞的分化
TSLP可以诱导原代髓样mDC细胞成熟，成熟的mDC细胞高表达OX40配体，OX40配体可以与天然CD4 +T细胞表面的OX40结合，进而使天然CD4 +T分化成Th2细胞，产生IL-4/IL-5/IL-13等免疫应答相关因子，使机体发生Th2炎症反应。本测试例用来检测本披露所得抗体可以阻断TSLP诱导的Th2细胞的分化。
采用磁珠分选的方法(CD1c(BDCA-1)+树突状细胞分离试剂盒，Miltenyi Biotec)从人外周血单核细胞(PBMC)中分离纯化初始髓样DC，将得到的mDC种植于96孔细胞培养板内，梯度稀释的抗体样品和重组表达的人TSLP(huTSLP-his，终浓度50ng/mL)预先孵育(37℃)45分钟左右，再分别加入到含有mDC的各细胞培养孔内，37℃培养24小时。收集刺激成熟的mDC，用PBS洗两次。用磁珠分离法(Myltenyi，Biotec)从PBMC中提取CD4 +CD45RA +天然T细胞。将分离得到的天然T细胞和成熟的mDC以5:1的比例混合种植于96孔细胞培养板内，共同培养6天。收集细胞，种植于anti-CD3(10μg/mL)预包被的96孔板内，并加入anti-CD28(1μg/mL)，再刺激分化的T细胞，培养24小时，最后收集细胞培养上清。ELISA检测上清中细胞分泌的Th2相关的细胞因子。IL-4和IL-5细胞因子用的是R&D的ELISA试剂盒检测，TNF-α和IL-13用欣博盛的ELISA试剂盒检测。结果如图5A-5D所示；其中mDC+TSLP+T细胞组和T细胞组没有加入抗体。
结果显示，本披露所得抗体可以显著抑制Th2细胞因子IL-4、IL-5、IL-13和TNF-α的产生，表明本披露所得抗体可以阻断TSLP诱导的Th2细胞的分化。
第三部分制剂
制剂制备与检测过程中使用的设备及结果计算方法如下：
SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。
SEC单体含量百分比SEC％＝A单体/A总*100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和)
SEC测定用仪器：安捷伦1260；柱子：waters，XBrige
Figure PCTCN2021132037-appb-000142
SEC(300×7.8mm，3.5μm)
CE(NR)毛细管凝胶电泳：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
非还原CE纯度百分比CE-(NR)％＝A主峰/A总*100％(A主峰为样品中主峰的峰面积，A总为所有峰面积之和)
CE测定用仪器：Beckman型号plus800
iCIEF成像毛细管等电聚焦电泳：
根据蛋白质等电点pI不同进行分离的技术。
iCIEF中性峰含量百分比iCIEF％＝中性峰面积/总面积*100％(总面积为酸性峰、中性峰和碱性峰面积之和)。
iCIEF测定所用仪器厂家simple protein，型号muarice。
IEC离子交换色谱：
以离子交换树脂为固定相，通过增加流动相中盐离子浓度，按照带电蛋白与固定离子基团的亲和力由弱到强的顺序而依次将蛋白洗脱出来的技术。
IEC中性峰含量百分比＝中性峰面积/总面积*100％(总面积为酸性峰、中性峰和碱性峰面积之和)
IEC测定所用仪器厂家Agilent，型号1260Bio。
渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM815。
蛋白浓度测定：
蛋白浓度测定仪器：紫外可见分光光度计，型号：Nano Drop oneC。
实施例2-1制剂缓冲体系与pH值的筛选
使用以下不同缓冲液，配制含有100mg/mL hu179-33抗体，0.1mg/mL聚山梨酯80(PS80)的制剂。采用超滤置换方法将抗体置换至下列各缓冲液中。
1. 20mM琥珀酸-琥珀酸钠(SA)，pH 5.0；
2. 20mM SA，pH 5.5；
3. 20mM柠檬酸-柠檬酸钠(CA)，pH 5.0；
4. 20mM CA，pH 5.5；
5. 20mM组氨酸-盐酸盐(His-HCl)，pH 5.5；
6. 20mM His-HCl，pH 6.0；
7. 20mM His-HCl，pH 6.5；
8. 20mM组氨酸-醋酸盐(His-AA)，pH 5.5；
9. 20mM His-AA，pH 6.0；
10. 20mM His-AA，pH 6.5；
11. 20mM磷酸盐(PB)，pH 7.0；
12. 20mM PB，pH 7.5；
13. 20mM三(羟甲基)氨基甲烷-盐酸盐(TRIS)，pH 7.5。
将每种制剂分别过滤、灌装、加塞、轧盖，取样品进行高温稳定性(40℃)、振摇(25℃，300rpm)和-35℃至2-8℃冻融5次循环(FT5C)的强制降解实验。从外观、SEC单体、非还原CE单体(CE(NR))和iCIEF中性峰方面考察抗体在各缓冲体液中的稳定性，结果见下表38。
表38缓冲液和pH筛选的稳定性结果
Figure PCTCN2021132037-appb-000143
Figure PCTCN2021132037-appb-000144
Figure PCTCN2021132037-appb-000145
备注：表中“D”表示天，例如D3表示3天，“M”表示月，例如M1表示1个月；“0h”表示实验开始时；“△”表示变化量，“FT5C”表示冻融5次，“N/A”表示未检测；下同。
结果分析如下：
外观显示，抗体在CA pH 5.0、CA pH 5.5缓冲体系、PB pH 7.0、PB pH 7.5缓冲体系和TRIS pH7.5缓冲体系中，在实验开始时和反复冻融之后，外观显示有乳光。振摇7天后，外观并未改善或出现颗粒或浑浊，说明抗体在上述缓冲液中稳定性不佳。而抗体在SA(pH5.0、pH 5.5)、His-HCl(pH5.5、pH 6.0和pH 6.5)和His-AA(pH5.5、pH 6.0和pH 6.5)缓冲体系中，在反复冻融、振摇及40℃放置1个月的强制降解条件下，制剂的外观依然澄清透明；仅His-HCl pH6.5缓冲体系在40℃放置1个月后出现悬浮颗粒，说明抗体在SA、His-HCl，pH5.0-6.0和His-AA，pH5.0-6.5的缓冲体系中比较稳定。
SEC和非还原CE数据显示：在反复冻融和振摇7天条件下，所有处方的SEC单体、CE(NR)单体和iCIEF均无明显变化；但在40℃M1条件下，SA pH5.0缓冲体系中，SEC单体下降约6.28％，CE(NR)单体下降8.98％；SA pH5.5缓冲体系中，SEC单体下降约5.55％，CE(NR)单体下降8.44％；而His-HCl和His-AA缓冲体中，SEC单体下降约3.78-4.72％、CE(NR)单体下降约4.65-7.43％，下降幅度小于SA pH5.0、SA pH5.5缓冲体系。说明抗体在His-HCl和His-AA体系中稳定性优于在SA缓冲体系的稳定性。
综上，抗TSLP抗体的缓冲体系优选为His-AA和His-HCl体系，SA体系次之；pH为5.0-6.5，优选为pH 5.5-6.5。
实施例2-2糖种类筛选
制备分别含有蔗糖、海藻糖、山梨糖醇和甘露醇的20mM His-AA pH6.0缓冲液、0.4mg/mL PS80和100mg/mL的hu179-33抗体制剂。将每种制剂分别过滤、灌装、加塞、轧盖。 将样品进行高温稳定性研究(40℃)、振摇(25℃，300rpm)和冻融5次循环(-35℃-2～8℃)强制降解实验，考察不同的糖对制剂稳定性的影响，结果见下表39。
表39糖种类筛选结果
Figure PCTCN2021132037-appb-000146
注：表格中海藻糖的含量是以α,α-二水合海藻糖的含量计量的。
结果显示，糖种类及浓度对各处方的稳定性的影响无明显差异，即蔗糖、海藻糖、山梨糖醇和甘露醇均可作为TSLP抗体制剂的稳定剂。
实施例2-3表面活性剂种类及浓度筛选
制备包含20mM组氨酸-醋酸盐缓冲液pH 6.0、70mg/mL蔗糖、100mg/mL的hu179-33抗体和不同聚山梨酯的制剂。具体处方如下表40所示：
表40聚山梨酯种类及浓度
Figure PCTCN2021132037-appb-000147
将每种制剂分别过滤、灌装、加塞、轧盖。取样品进行高温稳定性(40℃)、振摇(25℃，300rpm)和冻融5次循环(-35℃-2～8℃)强制降解实验，PS80和PS20对制剂的稳定性的影响，结果见下表41。
表41聚山梨酯种类筛选结果
Figure PCTCN2021132037-appb-000148
Figure PCTCN2021132037-appb-000149
实验结果显示，不同种类的聚山梨酯均可用于本披露中的抗体制剂；不同浓度的聚山梨酯20或80对TSLP抗体制剂的稳定性影响无差异。
实施例2-4制剂的稳定性研究
制备包含20mM His-AA pH 6.0，100mg/mL hu179-33抗体，70mg/mL蔗糖，和0.8mg/mL PS80的制剂。无菌过滤、罐装。将样品进行高温稳定性研究(40℃)、振摇(25℃，300rpm)和冻融5次循环(-35℃-2～8℃)研究的强制降解实验，从SEC、非还原CE和iCIEF方面考察制剂的稳定性，结果见下表42。
表42制剂稳定性结果
放置条件 SEC％ CE(NR)％ iCIEF％
0h 98.2 94.8 59.9
FT5C 97.8 94.7 59.3
振摇D10 97.4 94.7 60.8
40℃M1 93.6 88.7 39.3
结果显示，在冻融和振摇条件下，各检测项结果无明显变化；40℃放置1个月后，SEC、CE及iCIEF的变化均在正常的稳定性变动范围内，因此包含0.8mg/mL PS80的抗体制剂的非常稳定。
为了考察制剂的长期稳定性，将包含20mM His-AA pH6.0，100mg/mL hu179-33抗体，70mg/mL蔗糖，和0.8mg/mL PS80的制剂放置4℃，3个月后，检测制剂的稳定性，结果见下表43。
表43 4℃稳定性实验结果
时间 外观 SEC单体％ IEC中性峰％ CE(NR)％
0h 澄明 98.7 59.8 96.4
4℃M3 澄明 97.8 58.8 97.0
结果显示：上述制剂在4℃放置3个月后，各检测指标几乎无变化，说明制剂的稳定良好。
实施例2-5稳定剂的筛选
制备包含20mM组氨酸-醋酸盐缓冲液，pH6.0、0.8mg/mL PS80、100mg/mL的hu179- 33抗体和不同稳定剂的制剂，具体处方如下：
1. 30mg/mL蔗糖和30mM组氨酸(His)；
2. 30mg/mL蔗糖和30mM色氨酸(Trp)；
3. 30mg/mL蔗糖和30mM甲硫氨酸(Met)；
4. 70mg/mL蔗糖和10mM EDTA；
5. 70mg/mL蔗糖、20mM甲硫氨酸和10mM EDTA。
将每种制剂分别过滤、灌装、加塞、轧盖。取样品进行高温稳定性(40℃)研究，考察不同稳定剂对抗体制剂的稳定性的影响，具体结果见下表44。
表44不同稳定剂的实验结果
Figure PCTCN2021132037-appb-000150
实验结果显示，组氨酸、色氨酸、甲硫氨酸和EDTA以及两者混合也可以抑制抗体聚集和降解，但对抗体的稳定性影响不是很大。因此，本披露中的制剂也可以选择性的添加上述成分。
实施例2-6 EDTA浓度筛选
制备包含20mM组氨酸-醋酸盐缓冲液pH 6.0、70mg/mL蔗糖、0.8mg/mL PS80、100mg/mL的hu179-33抗体和不同浓度EDTA的制剂，具体处方设计见下表45。
表45 EDTA浓度筛选处方表
Figure PCTCN2021132037-appb-000151
Figure PCTCN2021132037-appb-000152
将每种制剂分别过滤、灌装、加塞、轧盖。取样品进行高温稳定性(40℃)研究，考察制剂的外观、SEC、非还原CE和iCIEF，实验结果见下表46。
表46 EDTA浓度筛选结果
Figure PCTCN2021132037-appb-000153
实验结果显示，0.37mM-10mM EDTA也可抑制抗体聚集和降解，但对制剂的稳定性影响不大。因此，本披露中的制剂也可以选择性的添加EDTA成分。
实施例2-7高浓度制剂制备
制备包含20mM His-AA pH 6.0、150mg/mL hu179-33抗体、70mg/mL蔗糖和0.4mg/mL PS80的制剂，无菌过滤、罐装。将样品进行高温稳定性研究(40℃)、振摇(25℃，300rpm)和冻融5次循环(-35℃-2～8℃)强制降解试验，考察制剂稳定性，实验结果见下表47。
表47高浓度制剂稳定性
Figure PCTCN2021132037-appb-000154
Figure PCTCN2021132037-appb-000155
实验结果显示，包含抗体浓度为150mg/mL制剂的稳定性仍然良好。
虽然以上描述了本披露的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本披露的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本披露的保护范围由所附权利要求书限定。

Claims (19)

  1. 一种药物组合物，其包含抗TSLP抗体和缓冲剂，其中所述的缓冲剂为
    组氨酸盐缓冲剂或琥珀酸盐缓冲剂；优选为组氨酸-醋酸盐缓冲剂或组氨酸-盐酸盐缓冲剂。
  2. 如权利要求1所述的药物组合物，其中所述抗TSLP抗体包含重链可变区和轻链可变区，其中：
    i)所述重链可变区包含分别如SEQ ID NO:26、SEQ ID NO:94和SEQ ID NO:28所示的HCDR1、HCDR2和HCDR3；和
    所述轻链可变区包含分别如SEQ ID NO:29、SEQ ID NO:113和SEQ ID NO:31所示的LCDR1、LCDR2和LCDR3；
    ii)所述重链可变区包含分别如SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22所示的HCDR1、HCDR2和HCDR3；和
    所述轻链可变区包含分别如SEQ ID NO:70、SEQ ID NO:24和SEQ ID NO:25所示的LCDR1、LCDR2和LCDR3；
    iii)所述重链可变区包含分别如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:45所示的HCDR1、HCDR2和HCDR3；和
    所述轻链可变区包含分别如SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:54所示的LCDR1、LCDR2和LCDR3；或
    iv)重链可变区包含分别如SEQ ID NO:32、SEQ ID NO:33和SEQ ID NO:34所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区包含分别如SEQ ID NO:35、SEQ ID NO:36和SEQ ID NO:37所示的LCDR1、LCDR2和LCDR3；
    优选地，其中所述的抗TSLP抗体包含如下任一项所示的重链可变区和轻链可变区：
    a)如SEQ ID NO:97所示的重链可变区序列和如SEQ ID NO:119所示的轻链可变区；
    b)如SEQ ID NO:69所示的重链可变区序列和如SEQ ID NO:74所示的轻链可变区；
    c)如SEQ ID NO:50所示的重链可变区序列和如SEQ ID NO:52所示的轻链可变区；或
    d)如SEQ ID NO:132所示的重链可变区序列和如SEQ ID NO:125所示的轻链可变 区；
    更优选地，所述的抗TSLP抗体包含：
    a)如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
    b)如SEQ ID NO:137所示的重链和如SEQ ID NO:138所示的轻链；
    c)如SEQ ID NO:135所示的重链和如SEQ ID NO:136所示的轻链；或
    d)如SEQ ID NO:141所示的重链和如SEQ ID NO:142所示的轻链。
  3. 如权利要求1或2所述的药物组合物，其中所述缓冲剂的浓度为5mM-50mM，优选为10mM-30mM，更优选为约20mM。
  4. 如权利要求1-3任一项所述的药物组合物，其中所述抗TSLP抗体的浓度为1mg/mL-150mg/mL，优选为100mg/mL-150mg/mL，更优选为约100mg/mL。
  5. 如权利要求1-4任一项所述的药物组合物，其中所述药物组合物还包含表面活性剂，所述表面活性剂优选为聚山梨酯，更优选为聚山梨酯80或聚山梨酯20，最优选为聚山梨酯80。
  6. 如权利要求5所述的药物组合物，其中所述表面活性剂的浓度为0.01mg/mL-1.0mg/mL，优选为0.1mg/mL-0.8mg/mL，更优选为约0.8mg/mL。
  7. 如权利要求1-6任一项所述的药物组合物，其中所述药物组合物还包含稳定剂，其中所述稳定剂选自糖、氨基酸和EDTA中的一种或更多种；
    优选地，其中所述糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种；和/或，其中所述氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种；
    更优选地，其中所述稳定剂为蔗糖。
  8. 如权利要求7所述的药物组合物，其中所述稳定剂为糖，其浓度为30mg/mL-100mg/mL，优选为50mg/mL-70mg/mL，更优选为约70mg/mL。
  9. 如权利要求8所述的药物组合物，其中所述稳定剂还包含氨基酸，其浓度为5mM-50mM，优选为10mM-30mM，更优选为30mM。
  10. 如权利要求8或9所述的药物组合物，其中所述稳定剂还包括EDTA，其浓度为0.1mM-10mM，优选为0.37mM-10mM。
  11. 如权利要求7-10任一项所述的药物组合物，其中所述的稳定剂为：
    i)30mg/mL-70mg/mL的糖和10mM-30mM的氨基酸；
    ii)30mg/mL-70mg/mL的糖和0.37mM-10mM的EDTA；或
    iii)30mg/mL-70mg/mL的糖、10mM-30mM的氨基酸和0.37mM-10mM的EDTA；
    其中所述糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种；和/或
    其中所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种；
    优选地，所述稳定剂为：
    i)30mg/mL的蔗糖和30mM的组氨酸、色氨酸或甲硫氨酸；
    ii)70mg/mL的蔗糖和0.37mM-10mM的EDTA；或
    iii)70mg/mL的蔗糖、20mM甲硫氨酸和0.37mM-10mM的EDTA。
  12. 如权利要求1-11任一项所述的药物组合物，其中所述药物组合物的pH为5.0-6.5，优选为5.5-6.5，更优选为约6.0-6.3。
  13. 如权利要求1-12任一项所述的药物组合物，其包含如下组分：
    (a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)50mg/mL-70mg/mL的糖；其中所述药物组合物的pH为5.5-6.5；或
    其中所述药物组合物包含如下组分：
    (a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)30mg/mL-70mg/mL的糖和10mM-30mM的氨基酸；其中所述药物组合物的pH为5.5-6.5；或
    其中所述的药物组合物包含如下组分：
    (a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)30mg/mL-70mg/mL的糖和0.37mM-10mM的EDTA；其中所述药物组合物的pH为5.5-6.5；或
    其中所述的药物组合物包含如下组分：
    (a)100mg/mL-150mg/mL的所述抗TSLP抗体；(b)0.1mg/mL-0.8mg/mL的聚山梨酯；(c)10mM-30mM的组氨酸盐缓冲剂；和(d)30mg/mL-70mg/mL的糖、10mM-30mM的氨基酸和0.37mM-10mM的EDTA；其中所述药物组合物的pH为5.5-6.5；
    其中，所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种；所述的氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种；所述的聚山梨酯选自聚山梨酯80和聚山梨酯20；
    优选地，其中所述药物组合物包含如下组分：
    (a)约100mg/mL的抗TSLP抗体；(b)约0.8mg/mL的聚山梨酯80；(c)20mM组氨酸-醋酸盐缓冲剂；和(d)70mg/mL的蔗糖，其中所述药物组合物的pH为6.0-6.3。
  14. 一种药物组合物，其包含：
    (a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)0.1mg/mL-0.8mg/mL的聚山梨酯20或80；(c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和(d)50mg/mL-70mg/mL的海藻糖、蔗糖、山梨糖醇或甘露醇；其中所述药物组合物的pH为5.5-6.5；
    优选地，所述药物组合物包含如下组分：
    (a)约100mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；(b)约0.8mg/mL的聚山梨酯80；(c)约20mM组氨酸-醋酸盐缓冲剂；和(d)约70mg/mL的蔗糖；其中所述药物组合物的pH为约6.0-6.3。
  15. 一种药物组合物，其包含：
    (a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
    (b)0.1mg/mL-0.8mg/mL的聚山梨酯；
    (c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和
    (d)30mg/mL-70mg/mL的糖和10mM-30mM氨基酸；或
    所述药物组合物包含：
    (a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
    (b)0.1mg/mL-0.8mg/mL的聚山梨酯；
    (c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和
    (d)30mg/mL-70mg/mL的糖和0.37mM-10mM的EDTA；或
    所述药物组合物包含：
    (a)100mg/mL-150mg/mL的抗TSLP抗体，其中所述的抗TSLP抗体包含如SEQ ID NO:139所示的重链和如SEQ ID NO:140所示的轻链；
    (b)0.1mg/mL-0.8mg/mL的聚山梨酯；
    (c)10mM-30mM的组氨酸-醋酸盐缓冲剂；和
    (d)30mg/mL-70mg/mL的糖和0.37mM-10mM的EDTA和10mM-30mM氨基酸；
    其中：
    所述的糖选自海藻糖、蔗糖、山梨糖醇和甘露醇中的一种或多种；
    所述氨基酸选自组氨酸、色氨酸和甲硫氨酸中的一种或多种；
    所述的聚山梨酯选自聚山梨酯20和聚山梨酯80；并且
    所述药物组合物的pH为5.5-6.5；优选为约6.0-6.3。
  16. 一种含抗TSLP抗体的冻干制剂，所述冻干制剂通过将如权利要求1-15任一项所述的药物组合物经冷冻干燥获得。
  17. 制备如权利要求1-15任一项所述的药物组合物的方法，所述方法包括将抗TSLP抗体原液经缓冲液置换的步骤。
  18. 一种制品，其包括容器，所述容器中装有如权利要求1-15任一项所述的药物组合物或如权利要求16所述的冻干制剂。
  19. 一种治疗疾病或病症的方法，所述方法包括向受试者施用治疗有效量的如权利要求1-15任一项所述的药物组合物，或如权利要求16所述的冻干制剂，其中所述疾病或病症选自过敏性疾病、癌症和免疫性疾病；
    所述过敏性疾病选自：哮喘、特发性肺纤维化、特应性皮炎、过敏性结膜炎、变应性鼻炎、过敏性鼻窦炎、荨麻疹、内瑟顿综合征、嗜酸性粒细胞性食管炎、食物过敏、过敏性腹泻、嗜酸性粒细胞性胃肠炎、过敏性支气管肺曲霉病、过敏性真菌鼻窦炎和慢性瘙痒；
    所述癌症选自：乳腺癌、结肠癌、肺癌、卵巢癌、前列腺癌；
    所述免疫性疾病选自：类风湿性关节炎、慢性阻塞性肺病、全身性硬化、多发性硬化症、瘢痕瘤、溃疡性结肠炎、鼻息肉病、慢性嗜酸性粒细胞性肺炎、嗜酸性粒细胞性支气管炎、腹腔病、Churg-Strauss综合征、嗜酸性粒细胞性肌痛综合征、高嗜酸粒细胞综合征、嗜酸性粒细胞性肉芽肿病伴随多血管炎、炎性肠病、硬皮病、间质性肺病、B型或C型慢性肝炎引发的纤维化、辐射诱发的纤维化和伤口愈合引发的纤维化；
    优选地，其中所述的疾病或病症与TSLP有关。
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WO2024146630A1 (zh) * 2023-01-06 2024-07-11 江苏恒瑞医药股份有限公司 一种tslp抗体治疗哮喘的方法
WO2024163978A2 (en) 2023-02-02 2024-08-08 Medimmune, Llc Treatment of chronic rhinosinusitis with anti-tslp antibody

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