WO2021115240A1 - 抗tslp抗体及其用途 - Google Patents

抗tslp抗体及其用途 Download PDF

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WO2021115240A1
WO2021115240A1 PCT/CN2020/134438 CN2020134438W WO2021115240A1 WO 2021115240 A1 WO2021115240 A1 WO 2021115240A1 CN 2020134438 W CN2020134438 W CN 2020134438W WO 2021115240 A1 WO2021115240 A1 WO 2021115240A1
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seq
sequence
antibody
cdr
antigen
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PCT/CN2020/134438
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English (en)
French (fr)
Inventor
肖亮
何进秋
薛彤彤
刘红水
王晶翼
顾红专
罗顺涛
戎一平
刘登念
何云
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四川科伦博泰生物医药股份有限公司
和铂医药(苏州)有限公司
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Application filed by 四川科伦博泰生物医药股份有限公司, 和铂医药(苏州)有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to KR1020227013655A priority Critical patent/KR20220113673A/ko
Priority to US17/771,271 priority patent/US20220363781A1/en
Priority to JP2022524138A priority patent/JP2023505408A/ja
Priority to MX2022004919A priority patent/MX2022004919A/es
Priority to CA3155759A priority patent/CA3155759A1/en
Priority to CN202080074409.1A priority patent/CN114729037A/zh
Priority to EP20898089.6A priority patent/EP4047019A4/en
Priority to AU2020400221A priority patent/AU2020400221A1/en
Publication of WO2021115240A1 publication Critical patent/WO2021115240A1/zh

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
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    • C07K2317/00—Immunoglobulins specific features
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    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/526—CH3 domain
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/53—Hinge
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines

Definitions

  • the present invention belongs to the field of therapeutic monoclonal antibodies. More specifically, the present invention relates to an antibody against TSLP, and also relates to the use of the antibody in the treatment of diseases.
  • Thymic stromal lymphopoietin is an inflammatory cytokine similar to IL7.
  • TSLP responds to microorganisms, physical damage or inflammatory cytokines (such as IL-1 ⁇ and TNF), and is mainly secreted by epithelial cells such as skin, lung, thymus, and gastrointestinal tract. Under pathological conditions such as inflammation, stromal cells, keratinocytes, Dendritic cells (DC) and mast cells can also secrete TSLP.
  • TSLP plays an important role in the initial triggering of allergic and adaptive airway inflammation.
  • TSLP Compared with healthy controls, TSLP is highly expressed in the airways of asthmatic patients, and its level is directly related to the expression of TH2 cytokines and chemokines, and the severity of the disease.
  • TSLP can induce the maturation of dendritic cells (DC) and up-regulate the expression of OX40L.
  • DC dendritic cells
  • OX40-OX40L participates in the polarization of TH2 cells induced by initial T cells, which release IL-4, IL-5, IL-13, etc. after differentiation
  • Cytokines cause the infiltration of mast cells and eosinophils and a series of allergic inflammatory reactions, which cause pathological changes in the airway, leading to asthma attacks.
  • TSLP can effectively activate mast cells, natural killer T (NKT) cells, produce IL-13 and other TH2 cytokines, and aggravate the occurrence and development of airway inflammation.
  • NKT natural killer T
  • the receptor of TSLP is a heterodimeric receptor complex composed of IL-7R ⁇ and a unique TSLPR chain (CRFL2).
  • the binding of TSLP heterodimeric receptor causes activation of STAT5 and cell proliferation.
  • DC cells have high expression of TSLPR and IL-7R ⁇ .
  • the inventors first developed a chimeric antibody with excellent properties, which can bind to human TSLP. On this basis, the inventors conducted research and modification on the chimeric antibody, and developed a fully human antibody of the chimeric antibody.
  • the fully human antibody of the present invention has substantially the same (or even better) biological functions as the chimeric antibody. Not only has a strong affinity for TSLP, but also can effectively block the proliferation effect of TSLP on Ba/F3 cells, as well as the ability of TSLP to activate PBMC and secrete cytokines.
  • the present invention further relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and its use in the preparation of medicines for the prevention and/or treatment of asthma, allergic inflammation, allergic reactions or autoimmune diseases .
  • the antibody of the present invention has a very high degree of humanization, even a fully human antibody, so that it can be safely administered to human subjects without triggering an immunogenic response. Therefore, the antibody of the present invention has great clinical value.
  • the present invention provides an antibody or antigen-binding fragment thereof that binds TSLP, said antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDR):
  • the antibody or antigen-binding fragment thereof comprises CDR-H1 or a variant of its sequence, CDR-H2 or a variant of its sequence, and CDR-H1 contained in the VH shown in SEQ ID NO:17. H3 or its sequence variants; and/or CDR-L1 or its sequence variants, CDR-L2 or its sequence variants, and CDR-L3 or its sequence variants contained in the VL shown in SEQ ID NO: 18 Variants.
  • the antibody or antigen-binding fragment thereof includes CDR-H1 or a variant of CDR-H2 or a sequence of CDR-H2 contained in the VH shown in SEQ ID NO: 30, and a variant of CDR-H2 or a sequence of CDR-H1 contained in the VH shown in SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof includes CDR-H1 or a variant of CDR-H2 or a sequence of CDR-H2 contained in the VH shown in SEQ ID NO: 40, and a variant of CDR-H2 or a sequence of CDR-H2 contained in the VH shown in SEQ ID NO: 40. H3 or its sequence variants; and/or CDR-L1 or its sequence variants, CDR-L2 or its sequence variants, and CDR-L3 or its sequence variants contained in the VL shown in SEQ ID NO: 41 Variants.
  • the antibody or antigen-binding fragment thereof comprises CDR-H1 or a variant of CDR-H2 or a sequence of CDR-H2 contained in the VH shown in SEQ ID NO:53, and a variant of CDR-H2 or a sequence thereof, and H3 or a variant of its sequence; and/or a variant of CDR-L1 or its sequence, a variant of CDR-L2 or its sequence, and a variant of CDR-L3 or its sequence contained in the VL shown in SEQ ID NO: 54 Variants.
  • the antibody or antigen-binding fragment thereof includes CDR-H1 or a variant of CDR-H2 or a sequence of CDR-H2 contained in the VH shown in SEQ ID NO: 68, and a variant of CDR-H2 or a sequence of CDR-H1 contained in the VH shown in SEQ ID NO: 68. H3 or a variant of its sequence; and/or a variant of CDR-L1 or its sequence, a variant of CDR-L2 or its sequence, and a variant of CDR-L3 or its sequence contained in the VL shown in SEQ ID NO: 69 Variants.
  • the variant of the sequence has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the source CDR. CDR.
  • the substitutions are conservative substitutions.
  • the CDR is defined according to the AbM, Chothia, Kabat or IMGT numbering system.
  • the VH and/or VL of the antibody or antigen-binding fragment thereof includes a framework region (FR) from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof binds human TSLP and/or monkey TSLP.
  • the present invention provides an antibody or antigen-binding fragment thereof capable of binding TSLP, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) and/or a light chain variable region (VL) .
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof of the present invention comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:
  • the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 3 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3).
  • the substitution, deletion or addition of CDR-H2 is SEQ ID NO: 5 or compared with the substitution, deletion or addition of one or several amino acids (for example, substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) comprising the following 3 CDRs:
  • the sequence is SEQ ID NO: 6 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids CDR-L1 of the sequence of the substitution, deletion or addition, the sequence is SEQ ID NO: 7 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition the sequence is SEQ ID NO: 8 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 19 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3).
  • the CDR-H1 of the sequence of amino acid substitution, deletion or addition, the sequence is SEQ ID NO: 20 or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 21 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) that contains the following 3 CDRs: the sequence is SEQ ID NO: 22 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of the substitution, deletion or addition, the sequence is SEQ ID NO: 23 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 24 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 32 or compared with the substitution, deletion or addition of one or several amino acids (for example, 1, 2, or 3)
  • the CDR-H1 of the sequence of the substitution, deletion or addition of amino acids the sequence is SEQ ID NO: 33 or compared with the substitution, deletion or addition of one or several amino acids (for example, 1, 2, or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 34 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) comprising the following 3 CDRs:
  • the sequence is SEQ ID NO: 35 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of the substitution, deletion or addition, the sequence is SEQ ID NO: 23 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 24 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • the heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 42 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3
  • the CDR-H1 of the sequence of amino acid substitutions, deletions or additions, the sequence is SEQ ID NO: 43 or compared with the substitution, deletion or addition of one or several amino acids (for example, 1, 2 or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 44 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) that contains the following 3 CDRs: the sequence is SEQ ID NO: 45 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of substitution, deletion or addition, the sequence is CDR-L2 of SEQ ID NO: 46, or the substitution, deletion or addition of one or several amino acids (for example, 1, 2, or 3)
  • the sequence of amino acid substitution, deletion or addition) the sequence is SEQ ID NO: 47 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 55 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 CDR-H1 of the sequence of amino acid substitutions, deletions or additions, the sequence is CDR-H2 of SEQ ID NO: 56, or compared with the CDR-H2 of one or several amino acid substitutions, deletions or additions (for example, one, two Or 3 amino acid substitutions, deletions or additions) sequence, the sequence is SEQ ID NO: 57 or compared with one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions) , Deleted or added) CDR-H3; and/or,
  • a light chain variable region comprising the following 3 CDRs:
  • the sequence is SEQ ID NO: 58 or compared with it, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of substitution, deletion or addition the sequence is SEQ ID NO: 59 or compared with the substitution, deletion or addition of one or several amino acids (for example, substitution of 1, 2, or 3 amino acids.
  • the CDR-L2 of the sequence of the deletion or addition the sequence is SEQ ID NO: 60 or has one or a few amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added).
  • the antibody or antigen-binding fragment thereof of the present invention comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof of the present invention comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the AbM numbering system:
  • the heavy chain variable region (VH) comprising the following 3 CDRs: the sequence is SEQ ID NO: 9 or compared with it, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3).
  • the CDR-H1 of the sequence of amino acid substitution, deletion or addition, the sequence is SEQ ID NO: 10 or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 11 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) that contains the following 3 CDRs: the sequence is SEQ ID NO: 12 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of substitution, deletion or addition, the sequence is SEQ ID NO: 13 or compared with the substitution, deletion or addition of one or several amino acids (for example, substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 8 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 25 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3
  • the CDR-H1 of the sequence of the substitution, deletion or addition of amino acids the sequence is SEQ ID NO: 26 or compared with the substitution, deletion or addition of one or several amino acids (for example, 1, 2 or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 27 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) that contains the following 3 CDRs: the sequence is SEQ ID NO: 28 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of the substitution, deletion or addition, the sequence is SEQ ID NO: 29 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 24 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 36 or compared with it, it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3).
  • the CDR-H1 of the sequence of amino acid substitution, deletion or addition, the sequence is SEQ ID NO: 37 or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 38 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) that contains the following 3 CDRs the sequence is SEQ ID NO: 39 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of the substitution, deletion or addition, the sequence is SEQ ID NO: 29 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 24 or has one or several amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • the heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 48 or compared with the substitution, deletion or addition of one or several amino acids (for example, 1, 2, or 3)
  • the CDR-H1 of the sequence of amino acid substitution, deletion or addition, the sequence is SEQ ID NO: 49 or it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids).
  • the light chain variable region (VL) comprising the following 3 CDRs: the sequence is SEQ ID NO: 51 or compared with it has one or a few amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids)
  • the CDR-L1 of the sequence of the substitution, deletion or addition, the sequence is SEQ ID NO: 52 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 47 or has one or a few amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added);
  • the heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 61 or compared with the substitution, deletion or addition of one or several amino acids (for example, 1, 2, or 3)
  • the CDR-H1 of the sequence of amino acid substitution, deletion or addition, the sequence is SEQ ID NO: 62 or compared with it has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acids)
  • the CDR-H2 of the sequence of the substitution, deletion or addition of the sequence is SEQ ID NO: 63 or compared with the substitution, deletion or addition of one or several amino acids (for example, the substitution of 1, 2, or 3 amino acids) , Deleted or added) CDR-H3; and/or,
  • the light chain variable region (VL) that contains the following 3 CDRs: the sequence is SEQ ID NO: 64 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2, or 3 amino acids).
  • the CDR-L1 of the sequence of substitution, deletion or addition, the sequence is SEQ ID NO: 65 or compared with the substitution, deletion or addition of one or several amino acids (for example, substitution of 1, 2, or 3 amino acids,
  • the CDR-L2 of the sequence of the deletion or addition, the sequence is SEQ ID NO: 60 or has one or a few amino acid substitutions, deletions, or additions (for example, 1, 2, or 3 amino acid substitutions, deletions, or CDR-L3 of the sequence added).
  • the antibody or antigen-binding fragment thereof of the present invention comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein CDRs are defined by the AbM numbering system:
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof of the present invention comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein it is the same as the aforementioned CDR defined by IMGT or AbM
  • at least one CDR in the variable region of the heavy chain (VH) and/or the variable region of the light chain (VL) contains a mutation, which is a substitution, deletion or addition of one or several amino acids, or any combination thereof ( For example, 1, 2, or 3 amino acid substitutions, deletions or additions or any combination thereof).
  • substitutions described in the present invention are conservative substitutions.
  • the VH of the antibody or antigen-binding fragment thereof of the present invention comprises a framework region (FR) derived from the heavy chain variable region (VH) of a human immunoglobulin, and/or the antibody or antigen thereof
  • the VL of the binding fragment contains the framework region (FR) derived from the light chain variable region (VL) of human immunoglobulin. Therefore, in certain embodiments, the antibodies of the invention or antigen-binding fragments thereof are humanized. In certain embodiments, the antibodies of the invention or antigen-binding fragments thereof are fully human.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • a human immunoglobulin heavy chain framework region or a variant thereof which has a conservative substitution of up to 20 amino acids (e.g., up to 20, Conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids );and / or
  • the degree of humanization of the antibody or antigen-binding fragment thereof of the present invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH The heavy chain variable region (VH), which comprises an amino acid sequence selected from the following:
  • VL The light chain variable region (VL), which comprises an amino acid sequence selected from the following:
  • the antibody or antigen-binding fragment thereof of the present invention comprises the VH shown in SEQ ID NO: 1, and/or the VL shown in SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the VH shown in SEQ ID NO: 17, and/or the VL shown in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the VH shown in SEQ ID NO: 30, and/or the VL shown in SEQ ID NO: 31.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the VH shown in SEQ ID NO: 40, and/or the VL shown in SEQ ID NO: 41.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the VH shown in SEQ ID NO:53, and/or the VL shown in SEQ ID NO:54.
  • the antibody or antigen-binding fragment thereof of the present invention comprises the VH shown in SEQ ID NO: 68, and/or the VL shown in SEQ ID NO: 69.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • VH and VL heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) are independently associated with (a) to (f ) VH and VL in any group are respectively at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% , At least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or
  • the substitution is a conservative substitution.
  • the heavy chain of the antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, which is similar to the wild-type sequence from which it is derived.
  • CH heavy chain constant region
  • conservative substitutions of up to 50 amino acids e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids ;
  • conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids For example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).
  • the light chain of the antibody or antigen-binding fragment thereof of the present invention comprises the light chain constant region (CL) of human immunoglobulin or a variant thereof, which is similar to the wild-type sequence from which it is derived.
  • CL light chain constant region
  • conservative substitutions of up to 50 amino acids e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids ;
  • conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids For example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).
  • the constant region is altered, for example mutated, to modify the properties of the anti-TSLP antibody molecule (e.g., to alter one or more of the following properties: Fc receptor binding, antibody glycosylation, cysteine residues Number of bases, effector cell function or complement function).
  • Functional changes can be produced by replacing at least one amino acid residue in the constant region of the antibody with different residues, for example, changing the affinity of the antibody for the effector ligand (such as FcR or complement C1q), thereby changing the effector function (such as reducing ).
  • the Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis (ADCP), CDC and so on.
  • the antibody or antigen-binding fragment thereof of the present invention has a heavy chain constant region (Fc), which is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. Region; particularly selected from the heavy chain constant region of, for example, IgG1, IgG2, IgG3, and IgG4, more particularly selected from the heavy chain constant region of IgG1 (for example, human IgG1).
  • the human IgG1 heavy chain constant region is shown in SEQ ID NO: 14.
  • the antibody or antigen-binding fragment thereof of the present invention has a light chain constant region, which is selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (e.g., a human kappa light chain constant region).
  • the antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region. In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 constant region shown in uniprot ID P01857 (SEQ ID NO: 74).
  • the antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region (for example, SEQ ID NO: 74) or a variant thereof.
  • the variant is located at positions 234, 235, 237, 265, 297, 331, 329, and 434 have mutations in at least one site.
  • the variant contains at least one of the following mutations: L234A, L235A, D265A, N297A, L234F, L235E, P331S, P329G, N434A, N434Y, N434F, N434W, N434S, N434G, N434H, N434Q.
  • the variant contains at least one of the following mutations: L234A, L235A, G237A, N434A.
  • the IgG1 heavy chain constant region variant comprises L234A, L235A, and G237A. In some embodiments, the IgG1 heavy chain constant region variant comprises mutations L234A, L235A, G237A, and N434A.
  • the antibody or antigen-binding fragment thereof comprises the CH shown in SEQ ID NO: 14 or a variant thereof, which has conservative substitutions of up to 20 amino acids compared with SEQ ID NO: 14 (for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Conservative substitutions of amino acids), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least compared with SEQ ID NO: 14 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the variant contains N297A and/or N434A. In some embodiments, the variant contains N434A.
  • the antibody or antigen-binding fragment thereof comprises the CH shown in SEQ ID NO: 15 or a variant thereof, which has conservative substitutions of up to 20 amino acids compared to SEQ ID NO: 15 (for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Conservative substitutions of amino acids), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least compared with SEQ ID NO: 15 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the antibody or antigen-binding fragment thereof comprises a human IgG4 heavy chain constant region (for example, SEQ ID NO: 75) or a variant thereof.
  • the variant is located at position 228 and/or 434 At least one site was mutated.
  • the mutant comprises S228P and/or N434A.
  • the human IgG4 heavy chain constant region variant comprises S228P and N434A.
  • the antibody or antigen-binding fragment thereof comprises the CH shown in SEQ ID NO: 70 or a variant thereof, which has conservative substitutions of up to 20 amino acids compared to SEQ ID NO: 70 (for example, conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Conservative substitutions of amino acids), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least compared with SEQ ID NO: 70 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof.
  • the light chain constant region comprises a kappa light chain constant region.
  • the light chain constant region comprises the light chain constant region (CL) shown in SEQ ID NO: 16 or a variant thereof, and the variant has at most 20 amino acids compared to SEQ ID NO: 16 Conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least compared with SEQ ID NO: 16 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 14 and a light chain constant region (CL) shown in SEQ ID NO: 16.
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 15 and a light chain constant region (CL) shown in SEQ ID NO: 16.
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) shown in SEQ ID NO: 70 and a light chain constant region (CL) shown in SEQ ID NO: 16.
  • CH heavy chain constant region
  • CL light chain constant region
  • the aforementioned site mutation causes the antibody or antigen-binding fragment to have no or reduced ADCP, ADCC and/or CDC activity compared to the corresponding antibody or antigen-binding fragment containing the constant region of a human IgG4 or IgG4 heavy chain.
  • the above-mentioned site mutation causes the antibody or antigen-binding fragment to have no or reduced ADCP, ADCC and/or CDC activity compared to the corresponding antibody or antigen-binding fragment without the mutation or substitution.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • a light chain which comprises an amino acid sequence selected from:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • a light chain which comprises an amino acid sequence selected from:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • a light chain which comprises an amino acid sequence selected from:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • a light chain which comprises an amino acid sequence selected from:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • a light chain which comprises an amino acid sequence selected from:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the invention comprises:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • a light chain which comprises an amino acid sequence selected from:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody of the present invention includes: a heavy chain including the VH shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and, including The VL shown in SEQ ID NO: 2 and the light chain of the light chain constant region (CL) shown in SEQ ID NO: 16.
  • the antibody of the present invention includes: a heavy chain including the VH shown in SEQ ID NO: 17 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and, including The VL shown in SEQ ID NO: 18 and the light chain of the light chain constant region (CL) shown in SEQ ID NO: 16.
  • the antibody of the present invention includes: a heavy chain including the VH shown in SEQ ID NO: 30 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and, including The VL shown in SEQ ID NO: 31 and the light chain of the light chain constant region (CL) shown in SEQ ID NO: 16.
  • the antibody of the present invention includes: a heavy chain including the VH shown in SEQ ID NO: 40 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and, including The VL shown in SEQ ID NO: 41 and the light chain of the light chain constant region (CL) shown in SEQ ID NO: 16.
  • the antibody of the present invention includes: a heavy chain including the VH shown in SEQ ID NO: 53 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and, including The VL shown in SEQ ID NO: 54 and the light chain of the light chain constant region (CL) shown in SEQ ID NO: 16.
  • the antibody of the present invention includes: a heavy chain including the VH shown in SEQ ID NO: 68 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and, including The VL shown in SEQ ID NO: 69 and the light chain of the light chain constant region (CL) shown in SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain
  • the heavy chain includes:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • sequence shown in (i) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, or at least 99% sequence identity;
  • the light chain includes:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • sequence shown in (vi) and (iv) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, or at least 99% sequence identity;
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain
  • the heavy chain includes:
  • substitutions, deletions or additions of one or several amino acids or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof; or
  • sequence shown in (i) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, or at least 99% sequence identity;
  • the light chain includes:
  • amino acid substitutions, deletions or additions or any combination thereof e.g. up to 50, up to 45, up to 40, up to 35, up to 30 , Up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example, 1, 2, 3, 4, 5, 6 , 7, 8, 9, or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • sequence shown in (vi) and (iv) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98%, or at least 99% sequence identity;
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibodies of the invention are chimeric antibodies, humanized antibodies or fully human antibodies.
  • the antibody or antigen-binding fragment thereof of the present invention is selected from scFv, Fab, Fab', (Fab') 2 , Fv fragment, disulfide-linked Fv (dsFv), diabody.
  • the antibody molecule or antigen-binding fragment thereof of the present invention may exhibit at least one of the following properties:
  • Binding TSLP for example, human TSLP
  • a KD of less than about 50 nM, for example, less than about 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.1 nM, 1 pM, 0.1 pM or lower; the KD may be known in the art Measured by the technology, for example, measured by Fortibio or ELISA;
  • EC50 binds TSLP (for example, human TSLP); the EC50 can be measured by techniques known in the art, for example, by flow cytometry, ELISA such as affinity ELISA technology or cell competition ELISA technology.
  • IC50 of 0.1pM or less inhibits the binding of TSLP to IL7R ⁇ /TSLPR; the IC50 is measured by ELISA technology;
  • Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5;
  • the isoelectric point (PI) is about 6.5 to about 8.5, such as about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.9, about 8.0, about 8.2 or about 8.5.
  • the antibody of the present invention has good affinity with FcRn.
  • the KD(M) value of affinity with FcRn is on the order of 10-9 .
  • the antibody of the present invention has a longer half-life in vivo.
  • the antibody of the present invention also has good hydrophilicity.
  • the hydrophobic time of the antibody of the present invention is between 8 minutes and 14 minutes as detected by a chromatographic column.
  • the antibody or antigen-binding fragment thereof of the present invention may be derivatized, for example, linked to another molecule (for example, another polypeptide or protein).
  • another molecule for example, another polypeptide or protein.
  • derivatization eg, labeling
  • the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms.
  • the antibody of the present invention or its antigen-binding fragment can be linked (by chemical coupling, gene fusion, non-covalent linkage or other means) to one or more other molecular groups, such as another antibody (for example, to form a bispecific Antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of antibodies or antigen-binding fragments to another molecule (for example, avidin or polyhistidine tag).
  • another antibody for example, to form a bispecific Antibodies
  • detection reagents for example, to form a bispecific Antibodies
  • pharmaceutical reagents for example, to form a bispecific Antibodies
  • proteins or polypeptides capable of mediating the binding of antibodies or antigen-binding fragments to another molecule (for example, avidin or polyhistidine tag).
  • bispecific antibody is produced by cross-linking two or more antibodies (belonging to the same type or different types).
  • Methods for obtaining bispecific antibodies are well known in the art, and examples thereof include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods), or genetic engineering methods.
  • Another type of derivatized antibody is a labeled antibody.
  • the antibody or antigen-binding fragment thereof of the present invention can be linked to a detectable label.
  • the detectable label of the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical or chemical means.
  • Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radioactive nuclear (E.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes for example, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.
  • radioactive nuclear E.g., 3H, 125I, 35S, 14C or 32P
  • radioactive labels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting reaction products produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers.
  • such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • the detectable label as described above can be connected to the antibody or antigen-binding fragment thereof of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the antibody or antigen-binding fragment thereof of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or sugar groups. These groups can be used to improve the biological properties of antibodies, for example to increase serum half-life.
  • chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or sugar groups.
  • one aspect of the present invention provides a conjugate comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention and a coupling portion
  • the coupling portion may be the aforementioned detectable label, such as a radioisotope , Fluorescent substances, luminescent substances, colored substances or enzymes.
  • the coupling moiety can also be a therapeutic agent.
  • the present invention provides a multispecific antibody, the multispecific antibody comprising a first antibody or fragments thereof, and another antibody or fragments thereof, or antibody analogs, wherein the first antibody or Fragments thereof, additional antibodies or fragments thereof, or antibody analogs retain their original binding specificity.
  • the first antibody or fragment thereof is any TSLP-binding (monoclonal) antibody or antigen-binding fragment thereof of the present invention.
  • antibody mimetic refers to the same specific binding to an antigen as an antibody, but without the structure of an antibody. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa, such as ankyrin repeat protein (DARPin) and fynomer.
  • the designed ankyrin repeat protein can be linked to IgG antibody, scFv-Fc antibody fragment or a combination thereof, as described in CN104341529A.
  • the anti-IL-17a fynomer is fused with an anti-IL-6R antibody to produce a bispecific fusion polypeptide, as described in WO2015141862A1.
  • the multispecific antibody is formed by coupling a first antibody or antigen-binding fragment thereof with other antibodies or antigen-binding fragments or antibody analogs thereof, and wherein each antibody or antigen-binding fragment or antibody thereof The analog retains its original binding specificity, and the first antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof according to the present invention.
  • the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • the antibody of the present invention can be prepared by various methods known in the art, for example, obtained by genetic engineering recombination technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into the expression vector and then transfected into the host cell. Then, the transfected host cell is cultured under specific conditions, and the antibody of the present invention is expressed.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J.Biochem.Biophys.Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium.
  • the present invention provides an isolated nucleic acid molecule comprising an antibody or antigen-binding fragment thereof encoding the present invention, or a heavy chain variable region and/or light chain variable region, or one or Nucleotide sequence of multiple CDRs.
  • the nucleotide sequence can be replaced based on the codon degeneracy.
  • the nucleotide sequence is codon optimized.
  • the isolated nucleic acid molecule of the present invention comprises: (i) the first nucleic acid and the second nucleic acid respectively encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment thereof of the present invention; Nucleic acid, or (ii) the first nucleic acid encoding the heavy chain variable region and the heavy chain constant region of the antibody or antigen-binding fragment thereof of the present invention, and the second nucleic acid of the light chain variable region and the light chain constant region, or (iii) The first nucleic acid and the second nucleic acid respectively encoding the heavy chain and the light chain of the antibody or antigen-binding fragment thereof of the present invention.
  • the first nucleic acid and the second nucleic acid comprise a degenerate sequence or a nucleic acid having substantially the same sequence as any of the first nucleic acid and the second nucleic acid in (i)-(iii) above.
  • the degenerate sequence or substantially the same sequence refers to at least about 85%, 90%, 95%, 99% or more than the nucleic acid molecule in (i)-(iii) Sequences of sequence identity or sequences with one or more nucleotide substitutions, or sequences that differ by no more than 3, 6, 15, 30, or 45 nucleotides.
  • a vector (such as a cloning vector or an expression vector) is provided, which comprises the isolated nucleic acid molecule of the present invention.
  • the vectors of the present invention are, for example, plasmids, cosmids, bacteriophages, lentiviruses and the like.
  • the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present invention in a subject (e.g., mammal, e.g., human).
  • a host cell which contains the isolated nucleic acid molecule of the present invention or the vector of the present invention.
  • the host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli).
  • Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include but are not limited to Sf9 cells.
  • the host cell of the present invention is a mammalian cell, such as CHO (e.g. CHO-K1, CHO-S, CHO DXB11, CHO DG44).
  • a method for preparing the antibody or antigen-binding fragment thereof of the present invention which includes culturing the host cell of the present invention under conditions that allow the expression of the antibody or antigen-binding fragment thereof, and obtaining the host cell from the cultured host cell.
  • the antibody or antigen-binding fragment thereof is recovered from the culture.
  • a pharmaceutical composition which comprises the antibody or antigen-binding fragment thereof, nucleic acid, carrier, host cell, multispecific antibody, and/or conjugate of the present invention, and a pharmaceutically acceptable carrier and / Or excipients.
  • the pharmaceutical composition of the present invention comprises the antibody of the present invention or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition of the present invention comprises the host cell of the present invention, and a pharmaceutically acceptable carrier and/or excipient, wherein the host cell comprises the isolated nucleic acid molecule as described above or Carrier.
  • the pharmaceutical composition of the present invention comprises the multispecific antibody of the present invention, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition of the present invention comprises the conjugate of the present invention and a pharmaceutically acceptable carrier and/or excipient.
  • the antibody or antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody or conjugate in the pharmaceutical composition of the present invention is used to produce at least one of the following biological activities in a subject:
  • Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5;
  • the antibody or antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody or conjugate in the pharmaceutical composition of the present invention can inhibit or block the binding of TSLP to TSLPR/IL7R ⁇ .
  • the combination of TSLP and TSLPR/IL7R ⁇ can cause many allergic inflammatory diseases, including allergic diseases and non-allergic diseases.
  • asthma including severe asthma
  • idiopathic pulmonary fibrosis atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilia Esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, COPD, systemic sclerosis, Keloids, ulcerative colitis, chronic sinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; celiac disease, such as eosinophilic gastroenteritis, Churg-Strauss syndrome; eosinophil-related Gastrointestinal diseases, such as eosinophilia/eosinophilic granuloma with polyangiitis,
  • TSLP and TSLPR/IL7R ⁇ are also associated with autoimmune diseases. Therefore, the antibody or its antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody or conjugate in the composition of the present invention can prevent or treat autoimmune diseases such as diabetes, myasthenia gravis, gastritis, pemphigus Sores, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid diseases.
  • autoimmune diseases such as diabetes, myasthenia gravis, gastritis, pemphigus Sores, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid diseases.
  • the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent are provided as separate components or as components of the same composition. Therefore, the antibody or antigen-binding fragment thereof of the present invention and the additional pharmaceutically active agent can be combined or separately administered simultaneously or sequentially.
  • the pharmaceutical composition may also include additional pharmaceutically active agents.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered alone or in combination with other active agents.
  • the anti-TSLP antibody or antigen-binding fragment thereof and one or more other active agents can be administered separately, simultaneously or sequentially.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with any suitable immunosuppressive agent, including but not limited to anti-inflammatory drugs, especially inhaled, intranasal or parenteral corticosteroids, such as budesonide, beclamethasone dipropionate, fluticasone propionate, ciclesonide, trichosone furoate, fluticasone furoate, fluticasone propionate, budesonide, ciclesonide, beclomethasone dipropionate, mometasone furoate, triturate Annaid and prednisolone.
  • anti-inflammatory drugs especially inhaled, intranasal or parenteral corticosteroids, such as budesonide, beclamethasone dipropionate, fluticasone propionate, ciclesonide, trichosone furoate, fluticasone furoate, fluticasone propionate, budesonide, cicle
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with a fixed dose of inhaled corticosteroid. Such as a fixed-dose combination with fluticasone furoate or fluticasone propionate.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be combined with a non-steroidal glucocorticoid receptor agonist; LTD4 antagonist or LTB4 antagonist, including montelukast, pranlukast, Zallukast, enclave, etc.; A2A agonist; A2B antagonist; dopamine receptor agonist; or, PDE4 inhibitor combination for administration.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with pirfenidone or nintedanib or an avB6 antagonist.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with bronchodilator drugs.
  • bronchodilator drugs Such as ⁇ -2 adrenergic receptor agonist and/or muscarinic antagonist combined administration.
  • Suitable beta-2 adrenoceptor agonists include vilanterol, salmeterol, salbutamol, formoterol, salmeterol, fenoterol, camoterol, tanbuterol, naminol Terbuterol, Clenbuterol, Pirbuterol, Fludentrol, Riputerol, Bambuterol, Indacaterol, Terbutaline, and their salts.
  • Suitable muscarinic antagonists include umeclidinium, tiotropium, glycopyrrolate, ipratropium, and their salts such as the hydrobromide of umeclidinium.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with a fixed dose of ⁇ -2 adrenergic receptor agonist and/or muscarinic antagonist, such as vilante A fixed-dose combination of roltriphenylacetate and umeclidinium bromide, or a dual combination with vilanterol triphenylacetate and umeclidinium bromide.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered with a combination of one or more bronchodilators and inhaled steroids.
  • Such combinations may include dual combinations such as fluticasone furoate and vilanterol triphenylacetate, fluticasone furoate and umeclidinium bromide, fluticasone propionate and salmeterol, budesonide and formoterol, mometasone And formoterol, and triple treatments such as fluticasone furoate, vilanterol triphenylacetate and umeclidinium bromide.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with a fixed dose of inhaled corticosteroids and one or more bronchodilators, such as fluticasone furoate and vilante Rostriphenylacetate, or fluticasone propionate and salmeterol, or fluticasone furoate and umeclidinium bromide, or a fixed-dose combination of fluticasone furoate, vilanterol triphenylacetate and umeclidinium bromide.
  • bronchodilators such as fluticasone furoate and vilante Rostriphenylacetate, or fluticasone propionate and salmeterol, or fluticasone furoate and umeclidinium bromide, or a fixed-dose combination of fluticasone furoate, vilanterol triphenylacetate and umeclidinium bromide.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with antagonists of cytokine receptors, such as CCR-1, CCR-3, CCR-4, CCR-5, CCR- 6. Antagonists of CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5.
  • antagonists of cytokine receptors such as CCR-1, CCR-3, CCR-4, CCR-5, CCR- 6.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with other cytokine or cytokine receptor antibodies, such as anti-IgE antibody, anti-IL31 antibody, anti-IL31R antibody, anti-IL13 antibody , Anti-endoglin antibody, anti-IL1b antibody, another anti-TSLP antibody or anti-hTSLPR antibody or a combination thereof.
  • cytokine or cytokine receptor antibodies such as anti-IgE antibody, anti-IL31 antibody, anti-IL31R antibody, anti-IL13 antibody , Anti-endoglin antibody, anti-IL1b antibody, another anti-TSLP antibody or anti-hTSLPR antibody or a combination thereof.
  • the anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with the following: anti-leukotriene antagonists such as montelukast, zafirukast and pranomilast; PDE4 inhibitors such as roflumilast; Xanthene; anti-IgE antibody; IL-13 antagonist; IL-6 antagonist and antagonist of IL-1, IL-33, IL-25 or TNF- ⁇ .
  • anti-leukotriene antagonists such as montelukast, zafirukast and pranomilast
  • PDE4 inhibitors such as roflumilast
  • Xanthene anti-IgE antibody
  • IL-13 antagonist anti-IgE antibody
  • IL-6 antagonist antagonist of IL-1, IL-33, IL-25 or TNF- ⁇ .
  • anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be combined with antihistamine or antitussive drugs, such as cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratadine, Loratadine, ketamine, and fexonadine hydrochloride, activastine, astemizole, azelastine, ebastine, epinadine, mizolastine, and tefenadine were administered in combination.
  • antihistamine or antitussive drugs such as cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratadine, Loratadine, ketamine, and fexonadine hydrochloride, activastine, astemizole, azelastine, ebastine, epinadine, mizolastine, and tefen
  • the above-mentioned TSLP binding antibody or antigen-binding fragment thereof is administered in combination with one or more other active agents.
  • the other active agents can be selected from but not limited to: immunosuppressants (such as corticosteroids, non-steroids) Glucocorticoid receptor agonist, leukotriene D4 antagonist, leukotriene B4 antagonist, A2A agonist, A2B antagonist, dopamine receptor agonist, pirfenidone nintedanib, or avB6 antagonist) , Bronchodilators (such as ⁇ -2 adrenergic receptor agonists, muscarinic antagonists, short-acting ⁇ 2 receptor agonists, long-acting ⁇ 2 receptor agonists, short-acting anticholinergic drugs, methylxanthines Class drugs, long-acting anticholinergic drugs), other cytokine or cytokine receptor antagonists or antibodies (e.g.
  • IL-13 antagonist IL-6 antagonist, IL-1, IL-33, IL-25 Or antagonist of TNF- ⁇ , anti-IgE antibody, anti-IL31 antibody, anti-IL31R antibody, anti-IL13 antibody, anti-endoglin antibody, anti-IL1b antibody, another anti-TSLP antibody or anti-hTSLPR antibody), antibiotics, radiotherapy, Leukotriene antagonists (e.g. montelukast, zalukast or pranlukast), PDE4 inhibitors (e.g. roflumilast, xanthene), antihistamines or antitussive drugs.
  • Leukotriene antagonists e.g. montelukast, zalukast or pranlukast
  • PDE4 inhibitors e.g. roflumilast, xanthene
  • antihistamines or antitussive drugs e.g. montelukast, zalukast or
  • the pharmaceutical composition is administered simultaneously, separately or sequentially with the other treatment, such as before, simultaneously or after the additional pharmaceutically active agent.
  • the antibody or antigen-binding fragment, nucleic acid, vector, host cell, conjugate or multispecific antibody of the present invention is provided for (1) inhibiting or blocking the binding of TSLP to TSLPR/IL7R ⁇ , (2 ) Down-regulate or eliminate the activity of TSLP; (3) down-regulate or block the expression of OX40L; (4) inhibit or block the activation and/or proliferation of mast cells, DC, and NKT cells induced by TSLP; (5) inhibit or block TSLP-induced secretion of osteoprotegerin (OPG), (6) inhibit or block TSLP-induced secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5; and/or (7) Prevent or treat allergic diseases, allergic reactions or autoimmune diseases.
  • OPG osteoprotegerin
  • the use of the antibody or antigen-binding fragment, nucleic acid, vector, host cell, conjugate or multispecific antibody of the present invention in the preparation of a medicine is provided, and the medicine is used for:
  • Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5 induced by TSLP;
  • the host cell of the present invention when used to prepare a medicine, contains the isolated nucleic acid molecule or vector as described above.
  • the antibody or antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody or conjugate of the present invention is used to prepare a drug
  • the drug is used in a subject (e.g., Humans) prevent and/or treat asthma, allergic inflammation, allergic reactions or autoimmune diseases.
  • the subject is a mammal, including non-human mammals and humans. In certain embodiments, the subject is a human.
  • the antibodies or antigen-binding fragments, nucleic acids, vectors, host cells, multispecific antibodies, conjugates, or drugs of the present invention are used to prevent and/or treat allergic inflammatory diseases, Including allergic diseases and non-allergic diseases.
  • asthma including severe asthma
  • idiopathic pulmonary fibrosis atopic dermatitis (AD)
  • allergic conjunctivitis allergic rhinitis
  • AR allergic conjunctivitis
  • NS Netherton syndrome
  • EOE eosinophilia Esophagitis
  • food allergy allergic diarrhea
  • eosinophilic gastroenteritis allergic bronchopulmonary aspergillosis
  • ABPA allergic fungal sinusitis
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • Systemic sclerosis keloids
  • ulcerative colitis chronic sinusitis (CRS) and nasal polyps
  • chronic eosinophilic pneumonia eosinophilic bronchitis
  • celiac disease such as eosinophilic gastroenteritis, Churg-Strauss syndrome
  • Eosinophil-related gastrointestinal diseases such as eosinophilia/eosinophilic
  • the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies or conjugates of the present invention are used to prevent and/or treat diseases related to autoimmunity.
  • the disease includes, but is not limited to: hyperthyroidism, diabetes, myasthenia gravis, ulcerative colitis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, erythema Lupus, rheumatoid arthritis, etc.
  • the present invention provides a method for (1) inhibiting or blocking the binding of TSLP to TSLPR/IL7R ⁇ in vivo or in vitro, (2) down-regulating or eliminating the activity of TSLP; (3) down-regulating or blocking the expression of OX40L; (4) Inhibit or block the secretion of osteoprotegerin (OPG) induced by TSLP; (5) Inhibit or block the secretion of Th2 cytokines; (6) Inhibit or block TSLP-induced mast cells, DC, NKT
  • OPG osteoprotegerin
  • the method for at least one of cell activation and/or proliferation comprising: applying to the cell or subject any one of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, Conjugate, or pharmaceutical composition.
  • an additional pharmaceutically active agent is applied.
  • the subject is a mammal, including non-human mammals and humans; preferably, the subject is a human.
  • the present invention provides a method for preventing and/or treating asthma, allergic reactions, allergic inflammation, or autoimmune diseases in a subject, the method comprising: The subject administers an effective amount of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, conjugate or pharmaceutical composition of the present invention.
  • the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, conjugates and pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions , Emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections and concentrated solutions for injections), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the pharmaceutical composition of the present invention should be sterile and stable under the conditions of production and storage, and can be prepared as an injection.
  • the antibody or antigen-binding fragment thereof of the present invention may be present in a pharmaceutical composition in a unit dosage form for ease of administration.
  • the pharmaceutical composition of the present invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody or conjugate of the present invention.
  • “Prophylactically effective amount” refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease.
  • “Therapeutically effective amount” refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who has already suffered from the disease, for example, 0.1 mg/ml to 5000 mg/ml.
  • the subject may be a mammal (including non-human mammals and humans), such as humans.
  • the antibody or antigen-binding fragment thereof of the present invention can bind to TSLP, and thus can be used to detect the presence or level of TSLP in a sample.
  • the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention bears a detectable label.
  • the kit further includes a second antibody, which specifically recognizes the antibody of the present invention or an antigen-binding fragment thereof.
  • the second antibody further includes a detectable label.
  • the detectable label may be any substance that can be detected by fluorescence, spectroscopy, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferable that such a label can be applied to immunological detection (for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).
  • immunological detection for example, enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.
  • Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3H, 125I, 35S, 14C or 32P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE) , Texas red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.
  • radionuclides For example, 3H, 125I, 35S, 14C or 32P
  • fluorescent dyes for example, flu
  • radioactive labels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting reaction products produced by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing colored markers.
  • the detectable label as described above can be attached to the recombinant protein of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the present invention provides a method for detecting the presence or level of TSLP in a sample, which includes the step of using the antibody or antigen-binding fragment thereof of the present invention.
  • the antibody or antigen-binding fragment thereof of the present invention also bears a detectable label.
  • the method further comprises using a reagent with a detectable label to detect the antibody or antigen-binding fragment thereof of the present invention.
  • the method can be used for diagnostic purposes or non-diagnostic purposes (for example, for TSLP-TSLP/IL-7Ra pathway research, drug screening, histochemical analysis, etc.).
  • the sample used for non-diagnostic purposes is a cell sample, such as a cell line or an ex vivo cell culture.
  • the present invention provides a method for detecting the presence or level of TSLP in a sample, the method comprising under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof of the present invention and TSLP , Contacting the sample with the antibody or antigen-binding fragment thereof, and detecting the formation of the complex.
  • the present invention provides a method for diagnosing asthma, allergic inflammation, allergic reaction or autoimmune disease in a subject, comprising
  • TSLP levels indicate the presence of asthma, allergic inflammation, allergic reactions, or autoimmune diseases.
  • the subject is a mammal, including non-human mammals and humans.
  • the subject is a human.
  • the allergic inflammation, allergic reaction or autoimmune disease is as described above.
  • the present invention provides that the antibody or antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody, conjugate or pharmaceutical composition of the present invention is used for the diagnosis of asthma, allergies. Use in drugs or kits for inflammation, allergic reactions or autoimmune diseases.
  • the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or level of TSLP in a sample in another aspect, provides a diagnostic or therapeutic kit, which includes one or more of the following materials: the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, Conjugate or pharmaceutical composition.
  • the diagnostic or therapeutic kit further includes instructions for use.
  • the antibody or antigen-binding fragment of the present invention has high binding affinity to TSLP and has good specificity. Therefore, the antibodies or antigen-binding fragments of the present invention are suitable for preventing and/or treating asthma, allergic inflammation, allergic reactions or autoimmune diseases.
  • the fully human antibody of the present invention retains a high degree of human origin, so that it can be safely administered to human subjects without triggering an immunogenic response. Therefore, the antibody or antigen-binding fragment of the present invention has great clinical value.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair has a light chain (LC) and a heavy chain (HC)).
  • Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL. Constant domains are not directly involved in the binding of antibodies and antigens, but exhibit a variety of effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (for example, effector cells) and classical complement The combination of the first component (C1q) of the system.
  • VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form an antigen binding site.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDR contained in the antibody or antigen-binding fragment thereof of the present invention is preferably determined by the Kabat, Chothia, or AbM or IMGT numbering system.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • germline antibody gene is a gene encoding immunoglobulin expressed by non-lymphocytes, which has not undergone the genetic rearrangement and maturation process leading to the expression of specific immunoglobulins.
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or The full-length antibody competes for specific binding to the antigen, which is also referred to as the "antigen-binding portion".
  • an antibody such as a polypeptide fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or The full-length antibody competes for specific binding to the antigen, which is also referred to as the "antigen-binding portion".
  • Recombinant DNA technology can be used. Or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDR) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (such as technology from Ablynx), domain antibodies (such as technology from Domantis), and such A polypeptide comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • full-length antibody means an antibody composed of two “full-length heavy chains” and two “full-length light chains.”
  • full-length heavy chain refers to a polypeptide chain that consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), and a heavy chain in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • HR hinge region
  • heavy chain constant region CH3 domain are composed; and, when the full-length antibody is of the IgE isotype, it optionally also includes the heavy chain constant region CH4 domain.
  • the "full-length heavy chain” is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction.
  • a "full-length light chain” is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction.
  • the two pairs of full-length antibody chains are connected by a disulfide bond between CL and CH1 and a disulfide bond between the HR of the two full-length heavy chains.
  • the full-length antibody of the present invention can be from a single species, such as human; it can also be a chimeric antibody or a humanized antibody.
  • the full-length antibody of the present invention includes two antigen binding sites formed by a pair of VH and VL respectively, and the two antigen binding sites specifically recognize/bind the same antigen.
  • the term “Fd fragment” means an antibody fragment composed of VH and CH1 domains
  • the term “dAb fragment” means an antibody fragment composed of VH domains (Ward et al., Nature 341:544 546 (1989))
  • the term “Fab fragment” means an antibody fragment composed of VL, VH, CL and CH1 domains
  • the term “F(ab') 2 fragment” means a fragment comprising a disulfide bridge connected by a hinge region The antibody fragment of two Fab fragments
  • the term “Fab'fragment” means the fragment obtained by reducing the disulfide bond connecting the two heavy chain fragments in the F(ab') 2 fragment, consisting of a complete light chain and heavy chain
  • the Fd fragment (consisting of the VH and CH1 domains) is composed.
  • Fv fragment means an antibody fragment composed of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen binding site. It is generally believed that the six CDRs confer the antigen binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind antigen, although its affinity may be lower than the complete binding site.
  • Fc fragment means that the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain are combined by disulfide bonds. Antibody fragments. The Fc fragment of an antibody has many different functions, but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker (see, for example, Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • GGGGS linker having an amino acid sequence
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol.
  • di-scFv refers to an antibody fragment formed by linking two scFvs.
  • the term "diabody” means that its VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains of the same chain, Thereby forcing the domain to pair with the complementary domain of the other chain and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).
  • antibody mimetic means that it specifically binds to the antigen like an antibody, but does not have an antibody structure. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
  • DARPin and fynomer are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa.
  • the designed ankyrin repeat protein (DARPin) can be linked to IgG antibody, scFv-Fc antibody fragment or a combination thereof, such as CN104341529A.
  • the anti-IL-17a fynomer binds to the anti-IL-6R antibody, such as WO2015141862A1.
  • Each of the above-mentioned antibody fragments maintains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of antibodies e.g., the aforementioned antibody fragments
  • a given antibody e.g., the antibody provided by the present invention
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the terms “monoclonal antibody”, “monoclonal antibody”, and “mAb” have the same meaning and are used interchangeably, referring to a group of highly homologous antibody molecules (that is, unless they may occur spontaneously). In addition to natural mutations, an antibody in a group of identical antibody molecules.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • the modifier "monoclonal” only indicates that the antibody is characterized as being obtained from a group of highly homologous antibodies, and cannot be understood as requiring any specific method to prepare the antibody.
  • chimeric antibody refers to an antibody whose light chain or/and part of its heavy chain is derived from an antibody (which may be derived from a specific species or belong to a certain species). Specific antibody class or subclass), and another part of the light chain or/and heavy chain is derived from another antibody (which can be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case , It still retains the binding activity to the target antigen (Cabilly et al., US Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)).
  • the expected properties of the antibodies of the present invention include: (1) inhibit or block the binding of TSLP to TSLPR/IL7R ⁇ ; (2) down-regulate or eliminate the activity of TSLP; (3) down-regulate or block the expression of OX40L; (4) ) Inhibit the secretion of Th2 cytokines; (5) Prevent or treat allergic inflammation.
  • the humanized antibody of the present invention retains one or more of the aforementioned expected properties of the parent antibody (human antibody or mouse-human chimeric antibody).
  • transgenic animals can also be used, which can produce no endogenous immunoglobulin after immunization, and can produce a complete human antibody library (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci.
  • degree of humanization is an index used to evaluate the number of non-human amino acid residues in a humanized antibody.
  • the degree of humanization of a humanized antibody can be predicted by the IMGT website DomainGapAlign to predict the homology of the variable region sequence with the human V domain.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen to which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the dissociation equilibrium constant (KD) of the interaction.
  • KD dissociation equilibrium constant
  • the term “KD” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -8 M, for example, less than about 10 -8 M, 10 -9 M, A KD of 10 -10 M or 10 -11 M or less binds the antigen.
  • KD ⁇ 10 ⁇ 10 -8 M the antibody or antigen-binding fragment thereof of the present invention is considered to specifically bind TSLP.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • Both the "binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio of kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). Any effective method can be used to measure KD, kon and kdis values.
  • a bioluminescence interferometry method e.g., ForteBio Octet method
  • ForteBio Octet method can be used to measure the dissociation constant.
  • surface plasmon resonance technology such as Biacore
  • Kinexa can also be used to measure the dissociation constant.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • the molecules are the same at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • the Needleman and Wunsch (J MoI Biol.48:444-453(1970)) algorithms in the GAP program integrated into the GCG software package can be used, and the Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences .
  • The% identity between two amino acid sequences can also be determined by the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)).
  • a sequence with "% identity” retains important biological activities of the sequence from which it is compared or derived, such as antibody binding specificity.
  • a sequence with one or several amino acid substitutions, deletions or additions or any combination thereof retains the important biological activity of the sequence from which it is compared or derived, such as antibody binding specificity.
  • a nucleotide sequence with "% identity” or a nucleotide sequence with a difference of no more than 3, 6, 15, 30 or 45 nucleotides can achieve the function similar to the nucleotide sequence from which it is compared or from the source, as shown The expressed protein can specifically bind to the same antigen or molecule.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including The ability to form covalent bonds or hydrogen bonds, etc.) is replaced by residues. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamate), uncharged polar side chains (e.g., glycine , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine Acid, proline, phenylalanine, methionine), beta branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999) ; And Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • amino acids are usually represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala
  • arginine can be represented by R or Arg
  • glycine can be represented by G or Gly
  • glutamine can be represented by Q or Gln.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancement Agents, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include but are not limited to sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugar, NaCl and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, paraben, chlorobutanol, phenol, sorbic acid and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredients in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , Lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate).
  • prevention means to prevent or delay the occurrence of a disease or condition or symptom (for example, asthma, allergic inflammation, allergic reaction, or autoimmune disease) in a subject.
  • treatment refers to a method performed in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, narrowing the scope of the disease, stabilizing (ie, no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the disease State, and relief of symptoms (whether partial or full), whether detectable or undetectable.
  • treatment can also refer to prolonging survival compared to expected survival (if not receiving treatment).
  • the term "subject” refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human.
  • the subject e.g., human
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., asthma, allergic inflammation, allergic reaction, or autoimmune disease
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, prevent, or delay the disease (e.g., asthma, allergic inflammation, allergic reaction, or autoimmune disease).
  • the amount of the occurrence of sexual diseases); the effective amount for the treatment of the disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who has already suffered from the disease. It is completely within the abilities of those skilled in the art to determine such an effective amount.
  • the effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient’s own immune system, the patient’s general conditions such as age, weight and sex, the way the drug is administered, and other treatments that are administered at the same time and many more.
  • immune cell includes cells that have hematopoietic origin and play a role in an immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes , Macrophages, eosinophils, mast cells, basophils and granulocytes.
  • immune response refers to immune cells (such as lymphocytes, antigen-presenting cells, phagocytes or granulocytes) and soluble macromolecules (including antibodies, cytokines) produced by immune cells or the liver. , And complement), which results in the selective damage, destruction or destruction of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues under autoimmune or pathological inflammation Cleared from the human body.
  • antigen-specific T cell response refers to an immune response generated by a T cell, and the response is generated when the T cell is stimulated by the T cell specific antigen.
  • Non-limiting examples of the response produced by T cells upon antigen-specific stimulation include the proliferation of T cells and the production of cytokines such as IL-2.
  • effector function refers to those biological activities that can be attributed to the Fc region of an antibody (natural sequence Fc region or amino acid sequence variant Fc region), and which vary with the antibody The same type varies.
  • pharmaceutically acceptable means that when the molecular body, molecular fragment or composition is properly administered to animals or humans, they will not produce adverse, allergic or other adverse reactions.
  • pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, and the like.
  • the antibody of the present invention can specifically recognize/bind TSLP with high affinity, inhibit or block the combination of TSLP and TSLPR/IL7RR, and can inhibit or block the proliferation of TSLP on Ba/F3 cells in vitro/in vivo Effect, blocking the ability of TSLP to activate PBMC and secrete cytokines.
  • the antibody of the present invention can inhibit or block the activation and/or proliferation of mast cells, DC, and NKT cells induced by TSLP, inhibit or block TSLP-induced OX40L expression, osteoprotegerin (OPG) secretion, or TSLP-induced
  • OX40L osteoprotegerin
  • TSLP-induced OX40L expression TSLP-induced OX40L expression
  • OPG osteoprotegerin
  • TSLP-induced TSLP-induced The secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5, therefore, the antibody of the present invention has the potential for preventing and/or treating asthma, other allergic reactions or autoimmune diseases.
  • the antibodies of the present invention are fully human antibodies and can be safely administered to subjects without triggering an immunogenic response. Therefore, the antibody of the present invention has great clinical value.
  • FR Antibody framework region amino acid residues other than CDR residues in the variable region of an antibody
  • Kabat The immunoglobulin comparison and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991 ).
  • Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classic rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. People (1989) Nature 342:878-883).
  • IMGT is based on the international immunogenetics information system (The international ImMunoGeneTics information) initiated by Lefranc et al. (IMGT)) for the numbering system, please refer to Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • Figure 2A Detection of the activity of chimeric antibody 25A5C5 in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 2B Detection of the activity of chimeric antibodies 27C2B6 and 37C2D10 in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 2C Detection of the activity of chimeric antibodies 43B1A8 and 90H3H11 in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 3A Detection of the binding affinity of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG, 37C2D10-hIgG to human TSLP protein
  • Figure 3B Detection of the binding affinity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG to human TSLP protein
  • Figure 4A Detection of the activity of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG and 37C2D10-hIgG in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 4B Detection of the activity of recombinant fully human antibody 43B1A8-hIgG in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 4C Detection of the activity of recombinant fully human antibody 90H3H11-hIgG in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 5 Detection of the activity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG in inhibiting the secretion of TARC from PBMC
  • Figure 7A Detection of the activity of recombinant fully human antibody 43B1-H2L2 in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 7B Detection of the activity of recombinant fully human antibody 43B1-H6L1 in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • Figure 8 Detection of the activity of recombinant fully human antibody 43B1-H2L2 in inhibiting the secretion of MDC cytokines from PBMC cells
  • Figure 9 Pharmacokinetic analysis of recombinant fully human antibody 43B1-H2L2 in cynomolgus monkeys
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995.
  • Those skilled in the art know that the embodiments describe the present invention by way of example, and are not intended to limit the scope of protection claimed by the present invention.
  • human TSLP or monkey TSLP in E. coli or mammalian cells.
  • the amino acid sequence of human TSLP refers to NP_149024.1 in NCBI's protein database.
  • the amino acid sequence of cynomolgus monkey (Macaca fascicularis) TSLP refers to XP_005557555.1 in the NCBI protein database.
  • the antigen used in this application includes TSLP expressed in a modified form, for example, 6 consecutive histidines are expressed as a tag (TSLP-His) fused to the C-terminus of the TSLP sequence.
  • the above sequence of human or monkey TSLP is codon optimized by GenScript, synthesized on an expression vector (ie a plasmid containing the complete coding sequence of human TSLP), and expressed and purified in E. coli or HEK293F cells of mammalian cells .
  • the natural receptor of TSLP is a heterodimer, composed of human TSLPR and human IL7R alpha (IL7R ⁇ ) subunits.
  • the sequence reference of human TSLPR (Uniprot: Q9HC73.1); the sequence reference of human IL7R alpha subunit (GenBank: AAR08908.1).
  • the human hIL7R ⁇ -hTSLPR-hFc fusion protein is a series of human IL7R ⁇ extracellular region (E21-D239), human TSLPR receptor extracellular region (Q23-K231) and human IgG1Fc region partial coding sequence (Hinge-CH2-CH3) in series Composed together. Through codon optimization, it is constructed on pLVX expression vector. The pLVX vector was used to establish a stable expression cell line of HEK293F, and finally purified to obtain the hIL7R ⁇ -hTSLPR-hFc fusion protein.
  • the cell line was identified by flow cytometry (flow cytometry: Beckman, CytoFlex; detection antibody APC-anti-human TSLPR (Biolegend), APC-anti-human IL7R ⁇ (Biolegend)) to confirm that the monoclonal cells correctly express human TSLPR And human IL7R ⁇ .
  • flow cytometry flow cytometry: Beckman, CytoFlex; detection antibody APC-anti-human TSLPR (Biolegend), APC-anti-human IL7R ⁇ (Biolegend)
  • flow cytometry results show that Ba/F3-hTSLPR-hIL7R ⁇ is a monoclonal cell line that expresses these two genes (close to 100%) and has good uniformity, which can be used for subsequent experiments.
  • Example 3 Mouse immunity and hybridoma fusion
  • Fully human transgenic mouse H2L2 (and Platinum Medicine) was used for immunization, and human TSLP expressed in E. coli (NP_149024.1), human TSLP expressed in mammalian cells (NP_149024.1), and monkey TSLP expressed in mammals ( XP_005557555.1), and a plasmid containing the complete coding sequence of human TSLP for multiple immunizations.
  • a booster immunization is carried out every two weeks for a total of 5-6 times.
  • the serum titer of anti-human TSLP antibody is detected by ELISA every two weeks (see Example 4.1). After multiple rounds of immunization and the best titer is selected according to the titer, the following protocol is used to fuse to generate hybridomas .
  • the same number of SP2/0 myeloma cells were added and mixed.
  • the cell mixture was washed and resuspended in the electrofusion buffer, and after fusion with the BTX-ECM2001 electrofusion instrument, the cell suspension was immediately transferred from the fusion chamber to the complete fusion medium, and incubated at 37°C for 1 hour.
  • the 96-well plate was plated at a density of 2 ⁇ 10 4 cells per well. After 5 days of culture, the complete fusion medium was used to change the medium, and the supernatant was taken for hybridoma screening at 7-10 days.
  • the soluble human TSLP-His protein was diluted to 1 ⁇ g/ml in 1xCBS coating solution, added to a 96-well plate, and incubated overnight at 4°C.
  • the 96-well plate was washed with PBST, and the plate was blocked with blocking solution (PBS+2% BSA) at 37°C for 2 hours.
  • blocking solution PBS+2% BSA
  • Add goat anti-rat IgG-HRP to the 96-well plate, incubate the plate in a 37°C incubator for 1 hour, wash and read the OD at 450 nm.
  • the monkey TSLP-His protein was diluted to 1 ⁇ g/ml in the CBS coating solution, added to a 96-well plate, and incubated overnight at 4°C.
  • the 96-well plate was washed with PBST, and the plate was blocked with blocking solution PBS+2% BSA at 37°C for 2 hours.
  • Add goat anti-rat IgG-HRP to the 96-well plate, incubate the plate in a 37°C incubator for 1 hour, wash and read the OD at 450 nm.
  • the hybridoma supernatant or purified antibody blocks the binding of human TSLP-His to the chimeric receptor IL7R ⁇ -TSLPR-hFc.
  • the soluble hIL7R ⁇ -hTSLPR-hFc protein was diluted in 1xCBS coating solution, added to a 96-well plate, and incubated at 4°C overnight.
  • the 96-well plate was washed with PBST, and the plate was blocked with blocking solution PBS+2% BSA at 37°C for 2 hours.
  • Add hTSLP-His protein (+/-) recombinantly expressed by mammalian cells to the plate, and add hybridoma supernatant or add the same volume of blocking solution, and incubate the plate in a 37°C incubator for 2 hours.
  • Add mouse anti-His-HRP to the 96-well plate, incubate the plate in a 37°C incubator for 1 hour, wash and read the OD at 450 nm.
  • the hybridomas with strong inhibition rate are selected as candidate clones.
  • Control antibody expression Refer to the chEMBL database (ID: CHEMBL3707229) for the control antibody sequence.
  • the base sequences of the heavy and light chains of the control antibody were synthesized in the pTT5 expression vector, and transiently transfected and expressed by CHO-S cells (purchased from Thermo), and then affinity purified by Protein A (MabSelect SuRe, GE), Obtain a control antibody.
  • hybridoma single clones were cultured without serum to obtain 50 mL of supernatant, and then purified by Protein A (MabSelect SuRe, GE) to obtain hybridoma chimeric antibodies. Because the H2L2 mouse antibody constant region is genetically modified into a rat constant region, the purified antibody is a chimeric antibody with a rat Fc in the fully human variable region.
  • the purified antibodies were quantified by spectrophotometry, and chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8 and 90H3H11 were obtained.
  • Example 6 ELISA detection of anti-TSLP chimeric antibody and TSLP affinity
  • ELISA method was used to detect the affinity of chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8 and 90H3H11 to human or monkey TSLP.
  • the specific method is briefly described as follows: Coat human TSLP-His or monkey TSLP-His antigen into a 96-well plate. After overnight at 4°C, add antibodies with different concentrations of gradient dilutions respectively. After incubating for 2 hours, add goat anti-rat Fc -HRP secondary antibody, after incubating for 1 hour, the absorbance value of A450nm wavelength is detected by the microplate reader.
  • test results are shown in Table 2.
  • the affinity of human TSLP 25A5C5 and 27C2B6 chimeric antibodies and control antibodies are basically the same, 43B1A8 and 90H3H11 antibodies are higher than the control antibodies.
  • 25A5C5 and 90H3H11 antibodies have almost no binding to monkey TSLP; 37C2D10, 27C2B6 and 43B1A8 all bind strongly to monkey TSLP.
  • Example 7 Competitive ELISA detection of anti-TSLP chimeric antibody blocking TSLP/hIL7R ⁇ -hTSLPR-hFc binding activity
  • Human TSLP activates downstream signaling pathways by binding to human hIL7R ⁇ -hTSLPR-hFc heterodimer receptor.
  • a competitive ELISA method was used to detect the activity of the chimeric antibody to block the binding of the chimeric receptor hIL7R ⁇ -hTSLPR-hFc to the antigen.
  • Example 8 Detection of Ba/F3-hTSLPR-hIL7R ⁇ cell proliferation inhibition by anti-TSLP chimeric antibody
  • the activity of the anti-TSLP chimeric antibody was detected by the Ba/F3-hTSLPR-hIL7R ⁇ cell proliferation inhibition method.
  • Ba/F3-hTSLPR-hIL7R ⁇ cell surface expresses hTSLPR and hIL7R ⁇ receptor proteins.
  • the dimer of the extracellular segment of these two receptor proteins can bind to human TSLP, and the intracellular segment can further signal transduction and activate intracellular STAT5. Phosphorylate and promote cell proliferation of Ba/F3-hTSLPR-hIL7R ⁇ .
  • the five chimeric antibodies can significantly inhibit the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells.
  • Example 9 Amplification of variable region of anti-TSLP chimeric antibody
  • the hybridoma cells were cultured to about 2 ⁇ 10 6 cells, and the hybridoma cells were lysed with TRIzol reagent (Thermo Fisher Sci.Cat#15596026) to extract RNA and synthesized with a cDNA reverse transcription kit (Thermo Fisher Sci.Cat#18080-200) The first strand cDNA.
  • TRIzol reagent Thermo Fisher Sci.Cat#15596026
  • a cDNA reverse transcription kit Thermo Fisher Sci.Cat#18080-200
  • the first strand cDNA Refer to the IMGT and AbM methods, as well as the sequence analysis of all murine antibodies, select regions with high homology to design multiple pairs of upstream primers for the variable region, and use CH1 homologous sequences for the downstream primers to obtain the antibody by PCR amplification by means of a primer pool.
  • PCR products are purified by DNA purification kit (Qiagen, Cat#28104) and cloned into pTT-5 vector. About 10 clones are picked for each ligation reaction and sequenced to obtain variable regions Sequence, and further analyzed by IMGT and AbM database.
  • amino acid sequences of the light chain variable region of 25A5C5, 27C2B6, 37C2D10, 43B1A8 and 90H3H11 were connected to the amino acid sequence of the light chain ⁇ constant region (SEQ ID NO: 16), and the amino acid sequence of the heavy chain variable region was connected to the IgG1 heavy chain constant region.
  • the amino acid sequence (SEQ ID NO: 14) is connected, and the nucleotide sequence corresponding to the sequence is constructed into the pTT5 vector.
  • the pTT5 vector corresponding to the heavy and light chains of each recombinant fully human antibody was transfected into CHO-S (purchased from Thermo) at the same time, and the supernatant was purified by using Protein A (MabSelect SuRe, GE).
  • the recombinant fully human antibodies were named 25A5C5-hIgG, 27C2B6-hIgG, 37C2D10-hIgG, 43B1A8-hIgG and 90H3H11-hIgG, and the protein content was quantified by spectrophotometry.
  • Example 11 Detection of dynamic affinity of anti-TSLP fully human antibody to TSLP
  • Fortibio is a commonly used dynamic affinity detection device to detect the dynamic affinity of anti-TSLP fully human antibodies with TSLP.
  • the brief method is as follows: the human or monkey TSLP antigen is serially diluted with PBST to obtain 100 nM, 50 nM, 25 nM, 12.5 ⁇ M, 6.25 nM, 3.125 nM, 1.5625 nM and 0 nM.
  • ProA biosensor Pall Life Sciences
  • the recombinant fully human antibody was diluted to 5 ⁇ g/mL with PBST and immobilized on the ProA sensor.
  • the sensor with immobilized antibody was equilibrated in PBST buffer for 60 s to obtain a baseline, then transferred to the antigen diluent for binding for 60 s, and then dissociated in PBST for 180 s. After one analysis cycle, the sensor is regenerated with 10mM Gly (pH 1.5).
  • Date analysis version 11.0 Pall
  • Kd rate constants of association
  • KD dissociation equilibrium constant
  • 25A5C5-hIgG, 43B1A8-hIgG and 90H3H11-hIgG bind human TSLP with a lower KD value than the control antibody, showing stronger affinity, consistent with the results of affinity ELISA.
  • 25A5C5-hIgG has no affinity; while 37C2D10-hIgG, 90H3H11-hIgG and 43B1A8-hIgG show good binding, with affinities ranging from 10 -8 M to 10 -9 M.
  • Example 12 Ba/F3-hTSLPR-hIL7a cell proliferation inhibition by recombinant fully human antibody
  • Recombinantly expressed fully human antibody using Ba/F3-hTSLPR-hIL7a cell proliferation inhibition method to detect the activity of recombinant fully human antibody.
  • Ba/F3-hTSLPR-hIL7a cell proliferation inhibition method to detect the activity of recombinant fully human antibody.
  • Table 8 Ba/F3-hTSLPR-hIL7R ⁇ cell proliferation inhibitory activity of recombinant fully human antibodies
  • Example 13 Detection of the activity of recombinant fully human antibodies in inhibiting the secretion of thymus activation regulator (TARC) from PBMC induced by human TSLP
  • PBMC contains CD11 + DC cells.
  • TSLP can bind to the receptor TSLPR/IL7R ⁇ , activate CD11 + DC cells, up-regulate OX40L and promote CD11 + DC cells to secrete Th2 cytokines (such as TARC and CCL22).
  • Anti-TSLP antibodies can block the binding of TSLP to TSLPR/IL7R ⁇ on the surface of DC cells, thereby blocking the activation of DC cells and the secretion of Th2 cytokines by DC cells.
  • the brief method is as follows: Use Ficoll separation solution (GE) to separate human peripheral blood PBMC, incubate hTSLP-His(+/-) with recombinant fully human antibody or control antibody for 30 minutes at room temperature, and add 2 ⁇ 10 5 PBMC cells/well, incubated for 48 hours. The supernatant was harvested and analyzed for human TARC production by ELISA, and the inhibition of TARC secretion by the hybridoma supernatant or purified antibody was determined. TARC ELISA kit (Yiqiao Shenzhou) was used to detect the level of TARC in the supernatant.
  • GE Ficoll separation solution
  • Example 14 Determination of the Tm value of the recombinant fully human antibody.
  • the Tm value of anti-TSLP antibody was measured by DSF (Differential Fluorescence Scanning Technology) method.
  • the specific experimental steps are as follows. Mix 12.5 ⁇ l 40 ⁇ SYPRO Orange dye (purchased from Lifetechnologies, catalog number: 56651), 5 ⁇ l volume of 1mg/ml fully human TSLP antibody (diluted in PBS) and 7.5 ⁇ l sterile water in the EP tube , Add the above sample mixture to the Q-PCR system (AB Applied Biosystems ABI, 7500) for reaction, Q-PCR parameter setting: Target (ROX), program (25°C, 3min; 1% rate, 95°C; 95°C, 2min). The results are imported into Graph Prism software to calculate the V50 value.
  • Q-PCR system AB Applied Biosystems ABI, 7500
  • the Tm value (74.72°C) of 43B1A8-hIgG and the Tm value (72.58°C) of 90H3H11-hIgG are higher than the Tm (66.47°C) of the control antibody. Better thermal stability.
  • Example 15 Determination of the hydrophobicity of recombinant fully human antibodies
  • the hydrophobicity comparison was analyzed by Agilent 1260 HPLC combined with TOSOH Tskgel Buty-NPR (2.5) column. In order to compare the difference in hydrophobicity of the three antibodies, the three antibodies were directly injected for analysis. Mobile phase A: 1.5M (NH 4 ) 2 SO 4 ; mobile phase B: 25 mM Na 2 HPO 4 (pH 7.0) + 25% IPA. The retention times of the three antibodies are shown in Table 11. The longer the time, the stronger the hydrophobic nature of the antibody.
  • hydrophilicity of 90H3H11-hIgG is equivalent to that of the control antibody; the hydrophilicity of 43B1A8-hIgG is better than that of the control antibody, showing process development and antibody aggregation Will be better than the control antibody.
  • Example 16 Obtaining of recombinantly modified fully human antibodies 43B1-H6L1 and 43B1-H2L2
  • the half-life of an antibody in vivo is closely related to its isoelectric point, affinity with FcRn, glycosylation modification and immunogenicity.
  • affinity of FcRn was enhanced by the amino acid sequence modification of the heavy and light chain antibodies.
  • Modification scheme 1 hIgG1Fc (SEQ ID NO: 14) is replaced with IgG4 (SEQ ID NO: 70) with 2 amino acid mutations, and the mutation feature is that the 434th amino acid (Eu numbering system) of IgG4 is changed from N to A, the 228th The amino acid at position mutated from S to P (Eu numbering system).
  • Modification scheme 2 The amino acid at position 434 (Eu numbering system) of hIgG1Fc (SEQ ID NO: 14) is replaced by N with A to obtain SEQ ID NO: 15; and the 16th R of FR1 of the heavy chain of 43B1A8-hIgG is mutated to G (Chothia numbering system); and the 79th R mutation of FR3 of the light chain of 43B1A8-hIgG is Q (Chothia numbering system).
  • the antibodies obtained by the first and second modifications were named 43B1-H6L1 and 43B1-H2L2.
  • the heavy chain variable region sequence of 43B1-H2L2 is SEQ ID NO: 68; the heavy chain constant region sequence is SEQ ID NO: 15; the light chain variable region sequence is SEQ ID NO: 69; the light chain constant region sequence is SEQ ID NO: 16.
  • the heavy chain variable region sequence of 43B1-H6L1 is SEQ ID NO: 40; the heavy chain constant region sequence is SEQ ID NO: 70; the light chain variable region sequence is SEQ ID NO: 41; the light chain constant region sequence is SEQ ID NO: 16.
  • Antibody Heavy chain Heavy chain variable region Heavy chain constant region Light chain Light chain variable region Light chain constant region 43B1A8-hIgG 66 40 14 67 41 16 43B1-H2L2 71 68 15 72 69 16 43B1-H6L1 73 40 70 67 41 16
  • the heavy and light chains of the above-mentioned two modified molecules, 43B1-H2L2 and 43B1-H6L1 were constructed respectively to construct pTT5 expression vectors, and the plasmids were extracted and transfected into CHO-S cells (purchased from Thermo). After about 10 days After culturing, the cell supernatant was purified by Protein A (MabSelect SuRe, GE), and the purified recombinant fully human antibody was used to quantify the protein content by spectrophotometry.
  • Example 17 Detection of the dynamic affinity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 with TSLP
  • Example 18 Isoelectric point (PI) detection of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2
  • the isoelectric focusing method was used to detect the isoelectric points of 43B1-H6L1, 43B1-H2L2 and 43B1A8-hIgG antibodies.
  • the brief method is as follows: The Maurice isoelectric focusing system of ProteinSimple is used in combination with its capillary cartridge for analysis. In order to compare the difference of the isoelectric points of the 3 antibodies, the 3 antibodies were diluted with water, and the detection system used a final concentration of 4% amphoteric electrolyte 3-10 (GE Company) to form a pH gradient. The results are shown in Table 14.
  • the isoelectric points of 43B1-H6L1, 43B1-H2L2 were lower than that of 43B1A8-hIgG by 1.3 and 0.4, respectively, indicating that the modification program was successful.
  • Example 19 Detection of the dynamic affinity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 with FcRn
  • the anti-TSLP antibody was serially diluted with PBST (pH 6.0) to obtain 200 nM, 100 nM, 50 nM, 25 nM, 12.5 ⁇ M, 6.25 nM, 3.125 nM, 1.5625 nM and 0 nM.
  • the SA biosensor (Pall Life Sciences) is pre-wetted with PBST (pH 6.0) buffer before use.
  • the biotin-labeled FcRn was diluted to 2.3 ⁇ g/mL and solidified on the SA sensor.
  • the immobilized FcRn sensor was equilibrated in PBST (pH 6.0) buffer for 60 s to obtain a baseline, then transferred to the antibody dilution solution for binding for 60 s, and then dissociated in PBST (pH 6.0) for 60 s. After one analysis cycle, the sensor was regenerated with PBST (pH 7.4).
  • PBST pH 7.4
  • Ka rate constants of association
  • Kd dissociation
  • the dynamic affinity KD of FcRn of 43B1-H6L1 and 43B1-H2L2 is less than that of the control antibody, indicating that the affinity of FcRn is approximately twice that of the control antibody.
  • Example 20 Determination of the activity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 in inhibiting the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells
  • the Ba/F3 cell proliferation inhibition method was used to detect the activity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2.
  • Methods Refer to the aforementioned Example 8, and analyze the activity EC50 of the antibody based on the data. The results are shown in Table 16 and Figures 7A and 7B.
  • 43B1-H2L2 and 43B1-H6L1 can inhibit the proliferation of Ba/F3-hTSLPR-hIL7R ⁇ cells caused by human TSLP, and the EC50 is equivalent to that of the control antibody, showing that the fully human antibody 43B1-H2L2 It is equivalent to 43B1-H6L1 in inhibiting the cell activity of TSLP.
  • Example 21 Recombinant fully human antibody inhibits human TSLP-induced PBMC secreting macrophage-derived chemokine (MDC) activity detection
  • TSLP can bind to the cell surface receptor TSLPR/IL7R ⁇ , activate DC cells, up-regulate OX40L and promote DC cells to secrete Th2 cytokines (such as TARC and MDC).
  • Th2 cytokines such as TARC and MDC.
  • Anti-TSLP antibodies can block the binding of TSLP to TSLPR/IL7R ⁇ on the surface of DC cells, thereby blocking the activation of DC cells and the secretion of Th2 cytokines by DC cells.
  • the brief method is as follows: Use Ficoll separation solution (GE) to separate human peripheral blood PBMC, incubate hTSLP-His(+/-) with recombinant fully human antibody or control antibody for 30 minutes at room temperature, and add 2 ⁇ 10 5 PBMC cells/well, incubate for 120 hours. The supernatant was harvested and analyzed for human MDC production by ELISA, and the inhibition of MDC secretion by the antibody was determined. MDC's ELISA kit (Raybiotech) was used to detect the level of TARC in the supernatant.
  • GE Ficoll separation solution
  • Example 22 Pharmacokinetic analysis of recombinant fully human antibodies in cynomolgus monkeys
  • the sequence of the 43B1-H2L2 candidate molecule in this application carries the N434A mutation.
  • the purpose is to affect its affinity with FcRn, thereby prolonging its drug metabolism half-life in the body. Therefore, this example uses subcutaneous injection of drugs to study 43B1-H2L2
  • the pharmacokinetics of the molecule in cynomolgus monkeys were compared with the pharmacokinetics of the control antibody.
  • the methods and results are as follows: Four cynomolgus monkeys (Macaca fascicularis, from Hainan Jingang Biotechnology Co., Ltd.) were selected and divided into two groups, one male and one male in each group. The subcutaneous injection dose is 5mg/kg.

Abstract

涉及抗TSLP的抗体或其抗原结合片段，编码它们的核酸分子，制备它们的方法。抗TSLP抗体或其抗原结合片段对TSLP具有高亲和力，能够有效结合TSLP并阻断TSLP对Ba/F3-hTSLPR-hIL7Rα细胞的增殖效应，以及阻断TSLP对PBMC的激活、分泌细胞因子的能力。同时进一步涉及包含所述抗体或其抗原结合片段的药物组合物，以及其在制备用于预防和/或治疗哮喘、变态反应性炎症、过敏反应或自身免疫性疾病药物中的用途。

Description

抗TSLP抗体及其用途 技术领域
本发明属于治疗性单克隆抗体领域，更具体地，本发明涉及一种针对TSLP的抗体，还涉及所述抗体在治疗疾病中的用途。
技术背景
胸腺基质淋巴细胞生成素(Thymic stromal lymphopoietin，TSLP)是一种与IL7类似的炎症细胞因子。TSLP对微生物、物理损伤或炎症细胞因子(如IL-1β和TNF)等响应，主要由皮肤、肺、胸腺与胃肠道等上皮细胞分泌，在炎症等病理状况下，基质细胞、角质细胞、树突状细胞(DC)和肥大细胞等也能分泌TSLP。TSLP在过敏性和适应性气道炎症的初始触发中有重要作用。与健康对照组相比，TSLP在哮喘患者气道高表达，其水平与TH2细胞因子和趋化因子的表达，以及疾病的严重程度直接相关。TSLP可诱导树突状细胞(DC)成熟，上调OX40L的表达，OX40-OX40L的相互作用参与初始T细胞诱导的TH2细胞极化，其分化后释放IL-4、IL-5、IL-13等细胞因子，引起肥大细胞、嗜酸性粒细胞浸润及系列变态炎性反应，使气道发生病理改变，从而导致哮喘发作。TSLP可有效地激活肥大细胞、自然杀伤T(NKT)细胞，产生IL-13等TH2型细胞因子，加重气道炎症的发生发展。
TSLP的受体是由IL-7Rα和独特TSLPR链(CRFL2)所组成的异源二聚体受体复合物。TSLP异源二聚受体的结合引起STAT5的活化及细胞增殖。DC细胞具有高表达的TSLPR和IL-7Rα。
因此，发展具有高度的特异性和亲和力、低的毒副作用、优良的临床药效的抗TSLP抗体是迫切而必要的，这将给哮喘患者提供更多的用药选择。
发明内容
在本发明中，发明人首先开发了具有优良性质的嵌合抗体，其能够结合人TSLP。在此基础上，发明人又对该嵌合抗体进行了研究和改造，开发了该嵌合抗体的全人源抗体。本发明的全人源抗体具有与嵌合抗体基本上相同的(或甚至更优良的)生物学功能。不仅对于TSLP有强的亲和力，而且能够有效地阻断TSLP对Ba/F3细胞的增殖效应，以及阻断TSLP对PBMC的激活、分泌细胞因子的能力。本发明进一步涉及包含所述抗体或其抗原结合片段的药物组合物，以及其在制备药物中的用途，所述药物用于预防和/或治疗哮喘、变态反应性炎症、过敏反应或自身免疫疾病。
本发明的抗体具有极高的人源化程度，甚至是全人源抗体，从而可安全地施用给人受试者，而不引发免疫原性反应。所以本发明的抗体具有重大的临床价值。
本发明的抗体
在一个方面，本发明提供了一种结合TSLP的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含如下的互补决定区(CDR)：
(a)SEQ ID NO:1、17、30、40、53或68所示的重链可变区(VH)中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:2、18、31、41、54或69所示的轻链可变区(VL)中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的 变体；或
(b)SEQ ID NO:1所示的VH中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:2所示的VL中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:17所示的VH中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:18所示的VL中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:30所示的VH中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:31所示的VL中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:40所示的VH中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:41所示的VL中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:53所示的VH中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:54所示的VL中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:68所示的VH中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体以及CDR-H3或其序列的变体；和/或SEQ ID NO:69所示的VL中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体以及CDR-L3或其序列的变体。
在某些优选实施方案中，所述序列的变体为与其来源CDR相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的CDR。
在某些优选实施方案中，所述的置换为保守置换。
优选地，所述CDR根据AbM、Chothia、Kabat或IMGT编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段的VH和/或VL中包括来自人的免疫球蛋白的构架区(FR)。
在某些实施方案中，所述抗体或其抗原结合片段结合人TSLP和/或猴TSLP。
在一个方面，本发明提供了一种能够结合TSLP的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：重链可变区(VH)和/或轻链可变区(VL)。
在某些实施方案中，本发明的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：3或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：4或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加) 的序列的CDR-H2，序列为SEQ ID NO：5或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：6或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：7或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：8或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：19或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：20或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：21或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：22或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：23或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：24或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
(c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：33或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：34或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：35或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：23或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：24或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
(d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：42或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、 缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：43或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：44或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：45或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：46的CDR-L2或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，序列为SEQ ID NO：47或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
或
(e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：55或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：56的CDR-H2或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列，序列为SEQ ID NO：57或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：58或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：59或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：60或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3。
在某些实施方案中，本发明的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号系统定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：3的CDR-H1，序列为SEQ ID NO：4的CDR-H2，序列为SEQ ID NO：5的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：6的CDR-L1，序列为SEQ ID NO：7的CDR-L2，序列为SEQ ID NO：8的CDR-L3；
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：19的CDR-H1，序列为SEQ ID NO：20的CDR-H2，序列为SEQ ID NO：21的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：22的CDR-L1，序列为SEQ ID NO：23的CDR-L2，序列为SEQ ID NO：24的CDR-L3；
(c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：32的CDR-H1，序列为SEQ ID NO：33的CDR-H2，序列为SEQ ID NO：34的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：35的CDR-L1，序 列为SEQ ID NO：23的CDR-L2，序列为SEQ ID NO：24的CDR-L3；
(d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：42的CDR-H1，序列为SEQ ID NO：43的CDR-H2，序列为SEQ ID NO：44的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：45的CDR-L1，序列为SEQ ID NO：46的CDR-L2，序列为SEQ ID NO：47的CDR-L3；
或
(e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：55的CDR-H1，序列为SEQ ID NO：56的CDR-H2，序列为SEQ ID NO：57的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：58的CDR-L1，序列为SEQ ID NO：59的CDR-L2，序列为SEQ ID NO：60的CDR-L3。
在某些实施方案中，本发明的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：9或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：10或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：11或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：12或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：13或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：8或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：25或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：26或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：27或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：28或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：29或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：24或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
(c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：36或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：37或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：38或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：39或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：29或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：24或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
(d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：48或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：49或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：50或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：51或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：52或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L2，序列为SEQ ID NO：47或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3；
或
(e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：61或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H1，序列为SEQ ID NO：62或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H2，序列为SEQ ID NO：63或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-H3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：64或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L1，序列为SEQ ID NO：65或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添 加)的序列的CDR-L2，序列为SEQ ID NO：60或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的CDR-L3。
在某些实施方案中，本发明的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按AbM编号系统定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：9的CDR-H1，序列为SEQ ID NO：10的CDR-H2，序列为SEQ ID NO：11的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：12的CDR-L1，序列为SEQ ID NO：13的CDR-L2，序列为SEQ ID NO：8的CDR-L3；
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：25的CDR-H1，序列为SEQ ID NO：26的CDR-H2，序列为SEQ ID NO：27的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：28的CDR-L1，序列为SEQ ID NO：29的CDR-L2，序列为SEQ ID NO：24的CDR-L3；
(c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：36的CDR-H1，序列为SEQ ID NO：37的CDR-H2，序列为SEQ ID NO：38的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：39的CDR-L1，序列为SEQ ID NO：29的CDR-L2，序列为SEQ ID NO：24的CDR-L3；
(d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：48的CDR-H1，序列为SEQ ID NO：49的CDR-H2，序列为SEQ ID NO：50的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：51的CDR-L1，序列为SEQ ID NO：52的CDR-L2，序列为SEQ ID NO：47的CDR-L3；
或
(e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO：61的CDR-H1，序列为SEQ ID NO：62的CDR-H2，序列为SEQ ID NO：63的CDR-H3；和/或，包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO：64的CDR-L1，序列为SEQ ID NO：65的CDR-L2，序列为SEQ ID NO：60的CDR-L3。
在某些实施方案中，本发明的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中，与前述IMGT或AbM定义的CDR相比，所述重链可变区(VH)和/或轻链可变区(VL)中至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)。
优选地，本发明所述的置换为保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段的VH包含来源于人免疫球蛋白的重链可变区(VH)的构架区(FR)，和/或所述抗体或其抗原结合片段的VL包含来源于人免疫球蛋白的轻链可变区(VL)的构架区(FR)。因此，在某些实施方案中，本发明的抗体或其抗原结合片段是人源化的。在某些实施方案中，本发明的抗体或其抗原结合片段是全人源的。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)人免疫球蛋白的重链构架区或其变体，所述变体与其所源自的胚系抗体基因编码的氨基酸序列相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、 至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)；和/或
(b)人免疫球蛋白的轻链构架区或其变体，所述变体与其所源自的胚系抗体基因编码的氨基酸序列相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。
在某些实施方案中，本发明的抗体或其抗原结合片段的人源化程度为至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO：1、17、30、40、53或68所示的序列；
(ii)与SEQ ID NO：1、17、30、40、53或68所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与SEQ ID NO：1、17、30、40、53或68所示的序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
和/或
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO：2、18、31、41、54或69所示的序列；
(v)与SEQ ID NO：2、18、31、41、54或69所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与SEQ ID NO：2、18、31、41、54或69所示的序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO：1所示的VH，和/或,SEQ ID NO：2所示的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO：17所示的VH，和/或,SEQ ID NO：18所示的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO：30所示的VH，和/或,SEQ ID NO：31所示的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO：40所示的VH，和/或,SEQ ID NO：41所示的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO：53所示的VH，和/或,SEQ ID NO：54所示的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段包含SEQ ID NO：68所示的VH，和/或,SEQ ID NO：69所示的VL。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)SEQ ID NO：1所示序列的VH和SEQ ID NO：2所示序列的VL；
(b)SEQ ID NO：17所示序列的VH和SEQ ID NO：18所示序列的VL；
(c)SEQ ID NO：30所示序列的VH和SEQ ID NO：31所示序列的VL；
(d)SEQ ID NO：40所示序列的VH和SEQ ID NO：41所示序列的VL；
(e)SEQ ID NO：53所示序列的VH和SEQ ID NO：54所示序列的VL；
(f)SEQ ID NO：68所示序列的VH和SEQ ID NO：69所示序列的VL；
(g)重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)独立地与(a)至(f)任一组中所述的VH和VL相比分别具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或
(h)重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)与(a)至(f)任一组中所述的VH和VL分别相比，独立地具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)。优选地，所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有至多50个氨基酸的保守置换(例如至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。在某些实施方案中，本发明的抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有至多50个氨基酸的保守置换(例如至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。
在一些实施方案中，恒定区被改变，例如被突变，以修饰抗TSLP抗体分子的性质(例如改变下列中的一个或多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低)。抗体的Fc区介导几种重要的效应子功能，例如ADCC、吞噬作用(ADCP)、CDC等。
在某些实施方案中，本发明的抗体或其抗原结合片段具有重链恒定区(Fc)，其选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；特别地选自例如IgG1、IgG2、IgG3和IgG4的重链恒定区，更特别地选自IgG1(例如是人IgG1)的重链恒定区。在一些实施方案中，人IgG1重链恒定区如SEQ ID NO：14所示。在一些 实施方案中，本发明的抗体或其抗原结合片段具有轻链恒定区，其选自例如κ或λ轻链恒定区，优选κ轻链恒定区(例如人κ轻链恒定区)。
在一些实施方案中，所述抗体或其抗原结合片段包含人IgG1重链恒定区。在一些优选地实施方案中，所述抗体或其抗原结合片段包含uniprot ID P01857所示的人IgG1恒定区(SEQ ID NO：74)。
在一些实施方案中，所述抗体或其抗原结合片段包含人IgG1重链恒定区(例如SEQ ID NO：74)或其变体，按照EU编号系统，所述变体在位点234、235、237、265、297、331、329、434至少一个位点发生突变。在一些实施方案中，所述变体含有下列突变中至少一个:L234A、L235A、D265A、N297A、L234F、L235E、P331S、P329G、N434A、N434Y、N434F、N434W、N434S、N434G、N434H，N434Q。在一些实施方案中，所述变体含有下列突变中至少一个:L234A、L235A、G237A、N434A。在一些实施方案中，所述IgG1重链恒定区变体包含L234A、L235A和G237A。在一些实施方案中，所述IgG1重链恒定区变体包含突变L234A、L235A、G237A和N434A。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO：14所示的CH或其变体，所述变体与SEQ ID NO：14相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：14相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。按照EU编号系统，所述变体含有N297A和/或N434A。在一些实施方案中，所述变体含有N434A。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO：15所示的CH或其变体，所述变体与SEQ ID NO：15相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：15相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述抗体或其抗原结合片段包含人IgG4重链恒定区(例如SEQ ID NO：75)或其变体，按照EU编号系统，所述变体在位点228和/或434至少一个位点发生突变。
在一些实施方案中，所述突变体包含S228P和/或N434A。在一些实施方案中，所述人IgG4重链恒定区变体包含S228P和N434A。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO：70所示的CH或其变体，所述变体与SEQ ID NO：70相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：70相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述抗体或其抗原结合片段包含轻链恒定区或其变体。在一些实施方案中，所述轻链恒定区包含κ轻链恒定区。在一些实施方案中，所述轻链恒定区包含SEQ ID NO：16所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO：16相 比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：16相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO：14所示的重链恒定区(CH)和SEQ ID NO：16所示的轻链恒定区(CL)。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO：15所示的重链恒定区(CH)和SEQ ID NO：16所示的轻链恒定区(CL)。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO：70所示的重链恒定区(CH)和SEQ ID NO：16所示的轻链恒定区(CL)。
在一些实施方案中，上述位点突变使抗体或抗原结合片段与含有人IgG4或IgG4重链恒定区的相应抗体或抗原结合片段相比，没有或具有降低的ADCP、ADCC和/或CDC活性。
在一些实施方案中，上述位点突变使抗体或抗原结合片段与不具有所述突变或置换的相应抗体或抗原结合片段相比，没有或具有降低的ADCP、ADCC和/或CDC活性。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO：1所示的VH序列和SEQ ID NO：14、15或70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO：2所示的VL序列和SEQ ID NO：16所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO：17所示的VH序列和SEQ ID NO：14、15或70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO：18所示的VL序列和SEQ ID NO：16所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO：30所示的VH序列和SEQ ID NO：14、15或70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO：31所示的VL序列和SEQ ID NO：16所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组 合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO：40所示的VH序列和SEQ ID NO：14、15或70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO：41所示的VL序列和SEQ ID NO：16所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO：53所示的VH序列和SEQ ID NO：14、15或70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个 或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO：54所示的VL序列和SEQ ID NO：16所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO：68所示的VH序列和SEQ ID NO：14、15或70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO：69所示的VL序列和SEQ ID NO：16所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体包含：包括SEQ ID NO：1所示的VH和SEQ ID NO：14、15或70所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：2所示的VL和SEQ ID NO：16所示的轻链恒定区(CL)的轻链。
在某些实施方案中，本发明的抗体包含：包括SEQ ID NO：17所示的VH和SEQ ID NO：14、15或70所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：18所示的VL和SEQ ID NO：16所示的轻链恒定区(CL)的轻链。
在某些实施方案中，本发明的抗体包含：包括SEQ ID NO：30所示的VH和SEQ ID NO：14、15或70所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：31所示的VL和SEQ ID NO：16所示的轻链恒定区(CL)的轻链。
在某些实施方案中，本发明的抗体包含：包括SEQ ID NO：40所示的VH和SEQ ID NO：14、15或70所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：41所示的VL和SEQ ID NO：16所示的轻链恒定区(CL)的轻链。
在某些实施方案中，本发明的抗体包含：包括SEQ ID NO：53所示的VH和SEQ ID NO：14、15或70所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：54所示的VL和SEQ ID NO：16所示的轻链恒定区(CL)的轻链。
在某些实施方案中，本发明的抗体包含：包括SEQ ID NO：68所示的VH和SEQ ID NO：14、15或70所示的重链恒定区(CH)的重链，和，包括SEQ ID NO：69所示的VL和SEQ ID NO：16所示的轻链恒定区(CL)的轻链。
在某些实施方案中，本发明的抗体或其抗原结合片段包含重链和轻链，
所述重链包含：
(i)SEQ ID NO：66或73所示的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
所述轻链包含：
(iv)SEQ ID NO：67所示的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体或其抗原结合片段包含重链和轻链，
所述重链包含：
(i)SEQ ID NO：71所示的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
所述轻链包含：
(iv)SEQ ID NO：72所示的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本发明的抗体是嵌合抗体，人源化抗体或全人源抗体。在某些实施方案中，本发明的抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’) 2、Fv片段、二硫键连接的Fv(dsFv)、双抗体(diabody)。
在某些实施方案中，本发明的抗体分子或其抗原结合片段可以显示出以下性质中的至少一种：
(a)以小于约50nM，例如小于约40nM、30nM、20nM、10nM、1nM、0.1nM、1pM、0.1pM或更低的KD结合TSLP(例如，人TSLP)；所述KD可以通过本领域公知的技术测得，例如通过Fortibio或ELISA测得；
(b)以小于约50nM，例如小于约40nM、30nM、20nM、10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.01nM、1pM、0.1pM或更小的EC50结合TSLP(例如，人TSLP)；所述EC50可以通过本领域公知的技术测得，例如通过流式细胞技术、ELISA如亲和力ELISA技术或细胞竞争ELISA技术测得；
(c)以小于约50nM，例如约50nM、20nM、10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.01nM、1pM、0.1pM或更小的IC50抑制TSLP与IL7Rα/TSLPR的结合；所述IC50通过ELISA技术测得；
(d)抑制或阻断TSLP诱导的OX40L表达；
(e)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖；
(f)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌；
(g)抑制或阻断Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌；
(h)良好的与FcRn结合的亲和力；
(i)等电点(PI)为约6.5到约8.5，如约6.5、约7.0、约7.1、约7.2、约7.3、约7.4、约7.5、约7.7、约7.9、约8.0、约8.2或约8.5。
此外，本发明的抗体具有良好的与FcRn的亲和力。在某些实施方案中，与FcRn亲和力的KD(M)值在10 -9级别。本发明的抗体具有更长的体内半衰期。本发明的抗体亦具有良好的亲水性。在某些实施方案中，通过色谱柱检测，本发明的抗体的疏水时间在8分钟到14分钟之间。
抗体衍生物
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会对其与TSLP(特别是人TSLP)的结合产生不利影响。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
一种类型的衍生化抗体(例如，双特异性抗体)是通过交叉连接2个或更多个抗体(属于同一类型或不同类型)而产生的。获得双特异性抗体的方法是本领域公知的，其实例包括但不限于，化学交联法、细胞工程法(杂交瘤法)或基因工程法。
另一种类型的衍生化抗体是标记的抗体。例如，可以将本发明的抗体或其抗原结合片段连接至可检测的标记。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，
Figure PCTCN2020134438-appb-000001
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
此外，本发明的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清 半衰期。
因此，本发明的一个方面提供了一种缀合物，其包括本发明的单克隆抗体或其抗原结合片段以及偶联部分，所述偶联部分可为前述的可检测的标记，如放射性同位素、荧光物质、发光物质、有色物质或酶。所述偶联部分也可为治疗剂。
作为抗体的衍生物之一，本发明提供一种多特异性抗体，所述多特异性抗体包含第一抗体或其片段，和另外的抗体或其片段，或抗体类似物，其中第一抗体或其片段，另外的抗体或其片段，或抗体类似物保留其原始结合特异性。所述第一抗体或其片段是本发明的任一结合TSLP的(单克隆)抗体或其抗原结合片段。如本文中所使用的，“抗体类似物(antibody mimetic)”，指与抗体一样特异性结合抗原，但却没有抗体结构。它们通常是人工肽或蛋白质，摩尔质量约为3至20kDa，例如锚蛋白重复蛋白(DARPin)和fynomer。经设计的锚蛋白重复蛋白(DARPin)可以与IgG抗体、scFv-Fc抗体片段或其组合相连，如CN104341529A所述。抗IL-17a的fynomer与抗IL-6R抗体融合产生双特异性融合多肽，如WO2015141862A1所述。
在某些实施方案中，所述多特异性抗体由第一抗体或其抗原结合片段与其他抗体或其抗原结合片段或抗体类似物通过偶联形成，并且其中各抗体或其抗原结合片段或抗体类似物保持其原结合特异性，所述第一抗体或其抗原结合片段为本发明所述的抗体或其抗原结合片段。某些实施方案中，所述多特异性抗体为双特异性抗体或三特异性抗体或四特异性抗体。
抗体的制备
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’) 2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，在另一个方面，本发明提供了一种分离的核酸分子，包含编码本发明的抗体或其抗原结合片段、或其重链可变区和/或轻链可变区、或其一个或多个CDR的核苷酸序列。根据本领域已知的密码子简并性，在某些实施方案中，所述核苷酸序列是可以根据密码子简并性进行替换的。在某些实施方案中，所述核苷酸序列是密码子最优化的。
在某些实施方案中，本发明所述分离的核酸分子包含：(i)分别编码本发明的抗体或其抗原结合片段的重链可变区和轻链可变区的第一核酸和第二核酸，或(ii)分别编码本发明的抗体或其抗原结合片段的重链可变区和重链恒定区的第一核酸，和轻链可变区和轻链恒定区的第二核酸，或(iii)分别编码本发明的抗体或其抗原结合片段的重链和轻链的第一核酸和第二核酸。在某些实施方案中，所述第一核酸和第二核酸包含与上述(i)-(iii) 中任一第一核酸和第二核酸的简并序列或基本上相同序列的核酸。在某些实施方案中，所述简并序列或基本上相同序列指与(i)-(iii)中所述核酸分子相比具有至少大约85％、90％、95％、99％或更高序列同一性的序列或具有一个或更多个核苷酸取代的序列，或相差不超过3、6、15、30或45个核苷酸的序列。
另一方面，提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。在某些实施方案中，本发明的载体是例如质粒，粘粒，噬菌体，慢病毒等。在某些实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。
另一方面，提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、CHO DG44)。
另一方面，提供了制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
用途、治疗方法和药物组合物
本发明另一方面，提供了药物组合物，其包含本发明的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、和/或缀合物以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本发明的药物组合物包含本发明的抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本发明的药物组合物包含本发明的宿主细胞，以及药学上可接受的载体和/或赋形剂，其中所述宿主细胞包含如前所述的分离的核酸分子或载体。
在某些实施方案中，本发明的药物组合物包含本发明的多特异性抗体，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本发明的药物组合物包含本发明的缀合物以及药学上可接受的载体和/或赋形剂。
另一方面，本发明的药物组合物中的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物用于在受试者中产生至少一项以下生物学活性：
(1)抑制或阻断TSLP与TSLPR/IL7Rα结合；
(2)下调或消除TSLP的活性；
(3)下调或阻断OX40L的表达；
(4)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌；
(5)抑制或阻断Th2类细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌；
(6)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖。
本发明的药物组合物中的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物能够抑制或阻断TSLP与TSLPR/IL7Rα结合。TSLP与TSLPR/IL7Rα结合能够引起很多变态反应性炎症疾病，包括过敏性疾病和非过敏性疾病。这些疾病包括但不限于哮喘(包括严重哮喘)、特发性肺纤维化、特应性皮炎(AD)、 过敏性结膜炎、变应性鼻炎(AR)、Netherton综合征(NS)、嗜酸性食管炎(EoE)、食物过敏、过敏性腹泻、嗜酸性胃肠炎、过敏性支气管肺曲霉菌病(ABPA)、过敏性真菌性鼻窦炎、类风湿性关节炎、COPD、系统性硬化症、瘢痕疙瘩、溃疡性结肠炎、慢性鼻窦炎(CRS)和鼻息肉、慢性嗜酸性肺炎、嗜酸性支气管炎；腹腔疾病，如嗜酸性胃肠炎、Churg-Strauss综合征；嗜酸性粒细胞相关的胃肠道疾病，如嗜酸性粒细胞增多症/嗜酸性肉芽肿伴多血管炎、嗜酸性食管炎和炎症性肠病；荨麻疹、系统性肥大细胞增多症、皮肤肥大细胞增多症、反复发作的特发性血管性水肿。因此本发明的物组合物中的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物能够预防或治疗前述疾病。
TSLP与TSLPR/IL7Rα结合也与自身免疫性疾病相关。因此本发明的物组合物中的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物能够预防或治疗自身免疫性疾病如糖尿病、重症肌无力、胃炎、天疱疮、原发性胆汁性肝硬化、多发性硬化症、狼疮、结肠炎、类风湿、银屑病和甲状腺疾病等。
在某些实施方案中，在所述药物组合物中，本发明的抗体或其抗原结合片段与所述另外的药学活性剂作为分离的组分或作为同一组合物的组分提供。因此，本发明的抗体或其抗原结合片段与所述另外的药学活性剂可以联合或分开,同时或相继施用。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。
本发明的抗TSLP的抗体或其抗原结合片段可单独或与其它活性剂组合给药。抗TSLP的抗体或其抗原结合片段和一种或多种其它活性剂可分开、同时或相继给药。
本发明的抗TSLP的抗体或其抗原结合片段可与采用任何合适的免疫抑制剂，包括但不限于抗炎症药物组合给药，尤其是吸入、鼻内或肠胃外皮质类固醇，如布地缩松、beclamethasone dipropionate、丙酸氟地松、环索奈德，糠酸毛他松，糠酸氟替卡松、丙酸氟替卡松、布地奈德、环索奈德、二丙酸倍氯米松、糠酸莫米松、曲安奈德和泼尼松龙。在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可以与吸入皮质类固醇的固定剂量组合进行给药。如与糠酸氟替卡松或丙酸氟替卡松的固定剂量组合。在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可以与非类固醇类糖皮质激素受体激动剂；LTD4拮抗剂或LTB4拮抗剂，包括孟鲁司特、普仑司特、扎鲁司特、安可来等；A2A激动剂；A2B拮抗剂；多巴胺受体激动剂；或，PDE4抑制剂组合进行给药。在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可与吡非尼酮或尼达尼布或avB6拮抗剂组合给药。
本发明的抗TSLP的抗体或其抗原结合片段可与支气管舒张药物组合给药。如β-2肾上腺素受体激动剂和/或毒蕈碱拮抗剂组合给药。合适的β-2肾上腺素受体激动剂包括维兰特罗、沙美特罗、沙丁胺醇、福莫特罗、沙甲胺醇、非诺特罗、卡莫特罗、依坦特罗、那明特罗、克仑特罗、吡布特罗、氟丁特罗、瑞普特罗、班布特罗、茚达特罗、特布他林、以及它们的盐。合适的毒蕈碱拮抗剂包括芜地溴铵、噻托溴铵、格隆溴铵、异丙托铵、以及它们的盐如芜地溴铵的氢溴酸盐。在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可以与β-2肾上腺素受体激动剂和/或毒蕈碱拮抗剂的固定剂量组合进行给药，如与维兰特罗三苯乙酸盐、芜地溴铵的固定剂量组合，或与维兰特罗三苯乙酸盐和芜地溴铵的双重组合。
本发明的抗TSLP的抗体或其抗原结合片段可与一种或多种支气管扩张剂和吸入的类固醇的组合一起给药。这种组合可包括双重组合如糠酸氟替卡松和维兰特罗三苯乙酸盐、糠酸氟替卡松和芜地溴铵、丙酸氟替卡松和沙美特罗、布地奈德和福莫特罗、莫米松和福莫特罗，和三重治疗如糠酸氟替卡松、维兰特罗三苯乙酸盐和芜地溴铵。在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可以与吸入的皮质类固醇和一种或多种支气管扩张剂的固定剂量组合进行给药，如与糠酸氟替卡松和维兰特罗三苯乙酸盐，或丙酸氟替卡松和沙美特罗，或糠酸氟替卡松和芜地溴铵，或糠酸氟替卡松、维兰特罗三苯乙酸盐和芜地溴铵的固定剂量组合。
在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可以与细胞因子受体的拮抗剂组合给药，例如CCR-1、CCR-3、CCR-4、CCR-5、CCR-6、CCR-7、CCR-8、CCR-9和CCR10、CXCR1、CXCR2、CXCR3、CXCR4、CXCR5的拮抗剂。在一个实施方案中，本发明的抗TSLP的抗体或其抗原结合片段可以与其他细胞因子或细胞因子受体的抗体组合给药，例如抗IgE抗体，抗IL31抗体、抗IL31R抗体、抗IL13抗体，抗内皮糖蛋白抗体、抗IL1b抗体、另一抗TSLP抗体或抗hTSLPR抗体或其组合。
本发明的抗TSLP的抗体或其抗原结合片段可与以下组合给药：抗白三烯拮抗剂如孟鲁司特、扎鲁司特和普仑司特；PDE4抑制剂如罗氟司特；呫吨；抗IgE抗体,；IL-13拮抗剂；IL-6拮抗剂以及IL-1、IL-33、IL-25或TNF-α的拮抗剂。
本发明的抗TSLP的抗体或其抗原结合片段可与抗组胺或止咳药物，如盐酸西替利嗪、醋氨酚、富马酸氯马斯丁、异丙嗪、氯雷他定、地氯雷他定、可他敏和盐酸非索那丁、activastine、阿司咪唑、氮卓斯汀、依巴斯汀、依匹那丁、咪唑斯汀和tefenadine组合给药。
上文所述的TSLP结合抗体或其抗原结合片段与另外的一种或多种其他活性剂的组合给药，其他活性剂可选自但不限于：免疫抑制剂(例如皮质类固醇、非类固醇类糖皮质激素受体激动剂、白三烯D4拮抗剂、白三烯B4拮抗剂、A2A激动剂、A2B拮抗剂、多巴胺受体激动剂、吡非尼酮尼达尼布、或avB6拮抗剂)、支气管扩张剂(例如β-2肾上腺素受体激动剂、毒蕈碱拮抗剂、短效β2受体激动剂、长效β2受体激动剂、短效抗胆碱能药物、甲基黄嘌呤类药物、长效抗胆碱能药物)、其他细胞因子或细胞因子受体的拮抗剂或抗体(例如IL-13拮抗剂、IL-6拮抗剂、IL-1、IL-33、IL-25或TNF-α的拮抗剂、抗IgE抗体、抗IL31抗体、抗IL31R抗体、抗IL13抗体、抗内皮糖蛋白抗体、抗IL1b抗体、另一抗TSLP抗体或抗hTSLPR抗体)、抗生素、放射治疗、白三烯拮抗剂(如孟鲁司特、扎鲁司特或普仑司特)，PDE4抑制剂(如罗氟司特，呫吨)、抗组胺或止咳药物。
在某些实施方案中，所述药物组合物与其他治疗同时、分开或相继施用，如在另外的药学活性剂之前、同时或之后施用。
本发明另一方面，提供本发明的抗体或其抗原结合片段、核酸、载体、宿主细胞、缀合物或者多特异性抗体用于(1)抑制或阻断TSLP与TSLPR/IL7Rα结合，(2)下调或消除TSLP的活性；(3)下调或阻断OX40L的表达；(4)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖，(5)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌,(6)抑制或阻断TSLP诱导的Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5 的分泌；和/或(7)预防或治疗变态反应性疾病、过敏反应性或自身免疫病。
本发明另一方面，提供本发明的抗体或其抗原结合片段、核酸、载体、宿主细胞、缀合物或者多特异性抗体在制备药物中的用途，所述药物用于：
(1)抑制或阻断TSLP与TSLPR/IL7Rα结合，
(2)下调或消除TSLP的活性；
(3)下调或阻断OX40L的表达；
(4)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖，
(5)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌,
(6)抑制或阻断TSLP诱导的Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌；和/或
(7)预防或治疗变态反应性疾病、过敏反应性或自身免疫病。
在某些实施方案中，当本发明宿主细胞用于制备药物时，所述宿主细胞包含如前所述的分离的核酸分子或载体。
在某些实施方案中，当本发明的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物用于制备药物时，所述药物用于在受试者(例如人)中预防和/或治疗哮喘、变态反应性炎症、过敏反应或自身免疫性疾病。
在某些实施方案中，所述受试者是哺乳动物，包括非人哺乳动物和人。在某些实施方案中，所述受试者是人。
在某些实施方案中，本发明所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物、或者药物用于预防和/或治疗变态反应性炎症疾病，包括过敏性疾病和非过敏性疾病。这些疾病包括但不限于哮喘(包括严重哮喘)、特发性肺纤维化、特应性皮炎(AD)、过敏性结膜炎、变应性鼻炎(AR)、Netherton综合征(NS)、嗜酸性食管炎(EOE)、食物过敏、过敏性腹泻、嗜酸性胃肠炎、过敏性支气管肺曲霉菌病(ABPA)、过敏性真菌性鼻窦炎、类风湿性关节炎、慢性阻塞性肺病(COPD)、系统性硬化症、瘢痕疙瘩、溃疡性结肠炎、慢性鼻窦炎(CRS)和鼻息肉、慢性嗜酸性肺炎、嗜酸性支气管炎；腹腔疾病，如嗜酸性胃肠炎、Churg-Strauss综合征；嗜酸性粒细胞相关的胃肠道疾病，如嗜酸性粒细胞增多症/嗜酸性肉芽肿伴多血管炎、嗜酸性食管炎和炎症性肠病；荨麻疹、系统性肥大细胞增多症、皮肤肥大细胞增多症、反复发作的特发性血管性水肿。
在某些实施方案中，本发明所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物用于预防和/或治疗与自身免疫相关的疾病。在某些实施方案中，所述疾病包括但不限于：甲状腺功能亢进、糖尿病、重症肌无力、溃疡性结肠炎、胃炎、天疱疮、原发性胆汁性肝硬化、多发性硬化症、红斑狼疮、类风湿关节炎等。
在另一个方面，本发明提供了一种在体内或体外(1)抑制或阻断TSLP与TSLPR/IL7Rα结合，(2)下调或消除TSLP的活性；(3)下调或阻断OX40L的表达；(4)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌；(5)抑制或阻断分泌Th2类细胞因子的分泌；(6)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖中至少一项的方法，包括：施加给细胞或受试者任一项本发明所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物、或者药物组合物。
可选地，在施加所述抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物或药物组合物的同时、之前或之后，施加另外的药学活性剂。
在某些实施方案中，所述受试者是哺乳动物，包括非人哺乳动物和人；优选地，所述受试者是人。
因此，在另一个方面，本发明提供了一种在受试者中预防和/或治疗哮喘、过敏反应、变态反应性炎症或自身免疫性疾病的方法，所述方法包括向有此需要的受试者施用有效量的本发明所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物或者药物组合物。
本发明的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物和药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定，可制备成注射剂。
此外，本发明的抗体或其抗原结合片段可以以单位剂量形式存在于药物组合物中，以便于施用。
本发明的药物组合物可以包括“治疗有效量”或“预防有效量”的本发明的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体或者缀合物。“预防有效量”是指，足以预防、阻止或延迟疾病的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量，例如0.1mg/ml到5000mg/ml。
在本发明中，所述受试者可以为哺乳动物(包括非人哺乳动物和人)，例如人。
检测方法和试剂盒
本发明的抗体或其抗原结合片段能够结合TSLP，从而可用于检测TSLP在样品中的存在或其水平。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。在某些实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记。在一个优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。
在本发明中，所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，
Figure PCTCN2020134438-appb-000002
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。本发明中涵盖的标记物可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所 述的可检测的标记连接至本发明的重组蛋白，以降低潜在的位阻。
在另一个方面，本发明提供了检测TSLP在样品中的存在或其水平的方法，其包括使用本发明的抗体或其抗原结合片段的步骤。
在一个优选的实施方案中，本发明的抗体或其抗原结合片段还带有可检测的标记。
在另一个优选的实施方案中，所述方法还包括，使用带有可检测的标记的试剂来检测本发明的抗体或其抗原结合片段。
所述方法可以用于诊断目的，或者非诊断目的(例如，用于TSLP-TSLP/IL-7Ra途径研究、药物筛选、组化分析等)。在某些实施方案中，用于非诊断目的的样品是细胞样品，例如细胞系或离体细胞培养物。
在一个实施方案中，本发明提供了一种检测样品中TSLP的存在或其水平的方法，所述方法包括在允许本发明所述抗体或其抗原结合片段与TSLP之间形成复合物的条件下，使所述样品与所述抗体或其抗原结合片段接触，并且检测所述复合物的形成。
在另一个方面，本发明提供了一种诊断受试者中哮喘、变态反应性炎症、过敏反应或自身免疫性疾病的方法，包括
-将本发明所述的抗体或其抗原结合片段、多特异性抗体、或缀合物与来自受试者的样品在允许抗体或其抗原结合片段与TSLP之间形成复合物的条件下接触，
-检测所述复合物的形成，
其中与健康对照相比，TSLP水平升高指示存在哮喘、变态反应性炎症、过敏反应或自身免疫性疾病。
可选地，所述受试者是哺乳动物，包括非人哺乳动物和人。优选地，所述受试者是人。
优选地，所述变态反应性炎症、过敏反应或自身免疫性疾病如上所述。
在另一个方面，本发明提供了本发明所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物或者药物组合物在制备用于诊断哮喘、变态反应性炎症、过敏反应或自身免疫性疾病的药物或试剂盒中的用途。
在另一个方面，提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测TSLP在样品中的存在或其水平。在另一个方面，本发明提供了诊断性或治疗性试剂盒，其包括一个或多个以下物质：本发明所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物或者药物组合物。可选地，所述诊断性或治疗性试剂盒还包括使用说明书。
本发明的抗体或抗原结合片段与TSLP的结合亲和力高，且具有好的特异性。因此，本发明的抗体或抗原结合片段适用于预防和/或治疗哮喘、变态反应性炎症、过敏反应或自身免疫性疾病。本发明的全人源抗体保留具有很高的人源程度，从而可安全地施用给人受试者，而不引发免疫原性反应。因此，本发明的抗体或抗原结合片段具有重大的临床价值。
术语定义
在本文中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本发明所用的并在所附权利要求书中的单数形式“一个”和“所述”等包括复数涵义，除非上下文中清楚地另有指明。因此，单数词语“一个”的涵义包括了“一个或多个”。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。
在本文中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或，AbM或IMGT编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“胚系抗体基因”是非淋巴细胞表达的免疫球蛋白的编码基因，它没有经历导致表达特异免疫球蛋白的遗传学重排及成熟的成熟过程。本发明的各种实施方案所提供的一个优点来源于一种认识，那就是胚系抗体基因编码的氨基酸序列比成熟抗体基因编码的氨基酸序列更多地保留了动物物种个体的特征性的重要氨基酸序列结构。因此当被治疗性应用于该物种时，更少地被该物种识别为外源物质。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指抗体的多肽片段,例如全长抗体的多肽片段，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)、线性抗体(linear antibody)、纳米抗体(如技术来自Ablynx)、结构域抗体(如技术来自Domantis)、和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“全长抗体”意指，由两条“全长重链”和两条“全长轻链”组成的抗体。其中，“全长重链”是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、 重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域。优选地，“全长重链”是在N端到C端方向上由VH、CH1、HR、CH2和CH3组成的多肽链。“全长轻链”是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链。两对全长抗体链通过在CL和CH1之间的二硫键和两条全长重链的HR之间的二硫键连接在一起。本发明的全长抗体可以来自单一物种，例如人；也可以是嵌合抗体或人源化抗体。本发明的全长抗体包含分别由VH和VL对形成的两个抗原结合部位，这两个抗原结合部位特异性识别/结合相同的抗原。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’) 2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’) 2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc片段”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.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。在一些情况下，scFv的VH与VL之间还可以存在二硫键。如本文中所使用的，术语“di-scFv”是指，由两个scFv连接形成的抗体片段。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
如本文中所使用的，“抗体类似物(antibody mimetic)”，指与抗体一样特异性结合抗 原，但却没有抗体结构。它们通常是人工肽或蛋白质，摩尔质量约为3至20kDa。例如，锚蛋白重复蛋白(DARPin)和fynomer。经设计的锚蛋白重复蛋白(DARPin)可以与IgG抗体、scFv-Fc抗体片段或其组合相连，如CN104341529A。抗IL-17a的fynomer与抗IL-6R抗体结合，如WO2015141862A1。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用，是指来自一群高度同源的抗体分子(也即，除可能自发出现的自然突变外，一群完全相同的抗体分子)中的一个抗体。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(Cabilly et al.的美国专利4,816,567；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。
在本发明中，本发明抗体的预期性质包括：(1)抑制或阻断TSLP与TSLPR/IL7Rα结合；(2)下调或消除TSLP的活性；(3)下调或阻断OX40L的表达；(4)抑制Th2类细胞因子的分泌；(5)预防或治疗变态反应性炎症。本发明的人源化抗体保留了亲本抗体(人源抗体或鼠-人嵌合抗体)的上述预期性质中的一项或多项。
为制备人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。或者，还可以利用转基因动物，其能够在免疫后不产生内源性免疫球蛋白、并且能够产生完整人抗体库(参见例如，Jakobovits等，1993，Proc.Natl.Acad.Sci.USA 90：2551；Jakobovits等，1993，Nature 362：255-258；Bruggermann等，1993，Year in Immunology 7：33；和Duchosal等，1992，Nature 355：258；Lonberg等人(1994)Nature 368(6474):856-859；WO02/43478)。其他抗体人源化改造的方法还包括噬菌体展示技术(Hoogenboom等，1991，J.Mol.Biol.227：381；Marks等，J.Mol.Biol.1991，222：581-597；Vaughan等，1996，Nature Biotech 14：309)。
如本文中所使用的，术语“人源化程度”是用于评价人源化抗体中非人源氨基酸残基的数量的指标。人源化抗体的人源化程度例如可通过IMGT网站DomainGapAlign来预测可变区序列与人V结构域的同源性。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以用相互作用的解离平衡常数(KD)表示。在本文中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。解离平衡常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于约10 -8M，例如小于约10 -8M、10 -9M、10 -10M或10 -11M或更小的KD结合该抗原。在某些实施方案中，当KD≤10×10 -8M时，本发明的抗体或其抗原结合片段被认为特异性地结合TSLP。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用生物发光干涉测量法(例如，ForteBio Octet法)来测量解离常数。除此以外还可用表面等离子共振技术(例如Biacore)或Kinexa来测量解离常数。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。两个氨基酸序列之间的％同一性也可用E.Meyers和W.Miller(Comput.Appl.Biosci.，4：11-17(1988))的算法来确定。
如本文中所使用的，具有“％同一性”的序列保留与其比较或来源的序列的重要生物活性，如抗体结合特异性。具有一个或几个氨基酸的置换、缺失或添加或其任意组合的序列保留与其比较或来源的序列的重要生物活性，如抗体结合特异性。具有“％同一性”的核苷酸序列或相差不超过3、6、15、30或45个核苷酸的核苷酸序列能够实现与其比较或来源的核苷酸序列近似的功能，如所表达的蛋白都能特异地结合同一抗原或分子。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本文中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示；精氨酸可用R或Arg表示；甘氨酸可用G或Gly表示；谷氨酰胺可用Q或Gln表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇， 苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，哮喘、变态反应性炎症、过敏反应或自身免疫性疾病)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如非人灵长类哺乳动物或人。在某些实施方式中，所述受试者(例如人)患有哮喘、变态反应性炎症、过敏反应或自身免疫性疾病，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，哮喘、变态反应性炎症、过敏反应或自身免疫性疾病)有效量是指，足以预防，阻止，或延迟疾病(例如，哮喘、变态反应性炎症、过敏反应或自身免疫性疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞，例如淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
如本文中所使用的，术语“免疫应答”是指，免疫细胞(例如淋巴细胞、抗原呈递细胞、吞噬细胞或粒细胞)以及由免疫细胞或肝脏所产生的可溶性大分子(包括抗体、细胞因子、以及补体)的作用，该作用导致对侵入性病原体、被病原体感染的细胞或组织、癌细胞、或者在自身免疫或病理炎症情况下的正常人类细胞或组织的选择性损害、破坏或将它们从人体中清除。在本文中，术语“抗原特异性T细胞应答”指由T细胞产生的免疫应答，该应答产生于当该T细胞特异的抗原对该T细胞的刺激之时。由T细胞在抗原特异性刺激时产生的反应的非限制性实例包括T细胞的增殖以及细胞因子(例如IL-2)的产生。
如本文中所使用的，术语“效应子功能(effector function)”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体同种型而变化。
术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其他不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括糖类(如乳糖)、淀粉、纤维素及其衍生物、植物油、明胶、多元醇(如丙二醇)、海藻酸等。
发明的有益效果
与现有技术相比，本发明的技术方案具有以下有益效果：
(1)本发明的抗体能够特异性高亲和性地识别/结合TSLP，抑制或阻断TSLP与 TSLPR/IL7RR的结合，并且能够在体外/体内抑制或阻断TSLP对Ba/F3细胞的增殖效应，阻断TSLP对PBMC的激活、分泌细胞因子的能力。因此，本发明的抗体能够抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖，抑制或阻断TSLP诱导的OX40L表达、骨保护素(OPG)的分泌或TSLP诱导的Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌，因此，本发明的抗体具有用于预防和/或治疗哮喘、其他过敏性反应或自身免疫性疾病的潜力。
(2)具有良好的热稳定性、亲水性、等电点、及与FcRn亲和力。
(3)本发明的一些抗体是全人源抗体，可安全地施用给受试者而不引发免疫原性反应。因此，本发明的抗体具有重大的临床价值。
缩略词
CDR       免疫球蛋白可变区中的互补决定区
FR        抗体构架区：抗体可变区中除CDR残基以外的氨基酸残基
VH        抗体重链可变区
VL        抗体轻链可变区
IgG       免疫球蛋白G
AbM       AbM CDR定义方式来源于Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268–9272)，此定义方法整合了Kabat及Chothia两者的部分定义。
Kabat     由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
Chothia   由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
IMGT      基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information
Figure PCTCN2020134438-appb-000003
(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
mAb       单克隆抗体
EC50      产生50％功效或结合的浓度
IC50      产生50％抑制的浓度
ELISA     酶联免疫吸附测定
PCR       聚合酶链式反应
HRP       辣根过氧化物酶
TSLP      胸腺基质淋巴细胞生成素
TSLPR     胸腺基质淋巴细胞生成素受体
IL7Rα    白介素7受体alpha亚基
TARC      胸腺激活调节趋化因子
hFc       人IgG抗体Fc段
KD        解离平衡常数
CDR-H1    免疫球蛋白重链可变区中的互补决定区1
CDR-H2    免疫球蛋白重链可变区中的互补决定区2
CDR-H3    免疫球蛋白重链可变区中的互补决定区3
CDR-L1    免疫球蛋白轻链可变区中的互补决定区1
CDR-L2    免疫球蛋白轻链可变区中的互补决定区2
CDR-L3    免疫球蛋白轻链可变区中的互补决定区3
附图说明
图1：人TSLPR/IL7Rα双基因过表达Ba/F3细胞系检测
图2A：嵌合抗体25A5C5抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图2B：嵌合抗体27C2B6、37C2D10抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图2C：嵌合抗体43B1A8和90H3H11抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图3A：重组全人源抗体25A5C5-hIgG、27C2B6-hIgG、37C2D10-hIgG结合人TSLP蛋白的亲和力检测
图3B：重组全人源抗体43B1A8-hIgG和90H3H11-hIgG结合人TSLP蛋白的亲和力检测
图4A：重组全人源抗体25A5C5-hIgG、27C2B6-hIgG和37C2D10-hIgG抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图4B：重组全人源抗体43B1A8-hIgG抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图4C：重组全人源抗体90H3H11-hIgG抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图5：重组全人源抗体43B1A8-hIgG和90H3H11-hIgG抑制PBMC分泌TARC的活性检测
图6：重组全人源抗体43B1A8-hIgG和90H3H11-hIgG热稳定性检测(Tm)
图7A：重组全人源抗体43B1-H2L2抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图7B：重组全人源抗体43B1-H6L1抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
图8：重组全人源抗体43B1-H2L2抑制PBMC细胞分泌MDC细胞因子的活性检测
图9：重组全人源抗体43B1-H2L2分子在食蟹猴体内的药代动力学分析
序列信息
本发明涉及的序列的信息描述于下面的表中。
Figure PCTCN2020134438-appb-000004
Figure PCTCN2020134438-appb-000005
Figure PCTCN2020134438-appb-000006
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：抗原的制备
在大肠杆菌或者哺乳动物细胞中表达人TSLP或者猴TSLP。人TSLP的氨基酸序列参考NCBI的蛋白数据库中的NP_149024.1。食蟹猴(Macaca fascicularis)TSLP的氨基酸序列参考NCBI的蛋白数据库中的XP_005557555.1。本申请中使用的抗原包括以经修饰形式表达的TSLP，例如在TSLP序列的C末端融合表达6个连续的组氨酸作为标签(TSLP-His)。上述人或猴TSLP的序列经由金斯瑞公司进行密码子优化，合成于表达载体上(即含人TSLP完整编码序列的质粒)，并在大肠杆菌中，或者哺乳动物细胞HEK293F细胞中表达和纯化。
TSLP的天然受体是异源二聚体，由人TSLPR与人IL7R alpha(IL7Rα)亚基组 成。人TSLPR的序列参考(Uniprot:Q9HC73.1)；人IL7R alpha亚基的序列参考(GenBank:AAR08908.1)。人hIL7Rα-hTSLPR-hFc融合蛋白是将人IL7Rα胞外区(E21-D239)、人TSLPR受体的胞外区(Q23-K231)和人IgG1Fc区域的部分编码序列(Hinge-CH2-CH3)串联在一起组成。通过密码子优化，构建在pLVX表达载体上。并利用pLVX载体建立HEK293F的表达稳定细胞系，最终纯化获得hIL7Rα-hTSLPR-hFc的融合蛋白。
实施例2：人TSLPR/IL7Rα双基因过表达细胞系的构建与鉴定
2.1人TSLPR/IL7Rα双基因过表达细胞系的构建
为验证人TSLP抗体阻断人TSLP结合受体人TSLPR/IL7Rα的功能，将人TSLPR完整编码的氨基酸序列(基因编号：UniProtKB/Swiss-Prot:Q9HC73.1，由南京金斯瑞生物科技有限公司合成)和人IL7R alpha亚基的完整氨基酸编码序列(基因编号：GenBank:AAR08908.1，由南京金斯瑞生物科技有限公司合成)交由金斯瑞公司进行密码子优化，并分别克隆到慢病毒载体pLVX-IRES-puro和pLVX-IRES-zeocin上，并通过文献所述(Mohammadi Z etl.,Mol Biotechnol.2015Sep；57(9):793-800)慢病毒包装系统制备病毒的方法，获得的病毒感染小鼠原B细胞株Ba/F3细胞(购买自南京科佰)，通过嘌呤霉素+博莱霉素筛选及单克隆挑选，获得单克隆的Ba/F3-hTSLPR-hIL7Rα稳定细胞系。
2.2人TSLPR/IL7Rα双基因过表达Ba/F3细胞系检测
细胞系采用流式细胞仪(流式细胞仪：Beckman，CytoFlex；检测抗体APC-anti-human TSLPR(Biolegend)，APC-anti-human IL7Rα(Biolegend))进行鉴定，确定单克隆细胞正确表达人TSLPR和人IL7Rα。如图1所示，流式结果显示Ba/F3-hTSLPR-hIL7Rα是表达这2种基因(接近100％)、均一性较好的单克隆细胞系，可用于后续实验。
实施例3：小鼠免疫与杂交瘤融合
3.1小鼠免疫
采用全人源转基因小鼠H2L2(和铂医药)进行免疫，分别采用大肠杆菌表达的人TSLP(NP_149024.1)、哺乳动物细胞表达的人TSLP(NP_149024.1)、哺乳动物表达的猴TSLP(XP_005557555.1)，以及含人TSLP完整编码序列的质粒进行多次免疫。每两周进行一次加强免疫，共5-6次。免疫期间，每两周通过ELISA方法检测抗人TSLP抗体的血清效价(参见实施例4.1)，经过多轮免疫并根据效价情况选择最佳效价的小鼠使用以下方案融合以生成杂交瘤。
3.2融合方法
将小鼠脾脏和淋巴结单细胞悬液混合后，加入相同数量的SP2/0骨髓瘤细胞并混匀。细胞混合液经电融合缓冲液洗涤重悬，用BTX-ECM2001电融合仪融合后，立即将细胞悬液从融合舱室中转移至完全融合培养基中，37℃孵育1小时。按2×10 4个细胞每孔的密度进行96孔板铺板。培养5天后使用完全融合培养基换液，7-10天时取上清进行杂交瘤筛选。
实施例4：杂交瘤筛选
4.1人TSLP结合ELISA筛选
将可溶性的人TSLP-His蛋白在1xCBS包被液中稀释至1μg/ml，并加入到96孔板，并在4℃下孵育过夜。用PBST洗涤96孔板，并在37℃下用封闭液(PBS+2％BSA)将板封闭2小时。向板中加入杂交瘤上清液，或加入同等体积封闭液，在37℃培养箱中将板培育2小时。96孔板中加入山羊抗大鼠IgG-HRP，在37℃培养箱中将板孵育1小时，洗涤并在450nm下读取OD。
4.2猴TSLP结合ELISA筛选
将猴TSLP-His蛋白在CBS包被液中稀释至1μg/ml，并加入到96孔板，并在4℃下培育过夜。用PBST洗涤96孔板，并在37℃下用封闭液PBS+2％BSA将板封闭2小时。将板中加入杂交瘤上清液，或加入同等体积封闭液，在37℃培养箱中将板培育2小时。96孔板中加入山羊抗大鼠IgG-HRP，在37℃培养箱中将板培育1小时，洗涤并在450nm下读取OD。
4.3阻断人TSLP-His与嵌合受体IL7Rα-TSLPR-hFc结合ELISA筛选
根据以下方案测定杂交瘤上清液或纯化抗体阻断人TSLP-His与嵌合受体IL7Rα-TSLPR-hFc的结合。
将可溶性的hIL7Rα-hTSLPR-hFc蛋白在1xCBS包被液中稀释，并加入到96孔板，并在4℃下培育过夜。用PBST洗涤96孔板，并在37℃下，用封闭液PBS+2％BSA将板封闭2小时。向板中加入哺乳动物细胞重组表达的hTSLP-His蛋白(+/-)，并加入杂交瘤上清液或加入同等体积封闭液，在37℃培养箱中将板培育2小时。96孔板中加入小鼠抗His-HRP，在37℃培养箱中将板培育1小时，洗涤并在450nm下读取OD。选择抑制率强的杂交瘤作为候选克隆。
根据以上亲和ELISA和竞争ELISA的活性，选择阳性克隆采用有限稀释法进行亚克隆分选以获得亚克隆。最终获得能够与人TSLP-His、猴TSLP-His结合，同时能够阻断人TSLP-His与嵌合受体hIL7Rα-hTSLPR-hFc结合的杂交瘤亚克隆。如表1所示，5个单克隆均与人TSLP(hTSLP)和猴TSLP(cTSLP)有较强的结合能力，也均能阻断人TSLP与其受体hIL7Rα-hTSLPR-hFc的结合能力，抑制率超过60％。
表1抗TSLP杂交瘤亚克隆筛选
Figure PCTCN2020134438-appb-000007
实施例5：抗TSLP嵌合抗体制备
对照抗体表达：对照抗体序列参考chEMBL数据库(ID：CHEMBL3707229)。对照抗体的重链和轻链碱基序列合成在pTT5表达载体，并通过CHO-S细胞(购买自Thermo公司)瞬时转染与表达，然后通过Protein A(MabSelect SuRe,GE)进行亲和纯化，获得对照抗体。
将杂交瘤单克隆进行无血清培养，获得50mL上清，然后通过Protein A(MabSelect SuRe,GE)进行纯化获得杂交瘤嵌合抗体。因H2L2小鼠抗体恒定区通过基因改造为大鼠恒定区，所以纯化获得的抗体为全人源可变区携带大鼠Fc的嵌合抗体。纯化的抗体采用分光光度法定量，获得嵌合抗体25A5C5，27C2B6，37C2D10，43B1A8和90H3H11。
实施例6：抗TSLP嵌合抗体与TSLP亲和力ELISA检测
采用ELISA方法检测嵌合抗体25A5C5，27C2B6，37C2D10，43B1A8和90H3H11与人或猴TSLP亲和力。具体方法简述如下：包被人TSLP-His或猴TSLP-His抗原至96孔板中，4℃过夜后，分别加入不同浓度梯度稀释后的抗体，孵育2小时后，加入山羊抗大鼠Fc-HRP二抗，孵育1小时后，酶标仪检测A450nm波长的吸光值。
检测结果如表2所示，人TSLP的亲和力：25A5C5和27C2B6嵌合抗体和对照抗体基本相当，43B1A8和90H3H11抗体高于对照抗体。25A5C5和90H3H11抗体与猴TSLP几乎无结合；37C2D10、27C2B6以及43B1A8均与与猴TSLP较强结合。
表2抗TSLP嵌合抗体与TSLP亲和力
Figure PCTCN2020134438-appb-000008
实施例7：抗TSLP嵌合抗体阻断TSLP/hIL7Rα-hTSLPR-hFc结合活性的竞争ELISA检测
人TSLP通过与人hIL7Rα-hTSLPR-hFc异源二聚体受体结合，激活下游信号通路。采用竞争ELISA方式检测嵌合抗体阻断嵌合受体hIL7Rα-hTSLPR-hFc与抗原结合的活性。具体步骤参见实施例4.3，孔板中加入梯度稀释的嵌合抗体。结果如表3所示，5个嵌合抗体均能有效阻断人TSLP与人hIL7Rα-hTSLPR-hFc异源二聚体受体结合。
表3：抗TSLP嵌合抗体竞争ELISA活性
抗体 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 对照抗体
IC50(nM) 0.89 0.77 0.51 1.33 1.65 0.93
实施例8：抗TSLP嵌合抗体的Ba/F3-hTSLPR-hIL7Rα细胞增殖抑制检测
通过Ba/F3-hTSLPR-hIL7Rα细胞增殖抑制方法，检测抗TSLP嵌合抗体的活性。Ba/F3-hTSLPR-hIL7Rα细胞表面表达hTSLPR和hIL7Rα受体蛋白，这两个受体蛋白的胞外段二聚体可以和人TSLP结合，胞内段可以进一步信号传导，激活细胞内的STAT5的磷酸化，并促进Ba/F3-hTSLPR-hIL7Rα的细胞增殖。
具体如下：取适量Ba/F3-hTSLPR-hIL7Rα稳定细胞系离心，并采用1640+10％FBS培养基洗涤2次，以移除培养基中所用重组鼠IL3。在室温下于各孔中将hTSLP-His(+/-)与纯化的抗TSLP嵌合抗体/或对照抗TSLP抗体(对照抗体)一起培育30分钟。添加1.5×10 4个Ba/F3-hTSLPR-hIL7Rα细胞/孔并培育3天。每孔加入CCK8(RHINO BIO，QDY-003-D)，并在450nm下读取OD，数据导出并采用Prism Graphpad软件分析嵌合抗体对细胞增殖的抑制反应。
如图2A-2C及表4所示，5个嵌合抗体均能够明显的抑制Ba/F3-hTSLPR-hIL7Rα细胞的增殖。
表4：嵌合抗体阻断人TSLP诱导的Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性检测
抗体 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 对照抗体
EC50(nM) 0.11 1.22 0.28 0.10 0.25 0.05
实施例9：抗TSLP嵌合抗体的可变区扩增
杂交瘤细胞培养到2×10 6个左右，利用TRIzol试剂(Thermo Fisher Sci.Cat#15596026)裂解杂交瘤细胞提取RNA并采用cDNA反转录试剂盒(Thermo Fisher Sci.Cat#18080-200)合成第一链cDNA。参考IMGT和AbM的方法，以及所有鼠源抗体序列分析，选择同源性高的区域设计多对可变区上游引物，下游引物使用CH1同源序列，采用引物pool的方式PCR扩增得到抗体的轻、重链可变区，PCR产物通过DNA纯化试剂盒(Qiagen,Cat#28104)纯化，并克隆到pTT-5载体上，每一个连接反应大概挑取10个克隆进行测序，获得可变区序列，并进一步通过IMGT和AbM数据库进行分析。
表5：抗人TSLP嵌合抗体可变区及CDR氨基酸序列及其编号
Figure PCTCN2020134438-appb-000009
实施例10：重组抗体的表达、纯化及亲和结合活性检测
将25A5C5，27C2B6，37C2D10，43B1A8和90H3H11的轻链可变区氨基酸序列分别与轻链κ恒定区氨基酸序列(SEQ ID NO：16)连接，重链可变区氨基酸序列分别与IgG1重链恒定区氨基酸序列(SEQ ID NO：14)连接，将序列对应的核苷酸序列构建至pTT5载体。将每一重组全人源抗体重链和轻链对应的pTT5载体，同时转染到CHO-S(购买自Thermo公司)，通过利用Protein A(MabSelect SuRe,GE)进行纯化上清，纯化后的重组全人源抗体分别命名为25A5C5-hIgG，27C2B6-hIgG，37C2D10-hIgG，43B1A8-hIgG和90H3H11-hIgG，并采用分光光度法定量蛋白含量。
检测抗TSLP的重组全人源抗体与哺乳动物表达的重组人或猴TSLP-His亲和力。具体实验步骤简述如下：包被人TSLP-His或猴TSLP-His抗原至96孔板中，4℃过夜后，分别加入不同浓度梯度稀释后的抗体，孵育2小时后，加入HRP标记的山羊抗人Fc二抗，孵育1小时后，A450nm波长读数检测。
结果如图3A-3B所示，5个候选抗体均能够结合人TSLP。同时，如表6所示，37C2D10-hIgG，43B1A8-hIgG，90H3H11-hIgG的hTSLP亲和力强于对照抗体。25A5C5-hIgG，27C2B6-hIgG的hTSLP亲和力与对照抗体相当。27C2B6-hIgG，37C2D10-hIgG，43B1A8-hIgG与猴TSLP亲和力基本相当。
表6重组全人源抗体的人、猴TSLP的结合力
Figure PCTCN2020134438-appb-000010
实施例11：抗TSLP全人源抗体与TSLP动态亲和力的检测
采用Fortibio是常用的动态亲和力检测设备，通过其检测抗TSLP全人源抗体与TSLP的动态亲和力。简述方法如下：对人或猴TSLP抗原采用PBST进行系列稀释，得到100nM、50nM、25nM、12.5μM、6.25nM、3.125nM、1.5625nM和0nM。ProA生物传感器(Pall生命科学)在使用前用PBST缓冲液预湿。将重组全人源抗体用PBST稀释到5μg/mL并固化在ProA传感器上。然后将固化了抗体的传感器在PBST缓冲液中平衡60s以获得基线，然后转移到抗原稀释液结合60s，再在PBST中解离180s。在一个分析循环后，传感器用10mM Gly(pH 1.5)再生。使用Date analysis 11.0版(Pall)，用1：1模型分析，确定结合(Ka)和解离(Kd)速率常数并使用其来计算解离平衡常数(KD)。
如表7所示，25A5C5-hIgG、43B1A8-hIgG和90H3H11-hIgG结合人TSLP的KD值小于对照抗体，显示出更强的亲和力，与亲和ELISA结果一致。在猴TSLP结合方面，25A5C5-hIgG无亲和力；而37C2D10-hIgG、90H3H11-hIgG和43B1A8-hIgG显示有较好结合，亲和力在10 -8M到10 -9M级别。
表7：全人源抗体的动态亲和力分析
抗体 hTSLP亲和力 cTSLP亲和力
  KD(M) KD(M)
25A5C5-hIgG 5.58E-10 不结合
37C2D10-hIgG 1.76E-09 2.01E-08
43B1A8-hIgG 5.81E-10 1.02E-09
90H3H11-hIgG 6.04E-10 1.08E-08
对照抗体 8.00E-10 1.15E-09
实施例12：重组全人源抗体的Ba/F3-hTSLPR-hIL7a细胞增殖抑制
重组表达的全人源抗体，采用Ba/F3-hTSLPR-hIL7a细胞增殖抑制方法，检测重组全人源抗体的活性。方法参照前述实施例8。
结果如表8所示，27C2B6-hIgG和37C2D10-hIgG能抑制人TSLP引起的Ba/F3-hTSLPR-hIL7Rα细胞增殖。25A5C5-hIgG和90H3H11-hIgG的抑制人TSLP引起的Ba/F3-hTSLPR-hIL7Rα细胞增殖，与对照抗体基本相当。而43B1A8-hIgG的抑制活性则显著强于对照抗体，大约0.25倍，如图4A-C所示。
表8：重组全人源抗体的Ba/F3-hTSLPR-hIL7Rα细胞增殖抑制活性
Figure PCTCN2020134438-appb-000011
实施例13：重组全人源抗体抑制人TSLP诱导的PBMC分泌胸腺活化调节因子(TARC)的活性检测
通过抑制人TSLP诱导人PBMC细胞分泌TARC(一种Th2类细胞因子)评估全人源TSLP抗体在原代细胞上的功能活性。PBMC中含有CD11 +DC细胞，有研究显示TSLP可以与受体TSLPR/IL7Rα结合，并激活CD11 +DC细胞，上调OX40L并促进CD11 +DC细胞分泌Th2类细胞因子(如：TARC和CCL22)。抗TSLP的抗体能够阻断TSLP与DC细胞表面的TSLPR/IL7Rα结合，从而阻断对DC细胞的激活和DC细胞分泌Th2类细胞因子。
简述方法如下：使用Ficoll分离液(GE)分离人外周血PBMC，在室温下将hTSLP-His(+/-)与重组全人源抗体或对照抗体一起培育30分钟，并添加2×10 5PBMC细胞/孔，培育48小时。收获上清液并通过ELISA分析人类TARC产生，并且测定杂交瘤上清液或纯化抗体对TARC分泌的抑制。使用TARC的ELISA试剂盒(义翘神州)检测上清中的TARC水平。
结果如表9和图5显示，全人源抗体43B1A8-hIgG和90H3H11-hIgG的EC50均低于对照抗体，显示其抑制TSLP诱导的PBMC细胞分泌TARC的活性均高于对照抗体分子。
表9：重组全人源抗体抑制人TSLP诱导PBMC分泌TARC
抗体 43B1A8-hIgG 90H3H11-hIgG 对照抗体
EC50(nM) 0.44 0.42 0.51
实施例14：重组全人源抗体的Tm值的测定。
采用DSF(差示荧光扫描技术)方法测定抗TSLP抗体的Tm值。具体实验步骤如下，将12.5μl 40×SYPRO Orange dye(购自Life technologies公司，货号：56651)、5μl体积的1mg/ml全人源TSLP抗体(PBS稀释)以及7.5μl无菌水混合于EP管中，将上述样品混合物加入Q-PCR系统(AB Applied Biosystems ABI，7500)中反应，Q-PCR参数设置：Target(ROX)，程序(25℃，3min；1％速率，95℃；95℃，2min)。结果导入Graph Prism软件计算V50值。如图6及表10，43B1A8-hIgG的Tm值(74.72℃)和90H3H11-hIgG的Tm值(72.58℃)高于对照抗体的Tm(66.47℃)，可以明确本发明制备的全人源抗体具有更优的热稳定性。
表10：重组全人源TSLP抗体的Tm值
抗体 对照抗体 43B1A8-hIgG 90H3H11-hIgG
Tm(℃) 66.47 74.72 72.58
实施例15：重组全人源抗体的疏水性的测定
疏水性比较采用Agilent 1260 HPLC结合TOSOH Tskgel Buty-NPR(2.5)色谱柱进行分析。为比较3个抗体疏水性差异，将3个抗体直接进样进行分析。流动相A：1.5M(NH 4) 2SO 4；流动相B：25mM Na 2HPO 4(pH7.0)+25％IPA。三个抗体的保留时间见表11，时间越越长显示抗体的疏水性质越强，90H3H11-hIgG亲水性与对照抗体相当；43B1A8-hIgG亲水性优于对照抗体，显示工艺开发以及抗体聚集会优于对照抗体。
表11：重组全人源抗体的疏水性检测
抗体 对照抗体 43B1A8-hIgG 90H3H11-hIgG
保留时间(分钟) 13.93 12.2 13.61
实施例16：重组改造的全人源抗体43B1-H6L1和43B1-H2L2的获得
抗体体内半衰期与抗体的等电点、与FcRn的亲和力、糖基化修饰以及免疫原性等有密切的关系。为了延长43B1A8-hIgG抗体半衰期，通过重链及轻链抗体氨基酸序列改造增强FcRn的亲和力。
改造方案1：hIgG1Fc(SEQ ID NO：14)替换为含有2个氨基酸突变的IgG4(SEQ ID NO：70)，突变特征为IgG4的434位氨基酸(Eu编号系统)由N突变为A，第228位氨基酸由S突变为P(Eu编号系统)。
改造方案2：hIgG1Fc(SEQ ID NO：14)的434位氨基酸(Eu编号系统)由N替换为A得到SEQ ID NO：15；并且43B1A8-hIgG的重链的FR1的第16位R突变为G(Chothia编号系统)；并且43B1A8-hIgG的轻链的FR3的第79位R突变为Q(Chothia编号系统)。
将上述第1和第2种改造获得的抗体命名为43B1-H6L1和43B1-H2L2。
43B1-H2L2的重链可变区序列为SEQ ID NO：68；重链恒定区序列为SEQ ID NO： 15；轻链可变区序列为SEQ ID NO：69；轻链恒定区序列为SEQ ID NO：16。
43B1-H6L1的重链可变区序列为SEQ ID NO：40；重链恒定区序列为SEQ ID NO：70；轻链可变区序列为SEQ ID NO：41；轻链恒定区序列为SEQ ID NO：16。
表12：抗体43B1-H6L1和43B1-H2L2氨基酸序列及其编号(SEQ ID NO)
抗体 重链 重链可变区 重链恒定区 轻链 轻链可变区 轻链恒定区
43B1A8-hIgG 66 40 14 67 41 16
43B1-H2L2 71 68 15 72 69 16
43B1-H6L1 73 40 70 67 41 16
将上述43B1-H2L2和43B1-H6L1这两种改造分子的重链和轻链分别构建pTT5表达载体，提取质粒后，转染到CHO-S细胞中(购买自Thermo公司)，经过大约10天左右的培养后，细胞上清通过Protein A(MabSelect SuRe,GE)进行纯化，纯化后的重组全人源抗体采用分光光度法定量蛋白含量。
实施例17：重组全人源抗体43B1-H6L1和43B1-H2L2与TSLP动态亲和力的检测
采用Fortibio检测抗TSLP全人源抗体与TSLP的动态亲和力。方法参考上述实施例11。结果如表13所示，43B1-H6L1和43B1-H2L2人源抗体分子的hTSLP的动态亲和力不弱于对照抗体。
表13：改造全人源抗体分子的动态亲和力分析
Figure PCTCN2020134438-appb-000012
实施例18：重组全人源抗体43B1-H6L1和43B1-H2L2的等电点(PI)检测
采用等电聚焦的方法，对43B1-H6L1、43B1-H2L2和43B1A8-hIgG抗体的等电点进行检测。简述方法如下：采用ProteinSimple公司的Maurice等电聚焦系统，结合其毛细管卡盒进行分析。为比较3个抗体等电点差异，将3个抗体用水稀释，检测体系利用终浓度4％的两性电解质3-10(GE公司)形成pH梯度。结果见表14所示，43B1-H6L1、43B1-H2L2分别比43B1A8-hIgG的等电点下降1.3和0.4，说明改造方案成功。
表14：重组全人源抗体的等电点PI的比较
抗体 43B1-H2L2 43B1-H6L1 43B1A8-hIgG
等电点 8.2 7.3 8.6
实施例19：重组全人源抗体43B1-H6L1和43B1-H2L2与FcRn的动态亲和力的检测
采用Fortibio检测改造的抗TSLP全人源抗体与FcRn的动态亲和力。简述方法如下：对抗TSLP抗体采用PBST(pH6.0)进行系列稀释，得到200nM、100nM、50nM、25nM、12.5μM、6.25nM、3.125nM、1.5625nM和0nM。SA生物传感器 (Pall生命科学)在使用前用PBST(pH6.0)缓冲液预湿。将生物素标记的FcRn稀释到2.3μg/mL并固化在SA传感器上。然后将固化了FcRn的传感器在PBST(pH6.0)缓冲液中平衡60s以获得基线，然后转移到抗体稀释液结合60s，再在PBST(pH6.0)中解离60s。在一个分析循环后，传感器用PBST(pH7.4)再生。使用Date analysis 11.0版(Pall)，用1：1模型分析，确定结合(Ka)和解离(Kd)速率常数并使用其来计算解离平衡常数(KD)。
如表15所示，43B1-H6L1和43B1-H2L2的FcRn的动态亲和力KD小于对照抗体，显示FcRn的亲和力大约是对照抗体的2倍。
表15：改造全人源抗体分子的FcRn亲和力分析
Figure PCTCN2020134438-appb-000013
实施例20：重组全人源抗体43B1-H6L1和43B1-H2L2抑制Ba/F3-hTSLPR-hIL7Rα细胞增殖的活性测定
采用Ba/F3细胞增殖抑制方法，检测重组全人源抗体43B1-H6L1和43B1-H2L2的活性。方法参照前述实施例8，根据数据分析抗体的活性EC50。结果如表16和图7A、7B所示，43B1-H2L2和43B1-H6L1能抑制人TSLP引起的Ba/F3-hTSLPR-hIL7Rα细胞增殖，EC50均与对照抗体相当，显示全人源抗体43B1-H2L2和43B1-H6L1抑制TSLP的细胞活性的能力相当。
表16：重组改造全人源抗体分子的Ba/F3-hTSLPR-hIL7Rα细胞增殖抑制活性
Figure PCTCN2020134438-appb-000014
实施例21：重组全人源抗体抑制人TSLP诱导的PBMC分泌巨噬细胞来源趋化因子(MDC)的活性检测
通过抑制人TSLP诱导人PBMC细胞分泌MDC评估全人源TSLP抗体在原代细胞上的功能活性。PBMC中含有DC细胞，有研究显示TSLP可以与细胞表面受体TSLPR/IL7Rα结合，并激活DC细胞，上调OX40L并促进DC细胞分泌Th2类细胞因子(如：TARC和MDC)。抗TSLP的抗体能够阻断TSLP与DC细胞表面的TSLPR/IL7Rα结合，从而阻断对DC细胞的激活和DC细胞分泌Th2类细胞因子。
简述方法如下：使用Ficoll分离液(GE)分离人外周血PBMC，在室温下将hTSLP-His(+/-)与重组全人源抗体或对照抗体一起培育30分钟，并添加2×10 5PBMC细胞/孔，培育120小时。收获上清液并通过ELISA分析人类MDC产生，并且测定抗体对MDC分泌的抑制。使用MDC的ELISA试剂盒(Raybiotech)检测上清中的TARC水平。
结果如表17和图8所示，43B1-H2L2和对照抗体均能够明显的抑制TSLP诱导的MDC因子的产生，其EC50分别为7.84pM和5.63pM。
表17：重组全人源抗体抑制人TSLP诱导PBMC分泌MDC
抗体 43B1-H2L2 对照抗体
EC50(pM) 7.84 5.63
实施例22：重组全人源抗体在食蟹猴体内的药代动力学分析
本申请中43B1-H2L2候选分子的序列携带N434A突变，目的是为了影响其与FcRn的亲和力，从而延长其在体内的药物代谢半衰期，因此本实施例采用皮下注射药物的方式，研究了43B1-H2L2分子在食蟹猴体内药代动力学情况，并和对照抗体的药代动力学情况进行比较。方法和结果如下：选取食蟹猴(Macaca fascicularis，来自海南金港生物技术股份有限公司)4只，分为2组，每组雌雄各一只。皮下注射剂量为5mg/kg。分别在0点，给药后5分钟，30分钟，2小时，4小时，8小时，1天，2天，3天，4天，7天，10天，14天，21天，28天，35天，42天，49天，56天采集血样，室温放置血样1小时至凝血后，离心获得血清样本，血清样本冷冻于-80℃待测。ELISA测定血清中的抗体含量，结果分析如下：单次皮下给药的药代参数和曲线见表18和图9。这些结果显示43B1-H2L2抗体有更长的半衰期，血药总量AUC也更高，大约是对照抗体的一倍。
表18：重组全人源抗体的食蟹猴皮下给药的药代动力学研究
PK参数 t 1/2(h) T max(h) C max(ng/ml) AUC (0-t)(hr*ng/ml)
43B1-H2L2 549.3731 48 67373.4 47271741
对照抗体 267.3537 72 75870.56 25600470
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的保护范围由所附权利要求及其任何等同物给出。

Claims (26)

  1. 一种结合胸腺基质淋巴细胞生成素(TSLP)的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含如下的互补决定区(CDR)：
    (a)SEQ ID NO:1、17、30、40、53或68所示的重链可变区(VH)中含有的CDR-H1或其序列的变体、CDR-H2或其序列的变体、以及CDR-H3或其序列的变体；和/或
    (b)SEQ ID NO:2、18、31、41、54或69所示的轻链可变区(VL)中含有的CDR-L1或其序列的变体、CDR-L2或其序列的变体、以及CDR-L3或其序列的变体；
    优选地，所述序列的变体为与其来源CDR相比为具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的CDR；优选地，所述的置换为保守置换。
  2. 权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (1)VH和/或VL，其中按IMGT编号系统定义：
    (1a)所述VH包含序列为SEQ ID NO：3的CDR-H1，序列为SEQ ID NO：4的CDR-H2和序列为SEQ ID NO：5的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：6的CDR-L1，序列为SEQ ID NO：7的CDR-L2和序列为SEQ ID NO：8的CDR-L3；
    (1b)所述VH包含序列为SEQ ID NO：19的CDR-H1，序列为SEQ ID NO：20的CDR-H2和序列为SEQ ID NO：21的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：22的CDR-L1，序列为SEQ ID NO：23的CDR-L2和序列为SEQ ID NO：24的CDR-L3；
    (1c)所述VH包含序列为SEQ ID NO：32的CDR-H1，序列为SEQ ID NO：33的CDR-H2和序列为SEQ ID NO：34的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：35的CDR-L1，序列为SEQ ID NO：23的CDR-L2和序列为SEQ ID NO：24的CDR-L3；
    (1d)所述VH包含序列为SEQ ID NO：42的CDR-H1，序列为SEQ ID NO：43的CDR-H2和序列为SEQ ID NO：44的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：45的CDR-L1，序列为SEQ ID NO：46的CDR-L2和序列为SEQ ID NO：47的CDR-L3；
    或者
    (1e)所述VH包含序列为SEQ ID NO：55的CDR-H1，序列为SEQ ID NO：56的CDR-H2和序列为SEQ ID NO：57的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：58的CDR-L1，序列为SEQ ID NO：59的CDR-L2和序列为SEQ ID NO：60的CDR-L3；
    (2)VH和/或VL，其中按AbM编号系统定义：
    (2a)所述VH包含序列为SEQ ID NO：9的CDR-H1，序列为SEQ ID NO：10的CDR-H2和序列为SEQ ID NO：11的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：12的CDR-L1，序列为SEQ ID NO：13的CDR-L2和序列为SEQ ID NO：8的CDR-L3；
    (2b)所述VH包含序列为SEQ ID NO：25的CDR-H1，序列为SEQ ID NO：26的CDR-H2和序列为SEQ ID NO：27的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：28的CDR-L1，序列为SEQ ID NO：29的CDR-L2和序列为SEQ ID NO：24的CDR-L3；
    (2c)所述VH包含序列为SEQ ID NO：36的CDR-H1，序列为SEQ ID NO：37的CDR-H2和序列为SEQ ID NO：38的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：39的CDR-L1，序列为SEQ ID NO：29的CDR-L2和序列为SEQ ID NO：24的CDR-L3；
    (2d)所述VH包含序列为SEQ ID NO：48的CDR-H1，序列为SEQ ID NO：49的CDR-H2和序列为SEQ ID NO：50的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：51的CDR-L1，序列为SEQ ID NO：52的CDR-L2和序列为SEQ ID NO：47的CDR-L3；
    或者
    (2e)所述VH包含序列为SEQ ID NO：61的CDR-H1，序列为SEQ ID NO：62的CDR-H2和序列为SEQ ID NO：63的CDR-H3；和/或，
    所述VL包含序列为SEQ ID NO：64的CDR-L1，序列为SEQ ID NO：65的CDR-L2和序列为SEQ ID NO：60的CDR-L3；
    或
    (3)VH和/或VL，其中与(1a)、(1b)、(1c)、(1d)、(1e)或(2a)、(2b)、(2c)、(2d)、(2e)中任一所述的VH和/或VL相比，至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换为保守置换；
    优选地，所述抗体或其抗原结合片段结合人TSLP和/或猴TSLP。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)SEQ ID NO：1、17、30、40、53或68所示的VH，和/或，SEQ ID NO：2、18、31、41、54或69任一项所示的VL；
    (b)与(a)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(a)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；或者
    (c)与(a)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的VH；和/或，与(a)中的任一VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的VL；优选地，所述的置换是保守置换。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合 片段包含：
    (a)SEQ ID NO：1所示序列的VH和SEQ ID NO：2所示序列的VL；
    (b)SEQ ID NO：17所示序列的VH和SEQ ID NO：18所示序列的VL；
    (c)SEQ ID NO：30所示序列的VH和SEQ ID NO：31所示序列的VL；
    (d)SEQ ID NO：40所示序列的VH和SEQ ID NO：41所示序列的VL；
    (e)SEQ ID NO：53所示序列的VH和SEQ ID NO：54所示序列的VL；
    (f)SEQ ID NO：68所示序列的VH和SEQ ID NO：69所示序列的VL；
    (g)VH和VL，与(a)至(f)任一组中的VH和VL相比，其VH具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，其VL具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或者
    (h)VH和VL，与(a)至(f)任一组中的VH和VL相比，其VH具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)；和/或，其VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换是保守置换。
  5. 权利要求1-4任一项所述抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段是嵌合抗体、人源化抗体或全人源抗体。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含：
    (a)人免疫球蛋白的重链恒定区(CH)或其变体；和/或
    (b)人免疫球蛋白的轻链恒定区(CL)或其变体，
    其中，所述变体与其所源自的野生型序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或者，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)；优选地，所述的置换是保守置换；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；
    优选地，所述抗体或其抗原结合片段包含人IgG1重链恒定区；
    优选地，所述轻链恒定区是κ或λ轻链恒定区，更优选地，所述抗体或其抗原结合片段包含人κ轻链恒定区。
  7. 权利要求6所述的抗体或其抗原结合片段，其中，
    所述重链恒定区或其变体包括：
    (1)人IgG1重链恒定区或其变体，按照EU编号系统，所述变体在位点234、235、237、265、297、331、329、434至少一个位点发生突变；优选地，所述变体含有下列突变中至少一个:L234A、L235A、D265A、N297A、L234F、L235E、P331S、P329G、N434A、N434Y、N434F、N434W、N434S、N434G、N434H，N434Q；优选地，所述变体含有下列突变中至少一个：L234A、L235A、G237A、N434A；更优选地，所述IgG1变体的突变为L234A、L235A和G237A；更优选地，所述IgG1变体的突变为L234A、L235A、G237A和N434A；或
    (2)SEQ ID NO：14所示的CH或其变体，所述变体与SEQ ID NO：14相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：14相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；按照EU编号系统，所述变体含有N297A和/或N434A，优选地，所述变体含有N434A；
    (3)SEQ ID NO：15所示的CH或其变体，所述变体与SEQ ID NO：15相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：15相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或
    (4)人IgG4重链恒定区或其变体，按照EU编号系统，所述变体在位点228和434至少一个位点发生突变；优选地所述突变体包含S228P和/或N434A；优选地，所述突变体包含S228P和N434A；或
    (5)SEQ ID NO：70所示的CH或其变体，所述变体与SEQ ID NO：70相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：70相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；
    和/或
    所述轻链恒定区或其变体：
    (6)包含κ轻链恒定区；或
    (7)包含SEQ ID NO：16所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO：16相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO：16相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO：14所示的重链恒定区(CH)和SEQ ID NO：16所示的轻链恒定区(CL)；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO：15所示的重链恒定区(CH)和SEQ ID NO：16所示的轻链恒定区(CL)；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO：70所示的重链恒定区(CH)和SEQ ID NO：16所示的轻链恒定区(CL)；
    优选地，所述突变或置换使抗体或抗原结合片段与不具有所述突变或置换的相应抗体或抗原结合片段相比，没有或具有降低的ADCP、ADCC和/或CDC活性。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗体包括：
    (a)包括SEQ ID NO：1所示的VH和SEQ ID NO：14、15或70所示的CH的重链，和，包括SEQ ID NO：2所示的VL和SEQ ID NO：16所示的CL的轻链；
    (b)包括SEQ ID NO：17所示的VH和SEQ ID NO：14、15或70所示的CH的重链，和，包括SEQ ID NO：18所示的VL和SEQ ID NO：16所示的CL的轻链；
    (c)包括SEQ ID NO：30所示的VH和SEQ ID NO：14、15或70所示的CH的重链，和，包括SEQ ID NO：31所示的VL和SEQ ID NO：16所示的CL的轻链；
    (d)包括SEQ ID NO：40所示的VH和SEQ ID NO：14、15或70所示的CH的重链，和，包括SEQ ID NO：41所示的VL和SEQ ID NO：16所示的CL的轻链；
    (e)包括SEQ ID NO：53所示的VH和SEQ ID NO：14、15或70所示的CH的重链，和，包括SEQ ID NO：54所示的VL和SEQ ID NO：16所示的CL的轻链；
    (f)包括SEQ ID NO：68所示的VH和SEQ ID NO：14、15或70所示的CH的重链，和，包括SEQ ID NO：69所示的VL和SEQ ID NO：16所示的CL的轻链；
    或
    (g)重链和轻链，其中，与(a)-(f)任一组中的重链和轻链相比，所述重链具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性，和/或，所述轻链具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
  9. 权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)重链和轻链，
    所述重链包含：
    (i)SEQ ID NO：66或73所示的序列；
    (ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
    (iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
    所述轻链包含：
    (iv)SEQ ID NO：67所示的序列；
    (v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
    (vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换；
    或
    (b)重链和轻链，
    所述重链包含：
    (i)SEQ ID NO：71所示的序列；
    (ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
    (iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
    所述轻链包含：
    (iv)SEQ ID NO：72所示的序列；
    (v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
    (vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
    优选地，(ii)或(v)中所述的置换是保守置换。
  10. 权利要求1-9任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’) 2、Fv片段、二硫键连接的Fv(dsFv)和双抗体(diabody)。
  11. 权利要求1-10任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；
    优选地，所述抗体或其抗原结合片段带有可检测的标记，例如酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
  12. 权利要求1-11任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段具有以下特性中的至少一项：
    (a)以小于约50nM，例如小于约20nM、10nM、1nM、0.1nM、0.01nM、1pM、0.1pM或更低的K D结合TSLP(例如，人TSLP)；所述K D通过Fortibio或ELISA测得；
    (b)以小于约50nM，例如小于约20nM、10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.01nM、1pM、0.1pM或更小的EC50结合TSLP(例如，人TSLP)；所述EC50通过流式细胞技术或ELISA技术测得；
    (c)以小于约50nM，例如约50nM、20nM、10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM、0.01nM、1pM、0.1pM或更小的IC50抑制TSLP与IL7Rα/TSLPR的结合；所述IC50通过ELISA技术测得；
    (d)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖；
    (e)抑制或阻断TSLP诱导的OX40L表达；
    (f)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌；
    (g)抑制或阻断TSLP诱导的Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌；
    (h)良好的与FcRn结合的亲和力；
    (i)等电点(PI)为约6.5到约8.5，如约6.5，约7.0，约7.1，约7.2，约7.3，约7.4，约7.5，约7.7，约7.9，约8.0，约8.2，约8.5。
  13. 分离的核酸分子，其编码权利要求1-12任一项所述的抗体或其抗原结合片段。
  14. 载体，其包含权利要求13所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  15. 宿主细胞，其包含权利要求13所述的分离的核酸分子或权利要求14所述的载体。
  16. 制备权利要求1-12任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求15所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  17. 多特异性抗体，其包含权利要求1-12任一项所述的结合TSLP的抗体或其抗原结合片段，和另外的抗体或其片段或抗体类似物；
    优选地，所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。
  18. 缀合物，其包括权利要求1-12中任一项所述的抗体或其抗原结合片段以及偶联部分，其中所述偶联部分为可检测的标记，如放射性同位素、荧光物质、发光物质、有色物质或酶，或者所述偶联部分为治疗剂。
  19. 药物组合物，其包含权利要求1-12任一项所述的抗体或其抗原结合片段、权利要求13所述的分离的核酸分子、权利要求14所述的载体、权利要求15所述的宿主细胞、权利要求17所述的多特异性抗体、和/或权利要求18所述的缀合物，以及药学上可接受的载体和/或赋形剂。
  20. 权利要求19所述的药物组合物，其中所述药物组合物用于在受试者中引起至少一项以下生物学活性：
    (a)抑制或阻断TSLP与IL7Rα-TSLPR的结合，
    (b)下调或消除TSLP的活性,
    (c)下调或阻断OX40L表达，
    (d)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖，
    (e)抑制或阻断TSLP诱导的骨保护素(OPG)的分泌,
    (f)抑制或阻断TSLP诱导的Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌。
  21. 一种试剂盒，其包括权利要求1-12任一项所述的抗体或其抗原结合片段、和/或权利要求13所述的核酸、和/或权利要求14所述的载体、和/或权利要求15所述的宿主细胞、和/或权利要求17所述的多特异性抗体、和/或权利要求18所述的缀合物、和/或权利要求19或20所述的药物组合物，以及可选地包括使用说明书。
  22. 权利要求1-12任一项所述的抗体或其抗原结合片段、和/或权利要求13所述的核酸、和/或权利要求14所述的载体、和/或权利要求15所述的宿主细胞、和/或权利要求17所述的多特异性抗体、和/或权利要求18所述的缀合物、和/或权利要求19或20所述的药物组合物，在制备用于预防和/或治疗变态反应性炎症或自身免疫性疾病的药物中的用途；
    可选地，所述变态反应性炎症选自：哮喘、特发性肺纤维化、特应性皮炎(AD)、过敏性结膜炎、变应性鼻炎(AR)、Netherton综合征、嗜酸性食管炎(EOE)、食物过敏、过敏性腹泻、嗜酸性胃肠炎、过敏性支气管肺曲霉菌病(ABPA)、过敏性真菌性鼻窦炎、类风湿性关节炎、慢性阻塞性肺病(COPD)、系统性硬化症、瘢痕疙瘩、溃疡性结肠炎、慢性鼻窦炎(CRS)和鼻息肉、慢性嗜酸性肺炎、嗜酸性支气管炎、Churg-Strauss综合征、嗜酸性粒细胞增多症、嗜酸性肉芽肿伴多血管炎、炎症性肠病、荨麻疹、系统性肥大细胞增多症、皮肤肥大细胞增多症、反复发作的特发性血管性水肿中的至少一种；
    可选地，所述自身免疫性疾病选自:糖尿病、重症肌无力、胃炎、天疱疮、原发性胆汁性肝硬化、多发性硬化症、狼疮、结肠炎、类风湿、银屑病和甲状腺疾病；
    可选地，所述用途包括所述药物与另外的一种或多种治疗剂的联合施用，所述另外的治疗剂选自但不限于:免疫抑制剂(例如皮质类固醇、非类固醇类糖皮质激素受体激动剂、白三烯D4拮抗剂、白三烯B4拮抗剂、A2A激动剂、A2B拮抗剂、多巴胺受体激动剂、吡非尼酮尼达尼布、或avB6拮抗剂)、支气管扩张剂(例如β-2肾上腺素受体激动剂、毒蕈碱拮抗剂、短效β2受体激动剂、长效β2受体激动剂、短效抗胆碱能药物、甲基黄嘌呤类药物、长效抗胆碱能药物)、其他细胞因子或细胞因子受体的拮抗剂或抗体(例如IL-13拮抗剂、IL-6拮抗剂、IL-1、IL-33、IL-25或TNF-α的拮抗剂、抗IgE抗体、抗IL31抗体、抗IL31R抗体、抗IL13抗体、抗内皮糖蛋白抗体、抗IL1b抗体、另一抗TSLP抗体或抗hTSLPR抗体)、抗生素、放射治疗、白三烯拮抗剂(如孟鲁司特、扎鲁司特或普仑司特)，PDE4抑制剂(如罗氟司特，呫吨)、抗组胺或止咳药物；
    可选地，所述抗体或抗原结合片段与另外的治疗剂相继或同时使用。
  23. 一种在体内或体外(a)抑制或阻断TSLP与IL7Rα-TSLPR结合、(b)下调或消除TSLP的活性、(c)下调或阻断OX40L表达、(d)抑制或阻断TSLP诱导的肥大细胞、DC、NKT细胞的激活和/或增殖、(e)抑制或阻断TSLP诱导骨保护素(OPG)的分泌、(f)抑制或阻断TSLP诱导的Th2细胞因子如TARC、CCL22、IL-4、IL-13或IL-5的分泌中至少一项的方法，包括：
    施加有效量的权利要求1-12任一项所述的抗体或其抗原结合片段、权利要求13所述的核酸、权利要求14所述的载体、权利要求15所述的宿主细胞、权利要求17所述的多特异性抗体、权利要求18所述的缀合物或者权利要求19或20所述的药物组合物，
    可选地，在施加所述抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、缀合物或药物组合物的同时、之前或之后，施加另外的治疗剂。
  24. 一种用于在受试者中预防和/或治疗变态反应性炎症或自身免疫性疾病的方法，所述方法包括向有此需要的受试者施用有效量的权利要求19或20所述的药物组合物，其中所述受试者是哺乳动物；优选地，所述受试者是人，
    可选地，在施加所述药物组合物的同时、之前或之后，施加另外的治疗剂，
    可选地，所述变态反应性炎症选自：哮喘、特发性肺纤维化、特应性皮炎(AD)、过敏性结膜炎、变应性鼻炎(AR)、Netherton综合征、嗜酸性食管炎(EOE)、食物过敏、过敏性腹泻、嗜酸性胃肠炎、过敏性支气管肺曲霉菌病(ABPA)、过敏性真菌性鼻窦炎、类风湿性关节炎、COPD、系统性硬化症、瘢痕疙瘩、溃疡性结肠炎、慢性鼻窦炎(CRS)和鼻息肉、慢性嗜酸性肺炎、嗜酸性支气管炎、Churg-Strauss综合征、嗜酸性粒细胞增多症、嗜酸性肉芽肿伴多血管炎、炎症性肠病、荨麻疹、系统性肥大细胞增多症、皮肤肥大细胞增多症、反复发作的特发性血管性水肿中的至少一种；
    可选地，所述自身免疫性疾病选自:糖尿病、重症肌无力、胃炎、天疱疮、原发性胆汁性肝硬化、多发性硬化症、狼疮、结肠炎、类风湿、银屑病和甲状腺疾病；
    可选地，所述另外的治疗剂选自但不限于:免疫抑制剂(例如皮质类固醇、非类固醇类糖皮质激素受体激动剂、白三烯D4拮抗剂、白三烯B4拮抗剂、A2A激动剂、A2B拮抗剂、多巴胺受体激动剂、吡非尼酮尼达尼布、或avB6拮抗剂)、支气管扩张剂(例如β-2肾上腺素受体激动剂、毒蕈碱拮抗剂、短效β2受体激动剂、长效β2受体激动剂、短效抗胆碱能药物、甲基黄嘌呤类药物、长效抗胆碱能药物)、其他细胞因子或细胞因子受体的拮抗剂或抗体(例如IL-13拮抗剂、IL-6拮抗剂、IL-1、IL-33、IL-25或TNF-α的拮抗剂、抗IgE抗体、抗IL31抗体、抗IL31R抗体、抗IL13抗体、抗内皮糖蛋白抗体、抗IL1b抗体、另一抗TSLP抗体或抗hTSLPR抗体)、抗生素、放射治疗、白三烯拮抗剂(如孟鲁司特、扎鲁司特或普仑司特)，PDE4抑制剂(如罗氟司特，呫吨)、抗组胺或止咳药物。
  25. 一种检测样品中TSLP存在或其水平的方法，其包括使所述样品与权利要求1-12任一项所述抗体或其抗原结合片段在允许所述抗体或其抗原结合片段与TSLP之间形成复合物的条件下接触，和检测所述复合物的形成。
  26. 权利要求1-12任一项所述的抗体或其抗原结合片段、或权利要求13所述的核酸、或权利要求14所述的载体、或权利要求15所述的宿主细胞、或权利要求17所述的多特异性抗体、或权利要求18所述的缀合物、或者权利要求19或20所述的药物组合物在制备用于诊断变态反应性炎症或自身免疫性疾病的药物或试剂盒中的用途，
    可选地，所述变态反应性炎症选自：哮喘、特发性肺纤维化、特应性皮炎(AD)、过敏性结膜炎、变应性鼻炎(AR)、Netherton综合征、嗜酸性食管炎(EOE)、食物过敏、过敏性腹泻、嗜酸性胃肠炎、过敏性支气管肺曲霉菌病(ABPA)、过敏性真菌性鼻窦炎、类风湿性关节炎、COPD、系统性硬化症、瘢痕疙瘩、溃疡性结肠炎、慢性鼻窦炎(CRS)和鼻息肉、慢性嗜酸性肺炎、嗜酸性支气管炎、Churg-Strauss综合征、嗜酸性粒细胞增多症、嗜酸性肉芽肿伴多血管炎、炎症性肠病、荨麻疹、系统性肥大细胞增多症、皮肤肥大细胞增多症、 反复发作的特发性血管性水肿中的至少一种；
    可选地，所述自身免疫性疾病选自：糖尿病、重症肌无力、胃炎、天疱疮、原发性胆汁性肝硬化、多发性硬化症、狼疮、结肠炎、类风湿、银屑病和甲状腺疾病。
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