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WO2024152881A1 - 抗tslp抗体及其应用 - Google Patents

抗tslp抗体及其应用 Download PDF

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Publication number
WO2024152881A1
WO2024152881A1 PCT/CN2023/143708 CN2023143708W WO2024152881A1 WO 2024152881 A1 WO2024152881 A1 WO 2024152881A1 CN 2023143708 W CN2023143708 W CN 2023143708W WO 2024152881 A1 WO2024152881 A1 WO 2024152881A1
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Prior art keywords
seq
amino acid
antibody
acid sequence
antigen
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PCT/CN2023/143708
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English (en)
French (fr)
Inventor
焦娇
王茜
王佳星
秦汉
刘芳兵
朱行
汪德娟
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甘李药业股份有限公司
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Publication of WO2024152881A1 publication Critical patent/WO2024152881A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention relates to an anti-TSLP (thymic stromal lymphopoietin) antibody, a method for preparing the antibody, a pharmaceutical composition comprising the antibody, a kit, and use of the antibody in preparing a drug for treating and/or preventing thymic stromal lymphopoietin (TSLP) infection or a disease associated with TSLP.
  • TSLP thymic stromal lymphopoietin
  • Asthma is one of the most common chronic allergic diseases in the world, with more than 300 million patients worldwide. As of 2022, the prevalence of asthma in people aged 20 and above in my country is 4.2%, and the number of patients has reached 45.7 million. Asthma is manifested by paroxysmal cough, sputum, chest tightness and dyspnea, which can last for weeks or longer. In severe attacks, breathing is extremely difficult and even life-threatening.
  • the first category is interleukin, such as interleukin 4, interleukin 5, interleukin 7, and interleukin 13.
  • the second category is granulocyte macrophage colony stimulating factor (GM-CSF).
  • TSLP granulocyte macrophage colony stimulating factor
  • Thymic stromal lymphopoietin is a protein of the cytokine family. It plays an important role in the maturation of T cell populations by activating antigen presenting cells.
  • TSLP is mainly produced by non-hematopoietic cells, such as fibroblasts, epithelial cells and different types of stromal or stromal-like cells.
  • TSLP forms a ternary signaling complex with the thymic stromal lymphocyte receptor CRLF2 (TSLPR) and the IL-7R ⁇ chain. It then activates intracellular signals by activating the STATs and JAK2 pathways.
  • TSLP type 2 helper T cell
  • the TSLP receptor is a complex consisting of two parts, one is TSLPR and the other is IL-7 receptor ⁇ chain (IL-7R ⁇ ).
  • TSLPR can only form a high-affinity receptor complex when it works together with IL-7 receptor ⁇ chain (IL-7R ⁇ ).
  • IL-7R ⁇ IL-7 receptor ⁇ chain
  • Its affinity with TSLP itself is very low.
  • TSLP first binds to TSLPR with relatively low affinity, then recruits IL-7R ⁇ with high affinity for binding, and finally activates signaling pathways such as STAT5, leading to DC maturation and T cell differentiation.
  • glucocorticoids can reduce the expression of TSLP and TSLPR in asthmatic mice, change the function of DCs induced by TSLP, shift the antigen-induced Th2 response toward the Th1 response, reduce IL-4, TL-5, and IL-13, and increase the production of IL-10 and IFN-y. This effect depends on the downregulation of OX40L in DCs and the increase in IL-12 production. Research data on asthmatic patients show that the severity of asthmatic airway inflammation is positively correlated with the expression level of TSLP.
  • TSLP-TSLPR and downstream signaling molecules play an important role in the occurrence and development of allergic inflammatory diseases (such as allergic asthma).
  • the drugs available for the treatment of asthma include: Nucala (mepolizumab, targeting IL-5) from GlaxoSmithKline (GSK), Cinqair (reslizumab, targeting IL-5) from Teva, and biological therapies currently under development for the treatment of asthma, such as AstraZeneca's own benralizumab (targeting the IL-5 receptor ⁇ subunit [IL-5R ⁇ ]) and Sanofi's Dupixent (targeting IL-4/IL-13). All four of these therapies only target specific inflammatory molecules that drive asthma inflammation and are only suitable for certain types of severe asthma patients, i.e., subgroups of patients, such as eosinophilic asthma.
  • the Tezspire targeting TSLP
  • AstraZeneca and Amgen treats a much larger population than the aforementioned biological therapies currently on the market.
  • the purpose of the present invention is to provide anti-TSLP antibodies and applications thereof.
  • the present invention successfully obtains a TSLP-specific antibody having comparable or better properties than the previous TSLP-specific antibody Tezspire, including in vitro efficacy, drugability, and animal efficacy results.
  • the present invention provides an antibody or an antigen-binding fragment thereof, which specifically binds to TSLP, comprising a heavy chain variable region and/or a light chain variable region, wherein:
  • the heavy chain variable region comprises
  • HCDRs heavy chain complementary determining regions
  • HCDR1, HCDR2 and HCDR3 contained in the peptide segment with the amino acid sequence of SEQ ID NO: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86 or 88; or
  • the light chain variable region comprises,
  • LCDR1, LCDR2 and LCDR3 contained in a peptide segment with an amino acid sequence of SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89; or
  • the present invention provides an antibody or an antigen-binding fragment thereof, which specifically binds to thymic stromal lymphopoietin (TSLP).
  • TSLP thymic stromal lymphopoietin
  • the antibody or antigen-binding fragment thereof which specifically binds to thymic stromal lymphopoietin (TSLP), comprises:
  • HCDRs heavy chain complementary determining regions
  • the peptide segment with amino acid sequence of SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87, 89 contains three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3.
  • LCDRs light chain complementary determining regions
  • An antibody or antigen-binding fragment thereof that specifically binds to TSLP comprising:
  • HCDR3 comprises or consists of any one of the following amino acid sequences: SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 and 80, or the HCDR3 comprises An amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 or 80; and/or
  • a light chain complementary determining region LCDR3 said LCDR3 comprising or consisting of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 6, 18, 30, 42, 63 and 83, or said LCDR3 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 6, 18, 30, 42, 63 or 83.
  • HCDR1 a heavy chain complementary determining region HCDR1, said HCDR1 comprising or consisting of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 1, 13, 25, 37, 58 and 78, or said HCDR1 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 1, 13, 25, 37, 58 or 78; and/or
  • a light chain complementary determining region LCDR1 wherein the LCDR1 comprises or consists of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 4, 16, 28, 40, 61 and 81, or the HCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 4, 16, 28, 40, 61 or 81;
  • it further comprises:
  • HCDR2 a heavy chain complementary determining region HCDR2
  • said HCDR2 comprising or consisting of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 and 79, or said LCDR1 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 or 79; and/or
  • LCDR2 a light chain complementary determining region LCDR2
  • said LCDR2 comprising or consisting of any amino acid sequence in the group consisting of the following amino acid sequences: SEQ ID NO: 5, 17, 29, 41, 51, 62 and 82, or said LCDR2 comprising an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequences shown in SEQ ID NO: 5, 17, 29, 41, 51, 62 and 82.
  • the antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining regions (HCDRs) HCDR1, HCDR2 and HCDR3; and three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein:
  • HCDR1 comprises SEQ ID NO: 1, 13, 25, 37, 58 or 78, or consists thereof, or the HCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 1, 13, 25, 37, 58 or 78;
  • HCDR2 comprises SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 or 79, or consists thereof, or the HCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 2, 14, 26, 38, 49, 59, 70 or 79;
  • HCDR3 comprises SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 or 80, or consists thereof, or the HCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 3, 15, 27, 39, 50, 60, 71 or 80;
  • LCDR1 comprises SEQ ID NO: 4, 16, 28, 40, 61 or 81, or consists of them, or the LCDR1 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 4, 16, 28, 40, 61 or 81;
  • LCDR2 comprises SEQ ID NO: 5, 17, 29, 41, 51, 62 or 82, or consists of them, or the LCDR2 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO: 5, 17, 29, 41, 51, 62 or 82;
  • LCDR3 comprises SEQ ID NO:6, 18, 30, 42, 63 or 83, or is composed thereof, or the LCDR3 comprises an amino acid sequence having 1, 2 or 3 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in SEQ ID NO:6, 18, 30, 42, 63 or 83.
  • the antibody or antigen-binding fragment thereof comprises:
  • HCDRs heavy chain complementary determining regions
  • the amino acid sequence of the peptide is as shown in SEQ ID NO: 8, 20, 32, 44, 53, 65, 73 or 85, wherein the three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 are contained in the peptide;
  • LCDRs three light chain complementary determining regions
  • HCDRs heavy chain complementary determining regions
  • the amino acid sequence of the peptide is shown in SEQ ID NO: 10, 22, 34, 46, 55, 67, 75 or 87, wherein the three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 are contained in the peptide;
  • LCDRs three light chain complementary determining regions
  • HCDRs heavy chain complementary determining regions
  • the three light chain complementary determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 contained in the peptide segment with amino acid sequence as shown in SEQ ID NO:12, 24, 36, 48, 57, 69, 77 or 89.
  • the antibody or antigen-binding fragment thereof comprises:
  • HCDR1 comprising or consisting of SEQ ID NO: 1,
  • HCDR2 comprising or consisting of SEQ ID NO:2,
  • HCDR3 comprising or consisting of SEQ ID NO:3,
  • LCDR1 comprising or consisting of SEQ ID NO:4,
  • LCDR2 comprising or consisting of SEQ ID NO:5, and
  • LCDR3 comprising or consisting of SEQ ID NO:6; or
  • HCDR1 comprising or consisting of SEQ ID NO: 13
  • HCDR2 comprising or consisting of SEQ ID NO:14,
  • HCDR3 comprising or consisting of SEQ ID NO:15,
  • LCDR1 comprising or consisting of SEQ ID NO:16
  • LCDR2 comprising or consisting of SEQ ID NO:17, and
  • LCDR3 comprising or consisting of SEQ ID NO:18; or
  • HCDR1 comprising or consisting of SEQ ID NO: 25,
  • HCDR2 comprising or consisting of SEQ ID NO:26,
  • HCDR3 comprising or consisting of SEQ ID NO:27,
  • LCDR1 comprising or consisting of SEQ ID NO:28,
  • LCDR2 comprising or consisting of SEQ ID NO:29, and
  • LCDR3 comprising or consisting of SEQ ID NO:30; or
  • HCDR1 comprising or consisting of SEQ ID NO:37,
  • HCDR2 comprising or consisting of SEQ ID NO:38,
  • HCDR3 comprising or consisting of SEQ ID NO:39,
  • LCDR1 comprising or consisting of SEQ ID NO:40
  • LCDR2 comprising or consisting of SEQ ID NO:41, and
  • LCDR3 comprising or consisting of SEQ ID NO:42; or
  • HCDR1 comprising or consisting of SEQ ID NO:25,
  • HCDR3 comprising or consisting of SEQ ID NO:50,
  • LCDR1 comprising or consisting of SEQ ID NO:28,
  • LCDR2 comprising or consisting of SEQ ID NO:51, and
  • LCDR3 comprising or consisting of SEQ ID NO:30; or
  • HCDR1 comprising or consisting of SEQ ID NO:58,
  • HCDR2 comprising or consisting of SEQ ID NO:59,
  • HCDR3 comprising or consisting of SEQ ID NO:60,
  • LCDR1 comprising or consisting of SEQ ID NO:61
  • LCDR2 comprising or consisting of SEQ ID NO:62, and
  • LCDR3 comprising or consisting of SEQ ID NO:63; or
  • HCDR1 comprising or consisting of SEQ ID NO:25,
  • HCDR2 comprising or consisting of SEQ ID NO:70,
  • HCDR3 comprising or consisting of SEQ ID NO:71,
  • LCDR1 comprising or consisting of SEQ ID NO:28,
  • LCDR2 comprising or consisting of SEQ ID NO:51, and
  • LCDR3 comprising or consisting of SEQ ID NO:30; or
  • HCDR1 comprising or consisting of SEQ ID NO:78,
  • HCDR2 comprising or consisting of SEQ ID NO:79,
  • HCDR3 comprising or consisting of SEQ ID NO:80,
  • LCDR1 comprising or consisting of SEQ ID NO:81,
  • LCDR2 comprising or consisting of SEQ ID NO:82, and
  • LCDR3 comprising or consisting of SEQ ID NO:83.
  • the present invention also discloses an antibody or an antigen-binding fragment thereof, which specifically binds to TSLP. It contains:
  • a heavy chain variable region comprising: SEQ ID NO:7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86 or 88; or a heavy chain variable region comprising: SEQ ID NO:7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86 or 88 have an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical; or the heavy chain variable region consists of SEQ ID NO:: 7, 9, 11, 19, 21, 23, 31, 33, 35, 43, 45, 47, 52, 54, 56, 64, 66, 68, 72, 74, 76, 84, 86 or 88 , or consists of an amino acid sequence that is at least 80%,
  • a light chain variable region comprising: SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89; or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89; or a light chain variable region comprising: SEQ ID NO: 8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, NO:8, 10, 12, 20, 22, 24, 32, 34, 36, 44, 46, 48, 53, 55, 57, 65, 67, 69, 73, 75, 77, 85, 87 or 89
  • the antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region
  • the amino acid sequence pairs of the heavy chain variable region and the light chain variable region are selected from any one of the groups consisting of the following amino acid sequence pairs: SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46.
  • each CDR is according to the Kabat definition, the Chothia definition, the Abm definition and/or the Contact definition.
  • each CDR is defined according to the Kabat definition or the Chothia definition.
  • Kabat definition is used in the embodiments of the present invention.
  • the antibody or antigen-binding fragment thereof further comprises a constant region, and the constant region is derived from an IgG antibody, an IgM antibody, an IgA antibody, an IgD antibody or an IgE antibody.
  • the constant region is derived from an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
  • the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, the heavy chain constant region regularly comprising: SEQ ID NO: 90 an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 90; or the heavy chain constant region consists of SEQ ID NO: 90, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to sequence SEQ ID NO: 90; and/or
  • a light chain constant region comprising: SEQ ID NO:90, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:90; or the light chain constant region consists of SEQ ID NO:90, or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO:90.
  • the antibody or antigen-binding fragment thereof wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.
  • the antigen binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single chain antibody (preferably scFv) or (Fab') 2 , single domain antibody, diabody (dAb) or linear antibody.
  • the second aspect of the present invention provides a nucleic acid for encoding the antibody or antigen-binding fragment thereof described in any one of the first aspects.
  • the nucleic acid can be synthetic, recombinant or isolated. Due to the degeneracy of nucleic acid codes, multiple nucleic acids will encode the same amino acid and all are included here.
  • the third aspect of the present invention provides an expression vector comprising the nucleic acid described in the second aspect.
  • the fourth aspect of the present invention provides a host cell comprising the expression vector described in the third aspect.
  • the fifth aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, which comprises culturing the host cell described in the fourth aspect, and recovering the antibody or antigen-binding fragment expressed thereby from the culture.
  • the antibodies or antigen-binding fragments thereof described herein can be produced from hybridomas that secrete the antibodies, or from recombinantly produced cells that have been transformed or transfected with one or more genes encoding the antibodies or antigen-binding fragments thereof.
  • Antibodies or antigen-binding portions thereof are produced by culturing host cells under conditions that express nucleic acids to produce antibodies, followed by recovery of the antibodies.
  • Recombinant expression utilizes the construction of an expression vector comprising a polynucleotide encoding an antibody or its antigen binding portion thereof. Once the polynucleotide is obtained, a vector for producing the antibody can be produced by recombinant DNA techniques well known in the art.
  • the expression vector may include appropriate transcription and translation control signals. This can be accomplished using in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is an Escherichia coli cell, a yeast cell, an insect cell, a plant cell or a mammalian cell.
  • the host cell is a Chinese hamster ovary cell (CHO), a CHO cell variant, a 293 cell or a NSO cell.
  • the cell lines include VERO, BHK, HeIa, COS, MDCK, 293F, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, CRL7030 and HsS78Bst cells.
  • the cell lines used in the present invention include but are not limited to the above cell lines.
  • the antibody or antigen-binding portion of an antibody can be purified by any method known in the art for purification of immunoglobulin molecules, for example, by chromatography, centrifugation, differential solubility, or by any other standard technique for purification of proteins. Technique.
  • the sixth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of the antibodies or antigen-binding fragments thereof described in the first aspect, and a pharmaceutically acceptable excipient, for example, the antibody or antigen-binding fragment thereof is combined with water for injection, or with saline.
  • the seventh aspect of the present invention provides a kit comprising at least one of the antibodies or antigen-binding fragments thereof or homologs or derivatives thereof described in the first aspect.
  • the eighth aspect of the present invention provides use of the antibody or antigen-binding fragment thereof described in the first aspect or the pharmaceutical composition of the sixth aspect in the preparation of a medicament for treating and/or preventing a disease associated with TSLP.
  • the ninth aspect of the present invention provides the antibody or antigen-binding fragment thereof according to the first aspect or the pharmaceutical composition according to the sixth aspect, for treating and/or preventing diseases associated with TSLP.
  • the present invention also provides a method for treating and/or preventing a disease associated with TSLP, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment described in any one of the first aspect or the pharmaceutical composition described in the sixth aspect.
  • the above-mentioned disease associated with TSLP is selected from: asthma or dermatitis.
  • the antibodies of the present invention can bind to TSLP specifically and with high affinity, inhibit or block the binding of TSLP to TSLP/IL7R, and can inhibit or block the proliferation effect of TSLP on Ba/F3 cells in vitro, and block the ability of TSLP to activate PBMC and secrete cytokines. Therefore, the antibodies of the present invention have the potential to be used for the prevention and/or treatment of asthma, other allergic reactions or autoimmune diseases.
  • Tezepelumab Compared with the positive drug Tezepelumab, it has better thermal stability, pH stability, oxidation stability, solubility and affinity with TSLP.
  • the TSLP antibody Q61-F of the present invention has better anti-dermatitis and anti-asthma effects than the positive drug Tezepelumab.
  • Figure 1 The ability of different TSLP antibodies to block the binding of TSLP to TSLPR and TSLPR-IL7R complex.
  • Figure A shows the ability of different TSLP antibodies to block the binding of TSLP to TSLPR-Fc protein.
  • Figure B shows the experimental results of different TSLP antibodies blocking the binding of TSLP to TSLPR-IL-7R-Fc protein.
  • the horizontal axis is the concentration of TSLP antibody (unit: ⁇ g/mL), and the vertical axis is the inhibition rate.
  • Figure 2 Results of TSLP antibody inhibiting the proliferation of Ba/F3 cells.
  • the horizontal axis is the antibody concentration (unit: ⁇ g/mL), and the vertical axis is the inhibition rate.
  • FIG. 3 The results of TSLP antibody inhibition on the secretion of TARC (also known as CCL17) by PBMC cells.
  • the horizontal axis is the TSLP antibody concentration (unit: ⁇ g/mL), and the vertical axis is the secretion amount of TARC (unit: pg/mL).
  • Figure 4 Solubility of anti-TSLP antibodies measured by PEG precipitation.
  • Figure 6 Comparison of the body weight of mice in each drug-administered group, with the horizontal axis representing days (unit: day) and the vertical axis representing the body weight of mice (unit: g).
  • Figure 7 Comparison of ear thickness of mice in each drug-dosing group, with the horizontal axis representing days (unit: day) and the vertical axis representing mouse ear thickness (unit: ⁇ m).
  • Figure 8 Comparison of IgE levels in the serum of mice in each drug-treated group.
  • the horizontal axis represents the group, and the vertical axis represents the IgE level in the serum of mice (unit: ng/mL).
  • Figure 9 Comparison of the body weight of mice in each drug-treated group, with the horizontal axis representing the number of days (unit: day) and the vertical axis representing the change in body weight of mice.
  • PBS refers to Phosphate Buffer Solution.
  • PBST refers to the addition of Tween-20 to PBS solution.
  • BSA refers to bovine serum albumin.
  • BSA blocking solution is made from albumin.
  • TMB stands for 3,3',5,5'-Tetramethylbenzidine Solution.
  • IC50 refers to the antibody concentration that produces 50% inhibition.
  • ELISA refers to enzyme-linked immunosorbent assay.
  • TARC refers to thymic activation-regulated chemokine, also known as CCL17.
  • PEG refers to polyethylene glycol (PEG).
  • HIC hydrophobic interaction chromatography
  • Tm is the melting point of protein.
  • ND (Not Detected) means that the experimental data were not detected under the experimental conditions.
  • NA (Not Apply) is not applicable, which means that under the experimental conditions, the experimental data exceeds the detection limit of the instrument and is not applicable.
  • any polypeptide chain is described herein as having an amino acid sequence starting at the N-terminus and ending at the C-terminus.
  • antibody and antigen-binding portion thereof herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
  • antibody refers to an immunoglobulin molecule composed of four polypeptide chains, namely two heavy chains (H) and two light chains (L) interconnected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region.
  • the light chain constant region comprises one domain (CL1).
  • the VH and VL regions can be further divided into highly variable regions called complementarity determining regions (CDR(s)), interspersed with more conserved regions called framework regions (FR).
  • CDR(s) complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from the amino acid terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, of which the three CDRs of VH are HCDR1, HCDR2 and HCDR3, and the three CDRs of VL are LCDR1, LCDR2 and LCDR3.
  • the amino acid distribution of each domain is usually consistent with the following definition: Kabat Sequences of Proteins of Immunological Interest (Kabat protein sequences with immunological significance) (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196: 901-917 (1987); Chothia et al., Nature, 342: 878-883 (1989).
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • subclasses e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • the heavy-chain constant domains that correspond to the different types of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (such as a TSLP protein). It has been demonstrated that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody.
  • binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains; (ii) a F(ab')2 fragment, i.e., a bivalent fragment consisting of two F(ab)' fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the single-armed VL and VH domains of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) CDRs.
  • a Fab fragment i.e., a monovalent fragment consisting of the VL, VH, CL1, and CH1 domains
  • a F(ab')2 fragment i.e., a bivalent fragment consisting of two F(ab)' fragment
  • the two domains VL and VH of the Fv fragment are encoded by different genes, they can be connected together by a synthetic linker through a recombinant method to form a single connected chain, wherein the VL and VH regions are paired to form a monovalent molecule (called single-chain Fv (scFv)).
  • single-chain Fv single-chain Fv
  • Such single-chain antibodies are also included in the term "antigen-binding portion" of the antibody.
  • Other forms of single-chain antibodies, such as bispecific antibodies, are also included.
  • Different analyses can be used to determine or roughly estimate CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.
  • the Kabat definition is a standard for numbering residues in antibodies and is often used to determine CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000).
  • the Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the location of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989).
  • the AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group that simulates antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies” Oxford, UK; Oxford Molecular, Ltd.
  • AbM defines the use of known databases and ab initio methods to model antibody tertiary structure from primary sequence, such as those described in Samudrala et al., "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999).
  • the contact definitions are based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).
  • the antibody of the present invention is not limited to its origin, and can be an antibody from any animal such as a human antibody, a mouse antibody, a rat antibody, etc. It can also be a recombinant antibody such as a chimeric antibody or a humanized antibody. Preferably, it is a humanized antibody.
  • murine antibody refers to a monoclonal antibody against human TSLP prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with TSLP antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.
  • the murine anti-TSLP antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or a variant thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable regions of the heavy and light chains of mammals other than humans, such as mouse antibodies, with the constant regions of the heavy and light chains of human antibodies to reduce the immune response induced by mouse antibodies.
  • Chimeric antibodies can be prepared using known methods. For example, chimeric antibodies can be prepared by inserting the genes of hybridoma clones into appropriate vectors and then introducing them into hosts. Specifically, cDNA of the variable region (V) of the antibody is synthesized from the mRNA of the hybridoma using reverse transcriptase. When the DNA encoding the V region of the target antibody is obtained, it is connected to the DNA encoding the desired human antibody constant region (C region) and then inserted into an expression vector.
  • V variable region
  • the DNA encoding the antibody V region can be inserted into a DNA expression vector containing the human antibody C region. It is inserted into an expression vector so that it is expressed under the regulation of the expression regulatory region.
  • the host cell is transformed with the expression vector to express the chimeric antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by transplanting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences. It can overcome the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • amino acid mutation refers to an amino acid replacement, insertion, deletion or modification.
  • a mutation in one or more amino acid sites on a polypeptide fragment and its variants, wherein the variant can be obtained by replacing, inserting, deleting or modifying amino acids at one or more sites on the polypeptide.
  • 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, and the variant of the antibody obtained by conservatively substituting the amino acid fully retains the biological activity of its source 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 amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.).
  • Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains
  • nucleic acid refers to DNA or RNA. Nucleic acids can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence.
  • nucleic acids or fragments thereof mean that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide substitutions, insertions or deletions, the nucleotide sequence has nucleotide identity in at least about 80%, more preferably at least about 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases as calculated using any of the following sequence identity calculation programs such as FASTA, BLAST or Gap.
  • the term "substantially similar” or “substantially similar” means that two peptide sequences have at least 80% sequence identity, more preferably at least 80%, 85%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when optimally aligned using programs such as Gap or BESTFIT with default gap weights.
  • Non-identical residue positions may differ by amino acid substitutions, deletions or insertions, and more preferably, non-identical residue positions differ by conservative amino acid substitutions.
  • Constant amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino acid residue that is not replaced by an amino
  • the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution.
  • Methods for making such adjustments are well known to those skilled in the art.
  • groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl groups: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.
  • conservative substitutions can be any changes with positive values in the PAM250 logarithm-likelihood matrix disclosed by Gonnet et al. (1992) Science 256:1443-1445.
  • “Moderately conservative" substitutions are any changes with non-negative values in the PAM250 logarithm-likelihood matrix.
  • TSLP Thymic Stromal Lymphopoietin
  • IL-7 interleukin-7
  • DCs dendritic cells
  • TSLP includes variants, isoforms, homologs, orthologs and paralogs of TSLP.
  • vector is intended to refer to any molecule or entity (eg, nucleic acid, plasmid, phage or virus) used to transfer protein coding information into a host cell.
  • molecule or entity eg, nucleic acid, plasmid, phage or virus
  • expression vector refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or regulates the expression of one or more heterologous coding regions operably linked thereto.
  • An expression vector may include, but is not limited to, sequences that affect or regulate transcription, translation, and, if introns are present, sequences that affect RNA splicing of the coding region operably linked thereto.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including the progeny of said cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom (regardless of the number of passages).
  • the nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that screened or selected for in the original cell are included herein. middle.
  • transfection means the uptake of foreign or exogenous DNA by a cell.
  • a cell is "transfected” when the exogenous DNA is introduced into the cell membrane.
  • transfection techniques are well known in the art. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.
  • the techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.
  • antibody framework or "FR region” refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. In essence, it is a variable domain without CDRs.
  • binding refers to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody binds with an affinity (KD) of less than about 10-8 M, such as less than about 10-9 M, 10-10 M, 10-11 M, 10-12 M or less.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibodies of the present disclosure bind to TSLP with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10-8 M or 10-9 M.
  • KD dissociation equilibrium constant
  • the affinity of the antibodies for cell surface antigens in the present disclosure is determined by FACS or Biacore to determine the KD value.
  • Tm refers to the melting point of the protein, that is, the temperature at which the molar heat capacity reaches a maximum value, and is an important parameter for characterizing the thermal stability of antibodies.
  • thermal stability is measured by differential scanning calorimetry (DSC).
  • DSC differential scanning calorimetry
  • the typical feature of the heat map produced by DSC is the presence of three partially overlapping development regions. Each of these melting zones is associated with a specific melting temperature (or Tm value), which corresponds to the maximum temperature change rate of heat capacity within a given temperature zone.
  • Tm1 corresponds to the CH2 domain of Fc
  • the second Tm2 corresponds to Fab
  • Tm3 corresponds to the CH3 domain of Fc.
  • Tm values of the CH2 and CH3 domains do not vary as much as the Fab Tm value. Therefore, during developability assessment or formulation screening, the Tm2 (Fab) value is mainly referenced. The larger the Tm2 (Fab) value, the better the thermal stability of the protein.
  • treatment includes therapeutic treatment, prophylactic treatment, and use in reducing the risk of a subject developing a disease or other risk factors. Treatment does not require a complete cure of the disease, but includes embodiments in which symptoms are alleviated or potential risk factors are mitigated.
  • the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” “About” or “approximately” means within 1, 2, 3 or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.05% of a given value or range. Whenever the term “about” or “approximately” precedes the first value in a series of two or more values, it should be understood that the term “about” or “approximately” applies to each value in the series.
  • an effective amount refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and/or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes presented during the development of the condition.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related conditions of the present invention or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and/or delaying the progression of the target antigen-related condition of the present invention in a patient.
  • composition refers to a variety of preparations, including an effective amount of an antibody, a pharmaceutically acceptable carrier, a diluent or an excipient, and the preparation is in the form of a sterile liquid solution, a liquid suspension or a lyophilized form.
  • the antibodies of the present invention may be used as a composition for sole administration, or may be used in combination with other active agents.
  • kit refers to a box for containing reagents such as antibodies, detection chemicals, drug residues, virus species, etc.
  • the kit includes antibodies of the present invention, fragments, homologues, derivatives thereof, etc., such as conjugates with labels or cytotoxicity, and instructions for use of antibodies, conjugates that kill specific types of cells, etc.
  • the instructions may include guidance for using antibodies, conjugates, etc. in vitro, in vivo or ex vivo.
  • Antibodies can be in liquid form or solid, usually lyophilized.
  • the kit may contain other reagents of interest, such as buffers, reconstitution solutions, and other necessary components for the intended use.
  • the reagents are packaged in predetermined amounts and combined with instructions for their use, such as for therapeutic use or for performing diagnostic assays.
  • the kit may include a substrate and cofactors required by the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore).
  • a substrate precursor that provides a detectable chromophore or fluorophore
  • other additives such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. may also be included.
  • the relative amounts of multiple reagents can be changed to provide concentrates of reagent solutions, which provides user flexibility, saves space, saves reagents, etc. This Some reagents may also be provided as a dry powder, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagent having the appropriate concentration.
  • asthma refers to allergic asthma, non-allergic asthma.
  • allergic asthma refers to asthma triggered by one or more inhaled allergens.
  • Such patients have positive IgE fluorescent enzyme immunoassay (FEIA) levels to one or more allergens that trigger the asthmatic reaction.
  • FEIA fluorescent enzyme immunoassay
  • non-allergic asthma refers to patients who have low eosinophils, low Th2, or low IgE at diagnosis. Patients with “non-allergic asthma” test negative in the IgE fluorescent enzyme immunoassay (FEIA) to a panel of allergens that include region-specific allergens. In addition to low IgE, those patients often have low or no eosinophil counts and low Th2 counts at diagnosis.
  • FEIA fluorescent enzyme immunoassay
  • TSLP-related diseases refers to diseases including, but not limited to, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Nesseton syndrome, eosinophilic esophagitis, food allergies, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, rheumatoid arthritis, chronic obstructive pulmonary disease, Systemic sclerosis, multiple sclerosis, keloids, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, coeliac disease, Churg-Strauss syndrome, eosinophilic myalgia syndrome, hypereosinophilic syndrome,
  • TSLP protein sequence in the NCBI database full length 159aa, ACCESSION: AAH40592
  • the base sequence was optimized and the HindIII/SacII restriction site was used to insert into the pcDNA3.1-Myc-His vector to construct an expression plasmid for TSLP protein.
  • TSLP protein the amino acid sequence of which is shown in SEQ ID NO:92 (SEQ ID NO:92 MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRTTNKCLEQVSQLQGLWRRFNRPLLKQQ).
  • a BaF3 cell line expressing both human TSLP receptor and human IL7R ⁇ (TSLPR/IL7R ⁇ ) was constructed.
  • the target gene TSLPR/IL7R ⁇ was cloned into the target cell line by electroporation to form a stable high-expression cell line.
  • the human TSLPR and human IL7R ⁇ genes were cloned into the pcDNA3.1-Myc-His plasmid, and then the human TSLPR was cloned into the BaF3 cell line by electroporation, and selected and cultured for 1 week under 400 ⁇ g/ml G418 screening pressure.
  • Anti-human TSLP monoclonal antibodies were produced by immunizing mice.
  • the experiments used Balb/c mice, female, 6-8 weeks old (source: Vital River). After the mice were purchased, they were raised in the laboratory environment for 1 week with a 12/12 hour light/dark cycle. The adapted mice were immunized with recombinant protein, huTSLP-his (200 ⁇ g) and Alum adjuvant. After the 4th-5th immunization, the antibody titer in the mice was determined by ELISA. Mice with high antibody titers in the serum and titers tending to a plateau were selected. After being killed by cervical dislocation, spleen cells were taken and fused with myeloma cells. Hybridoma cells were obtained by fusing spleen lymphocytes with myeloma cells Sp2/0 cells using an optimized PEG-mediated fusion step. Hybridoma cells were screened using traditional ELISA and Biacore methods.
  • the CM5 chip with fixed Anti-mouse antibody was used to capture the supernatant of hybridoma cells, and then the sample TSLP protein was analyzed to determine the affinity between the antibody and the protein, and the antibody was preliminarily screened kinetically.
  • the specific operation steps are to set three cycles of Startup, then set capture (capture of antibodies in the supernatant) to 300s, 30 ⁇ L/min; analysis (analysis of samples) concentration to 200nM, binding time 120s, 30 ⁇ L/min; Dissociation time to 120s, Regeneration (10mM glycine-HCl) to 30 ⁇ L/min, 30s; then fill in the sample number and the protein concentration of the corresponding protein in the list, add the corresponding reagent according to the reagent position given by the program, cover the membrane, put the 96-well plate into the sample chamber, and start the program.
  • the monoclonal hybridoma cell lines with good activity were screened, and the hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted with TRNzol and reverse transcribed ( All-in-One First-Strand cDNA Synthesis SuperMix for PCR, Transgene, AT321-01).
  • the cDNA obtained by reverse transcription was amplified by PCR and sent to a sequencing company for sequencing.
  • the mouse anti-TSLP antibody was obtained by sequencing: Q46, Q61, Q75, Q78, Q112, Q148, Q170, Q187 sequence, and its variable region amino acid sequence is as follows:
  • variable region of a mouse antibody is fused with the constant region of a human antibody, and the resulting chimeric antibodies are screened.
  • sequence of the humanized constant region is as follows:
  • the Q61, Q187, Q78, Q75, Q46, Q148, Q112, and Q170 antibodies that have been screened for excellent in vitro and in vivo activity were humanized.
  • the humanization of mouse monoclonal antibodies is carried out according to the methods disclosed in many literatures in this field.
  • the mouse CDR sequence is transplanted to the human antibody variable region framework to overcome the heterologous reaction induced by the large amount of mouse protein components in the chimeric antibody.
  • the human antibody variable region framework sequence is then backmutated to obtain the final humanized molecule.
  • the humanized VH template of Q46 is IGHV1-69-2*01, and the humanized VL template is IGKV1-13*02.
  • the CDR of Q46 is transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q46 humanized antibody are as follows:
  • V24A in the table indicates that the amino acid V at position 24 of the humanized antibody is mutated back to A.
  • the VH and VL of the humanized antibody huQ61 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q46-F with a high degree of humanization and excellent activity was determined, and its reverse mutation site was V24A in the heavy chain, and the sequence is as follows:
  • the humanized VH template of Q61 is IGHV1-18*01, and the humanized VL template is IGKV1-27*01.
  • the CDR of Q61 is transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q61 humanized antibody are as follows:
  • M48I in the table indicates that the amino acid M at position 48 of the humanized antibody is mutated back to I.
  • the VH and VL of the humanized antibody huQ61 were subjected to single mutation and multiple mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q61-F with a high degree of humanization and excellent activity was determined, and its reverse mutation sites were Y95F in the heavy chain and F72Y in the light chain.
  • the sequence is as follows:
  • the humanized VH template of Q75 is IGHV1-46*01, and the humanized VL template is IGKV3-15*01.
  • the CDR of Q75 is transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q75 humanized antibody are as follows:
  • M48I in the table indicates that the amino acid M at position 48 of the humanized antibody is mutated back to I.
  • the VH and VL of the humanized antibody huQ75 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q75-F with a high degree of humanization and excellent activity was determined, and its reverse mutation sites were R72A in the heavy chain and F72Y in the light chain.
  • the sequence is as follows:
  • the humanized VH template of Q78 is IGHV1-18*01, and the humanized VL template is IGKV1-33*01.
  • the CDR of Q78 was transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q78 humanized antibody are as follows:
  • I37V means that the amino acid I at position 37 of the humanized antibody is mutated back to V.
  • VH and VL of the humanized antibody huQ78 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined. The affinity and cell activity of the expressed proteins were measured, and finally the humanized antibody with high degree of humanization and excellent activity was determined.
  • Q78-F, the reverse mutation sites are V71K in the heavy chain and F71Y in the light chain, and the sequence is as follows:
  • the humanized VH template of Q112 is IGHV1-69*10, and the humanized VL template is IGKV3-7*02.
  • the CDR of Q112 is transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q112 humanized antibody are as follows:
  • M48I in the table indicates that the amino acid M at position 48 of the humanized antibody is mutated back to I.
  • the VH and VL of the humanized antibody huQ112 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q112-F with a high degree of humanization and excellent activity was determined, and its reverse mutation site was light chain F72Y, and the sequence is as follows:
  • the humanized VH template of Q148 is IGHV2-70*04, and the humanized VL template is IGKV3-11*01.
  • the CDR of Q148 was transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q148 humanized antibody are as follows:
  • L4M means that the amino acid L at position 4 of the humanized antibody is mutated back to M.
  • the VH and VL of the humanized antibody Q148 were subjected to single mutation and multiple mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q148-F with a high degree of humanization and excellent activity was determined, and its reverse mutation site was light chain L4M, and the sequence is as follows:
  • the humanized VH template of Q170 is IGHV1-46*01, and the humanized VL template is IGKV1-37*01.
  • the CDR of Q170 was transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q170 humanized antibody are as follows:
  • V68A in the table indicates that the amino acid V at position 68 of the humanized antibody is mutated back to A.
  • the VH and VL of the humanized antibody Q170 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q170-F with a high degree of humanization and excellent activity was determined, and its reverse mutation sites were R72A in the heavy chain and F72Y in the light chain.
  • the sequence is as follows:
  • the humanized VH template of Q187 is 1T3F, and the template of humanized VL is 1T3F.
  • the CDR of Q187 is transplanted to the humanized template.
  • the variable region sequence obtained after transplantation is as follows:
  • the reverse mutation sites of Q187 humanized antibody are as follows:
  • I28T in the table indicates that the amino acid I at position 28 of the humanized antibody is mutated back to T.
  • the VH and VL of the humanized antibody Q187 were subjected to single mutation and multi-point mutation according to the mutation sites in the above table, and the constructed different reverse mutation heavy and light chains were combined, and the affinity and cell activity of the expressed proteins were measured. Finally, the humanized antibody Q187-F with a high degree of humanization and excellent activity was determined, and its reverse mutation sites were I28T, T30I and K74T in the heavy chain and I48V in the light chain.
  • the sequence is as follows:
  • the affinity of anti-TSLP humanized antibody binding to TSLP was determined by Biacore method.
  • the antibody was captured by CM5 chip with fixed Anti-mouse antibody through molecular interaction analyzer Biacore 8K, and then the sample (target protein) was analyzed to determine the affinity of antibody and protein, and the antibody was preliminarily screened kinetically.
  • the specific operation steps are to set three cycles of Startup, then set capture (capture antibody in supernatant) to 300s, 30 ⁇ L/min; analysis (analysis sample) concentration to 200nM, binding time to 120s, 30 ⁇ L/min; Dissociation time to 120s, Regeneration (10mM glycine-HCl) to 30 ⁇ L/min, 30s; then fill in the sample number and the protein concentration of the corresponding protein in the list, add the corresponding reagent according to the reagent position given by the program, cover the film, put the 96-well plate into the sample chamber, and start the program.
  • the results of the affinity of anti-TSLP humanized antibody binding to TSLP are shown in Table 10.
  • the humanized antibodies Q61-F, Q187-F, Q75-F and Q112-F obtained in the present invention all have affinities higher than nM (10 -10 ) level, can effectively bind to TSLP, and have a binding ability superior to that of the positive control drug Teze.
  • the Tm value of anti-TSLP antibody was determined by DSF (differential fluorescence scanning technology):
  • Antibody preparation Replace the antibody to be tested with D-PBS buffer and concentrate the antibody to 0.5 mg/mL. Filter through a 0.22 ⁇ m filter membrane and place at 4°C for testing.
  • Instrument preparation Use 40% Decon90 to clean the instrument, and increase the scanning temperature from 10°C to 60°C, with a heating rate of 180°C/h. Set a total of 9 cleaning programs. After cleaning, set 3 groups of water, and increase the scanning temperature from 10°C to 130°C, with a heating rate of 180°C/h. If the DSC curves of the three groups of water are consistent, the instrument cleaning program is completed and wait for sample measurement.
  • Sample loading and determination Set up the program and data storage path according to the above method. According to the program instructions, add 400 ⁇ L of the corresponding test solution to the 96-well plate. After the sample loading is completed, put the 96-well plate into the sample storage rack. Click Start to start the Tm value determination.
  • hydrophobicity of the antibody to be tested is measured by HPLC. Hydrophobicity can indirectly reflect the strength of the interaction between antibody molecules, thereby providing a reference for the aggregation and non-specific binding tendency of antibody molecules. Stronger hydrophobicity may indicate that the molecule is more likely to aggregate and non-specific binding occurs.
  • HIC Hydrophobic interaction chromatography
  • the stability of the antibodies to be tested was determined by HPLC, and the stability of different anti-TSLP antibodies under pH accelerated conditions and oxidation accelerated conditions were determined respectively.
  • PEG stock solution Dissolve the weighed PEG solid in PBS to prepare a 40% mass fraction (w/v) stock solution, and adjust the pH to 7.2.
  • the solubility is evaluated by observation or measuring OD 500 with a spectrophotometer.
  • the positive control antibody TEZE precipitated first, while the Q61-F humanized antibody precipitated at 8% PEG mass fraction; Q187-F precipitated at 7% PEG mass fraction, but was significantly better than the positive control antibody TEZE.
  • Example 10 Anti-TSLP humanized antibody blocking TSLP binding to TSLPR and TSLPR-IL7R complex
  • TSLP antibodies block the ability of TSLP to bind to TSLPR and TSLPR-IL7R complex.
  • TSLP binds to TSLPR and further recruits IL-7R to form a complex to exert its function.
  • In vitro competitive binding experiments were used to verify the ability of antibodies to block the binding of TSLP to TSLPR and TSLPR-IL7R complex.
  • the TSLP protein linked to Biotin was incubated with antibodies of different concentrations for 30 minutes, it was added to a 96-well plate coated with the TSLPR-IL7R complex, incubated at 37°C for 1 hour, and horseradish peroxidase-labeled Streptavidin was added to color it. After termination, the OD value was measured at 450nm.
  • Q61-F has a better ability to block the binding of TSLP to TSLPR and TSLPR-IL7R complex than the control antibody Teze.
  • Ba/F3 cells were electroporated to express human TSLPR and IL-7R proteins at the same time, and proliferate in dependence on TSLP.
  • the activity of anti-TSLP antibodies was measured by adding different concentrations of antibodies to bind to the TSLP factor added to the culture medium, thereby inhibiting the proliferation of BaF3 cells that depend on TSLP for growth.
  • TSLP factor and different concentrations of antibodies were added to a 96-well cell culture plate, and 1.5 ⁇ 10 4 cells were added to each well. After culturing for 2 days at 37°C in an incubator containing 5% CO 2 , CCK8 was added to detect the proliferation ability of the cells, and the OD value was measured at 450nm.
  • Q61-F has a better ability to inhibit the proliferation of Ba/F3 cells than the control antibody Teze.
  • TSLP antibodies inhibit the secretion of TARC (thymic and activation-regulated chemokine, also known as CCL17) by PBMC cells.
  • TARC can bind to the CCR4 receptor and mediate the occurrence of inflammation, cancer and autoimmune-related diseases.
  • TSLP can induce PBMC cells to produce chemokines such as TARC, but the binding of anti-TSLP antibodies to TSLP can inhibit the production of this chemokine, so the concentration detection of TARC can be used for in vitro efficacy evaluation experiments.
  • TSLP factors and different concentrations of antibodies were added to a 96-well cell culture plate, and 8 ⁇ 10 5 PBMC cells were added to each well.
  • the cells were cultured at 37°C in an incubator containing 5% CO 2 for 2 days, and the concentration of TARC in the cell supernatant was detected using the Human CCL17/TARC ELISA Kit. As shown in Figure 3, Q61-F and Q87-F have comparable ability to inhibit TARC secretion compared with TEZE.
  • mice were housed in IVC (individual ventilation cages) with sterile water available. Mice were fed twice daily (AM and PM) except on the study day. Before starting the study, the general health of the animals was assessed and body weights were recorded. The study was conducted after confirming the health of the mice.
  • AD Atopic dermatitis model construction: According to Figure 5, 25 ⁇ L 0.8% Oxazolone solution was evenly applied to the right ear and back of the mouse on day 0 to sensitize the mouse, and then 25 ⁇ L 0.4% Oxazolone solution was applied to the same part on days 7, 9, 11, 14, 16, 18, 21, 23, and 25 for stimulation. The mice were weighed twice a week after the start of the experiment and the thickness of the ears was measured. Subcutaneous administration began on the 6th day. The dosing regimen, frequency of administration, dosage and volume of each administration group are shown in Table 9. Blood was collected from the orbital venous plexus on the 26th day, and the IgE level in the serum was detected using the MOUSE IgE ELISA KIT kit.
  • mice were housed in IVC (individual ventilation cages) with sterile water available. Mice were fed twice daily (AM and PM) except on the study day. Before starting the study, the general health of the animals was assessed and body weights were recorded. The study was conducted after confirming the health of the mice.
  • G1 to G5 25 animals were randomly divided into 5 groups according to body weight on Day-3, marked as G1 to G5, with 5 animals in each group.
  • G1 was the non-modeling group
  • G2 to G5 were the modeling groups
  • G2 was the negative control group
  • G3 was the test product group 1
  • G4 was the test product group 2
  • G5 was the test product group 3.
  • each animal in group G1 was sensitized by intraperitoneal injection of 200 ⁇ L of PBS, and each animal in groups G2 to G5 was sensitized by intraperitoneal injection of 200 ⁇ L 200 ⁇ g/mL OVA.
  • days 21 to 25 for 5 consecutive days, animals in groups G2 to G5 were stimulated by inhalation of 2% OVA by atomization every day, 30 minutes each time.
  • the in vivo efficacy experiment was terminated on day 26, and alveolar lavage fluid, serum, and lung tissue were collected for subsequent in vitro testing.
  • FIG9 is a comparison of the body weight of mice in each drug administration group. The results show that during the entire duration of the drug efficacy experiment (26 days), the body weight of mice increased steadily, and the increase was the same as that of the control group. This indicated that the basic health status of the mice was normal (consistent with the daily observation that the mice were in good mental state and had normal appetite), and was not affected by modeling and drug administration.
  • Figure 10 is a comparison of the IgE content in the serum of mice in each drug administration group.
  • the results show that the serum IgE of the modeling group MC increased significantly compared with the NC group, indicating that the modeling was successful.
  • Q61-F and Q187-F antibodies the IgE level decreased, indicating that in the asthma model, the antibody can exert its efficacy, but the efficacy of Teze is not obvious, while Q61-F has a certain efficacy, indicating that the efficacy of Q61-F is better than that of the positive drug Teze.

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Abstract

提供了特异性结合TSLP的抗体或其抗原结合片段。所述特异性结合TSLP抗体或其抗原结合片段可以作为用于治疗或预防与TSLP相关的疾病的药物，尤其是哮喘和特应性皮炎疾病的药物。

Description

抗TSLP抗体及其应用 技术领域
本发明涉及抗TSLP(胸腺基质淋巴细胞生成素)抗体、制备所述抗体的方法、包含所述抗体的药物组合物、试剂盒及所述抗体在制备用于治疗和/或预防胸腺基质淋巴细胞生成素(TSLP)感染或与TSLP相关的疾病的药物中的用途。
背景技术
哮喘是全球最常见的慢性过敏性疾病之一，全球有3亿多患者。截至2022年，我国20岁及以上人群哮喘患病率为4.2％，患者人数达4570万。哮喘表现为发作性咳嗽、咳痰、胸闷及呼吸困难等，可持续数周或更长时间。严重发作时，呼吸极度困难，甚至会危及生命。目前市面上治疗哮喘比较热的靶点有三大类，第一类是白细胞介素，比如：白细胞介素4、白细胞介素5、白细胞介素7、白细胞介素13，第二类是粒细胞-巨噬细胞集落刺激因子(Granulocyte macrophage-colony stimulating factor,GM-CSF)，第三类是TSLP，TSLP被证明在哮喘疾病的发生发展中发挥重要作用，因此，开发靶向TSLP、阻断其信号通路的特异性抗体，具有非常重大的临床价值。
胸腺基质淋巴细胞生成素(Thymic stromal lymphopoietin，TSLP)是一种细胞因子家族的蛋白质。它通过激活抗原呈递细胞在T细胞群的成熟中发挥重要作用。TSLP主要由非造血细胞产生，如成纤维细胞、上皮细胞和不同类型的基质或基质样细胞。TSLP与胸腺基质淋巴细胞受体CRLF2(TSLPR)和IL-7Rα链形成三元信号复合物。然后通过激活STATs和JAK2途径激活细胞内信号。主要影响髓系细胞，诱导单核细胞释放T细胞吸引趋化因子，促进CD11c(+)树突状细胞的成熟。该蛋白促进2型辅助性T细胞(TH2)反应，参与各种炎症性疾病中的免疫反应，包括哮喘、过敏性炎症和慢性阻塞性肺疾病。因此TSLP被认为是治疗这类疾病的潜在治疗靶点。
TSLP受体是个复合物，包括两部分，一部分为TSLPR，另一部分为 IL-7受体α链(IL-7Rα)。TSLPR只有与IL-7受体α链(IL-7Rα)共同作用时才可形成高亲和力的受体复合物，其本身与TSLP亲和力非常低。TSLP先与TSLPR以相对较低的亲和力结合，然后以高亲和力招募IL-7Rα进行结合，最终以stat5等信号通路的激活，导致DC的成熟和T细胞的分化。
研究发现，糖皮质激素可降低哮喘鼠TSLP、TSLPR的表达，可改变TSLP诱导的DC功能，使抗原诱导Th2反应朝Th1反应偏移，IL-4、TL-5、IL-13减少，产生IL-10和IFN-y增加，此作用依赖于DC中OX40L的下调以及IL-12产量增加。哮喘患者的研究数据表明，哮喘气道炎症的严重程度与TSLP的表达水平呈正相关。
TSLP-TSLPR及下游信号分子对过敏性炎症疾病(如过敏性哮喘)的发生及发展过程具有重要作用。
目前，上市的能治疗哮喘的药物有：葛兰素史克(GSK)的Nucala(mepolizumab，靶向IL-5)，梯瓦的Cinqair(reslizumab，靶向IL-5)，以及目前正在开发治疗哮喘的生物疗法，例如阿斯利康自己的benralizumab(靶向IL-5受体α亚基[IL-5Rα])以及赛诺菲的Dupixent(靶向IL-4/IL-13)，所有这4种疗法仅靶向于驱动哮喘炎症的特定炎性分子，只适合某些类型的重症哮喘患者，即亚组患者，如嗜酸性粒细胞性哮喘。而由阿斯利康与安进联合开发的Tezspire(靶向TSLP)治疗人群远大于目前已上市前述的生物疗法。
因此迫切需要开发新的抗TSLP抗体药物，尤其是效果优良、价格便宜的药物。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供抗TSLP抗体及其应用。
本发明成功获得了与之前的TSLP特异性抗体Tezspire具有相当或更好特性的TSLP特异性抗体，这些特性包括体外药效、成药性和动物药效结果等。
在第一方面，本发明提供了一种抗体或其抗原结合片段，其特异性地结合于TSLP，其包含重链可变区和/或轻链可变区，其中：
1)所述重链可变区包含
(i)氨基酸序列为SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88的肽段内所含有的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3；或
(ii)相对于(i)中所述的三个重链互补决定区(HCDRs)的序列，在所述三个重链互补决定区(HCDRs)上共包含至少一个且不超过5个氨基酸突变(优选氨基酸置换，优选保守置换)的序列；
和/或
2)所述轻链可变区包含，
(i)氨基酸序列为SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89的肽段内所含有的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3；或
(ii)相对于(i)中所述的三个轻链互补决定区(LCDRs)的序列，在所述三个轻链互补决定区(LCDRs)上共包含至少一个且不超过5个氨基酸突变(优选氨基酸置换，优选保守置换)的序列。
本发明提供了一种抗体或其抗原结合片段，其特异性结合胸腺基质淋巴细胞生成素(Thymic stromal lymphopoietin，TSLP)，通过IC50测定，PEG沉降法，动物实验来表征本抗体优异的稳定性和体外成药性以及动物药效结果。
可选地，所述抗体或其抗原结合片段，其特异性结合胸腺基质淋巴细胞生成素(Thymic stromal lymphopoietin，TSLP)，其包含：
氨基酸序列为SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86、88的肽段内所含有的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3；和/或
氨基酸序列为SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87、89的肽段内所含有的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3。
抗体或其抗原结合片段，其特异性结合TSLP，其包含：
a)重链互补决定区HCDR3，所述HCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:3、15、27、39、50、60、71和80，或所述HCDR3包含 与如SEQ ID NO:3、15、27、39、50、60、71或80所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；和/或
b)轻链互补决定区LCDR3，所述LCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:6、18、30、42、63和83，或所述LCDR3包含与如SEQ ID NO:6、18、30、42、63或83所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列。
其进一步包含：
c)重链互补决定区HCDR1，所述HCDR1包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:1、13、25、37、58和78，或所述HCDR1包含与如SEQ ID NO:1、13、25、37、58或78所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；和/或
d)轻链互补决定区LCDR1，所述LCDR1包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:4、16、28、40、61和81，或所述HCDR2包含与如SEQ ID NO:4、16、28、40、61或81所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
优选地，其进一步包含：
e)重链互补决定区HCDR2，所述HCDR2包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:2、14、26、38、49、59、70和79,或所述LCDR1包含与如SEQ ID NO:2、14、26、38、49、59、70或79所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；和/或
f)轻链互补决定区LCDR2，所述LCDR2包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:5、17、29、41、51、62和82,或所述LCDR2包含与如SEQ ID NO:5、17、29、41、51、62和82所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列。
可选地，所述抗体或其抗原结合片段，其包含三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3；以及三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3，其中：
HCDR1包含SEQ ID NO:1、13、25、37、58或78、或由其组成,或所述HCDR1包含与如SEQ ID NO:1、13、25、37、58、或78所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
HCDR2包含SEQ I DNO:2、14、26、38、49、59、70或79、或由其组成，或所述HCDR2包含与如SEQ ID NO:2、14、26、38、49、59、70或79所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
HCDR3包含SEQ ID NO:3、15、27、39、50、60、71或80、或由其组成，或所述HCDR3包含与如SEQ ID NO:3、15、27、39、50、60、71或80所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
LCDR1包含SEQ ID NO:4、16、28、40、61或81、或由其组成，或所述LCDR1包含与如SEQ ID NO:4、16、28、40、61或81所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
LCDR2包含SEQ ID NO:5、17、29、41、51、62或82、或由其组成，或所述LCDR2包含与如SEQ ID NO:5、17、29、41、51、62或82所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
LCDR3包含SEQ ID NO:6、18、30、42、63或83、或由其组成，或所述LCDR3包含与如SEQ ID NO:6、18、30、42、63或83所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列。
可选地，所述的抗体或其抗原结合片段，其包含：
(1)氨基酸序列如SEQ ID NO:7、19、31、43、52、64、72或84所示的肽段内所含的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3，和
氨基酸序列如SEQ ID NO:8、20、32、44、53、65、73或85所示的肽段内所含的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3；
(2)氨基酸序列如SEQ ID NO:9、21、33、45、54、66、74或86所示的肽段内所含的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3，和
氨基酸序列如SEQ ID NO:10、22、34、46、55、67、75或87所示的肽段内所含的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3；
(3)氨基酸序列如SEQ ID NO:11、23、35、47、56、68、76或88所示的肽段内所含的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3，和
氨基酸序列如SEQ ID NO:12、24、36、48、57、69、77或89所示的肽段内所含的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3。
可选地，所述的抗体或其抗原结合片段，其包含：
1)包含SEQ ID NO:1或由其组成的HCDR1，
包含SEQ ID NO:2或由其组成的HCDR2，
包含SEQ ID NO:3或由其组成的HCDR3，
包含SEQ ID NO:4或由其组成的LCDR1，
包含SEQ ID NO:5或由其组成的LCDR2，和
包含SEQ ID NO:6或由其组成的LCDR3；或
2)包含SEQ ID NO:13或由其组成的HCDR1，
包含SEQ ID NO:14或由其组成的HCDR2，
包含SEQ ID NO:15或由其组成的HCDR3，
包含SEQ ID NO:16或由其组成的LCDR1，
包含SEQ ID NO:17或由其组成的LCDR2，和
包含SEQ ID NO:18或由其组成的LCDR3；或
3)包含SEQ ID NO:25或由其组成的HCDR1，
包含SEQ ID NO:26或由其组成的HCDR2，
包含SEQ ID NO:27或由其组成的HCDR3，
包含SEQ ID NO:28或由其组成的LCDR1，
包含SEQ ID NO:29或由其组成的LCDR2，和
包含SEQ ID NO:30或由其组成的LCDR3；或
4)包含SEQ ID NO:37或由其组成的HCDR1，
包含SEQ ID NO:38或由其组成的HCDR2，
包含SEQ ID NO:39或由其组成的HCDR3，
包含SEQ ID NO:40或由其组成的LCDR1，
包含SEQ ID NO:41或由其组成的LCDR2，和
包含SEQ ID NO:42或由其组成的LCDR3；或
5)包含SEQ ID NO:25或由其组成的HCDR1，
包含SEQ ID NO:49或由其组成的HCDR2，
包含SEQ ID NO:50或由其组成的HCDR3，
包含SEQ ID NO:28或由其组成的LCDR1，
包含SEQ ID NO:51或由其组成的LCDR2，和
包含SEQ ID NO:30或由其组成的LCDR3；或
6)包含SEQ ID NO:58或由其组成的HCDR1，
包含SEQ ID NO:59或由其组成的HCDR2，
包含SEQ ID NO:60或由其组成的HCDR3，
包含SEQ ID NO:61或由其组成的LCDR1，
包含SEQ ID NO:62或由其组成的LCDR2，和
包含SEQ ID NO:63或由其组成的LCDR3；或
7)包含SEQ ID NO:25或由其组成的HCDR1，
包含SEQ ID NO:70或由其组成的HCDR2，
包含SEQ ID NO:71或由其组成的HCDR3，
包含SEQ ID NO:28或由其组成的LCDR1，
包含SEQ ID NO:51或由其组成的LCDR2，和
包含SEQ ID NO:30或由其组成的LCDR3；或
8)包含SEQ ID NO:78或由其组成的HCDR1，
包含SEQ ID NO:79或由其组成的HCDR2，
包含SEQ ID NO:80或由其组成的HCDR3，
包含SEQ ID NO:81或由其组成的LCDR1，
包含SEQ ID NO:82或由其组成的LCDR2，和
包含SEQ ID NO:83或由其组成的LCDR3。
本发明还公开了一种抗体或其抗原结合片段，其特异性结合于TSLP， 其包含：
重链可变区，所述重链可变区包含：SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88；或与序列SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述重链可变区由SEQ ID NO：:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88组成，或由与序列SEQ ID NO::7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成；或所述重链可变区包含与SEQ ID NO：7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88相比，具有一个或多个(优选不超过10个、更优选不超过5个)的氨基酸突变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由其组成,优选地，所述氨基酸突变不发生在重链互补决定区；和/或
轻链可变区，所述轻链可变区包含：SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89；或与序列SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述轻链可变区由SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89组成，或由与序列SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成；或所述轻链可变区包含与SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89相比，具有一个或多个(优选不超过10个、更优选不超过5个)的氨基酸突变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，或由其组成，优选地，所述氨基酸突变不发生在轻链互补决定区。
可选地，所述抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区和轻链可变区的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:7和SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12、SEQ ID NO:19和SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24、SEQ ID NO:31和SEQ ID NO:32、SEQ ID NO:33和SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36、SEQ ID NO:43和SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46、SEQ ID NO:47和SEQ ID NO:48、SEQ ID NO:52和SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57、SEQ ID NO:64和SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67、SEQ ID NO:68和SEQ ID NO:69、SEQ ID NO:72和SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77、SEQ ID NO:84和SEQ ID NO:85、SEQ ID NO:86和SEQ ID NO:87、和SEQ ID NO:88和SEQ ID NO:89。
可选地，所述的抗体或其抗原结合片段，其中每个CDR根据Kabat定义、Chothia定义、Abm定义和/或Contact(接触)定义。
可选地，抗体或其抗原结合片段，其中每个CDR根据Kabat定义或Chothia定义。例如，本发明实施例中即采用Kabat定义方式。
可选地，所述的抗体或其抗原结合片段，其进一步包含恒定区，所述恒定区来自于IgG抗体、IgM抗体、IgA抗体、IgD抗体或IgE抗体，优选地，所述恒定区来自于IgG1抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
可选地，所述的抗体或其抗原结合片段，其进一步包含重链恒定区，所述重链很定期包含：SEQ ID NO：90与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述重链恒定区由SEQ ID NO:90组成，或由与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成；和/或
轻链恒定区，所述轻链恒定区包含：SEQ ID NO:90，或与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述轻链恒定区由SEQ ID NO:90组成，或由与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成。
可选地，所述的抗体或其抗原结合片段，其中所述抗体是鼠源抗体、嵌合抗体、人源化抗体或人抗体。
可选地，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(优选scFv)或(Fab’)2、单结构域抗体、双抗体(dAb)或线性抗体。
本发明第二方面提供一种核酸，其用于编码第一方面任一项所述抗体或其抗原结合片段。所述核酸可以为合成的、重组的或分离的。由于核酸密码的简并性，多种核酸将编码相同氨基酸并且所有都涵盖于此。
本发明第三方面提供了一种表达载体，其包含第二方面所述核酸。
本发明第四方面提供了一种宿主细胞，其包含第三方面所述表达载体。
本发明第五方面提供了生产抗体或其抗原结合片段的方法，其包含培养第四方面所述的宿主细胞，并从培养物中回收由此表达的抗体或抗原结合片段。
在此描述的抗体或其抗原结合片段可以从分泌抗体的杂交瘤中生产，或者从重组产生的细胞中生产，该细胞已经用编码抗体或其抗原结合片段的一种或多种基因进行转化或转染。通过在表达核酸以产生抗体的条件下培养宿主细胞，随后回收抗体来产生抗体或其抗原结合部分。
重组表达利用包含多核苷酸的表达载体的构建，该多核苷酸编码抗体或其抗原结合部分。一旦获得了多核苷酸，便可通过本领域中熟知的重组DNA技术生产用于产生抗体的载体。表达载体可以包括适当的转录和翻译控制信号。这可以使用体外重组DNA技术、合成技术和体内基因重组来完成。
可选地，所述宿主细胞为原核细胞或真核细胞。
可选地，所述宿主细胞为大肠杆菌细胞、酵母细胞、昆虫细胞、植物细胞或哺乳动物细胞。
可选地，所述宿主细胞为中国仓鼠卵巢细胞(CHO)、CHO细胞变体、293细胞或NSO细胞。所述细胞系包括VERO、BHK、HeIa、COS、MDCK、293F、293T、3T3、W138、BT483、Hs578T、HTB2、BT20和T47D、CRL7030以及HsS78Bst细胞。但本发明所用细胞系包括但不限于以上细胞系。
一旦抗体或抗体的抗原结合部分已经通过重组表达产生，就可以通过本领域中已知的用于免疫球蛋白分子的纯化的任何方法将其纯化，例如，通过色谱法、离心、差别溶解度，或者通过用于蛋白质的纯化的任何其他标准技 术。
本发明第六方面提供了一种药物组合物，其包含至少一种第一方面所述的任一种抗体或其抗原结合片段，和可药用赋形剂。例如，抗体或其抗原结合片段与注射用水组合，或与盐水组合。
本发明第七方面提供了一种试剂盒，其包含至少一种第一方面所述的任一种抗体或其抗原结合片段或其同源物、衍生物。
本发明第八方面提供了一种第一方面所述的抗体或其抗原结合片段或第六方面的药物组合物在制备用于治疗和/或预防与TSLP相关的疾病的药物中的用途。
本发明第九方面提供了一种第一方面所述的抗体或其抗原结合片段或第六方面的药物组合物，用于治疗和/或预防与TSLP相关的疾病。
本发明还提供了一种治疗和/或预防与TSLP相关的疾病的方法，所述方法包括施用治疗有效量的第一方面中任一项所述的抗体或抗原结合片段或第六方面的药物组合物。
上述与TSLP相关的疾病选自：哮喘或皮炎。
本发明的有益效果
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、高亲和性地结合TSLP，抑制或阻断TSLP与TSLP/IL7R的结合，并且能够在体外抑制或阻断TSLP对Ba/F3细胞的增值效应，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)与阳性药Tezepelumab相比，具有更优的热稳定性、pH稳定性、氧化稳定性、溶解度和与TSLP的亲和力。
3)本发明的TSLP抗体Q61-F相比于阳性药Tezepelumab，具有更好的抗皮炎和抗哮喘的作用。
附图说明
图1：不同的TSLP抗体阻断TSLP与TSLPR以及TSLPR-IL7R复合体结合的能力，A图是不同的TSLP抗体阻断TSLP与TSLPR-Fc蛋白结合的 实验结果，B图是不同的TSLP抗体阻断TSLP与TSLPR-IL-7R-Fc蛋白结合的实验结果。横轴为TSLP抗体的浓度(单位：μg/mL)，纵轴为抑制率。
图2：TSLP抗体抑制Ba/F3细胞增殖的结果。横轴为抗体浓度(单位：μg/mL)，纵轴为抑制率。
图3：TSLP抗体抑制对PBMC细胞分泌TARC(又称CCL17)的结果。横轴为TSLP抗体浓度(单位：μg/mL)，纵轴为TARC的分泌量(单位：pg/mL)。
图4：采用PEG沉淀法测定抗TSLP抗体溶解度。
图5：AD(特应性皮炎)模型图
图6：各给药组小鼠体重对比结果，横轴为天数(单位：天)，纵轴为小鼠体重(单位：g)。
图7：各给药组小鼠耳朵厚度对比结果，横轴为天数(单位：天)，纵轴为小鼠耳朵厚度(单位：μm)。
图8：各给药组小鼠血清中IgE含量对比结果，横轴为组别，纵轴为小鼠血清中IgE含量(单位：ng/mL)。
图9：各给药组小鼠体重对比结果，横轴为天数(单位：天)，纵轴为小鼠体重变化。
图10：各给药组小鼠血清中IgE含量对比结果，横轴为组别，纵轴为小鼠血清中IgE含量(单位：ng/mL)。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
缩略语
PBS是指磷酸盐缓冲溶液(Phosphate Buffer Solution)。
PBST是指PBS溶液中加入Tween-20。
BSA是指牛血清白蛋白(Bovine Serum Albumin)。BSA封闭液是由清白蛋白配制而成。
TMB是指3,3’,5,5’-四甲基联苯胺(3,3’,5,5’-Tetramethylbenzidine) 溶液。
IC50是指产生50％抑制的抗体浓度。
ELISA是指酶联免疫吸附测定。
TARC是指胸腺激活调节趋化因子，又称CCL17。
PEG是指聚乙二醇(polyethylene glycol，PEG)。
HIC是疏水色谱(Hydrophobic interaction chromatography，HIC)。
Tm是蛋白质熔点(Melting temperature)。
ND(Not Detected)是指在实验条件下，实验数据未检测。
NA(Not Apply)是不适用，指在实验条件下，实验数据超出仪器的检测限，不适用。
定义
除非另外指明，在此将任何多肽链描述为具有起始于N-末端并终止于C-末端的氨基酸序列。
以最宽泛的意义使用本文中的术语“抗体和其抗原结合部分”，其包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、和抗体片段，只要它们显示所希望的抗原-结合活性即可。
本文所用的术语“抗体”指由四条多肽链即由二硫键互联的两条重链(H)和两条轻链(L)组成的免疫球蛋白分子。每条重链包含重链可变区(HCVR或VH)和重链恒定区。重链恒定区包含CH1、CH2和CH3三个结构域。每条轻链包含轻链可变区(LCVR或VL)和轻链恒定区。轻链恒定区包含一个结构域(CL1)。VH和VL区可进一步分成被称为互补决定区(Complementarity determining region(s)，CDR(s))的高变区，其中散布着较保守的被称为框架区(framework region，FR)的区域。每个VH和VL由三个CDR和四个FR组成，从氨基酸末端到羧基末端按下列次序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4，其中VH的三个CDRs为HCDR1，HCDR2和HCDR3，VL的三个CDRs为LCDR1，LCDR2和LCDR3。每一结构域的氨基酸分配通常与如下定义一致：Kabat Sequences of Proteins of Immunological Interest(具有免疫学意义的Kabat蛋白序列)(National Institutes of Health，Bethesda，Md.(1987和1991))或Chothia&Lesk，J.Mol.Biol.，196：901-917(1987)；Chothia等，Nature，342：878-883(1989)。
存在五种主要的抗体类型：IgA、IgD、IgE、IgG和IgM，并且这些中的 一些可以进一步分为亚类(同种型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于免疫球蛋白的不同类型的重链恒定结构域分别被称为α、δ、ε、γ和μ。
本文所用的术语抗体的“抗原结合部分”(或简称“抗体部分”或“抗体片段”)指抗体中保留了与抗原(如TSLP蛋白)特异性结合能力的一个或多个片段。业已证明，抗体的抗原结合功能可由全长抗体的某些片段来实现。抗体的“抗原结合部分”这一术语所涵盖的结合片段包括(i)Fab片段，即由VL、VH、CL1和CH1结构域组成的单价片段；(ii)F(ab’)2片段，即由铰链区的二硫键连接的两个F(ab)’片段组成的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂VL和VH结构域组成的Fv片段；(v)由VH结构域组成的dAb片段；以及(vi)CDR。此外，尽管，Fv片段的两个结构域VL和VH是由不同的基因编码的，但它们可通过重组方法，由一种合成的接头连接在一起而成为单独的相连的链，其中VL和VH区配对而形成单价分子(称为单链Fv(scFv))。这样的单链抗体也涵盖在抗体的“抗原结合部分”的术语范围内。其他形式的单链抗体，如双特异抗体也涵盖在内。
可采用不同的分析来确定或粗略估计CDR区。所述方法实例包括但不限于Kabat定义、Chothia定义、AbM定义和接触定义(contact definition)。Kabat定义为用于在抗体中为残基编号的标准，通常用于确定CDR区。参见例如Johnson&Wu，Nucleic Acids Res.，28：214-8(2000)。Chothia定义与Kabat定义类似，但Chothia定义考虑某些结构环区的位置。参见例如Chothia等，J.Mol.Biol.，196：901-17(1986)；Chothia等，Nature，342：877-83(1989)。AbM定义使用由Oxford Molecular Group制作的模仿抗体结构的整合的计算机程序组。参见例如Martin等，Proc Natl Acad Sci(USA)，86：9268-9272(1989)；“AbMTM，A Computer Program for Modeling Variable Regions of Antibodies(AbMTM，用于为抗体可变区建模的计算机程序)”Oxford，UK；Oxford Molecular，Ltd。AbM定义用已知数据库和从头开始法(ab initio method)由一级序列建立抗体三级结构的模型，所述方法例如为在以下文献中所述的方法：Samudrala等，“Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach(用联合的分级方法从头开始预测蛋白结构)”PROTEINS，Structure，Function and Genetics Suppl.，3：194-198(1999)。 接触定义基于对有效的复合体晶体结构的分析。参见例如MacCallum等，J.Mol.Biol.，5：732-45(1996)。
本发明的抗体，对其来源没有限定，可以是人抗体、小鼠抗体、大鼠抗体等来自任何动物的抗体。还可以是嵌合抗体或人源化抗体等重组抗体。优选的是人源化抗体。
术语“鼠源抗体”是根据本领域知识和技能制备的针对人TSLP的单克隆抗体。制备时用TSLP抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。所述的鼠源抗TSLP抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”是指将人以外的哺乳动物、例如小鼠抗体的重链、轻链的可变区和人抗体的重链、轻链的恒定区的抗体融合而成的抗体，以减轻鼠源性抗体诱发的免疫应答反应。嵌合抗体可以利用已知的方法来制备。例如，由杂交瘤克隆体的基因，将其插入到适当的载体中，再将其导入宿主中，即可制备嵌合抗体。具体而言，使用逆转录酶由杂交瘤的mRNA合成抗体可变区(V)区的cDNA。得到编码目标抗体V区的DNA时，连接其与编码所期望的人抗体恒定区(C区)的DNA，再将其插入到表达载体中。或者，可以将编码抗体V区的DNA插入到包含人抗体C区的DNA表达载体中。将其插入到表达载体中，使之在表达调节区的调解下表达。接下来，利用该表达载体转化宿主细胞，可以使嵌合抗体表达。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
术语“氨基酸突变”是指氨基酸置换、插入、缺失或修饰。具体为多肽 与其变体之间，在多肽片段上某个或某些氨基酸位点上的突变，其中变体可以由多肽上某个或某些位点经置换、插入、缺失或修饰氨基酸获得。
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，通过保守置换氨基酸得到的抗体的变体充分地保留其来源序列的生物学活性。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。
术语“核酸”是指DNA或RNA。核酸可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“基本同一性”或“基本上相同”当其指核酸或其片段时，表示当以适当的核苷酸替换、插入或删除与另一个核酸(或其互补链)进行最佳比对时，用下述任何的序列同一性计算程序如FASTA、BLAST或Gap计算，在至少约80％，更优选在至少约80％，85％，91％，92％，93％，94％，95％，96％、97％、98％或99％的核苷酸碱基中具有核苷酸序列同一性。
当应用于多肽时，术语“基本相似”或“基本上相似”意为当以Gap或BESTFIT等程序用默认空位权重进行最佳比对时，两个肽序列至少具有80％的序列同一性，更优选的是至少具有80％，85％，91％，92％，93％，94％，95％，96％，97％，98％或99％的序列同一性。不相同的残基位置的区别可以为氨基酸的取代、删除或插入，更优选的是，不相同的残基位置的区别在于保守的氨基酸取代。“保守的氨基酸取代”是这样的取代，其中氨基酸残基被 另一个含有类似化学性质(如电荷或疏水性)的侧链(R基团)的氨基酸残基所取代。一般来说，保守的氨基酸取代不会从实质上改变蛋白质的功能性质。在两个或两个以上氨基酸序列由于保守的取代而不同的情况下，可向上调节序列同一性百分数或类似程度，以修正取代的保守性。进行这种调节的方法为本领域的专业人员所熟知。含有具有类似化学性质侧链的氨基酸群的实例包括(1)脂肪族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；(2)脂肪族-羟基族：丝氨酸和苏氨酸；(3)含酰胺侧链：天冬酰胺和谷氨酰胺；(4)芳香族侧链：苯丙氨酸、酪氨酸和色氨酸；(5)碱性侧链：赖氨酸、精氨酸和组氨酸；(6)酸性侧链：天冬氨酸和谷氨酸，以及(7)含硫侧链：半胱氨酸和甲硫氨酸。优选的保守的氨基酸取代群为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸、谷氨酸-天冬氨酸，以及天冬酰胺-谷氨酰胺。或者，保守的取代可以是Gonnet等人(1992)Science256:1443-1445所披露的PAM250对数-似然矩阵中具有正值的任何变化。“中度保守的”取代是PAM250对数-似然矩阵中具有非负值的任何变化。
术语“胸腺基质淋巴细胞生成素(Thymic Stromal Lymphopoietin,TSLP)”是四α-螺旋束I型细胞因子，也是响应促炎症刺激而产生的上皮细胞衍生的细胞因子，与白细胞介素-7(IL-7)密切相关，其通过刺激树突细胞(DC)起始变态反应，是调节人体免疫反应的重要因子。术语“TSLP”包括TSLP的变体、同种型、同系物、直系同源物和旁系同源物。
术语“载体”意指用于将蛋白编码信息转移到宿主细胞的任何分子或实体(例如核酸、质粒、噬菌体或病毒)。
术语“表达载体”或“表达构建体”是指适于转化宿主细胞并含有指导和/或调控一个或多个可操作地与其连接的异源编码区的表达的核酸序列的载体。表达载体可包括但不限于：影响或调控转录、翻译的序列；和若存在内含子，则影响可操作地与其连接的编码区的RNA剪接的序列。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换使用，是指向其中引入外源核酸的细胞，包括所述细胞的子代。宿主细胞包括“转化子”和“转化细胞”，其包括原代转化的细胞和从其衍生的子代(不考虑传代数)。子代的核酸含量可以不完全与母细胞相同，而是可以含有突变。具有与对于在最初细胞中筛选或选择的相同功能或生物活性的突变体子代包括在本文 中。
术语“转染”意指细胞吸收外来或外源DNA，当将所述外源DNA引入到细胞膜内时细胞就被“转染”了。多种转染技术在本领域是众所周知。参见例如Graham等，1973，Virology 52：456；Sambrook等，2001，Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)；Davis等，1986，Basic Methods in Molecular Biology(分子生物学基本方法)，Elsevier；Chu等，1981，Gene 13：197。所述技术可用于将一种或多种外源DNA部分引入到合适的宿主细胞中。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10-8M，例如大约小于10-9M、10-10M、10-11M、10-12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10-7M，例如小于大约10-8M或10-9M的解离平衡常数(KD)结合TSLP，例如，在本公开中抗体与细胞表面抗原的亲和力采用FACS或Biacore法测定KD值。
术语“Tm”是指蛋白质熔点，也就是摩尔热容达到最大值的温度，是表征抗体热稳定性的重要参数。传统上，热稳定性是通过差示扫描量热仪(DSC)来测量的。DSC所产生的热图的典型特征是存在三个部分重叠的展开区域。这些熔融区中的每一个都与特定的熔化温度(或Tm值)相关联，Tm值对应于给定温度区域内热容的最大温度变化率。在大多数情况下，第一个(最低温度)Tm1对应于Fc的CH2结构域，第二个Tm2对应于Fab，Tm3对应于Fc的CH3结构域。CH2和CH3结构域的Tm值变化不像Fab Tm值那么大。因此，在可开发性评估或配方筛选期间，主要参考Tm2(Fab)值。Tm2(Fab)值越大，证明蛋白的热稳定性越好。
术语“治疗”包括治疗性治疗、预防性治疗及在降低受治疗者发展疾病的风险或其他风险因素中的应用。治疗不需要完全治愈疾病，而是包括在其中减轻症状或减轻潜在风险因素的实施方案。
术语“约”或“大约”意指如由本领域普通技术人员所确定的具体值的可接受误差，其部分地取决于如何测量或确定所述值。在某些实施例中，术语“约” 或“大约”意指1个、2个、3个或4个标准偏差内。在某些实施例中，术语“约”或“大约”意指给定值或范围的30％、25％、20％、15％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.05％内。每当术语“约”或“大约”在一系列两个或更多个数值中的第一数值前面时，应了解术语“约”或“大约”适用于该系列中的每个数值。
术语“有效量”或“有效剂量”指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶抗原相关病症的进展。
术语“药物组合物”是指多种制备物的制剂。含有有效量的抗体、药学上可接受的载体、稀释剂或赋形剂，该制剂为无菌液体溶液、液体悬浮剂或冻干形式。
本发明的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。
术语“试剂盒”是指用于盛放抗体、检测化学成分、药物残留、病毒种类等试剂的盒子。例如所述的试剂盒包括本发明的抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的缀合物，以及抗体使用说明书、杀死特定类型细胞的缀合物等。该说明书可包括在体外、体内或离体使用抗体、缀合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它事宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这 些试剂也可以以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
如本文所用的术语“哮喘”是指过敏性哮喘、非过敏性哮喘。
如本文所用的术语“过敏性哮喘”是指由一种或多种吸入性过敏原引发的哮喘。此类患者对引发哮喘反应的一种或多种过敏原具有阳性的IgE荧光酶免疫分析(FEIA)水平。
通常，大部分过敏性哮喘与Th2型炎症有关。
术语“非过敏性哮喘”是指在诊断时具有低嗜酸细胞、低Th2或低IgE的患者。在IgE荧光酶免疫分析(FEIA)中，患有“非过敏性哮喘”的患者对包括地区特异性过敏原的一组过敏原的反应呈阴性。除低IgE的外，那些患者经常在诊断时具有低嗜酸细胞计数或无嗜酸细胞计数和低Th2计数。
术语“TSLP相关的疾病”是指包括但不限于哮喘、特发性肺纤维化、特应性皮炎、过敏性结膜炎、变应性鼻炎、过敏性鼻窦炎、荨麻疹、内塞顿综合征、嗜酸性粒细胞性食管炎、食物过敏、过敏性腹泻、嗜酸性粒细胞性胃肠炎、过敏性支气管肺曲霉病、过敏性真菌鼻窦炎、慢性瘙痒、癌症、乳腺癌、结肠癌、肺癌、卵巢癌、前列腺癌、类风湿性关节炎、慢性阻塞性肺病、全身性硬化、多发性硬化症、瘢痕瘤、溃疡性结肠炎、鼻息肉病、慢性嗜酸性粒细胞性肺炎、嗜酸性粒细胞性支气管炎、腹腔病、Churg-Strauss综合征、嗜酸性粒细胞性肌痛综合征、高嗜酸粒细胞综合征、嗜酸性粒细胞性肉芽肿病伴随多血管炎、炎性肠病、硬皮病、间质性肺病、B型或C型慢性肝炎引发的纤维化、辐射诱发的纤维化和伤口愈合引发的纤维化。
实施例1.TSLP重组蛋白的制备
根据NCBI数据库内的TSLP蛋白序列(全长159aa，ACCESSION:AAH40592)，进行碱基序列优化，利用HindIII/SacII酶切位点插入pcDNA3.1-Myc-His载体，构建TSLP蛋白的表达质粒，转染293F细胞后，培养并收集细胞表达的上清液，浓缩后经镍柱(Cytiva)纯化，得到TSLP蛋白，氨基酸序列如SEQ ID NO:92所示(SEQ ID NO:92MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRTTNKCLEQVSQLQGLWRRFNRPLLKQQ)。
实施例2.重组TSLP受体和IL7RA受体细胞系的构建和鉴定
为筛选可以阻断TSLP结合TSLP受体的抗体，构建了同时表达人TSLP受体和人IL7Rα(TSLPR/IL7Rα)的BaF3细胞株。采用电转方法将目的基因TSLPR/IL7Rα克隆至目的细胞株内形成稳定高表达细胞株。首先分别将人TSLPR和人IL7Rα基因克隆至pcDNA3.1-Myc-His质粒内，然后通过电转的方法将人TSLPR克隆至BaF3细胞株内，经400μg/ml G418筛选压力下选择培养1周。在此基础上进行第二轮感染，再将人IL7Rα基因克隆进去，用400μg/ml G418筛选两至三周。最后通过流式分选的方法，筛选出同时高表达TSLPR和IL7Rα的单克隆细胞株。
实施例3.抗人TSLP单克隆抗体的制备和筛选
免疫动物
抗人TSLP单克隆抗体通过免疫小鼠产生，实验用Balb/c小鼠，雌性，6-8周龄(来源：维通利华)。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节。将已适应环境的小鼠用重组蛋白，huTSLP-his(200μg)和Alum佐剂免疫。在第4-5次免疫以后，用ELISA方法确定小鼠中的抗体滴度，选择血清中抗体滴度高并且滴度趋于平台的小鼠，脱颈处死后，取脾细胞，与骨髓瘤细胞融合。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞进行融合得到杂交瘤细胞。用传统的ELISA方法和Biacore方法筛选出杂交瘤细胞。
使用ELISA方法和Biacore方法筛选杂交瘤细胞
ELISA方法
(1)将96孔板中加入100μL，1μg/mL的抗原蛋白溶液，4℃过夜包被；
(2)向已包被好靶蛋白TSLP的96孔检测板中加入100μL的细胞上清(或纯化抗体)；
(3)37℃生化培养箱孵育1h；
(4)用PBST作为洗液洗涤3遍，并拍去残留溶液；(5)加入用PBST+2％BSA的anti-mouse Fc或anti-human FC酶标二抗(按1：10000稀释)，每孔100μL；
(6)在37℃生化培养箱孵育1h；
(7)用PBST为洗液洗涤4遍拍去残留溶液；
(8)加入TMB单组分显色液，100μL/孔，避光显色8min左右；
(9)加入终止液2M H2SO4终止反应，50μL/孔；
(10)酶标仪读取OD450值。
Biacore方法
利用分子相互作用分析仪Biacore 8K，采用固定Anti-mouse antibody的CM5芯片捕获杂交瘤细胞上清液，再进分析样品TSLP蛋白，从而测定抗体与蛋白的亲和力，对抗体进行动力学初筛。具体操作步骤为设置三个循环的Startup，之后设置capture(捕获上清中的抗体)为300s，30μL/min；analysis(分析样品)浓度为200nM，结合时间120s，30μL/min；Dissociation time为120s，Regeneration(10mM glycine-HCl)为30μL/min，30s；之后在列表中填写样品编号，及相应蛋白的蛋白浓度，根据程序给定的试剂位置加入相应试剂后盖上膜，将96孔板放入样品舱内，开始程序即可。
杂交瘤亚克隆细胞株的测序
筛选得到活性好的单克隆杂交瘤细胞株，分别收集对数生长期杂交瘤细胞，用TRNzol提取RNA，并进行反转录(All-in-One First-Strand cDNA Synthesis SuperMix for PCR,Transgene,AT321-01)。将反转录得到的cDNA进行PCR扩增后送测序公司测序。经测序得到鼠源抗TSLP抗体：Q46、Q61、Q75、Q78、Q112、Q148、Q170、Q187序列，其可变区氨基酸序列如下：
表1鼠源TSLP抗体测序结果




制备嵌合抗体，将鼠源性抗体的可变区与人抗体的恒定区融合，并对其形成的嵌合抗体进行筛选。人源化恒定区的序列如下：
重链恒定区(SEQ ID NO:90)：
轻链恒定区(SEQ ID NO:91)：
实施例4.抗人TSLP单克隆抗体的人源化设计
为了降低鼠源抗体的免疫原性，将已筛选出的体内外活性优异的Q61、Q187、Q78、Q75、Q46、Q148、Q112、Q170抗体进行了人源化。鼠源单克隆抗体的人源化根据本领域许多文献公示的方法进行。简言之，将鼠的CDR序列移植到人的抗体可变区框架，克服嵌合抗体中由于携带大量鼠蛋白成分而诱导的异源性反应。然后对人抗体可变区框架序列进行回复突变，得到最终的人源化分子。4.1 Q46的人源FR区的选择和回复突变
(1)人源FR区的选择
Q46的人源化VH模板为IGHV1-69-2*01，人源化VL的模板为IGKV1-13*02，将Q46的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ46VH-CDR：
huQ46VL-CDR：
(2)人源化抗体的回复突变
Q46人源化抗体的回复突变位点如下表：
表2 Q46人源化抗体的回复突变
注：表中如V24A表示将人源化抗体24位氨基酸V回复突变为A。
对人源化抗体huQ61的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q46-F，其回复突变位点为重链V24A，序列如下：
Q46-F重链可变区VH序列：
Q46-F轻链可变区VL序列：
4.2 Q61的人源FR区的选择和回复突变
(1)人源FR区的选择
Q61的人源化VH模板为IGHV1-18*01，人源化VL的模板为IGKV1-27*01，将Q61的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ61VH-CDR：
huQ61VL-CDR：
(2)人源化抗体的回复突变
Q61人源化抗体的回复突变位点如下表：
表3 Q61人源化抗体的回复突变
注：表中如M48I表示将人源化抗体48位氨基酸M回复突变为I。
对人源化抗体huQ61的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q61-F，其回复突变位点为重链Y95F以及轻链F72Y，序列如下：
Q61-F重链可变区VH序列：
Q61-F轻链可变区VL序列：
4.3 Q75的人源FR区的选择和回复突变
(1)人源FR区的选择
Q75的人源化VH模板为IGHV1-46*01，人源化VL的模板为IGKV3-15*01，将Q75的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ75VH-CDR：
huQ75VL-CDR：
(2)人源化抗体的回复突变
Q75人源化抗体的回复突变位点如下表：
表4 Q75人源化抗体的回复突变
注：表中如M48I表示将人源化抗体48位氨基酸M回复突变为I。
对人源化抗体huQ75的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q75-F，其回复突变位点为重链R72A，轻链F72Y，序列如下：
Q75-F重链可变区VH序列：
Q75-F轻链可变区VL序列：
4.4 Q78的人源FR区的选择和回复突变
(1)人源FR区的选择
Q78的人源化VH模板为IGHV1-18*01，人源化VL的模板为IGKV1-33*01，将Q78的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ78VH-CDR：
huQ78VL-CDR：
(2)人源化抗体的回复突变
Q78人源化抗体的回复突变位点如下表：
表5 Q78人源化抗体的回复突变
注：表中如I37V表示将人源化抗体37位氨基酸I回复突变为V。
对人源化抗体huQ78的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体 Q78-F，其回复突变位点为重链V71K，轻链F71Y，序列如下：
Q78-F重链可变区VH序列：
Q78-F轻链可变区VL序列：
4.5 Q112的人源FR区的选择和回复突变
(1)人源FR区的选择
Q112的人源化VH模板为IGHV1-69*10，人源化VL的模板为IGKV3-7*02，将Q112的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ112VH-CDR：
hu12VL-CDR：
(2)人源化抗体的回复突变
Q112人源化抗体的回复突变位点如下表：
表6 Q112人源化抗体的回复突变
注：表中如M48I表示将人源化抗体48位氨基酸M回复突变为I。
对人源化抗体huQ112的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q112-F，其回复突变位点为轻链F72Y，序列如下：
Q112-F重链可变区VH序列：
Q112-F轻链可变区VL序列：
4.6 Q148的人源FR区的选择和回复突变
(1)人源FR区的选择
Q148的人源化VH模板为IGHV2-70*04，人源化VL的模板为IGKV3-11*01，将Q148的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ148VH-CDR：
huQ148VL-CDR：
(2)人源化抗体的回复突变
Q148人源化抗体的回复突变位点如下表：
表7 Q148人源化抗体的回复突变
注：表中如L4M表示将人源化抗体4位氨基酸L回复突变为M。
对人源化抗体Q148的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q148-F，其回复突变位点为轻链L4M，序列如下：
Q148-F重链可变区VH序列：
Q148-F轻链可变区VL序列：
4.7 Q170的人源FR区的选择和回复突变
(1)人源FR区的选择
Q170的人源化VH模板为IGHV1-46*01，人源化VL的模板为IGKV1-37*01，将Q170的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ170VH-CDR：
huQ170VL-CDR：
(2)人源化抗体的回复突变
Q170人源化抗体的回复突变位点如下表：
表8 Q170人源化抗体的回复突变

注：表中如V68A表示将人源化抗体68位氨基酸V回复突变为A。
对人源化抗体Q170的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q170-F，其回复突变位点为重链R72A，轻链F72Y，序列如下：
Q170-F重链可变区VH序列：
Q170-F轻链可变区VL序列：
4.8 Q187的人源FR区的选择和回复突变
(1)人源FR区的选择
Q187的人源化VH模板为1T3F，人源化VL的模板为1T3F，将Q187的CDR移植到人源模板上，移植后获得的可变区序列如下：
huQ187VH-CDR grafted：
huQ187VL-CDR grafted：
(2)人源化抗体的回复突变
Q187人源化抗体的回复突变位点如下表：
表9 Q187人源化抗体的回复突变
注：表中如I28T表示将人源化抗体28位氨基酸I回复突变为T。
对人源化抗体Q187的VH和VL按照上表的突变位点进行单一突变及多点突变，并对构建的不同的回复突变重轻链进行组合，将表达的蛋白进行亲和力及细胞活性的测定，最终确定人源化程度高、活性优异的人源化抗体Q187-F，其回复突变位点为重链I28T、T30I及K74T，轻链I48V，序列如下：
Q187-F重链可变区VH序列：
Q187-F轻链可变区VL序列：
实施例5.抗TSLP人源化抗体的亲和力测定
利用Biacore方法测定抗TSLP人源化抗体结合TSLP的亲和力。通过分子相互作用分析仪Biacore 8K，采用固定Anti-mouse antibody的CM5芯片捕获抗体，再进分析样品(靶点蛋白)，从而测定抗体与蛋白的亲和力，对抗体进行动力学初筛。具体操作步骤为设置三个循环的Startup，之后设置capture(捕获上清中的抗体)为300s，30μL/min；analysis(分析样品)浓度为200nM，结合时间120s，30μL/min；Dissociation time为120s，Regeneration(10mM glycine-HCl)为30μL/min，30s；之后在列表中填写样品编号，及相应蛋白的蛋白浓度，根据程序给定的试剂位置加入相应试剂后盖上膜，将96孔板放入样品舱内，开始程序即可。测定的抗TSLP人源化抗体结合TSLP的亲和力结果如表10所示。
表10抗TSLP人源化抗体结合TSLP的亲和力结果
本发明获得的人源化抗体Q61-F、Q187-F、Q75-F、Q112-F的亲和力均高于nM(10-10)级，可有效结合TSLP，结合能力优于阳性对照药Teze。
实施例6.测定抗TSLP人源化抗体的热稳定性
采用DSF(差示荧光扫描技术)方法测定抗TSLP抗体的Tm值：
(1)通过MicroCalTM VP Cap DSC system(Malvern Panalytical,Northampton,USA)对待测抗体Tm值进行测定
(2)抗体的准备：将待测抗体置换为D-PBS buffer，并将抗体浓缩至0.5mg/mL，0.22μm滤膜过滤后，置于4℃待测。
(3)仪器的准备：使用40％Decon90对仪器进行清洗，扫描温度从10℃升温至60℃，升温速率设置为180℃/h，共设置9组清洗程序。清洗结束，设置3组水，扫描温度从10℃升温至130℃，升温速率设置为180℃/h。如果三组水的DSC曲线一致，则仪器清洗程序完成，等待样品测定。
(4)程序的测定：设置三组buffer，扫描温度从10℃升温至130℃，升温速率设置为180℃/h，作为仪器校准的对照。Buffer、样品、14％decon和14％decon共同构成一组待测样品。Buffer及样品扫描温度从10℃升温至130℃，升温速率设置为180℃/h；14％decon清洗扫描温度从10℃升温至60℃，升温速率设置为180℃/h，并在个样品前设置clean before load。
(5)加样及测定：按照上述方法设定好程序并设置好数据存储路径。按照程序指示，在96深孔板中加入400μL对应的待测溶液。加样完成，将96孔板放入样品存储架子。点击Start，开始Tm值的测定。
人源化抗体Tm值测定的结果如表11所示。
表11.抗体Tm值测定
经测定，人源化抗体Q61-F及Q187-F在同样的浓度及测定条件下，Tm均高于阳性对照抗体Teze，且均大于70℃，热稳定性良好。
实施例7.测定抗TSLP人源化抗体的疏水性
通过HPLC对待测抗体疏水性进行测定。疏水性可以间接反应抗体分子间相互作用力的强弱，从而对抗体分子的聚集性、非特异性结合的倾向提供参考。较强的疏水性可能预示分子较容易聚集以及出现非特异性结合。疏水色谱HIC(Hydrophobic interaction chromatography，HIC)的实验步骤如下：
(1)平衡液相系统至系统稳定：按Waters e2695/LC-20AT型高效液相色谱仪标准操作规程开启仪器并完成溶剂管理系统的准备，先连接双通用超纯水以1.0mL/min的流速冲洗管路30分钟，再用A液(1.5M硫酸铵+25mM磷酸钠，pH＝6.8)以1.0mL/min的流速冲洗管路30min，降低流速到0mL/min，连接好所需色谱柱后用A液以0.5mL/min的流速平衡色谱柱到基线平稳，约30min。
(2)样品处理：使用PBS将样品稀释至浓度为2.0mg/mL。
(3)进样3个PBS后，进样2.0mg/mL的待测样品，进样体积为10.0μL
(4)用B液(75％25mM磷酸钠+25％异丙醇，pH＝6.8)对样品进行线性梯度洗脱。
(4)数据处理：根据样品保留时间进行疏水性分析。
(5)仪器及色谱柱处理：实验完成后用超纯水以0.5mL/min冲洗色谱柱60分钟，保存色谱柱，填写《HPLC使用记录》；再用20％甲醇溶液以0.2mL/min冲洗管路30分钟，关闭液相色谱。
HIC结果如表12所示，保留时间越短，说明分子的疏水性越弱。从结果可以看出，Q61-F的保留时间低于阳性对照抗体Teze，疏水性优于阳性药。
表12.抗体疏水性
实施例8.测定抗TSLP人源化抗体的稳定性
通过HPLC对待测抗体稳定性进行测定，分别测定pH加速条件和氧化加速条件下，不同的抗TSLP抗体的稳定性。
8.1测定pH加速条件下抗TSLP人源化抗体稳定性
测定酸性pH、极端酸性pH、碱性pH对抗TSLP人源化抗体稳定性影响的加速实验：将2.0mg的人源化抗体及阳性对照抗体的溶剂分别置换为50mM乙酸钠(乙酸调节pH＝5.5)、20mM的pH＝3.0的柠檬酸缓冲液、20mM的Tris、10mM的EDTA(NaOH调节pH＝9.0)，各自将总体积调节至1.0mL，将pH＝5.5的抗体样品置于40℃烘箱中孵育14天，将pH＝3.0的抗体样品置于室温避光放置24h，将pH＝9.0的抗体样品置于40℃烘箱中孵育7天，将各自的溶剂重新置换为PBS，测定样品的聚体和碎片变化，以考察不同pH对抗体稳定性的影响。结果如表13所示。
表13.pH加速条件下抗体稳定性

如表13所示，不同pH处理条件下，人源化抗体Q61-F与Q187-F的聚体和碎片化程度不明显，且略优于阳性药，说明在不同pH加速处理条件下，人源化抗体Q61-F与Q187-F具有较好的稳定性。
8.2测定氧化加速条件下抗TSLP抗体稳定性
在1.0mL浓度为2.0mg/mL的人源化抗体及阳性对照抗体中加入3.3μL的质量分数为30％的H2O2，于室温避光放置24h；加速完成后，将其缓冲液重新置换为PBS，放置于-20℃保存。之后，测定样品的聚体和碎片变化以及细胞活性变化，以考察氧化处理对抗体稳定性的影响。结果如表14所示。
表14.氧化加速条件下抗体稳定性
如表14所示，在氧化处理条件下，人源化抗体Q61-F与Q187-F的聚体 和碎片化程度不明显，优于阳性对照药，说明在氧化处理条件下，人源化抗体Q61-F与Q187-F具有较好的稳定性。
实施例9.抗TSLP人源化抗体的溶解度的测定
采用PEG沉淀法测定抗TSLP抗体溶解度，实验步骤如下：
(1)PEG原液的配置：将称量好的PEG固体溶解到PBS中，配置成40％质量分数(w/v)为40％的原液，调节pH至7.2。
(2)微孔板的高通量测定：将待测抗体用PBS稀释至10mg/mL，分别取150μL加入96孔板；将PEG用PBS稀释成质量分数为6％、8％、10％、12％、14％、16％、18％、20％、22％和24％的溶液，分别取150μL依次将入到对应抗体的孔中，充分吹打混匀，至抗体终浓度为5mg/mL，PEG终浓度分别为3％-12％。不同PEG浓度的混合液会出现不同程度的沉淀，室温放置15min。混合液的浊度反映了沉淀量的多少。通过观察或者分光光度计测定OD500评价溶解度的大小。从图4可以看出，在相同浓度抗体，相同缓冲体系及温度条件下，阳性对照抗体TEZE最先出现沉淀，而Q61-F人源化抗体在8％PEG质量分数下出现沉淀；Q187-F从7％PEG质量分数出现沉淀，但明显好于阳性对照抗体TEZE。由此可见，Q61-F及Q187-F溶解度大于TEZE。
实施例10.抗TSLP人源化抗体阻断TSLP与TSLPR以及TSLPR-IL7R复合体结合实验
不同的TSLP抗体阻断TSLP与TSLPR以及TSLPR-IL7R复合体结合的能力，TSLP通过与TSLPR结合，并进一步招募IL-7R，形成复合体发挥功能，利用体外竞争结合实验，验证抗体阻断TSLP与TSLPR以及TSLPR-IL7R复合体结合的能力。将连接了Biotin的TSLP蛋白与不同浓度的抗体共孵育30min后，添加到包被了TSLPR-IL7R复合体的96孔板中，37℃孵育1h，加入辣根过氧化物酶标记Streptavidin使其显色，终止后在450nm处测OD值。如图1和表15所示，Q61-F阻断TSLP与TSLPR以及TSLPR-IL7R复合体结合的能力优于对照抗体Teze。
表15.抗TSLP人源化抗体阻断TSLP与TSLPR以及TSLPR-IL7R复合体结合的IC50浓度

实施例11.抗TSLP人源化抗体抑制Ba/F3细胞增殖
电转Ba/F3细胞，使之同时表达人TSLPR及IL-7R蛋白，并依赖TSLP进行增殖。测量抗TSLP抗体的活性是通过加入不同浓度的抗体，使之与培养基中添加的TSLP因子结合，从而抑制需依赖TSLP而生长的BaF3细胞的增殖来体现。在96孔细胞培养板中加入TSLP因子和不同浓度的抗体，每孔再加入1.5×104个细胞，在37℃，含5％CO2的培养箱中培养2天后，加入CCK8检测细胞的增殖能力，在450nm处测OD值。如图2和表16所示，Q61-F抑制Ba/F3细胞增殖的能力优于对照抗体Teze。
表16.抗TSLP人源化抗体抑制Ba/F3细胞增殖的IC50浓度
实施例12.抗TSLP人源化抗体降低TARC含量
TSLP抗体抑制对PBMC细胞分泌TARC(胸腺和激活调节的趋化因子，又称CCL17)，TARC可以与CCR4受体结合，介导炎症，癌症以及自身免疫相关疾病的发生。TSLP能诱导PBMC细胞产生趋化因子，如TARC，但抗TSLP抗体与TSLP的结合可抑制该趋化因子的产生，因此TARC的浓度检测可用于体外药效评估实验。在96孔细胞培养板中加入TSLP因子和不同浓度的抗体，每孔再加入8×105个PBMC细胞，在37℃，含5％CO2的培养箱中培养2天，用Human CCL17/TARC ELISA Kit试剂盒检测细胞上清液中TARC的浓度的结果。如图3所示，Q61-F、Q87-F与TEZE相比，抑制TARC分泌能力相当。
实施例13.抗TSLP人源化抗体的抗皮炎药效实验
动物说明
物种：小鼠
品种：B-hIL4hIL4RA mice
年龄：6-8周
重量：18-20g
来源：百奥赛图
饲养
将小鼠养在IVC(独立通气笼盒)中，提供灭菌水任其取用。除研究日之外，每日喂养小鼠两次(AM和PM)。在开始研究之前，评估动物的总体健康状况并记录体重。确定小鼠健康后进行研究。
AD(Atopic dermatitis，特应性皮炎)模型构建：按照图5，在第0天在小鼠右耳及背部均匀涂抹25μL 0.8％Oxazolone(恶唑酮)溶液使小鼠致敏，随后第7,9,11,14,16,18,21,23,25天在同一部位涂抹25μL 0.4％Oxazolone溶液进行激发。实验开始后一周称重两次以及测量小鼠耳朵厚度，从第6天开始皮下给药，各给药组的给药方案、给药频率、给药剂量及体积如表9所示，在第26天眼眶静脉丛采血，利用试剂盒MOUSE IgE ELISA KIT(依科赛)检测血清中IgE水平。
表17各实验组给药方案、给药频率、给药剂量及体积
如图6小鼠体重变化图所示，在整个抗皮炎药效实验过程中，各给药组的小鼠体重持续增加，增加幅度基本一致，说明各给药组的小鼠健康状况正常，给药对小鼠没有毒副作用；如图7各给药组小鼠耳朵厚度的对比结果，模型对照组(MC)小鼠的耳朵厚度，相较于空白对照组(NC)在不断增加，肉眼也可观察到耳朵皲裂增厚的现象，而各给药组，尤其是Q61-F组，明显缓解了小鼠耳朵增厚的程度，且药效与Teze相当且略优；如图8中各给药组小鼠血清中总IgE浓度对比结果，造模前，各组小鼠的本底血清IgE水平相似，并且维持在较低水平。造模后，MC组血清IgE相较于NC组明显增加，经Teze、Q61-F及Q187-F抗体治疗后，IgE水平 均大幅降低，药效相当。以上表明在动物体内，Q61-F抗体具有优于阳性药TEZE的抗皮炎药效。
实施例14.抗TSLP人源化抗体的抗哮喘药效实验
动物说明
物种：小鼠
品种：B-hTSLP/hTSLPR mice
年龄：8周
重量：16-22g
来源：百奥赛图
将小鼠养在IVC(独立通气笼盒)中，提供灭菌水任其取用。除研究日之外，每日喂养小鼠两次(AM和PM)。在开始研究之前，评估动物的总体健康状况并记录体重。确定小鼠健康后进行研究。
25只动物在Day-3按照体重随机分为5组，分别标记为G1～G5，每组5只动物。其中G1为未造模组，G2～G5为造模组，G2为阴性对照组，G3为受试品1组，G4为受试品2组，G5为受试品3组。
分别在第0、7、14天，G1组的每只动物，腹腔注射200μL的PBS致敏，G2～G5组的每只动物，腹腔注射200μL 200μg/mL的OVA致敏。在第21-25天，连续5天，G2～G5组动物每天通过雾化的方式，吸入2％的OVA进行激发，每次30min。第26天结束体内药效实验，取肺泡灌洗液，血清，肺脏组织用于后续的体外检测。
表18各实验组给药方案、给药频率、给药剂量及体积
图9是各给药组小鼠体重的对比结果图，结果显示，在整个药效实验的持续过程当中(26天)，小鼠体重平稳增加，且增加幅度与对照组相同，说 明小鼠基本健康状况正常(与日常观察到的小鼠精神状态良好，食欲正常现象相吻合)，未因造模及给药受到影响。
图10是各给药组小鼠血清中IgE含量的对比结果图，结果显示，造模组MC血清IgE相较于NC组明显增加，说明造模成功。经Teze、Q61-F及Q187-F抗体治疗后，IgE水平有所降低，说明在哮喘模型中，抗体可发挥药效，但Teze药效不明显，而Q61-F体现出一定药效，说明Q61-F的药效优于阳性药Teze。

Claims (27)

  1. 抗体或其抗原结合片段，其特异性地结合于TSLP，其包含重链可变区和/或轻链可变区，其中：
    1)所述重链可变区包含
    (i)氨基酸序列为SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88的肽段内所含有的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3；或
    (ii)相对于(i)中所述的三个重链互补决定区(HCDRs)的序列，在所述三个重链互补决定区(HCDRs)上共包含至少一个且不超过5个氨基酸突变(优选氨基酸置换，优选保守置换)的序列；
    和/或
    2)所述轻链可变区包含，
    (i)氨基酸序列为SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89的肽段内所含有的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3；或
    (ii)相对于(i)中所述的三个轻链互补决定区(LCDRs)的序列，在所述三个轻链互补决定区(LCDRs)上共包含至少一个且不超过5个氨基酸突变(优选氨基酸置换，优选保守置换)的序列。
  2. 抗体或其抗原结合片段，其特异性结合TSLP，其包含：
    氨基酸序列为SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88的肽段内所含有的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3；和/或
    氨基酸序列为SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89的肽段内所含有的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3。
  3. 抗体或其抗原结合片段，其特异性结合TSLP，其包含：
    a)重链互补决定区HCDR3，所述HCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:3、15、27、39、50、60、71和80，或所述HCDR3包含与如SEQ ID NO:3、15、27、39、50、60、71或80所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨 基酸序列；和/或
    b)轻链互补决定区LCDR3，所述LCDR3包含下述氨基酸序列所构成的组中的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:6、18、30、42、63和83，或所述LCDR3包含与如SEQ ID NO:6、18、30、42、63或83所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列。
  4. 如权利要求3所述的抗体或其抗原结合片段，其进一步包含：
    c)重链互补决定区HCDR1，所述HCDR1包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:1、13、25、37、58和78，或所述HCDR1包含与如SEQ ID NO:1、13、25、37、58或78所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；和/或
    d)轻链互补决定区LCDR1，所述LCDR1包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:4、16、28、40、61和81，或所述HCDR2包含与如SEQ ID NO:4、16、28、40、61或81所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
    优选地，其进一步包含：
    e)重链互补决定区HCDR2，所述HCDR2包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:2、14、26、38、49、59、70和79,或所述LCDR1包含与如SEQ ID NO:2、14、26、38、49、59、70或79所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；和/或
    f)轻链互补决定区LCDR2，所述LCDR2包含下述氨基酸序列所构成的组的任一氨基酸序列或由下述氨基酸序列所构成的组中的任一氨基酸序列组成：SEQ ID NO:5、17、29、41、51、62和82,或所述LCDR2包含与如SEQ ID NO:5、17、29、41、51、62和82所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列。
  5. 抗体或其抗原结合片段，其包含三个重链互补决定区(HCDRs)HCDR1、
    HCDR2与HCDR3；以及三个轻链互补决定区(LCDRs)LCDR1、LCDR2 与LCDR3，其中：
    HCDR1包含SEQ ID NO:1、13、25、37、58或78、或由其组成,或所述HCDR1包含与如SEQ ID NO:1、13、25、37、58、或78所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
    HCDR2包含SEQ I DNO:2、14、26、38、49、59、70或79、或由其组成，或所述HCDR2包含与如SEQ ID NO:2、14、26、38、49、59、70或79所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
    HCDR3包含SEQ ID NO:3、15、27、39、50、60、71或80、或由其组成，或所述HCDR3包含与如SEQ ID NO:3、15、27、39、50、60、71或80所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
    LCDR1包含SEQ ID NO:4、16、28、40、61或81、或由其组成，或所述LCDR1包含与如SEQ ID NO:4、16、28、40、61或81所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
    LCDR2包含SEQ ID NO:5、17、29、41、51、62或82、或由其组成，或所述LCDR2包含与如SEQ ID NO:5、17、29、41、51、62或82所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列；
    LCDR3包含SEQ ID NO:6、18、30、42、63或83、或由其组成，或所述LCDR3包含与如SEQ ID NO:6、18、30、42、63或83所示的氨基酸序列相比具有1个、2个或3个氨基酸突变(优选氨基酸置换，优选保守置换)的氨基酸序列。
  6. 如权利要求1或2所述的抗体或其抗原结合片段，其包含：
    (1)氨基酸序列如SEQ ID NO:7、19、31、43、52、64、72或84所示的肽段内所含的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3，和
    氨基酸序列如SEQ ID NO:8、20、32、44、53、65、73或85所示的肽段内所含的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3；
    (2)氨基酸序列如SEQ ID NO:9、21、33、45、54、66、74或86所示的肽段内所含的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3，和
    氨基酸序列如SEQ ID NO:10、22、34、46、55、67、75或87所示的肽段内所含的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3；
    (3)氨基酸序列如SEQ ID NO:11、23、35、47、56、68、76或88所示的肽段内所含的三个重链互补决定区(HCDRs)HCDR1、HCDR2与HCDR3，和
    氨基酸序列如SEQ ID NO:12、24、36、48、57、69、77或89所示的肽段内所含的三个轻链互补决定区(LCDRs)LCDR1、LCDR2与LCDR3。
  7. 如权利要求1-6任一项所述的抗体或其抗原结合片段，其包含：
    1)包含SEQ ID NO:1或由其组成的HCDR1，
    包含SEQ ID NO:2或由其组成的HCDR2，
    包含SEQ ID NO:3或由其组成的HCDR3，
    包含SEQ ID NO:4或由其组成的LCDR1，
    包含SEQ ID NO:5或由其组成的LCDR2，和
    包含SEQ ID NO:6或由其组成的LCDR3；或
    2)包含SEQ ID NO:13或由其组成的HCDR1，
    包含SEQ ID NO:14或由其组成的HCDR2，
    包含SEQ ID NO:15或由其组成的HCDR3，
    包含SEQ ID NO:16或由其组成的LCDR1，
    包含SEQ ID NO:17或由其组成的LCDR2，和
    包含SEQ ID NO:18或由其组成的LCDR3；或
    3)包含SEQ ID NO:25或由其组成的HCDR1，
    包含SEQ ID NO:26或由其组成的HCDR2，
    包含SEQ ID NO:27或由其组成的HCDR3，
    包含SEQ ID NO:28或由其组成的LCDR1，
    包含SEQ ID NO:29或由其组成的LCDR2，和
    包含SEQ ID NO:30或由其组成的LCDR3；或
    4)包含SEQ ID NO:37或由其组成的HCDR1，
    包含SEQ ID NO:38或由其组成的HCDR2，
    包含SEQ ID NO:39或由其组成的HCDR3，
    包含SEQ ID NO:40或由其组成的LCDR1，
    包含SEQ ID NO:41或由其组成的LCDR2，和
    包含SEQ ID NO:42或由其组成的LCDR3；或
    5)包含SEQ ID NO:25或由其组成的HCDR1，
    包含SEQ ID NO:49或由其组成的HCDR2，
    包含SEQ ID NO:50或由其组成的HCDR3，
    包含SEQ ID NO:28或由其组成的LCDR1，
    包含SEQ ID NO:51或由其组成的LCDR2，和
    包含SEQ ID NO:30或由其组成的LCDR3；或
    6)包含SEQ ID NO:58或由其组成的HCDR1，
    包含SEQ ID NO:59或由其组成的HCDR2，
    包含SEQ ID NO:60或由其组成的HCDR3，
    包含SEQ ID NO:61或由其组成的LCDR1，
    包含SEQ ID NO:62或由其组成的LCDR2，和
    包含SEQ ID NO:63或由其组成的LCDR3；或
    7)包含SEQ ID NO:25或由其组成的HCDR1，
    包含SEQ ID NO:70或由其组成的HCDR2，
    包含SEQ ID NO:71或由其组成的HCDR3，
    包含SEQ ID NO:28或由其组成的LCDR1，
    包含SEQ ID NO:51或由其组成的LCDR2，和
    包含SEQ ID NO:30或由其组成的LCDR3；或
    8)包含SEQ ID NO:78或由其组成的HCDR1，
    包含SEQ ID NO:79或由其组成的HCDR2，
    包含SEQ ID NO:80或由其组成的HCDR3，
    包含SEQ ID NO:81或由其组成的LCDR1，
    包含SEQ ID NO:82或由其组成的LCDR2，和
    包含SEQ ID NO:83或由其组成的LCDR3。
  8. 抗体或其抗原结合片段，其特异性结合于TSLP，其包含：
    重链可变区，所述重链可变区包含：SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86 或88；或与序列SEQ ID NO:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述重链可变区由SEQ ID NO：:7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88组成，或由与序列SEQ ID NO::7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成；或所述重链可变区包含与SEQ ID NO：7、9、11、19、21、23、31、33、35、43、45、47、52、54、56、64、66、68、72、74、76、84、86或88相比，具有一个或多个(优选不超过10个、更优选不超过5个)的氨基酸突变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列或由其组成,优选地，所述氨基酸突变不发生在重链互补决定区；和/或
    轻链可变区，所述轻链可变区包含：SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89；或与序列SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述轻链可变区由SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89组成，或由与序列SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成；或所述轻链可变区包含与SEQ ID NO:8、10、12、20、22、24、32、34、36、44、46、48、53、55、57、65、67、69、73、75、77、85、87或89相比，具有一个或多个(优选不超过10个、更优选不超过5个)的氨基酸突变(优选氨基酸置换，更优选氨基酸保守置换)的氨基酸序列，或由其组成，优选地，所述氨基酸突变不发生在轻链互补决定区。
  9. 如权利要求8所述的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区和轻链可变区的氨基酸序列对选自由下述氨基酸序列对所构成的组中的任一组：SEQ ID NO:7和SEQ ID NO:8、SEQ ID NO:9 和SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12、SEQ ID NO:19和SEQ ID NO:20、SEQ ID NO:21和SEQ ID NO:22、SEQ ID NO:23和SEQ ID NO:24、SEQ ID NO:31和SEQ ID NO:32、SEQ ID NO:33和SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36、SEQ ID NO:43和SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46、SEQ ID NO:47和SEQ ID NO:48、SEQ ID NO:52和SEQ ID NO:53、SEQ ID NO:54和SEQ ID NO:55、SEQ ID NO:56和SEQ ID NO:57、SEQ ID NO:64和SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67、SEQ ID NO:68和SEQ ID NO:69、SEQ ID NO:72和SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75、SEQ ID NO:76和SEQ ID NO:77、SEQ ID NO:84和SEQ ID NO:85、SEQ ID NO:86和SEQ ID NO:87、和SEQ ID NO:88和SEQ ID NO:89。
  10. 如权利要求1、2或6任一项所述的抗体或其抗原结合片段，其中每个CDR根据Kabat定义方案、Chothia定义方案、Abm定义方案、IMGT定义方案和/或Contact(接触)定义方案定义。
  11. 如权利要求10所述的抗体或其抗原结合片段，其中每个CDR根据Kabat定义方案或Chothia定义方案定义。
  12. 权利要求1至11中任一项所述的抗体或其抗原结合片段，其进一步包含恒定区，所述恒定区来自于IgG抗体、IgM抗体、IgA抗体、IgD抗体或IgE抗体，优选地，所述恒定区来自于IgG1抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
  13. 权利要求1至11中任一项所述的抗体或其抗原结合片段，其进一步包含重链恒定区，所述重链很定期包含：SEQ ID NO：90与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述重链恒定区由SEQ ID NO:90组成，或由与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成；和/或
    轻链恒定区，所述轻链恒定区包含：SEQ ID NO:90，或与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或所述轻链恒定区由SEQ ID NO:90组成，或由与序列SEQ ID NO:90有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列组成。
  14. 权利要求1至11中任一项所述的抗体或其抗原结合片段，其中所述 抗体是鼠源抗体、嵌合抗体、人源化抗体或人抗体。
  15. 权利要求1至11中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段是选自以下的抗体片段：Fab、Fab’、Fab’-SH、Fv、单链抗体(优选scFv)或(Fab’)2、单结构域抗体、双抗体(dAb)或线性抗体。
  16. 核酸，所述核酸编码如权利要求1-15中任一项的抗体或其抗原结合片段。
  17. 表达载体，其包含如权利要求16所述的核酸。
  18. 宿主细胞，其包含如权利要求17所述的表达载体。
  19. 生产抗体或其抗原结合片段的方法，其包含培养如权利要求18所述的宿主细胞，并从培养物中回收由所述宿主细胞表达的抗体或其抗原结合片段。
  20. 如权利要求19所述的方法，其中所述宿主细胞为原核细胞或真核细胞。
  21. 如权利要求19或20所述的方法，所述宿主细胞为大肠杆菌细胞、酵母细胞、昆虫细胞、植物细胞或哺乳动物细胞。
  22. 如权利要求21所述的方法，所述宿主细胞为中国仓鼠卵巢细胞(CHO)、CHO细胞变体、293细胞或NSO细胞。
  23. 一种药物组合物，其包含至少一种权利要求1-15中任一项所述的抗体或其抗原结合片段、和可药用赋形剂。
  24. 一种试剂盒，其包含至少一种权利要求1-15中任一项所述的抗体或其抗原结合片段、同源物及其衍生物。
  25. 权利要求1-15中任一项的抗体或抗原结合片段或权23所述的药物组合物在制备用于治疗和/或预防与TSLP相关的疾病的药物中的用途；优选地，所述与TSLP相关的疾病选自：哮喘或皮炎。
  26. 权利要求1-15中任一项所述的抗体或抗原结合片段或权23所述的药物组合物，其用于治疗和/或预防与TSLP相关的疾病，其中所述与TSLP相关的疾病选自：哮喘或皮炎。
  27. 一种治疗和/或预防与TSLP相关的疾病的方法，所述的方法包括向受试者施用权利要求1-15中任一项所述的抗体或抗原结合片段或权23所述的药物组合物，其中所述与TSLP相关的疾病选自：哮喘或皮炎。
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