WO2022253147A1 - 抗胸腺基质淋巴细胞生成素(tslp)抗体及其用途 - Google Patents

抗胸腺基质淋巴细胞生成素(tslp)抗体及其用途 Download PDF

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WO2022253147A1
WO2022253147A1 PCT/CN2022/095806 CN2022095806W WO2022253147A1 WO 2022253147 A1 WO2022253147 A1 WO 2022253147A1 CN 2022095806 W CN2022095806 W CN 2022095806W WO 2022253147 A1 WO2022253147 A1 WO 2022253147A1
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antibody
seq
tslp
variable region
chain variable
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French (fr)
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屈向东
潘琴
都业杰
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启愈生物技术(上海)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biomedicine, relates to an anti-TSLP antibody, and also relates to the application of the TSLP antibody.
  • Human thymine stromal lymphopoietin is an IL-7-like, short-chain type I cytokine composed of four helical bundles connected by three pairs of intrachain disulfide bonds, and is a key factor in IL-2 cells.
  • a member of the factor family, the coding gene is located on chromosome 5q22.1. It conducts signal transduction by binding to heterodimeric receptors, which are composed of IL-7R ⁇ subunits and a unique component TSLPR with homology to common ⁇ receptor-like chains, belonging to the hematopoietic factor receptor Only when the receptor complex TSLPR ⁇ /IL-7R ⁇ acts together can it have high affinity.
  • TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine and tonsil, as well as airway smooth muscle cells, lung fibroblasts and stromal cells. These cells produce TSLP in response to pro-inflammatory stimuli, and TSLP drives allergic inflammatory responses through its activity on a variety of innate immune cells, including dendritic cells, monocytes, and mast cells .
  • TSLP combines with its specific receptors TSLPR and IL-7R ⁇ to form a ternary complex to initiate signal transduction.
  • phosphorylation of Janus kinase 1 (JAK1) and JAK2 activates signal transducer and transcription factor (STAT) 1, STAT3, and STAT5, initiates pro-inflammatory signals, promotes DC maturation and activation, and induces functional type II helper T cells (Th2), regulatory T cells (Treg), and T follicular helper cells (Tfh), which regulate inflammatory processes in the skin, lung, and intestinal mucosal barriers.
  • Th2 immunity in TSLPR-deficient mice was low, but Th1 immunity was normal or even enhanced, suggesting that the amount of TSLPR is an important factor affecting the development of allergy.
  • TSLP is critical in the maturation of antigen-presenting cells and hematopoietic cells.
  • TSLP is distributed in a variety of immune cells (including DC, type II innate lymphocytes (ILC2), T cells, B cells, natural killer T cells (NKT), Treg cells, eosinophils, neutrophils, mast cells and macrophages) and non-immune cells (platelets and sensory neurons).
  • TSLP acts on a variety of cell lines, especially myeloid DC, by forming the TSLP-TSLPR-IL-7R ⁇ complex.
  • TSLP can activate human peripheral blood CD11c+DC, upregulate major histocompatibility complex (MHC) class II, OX40 ligand (OX40L/CD134L, CD252), CD54, CD80, CD83 and CD86, and activation marker DC-LAMP Expressed to promote the differentiation of naive CD4+ T cells (Th0) into Th2 cells.
  • MHC major histocompatibility complex
  • OX40L/CD134L, CD252 OX40 ligand
  • CD54 CD54
  • CD80 CD83 and CD86
  • activation marker DC-LAMP Expressed to promote the differentiation of naive CD4+ T cells (Th0) into Th2 cells.
  • TSLP failed to stimulate myeloid DCs to produce Th1-polarizing cytokines IL-12, pro-inflammatory cytokines TNF- ⁇ , IL-1 ⁇ , and IL-6, which are key features of TSLP in constructing a Th2 immune microenvironment.
  • TSLP-DCs enhance Th2 immune responses by activating Th2 effector memory cells and hindering FOXP3+ Treg production.
  • TSLP interacts with CD4+T cells, CD8+T cells and Treg cells to further promote Th2 cell proliferation and activation.
  • TSLP can still activate mast cells, innate lymphocytes, epithelial cells, macrophages, etc., cooperate with epithelial cell chemokines such as IL-25 and IL-33, and jointly promote Th2 cytokines (IL-4, IL-5 and IL- 13) Generate. In addition, TSLP promotes eosinophil activation and chemotaxis.
  • TSLP significantly delayed the apoptosis of eosinophils in a concentration-dependent manner, and induced the production of IL-6 and chemokines CXCL8 and CXCL1 by up-regulating the expression of CD18 and intercellular adhesion factor 1 (ICAM1) and down-regulating L-selectin, resulting in eosinophils Inflammation of granulocytes.
  • IAM1 intercellular adhesion factor 1
  • TSLP can balance innate immunity and adaptive immunity by acting on myeloid and lymphoid cells, and plays an important role in initiating and promoting allergic inflammation mediated by Th2 cells.
  • TSLP is overexpressed in some autoimmune diseases and inflammatory diseases/conditions, such as atopic dermatitis, food allergy, allergic rhinitis, Netherton syndrome, asthma, indicating that this cytokine plays an important role in these allergic inflammatory diseases important role in the pathogenesis of
  • transgenic overexpression of TSLP in the skin or lung and ablation of the gene target effect of TSLP negative regulators results in an allergic inflammatory disease that closely resembles human atopic dermatitis or asthma.
  • Atopic dermatitis is a chronic inflammatory skin disease.
  • the skin-mucosal barrier defect is the fuse of the allergic process, and TSLP can be used as an "alarm" for the development of the allergic process.
  • AD skin lesions provide target sites for allergen invasion, and multiple pattern recognition receptors in epithelial cells are triggered to activate nuclear factor ⁇ B (NF- ⁇ B) and generate reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • Promotes TSLP, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-25, IL-33 and other inflammatory factors are released.
  • TSLP promotes immature DC to upregulate the expression of OX40L and Notch ligands, downregulate the level of IL-12, secrete Th2 cell chemokines (CCL17 and CCL22, etc.), and then stimulate the proliferation of innate lymphocytes and produce Th2 cytokines.
  • High levels of TSLP can also directly stimulate transient receptor potential kinin 1 (TRPA1) positive skin sensory neurons, triggering strong itching and aggravating skin lesions.
  • TRPA1 transient receptor potential kinin 1
  • TSLP facilitates the migration, maturation, and activation of Langerhans cells (LC), promotes DC polarization, and induces Th2 immunity in situ.
  • LC Langerhans cells
  • Th2 immunity is likely to act as the initiator of the allergic process and initiate the systemic Th2 immune response.
  • Intradermal injection of TSLP can induce food allergy in mice, including the expansion of systemic basophils, the increase of serum-specific IgE levels and the accumulation of mast cells in the intestine, and promote the inflammatory response of the digestive tract.
  • reducing TSLP levels or depleting basophils reduced susceptibility to intestinal food allergy. This suggests that the TSLP-basophil axis may be a potential therapeutic target for food allergy.
  • TSLP stimulates DC maturation and activation, promotes the proliferation and differentiation of CD4+Th0 cells into Th2 cells, and secretes a large number of Th2 cytokines in peripheral blood and esophagus.
  • the combination of TSLP and NKT cells TSLPR can significantly induce NKT cells to recruit esophageal epithelium and produce a large amount of IL-13, which mediates milk allergy.
  • TSLP plays an important role in the differentiation of Foxp3+Treg cells.
  • TSLP plays an important role in the differentiation of Foxp3+Treg cells.
  • TSLP is crucial for maintaining the stability of intestinal environment.
  • Allergic rhinitis is an allergic disease dominated by upper respiratory tract Th2 immunity, in which epithelial cell-derived histamine-mediated inflammation plays an important role, and epithelial cell-derived cytokines (such as IL-33, IL -25 and TSLP) are key to initiating and driving allergic immune responses.
  • epithelial cell-derived cytokines such as IL-33, IL -25 and TSLP
  • H4R histamine receptor 4
  • Th2 cytokines such as IL-4, IL-5, and IL-13 can further activate B cells, eosinophils, and mast cells to form a systemic Th2 immune advantage and stimulate the allergic process.
  • reducing TSLP levels by blocking H4R reduced allergic inflammation.
  • Asthma is a chronic disease characterized by airway inflammation, clinically characterized by recurrent wheezing, shortness of breath, chest tightness, and coughing.
  • Americans approximately 300 million people suffer from asthma.
  • Existing therapeutic drugs include bronchodilators, glucocorticoids, combination preparations of the two (Seretide, Symbicort), leukotriene modulators, long-acting cholinergic receptor antagonists (Tiotropium bromide) , IgE antibodies, etc., cannot control the condition of all asthmatic patients.
  • TSLP-TSLPR The role of TSLP-TSLPR is mainly completed through the JAK-STAT signaling pathway. Studies suggest that the upregulation of TSLP will combine with TSLPR on DC cells to cause JAK activation, recruit the transcription factor STAT5, cause downstream signal transduction, and eventually lead to the activation of DC cells.
  • DC cell activation exhibits upregulation of co-stimulatory molecules (such as CD80, CD40, CD86) and secretion of chemokines (TARC/CCL17, MDC/CCL22, and I-309/CCL1), thereby providing an incentive for Th0 to Th2 cell differentiation.
  • co-stimulatory molecules such as CD80, CD40, CD86
  • chemokines TARC/CCL17, MDC/CCL22, and I-309/CCL1
  • chemokines TARC/CCL17, MDC/CCL22, and I-309/CCL1
  • anti-cytokine (IL-4, IL-13, IL-5) drugs only target specific inflammatory molecules that drive asthma inflammation, and are only suitable for certain types of severe asthma patients, namely sub group of patients, such as eosinophilic asthma.
  • TSLP is significantly different from targets such as IL4 and IL5.
  • TSLP acts in the early upstream of the inflammatory cascade and may be applicable to a wide range of patients with severe uncontrolled asthma.
  • Anti-TSLP monoclonal antibody can effectively block the effect of TSLP/TSLPR, reverse airway inflammation, prevent tissue structural changes, reduce airway hyperresponsiveness (AHR) and TGF- ⁇ 1 levels in mite dust-induced mouse asthma model.
  • Anti-TSLP monoclonal antibody effectively reduced the expression of Th2 factors (IL-4, IL-5, etc.) in serum protein-induced asthma model in mice.
  • the safety of anti-TSLP monoclonal antibody has also been fully confirmed in monkeys.
  • the anti-TSLP monoclonal antibody in clinical research has shown a good objective response rate in early clinical trials, effectively relieving the symptoms of the tested patients.
  • TSLP TSLP-infiltrating myeloid cells
  • IL-1a factor IL-1a factor
  • TSLP can further induce tumor cells to express anti-apoptotic protein BCL-2, so that tumor cells can be prevented from death. Therefore, TSLP is critical for tumor survival.
  • Breast cancer cells had a higher mortality rate in TSLP-free tumors compared with TSLP-exposed tumors. Further studies showed that blocking TSLP in breast cancer models significantly inhibited the growth of breast tumors and prevented their lung metastasis. Additionally, blocking TSLP prevented the growth of not only breast cancer tumors, but also many other tumors in which TSLP is elevated, such as pancreatic cancer, cervical cancer, and multiple myeloma.
  • an antibody binding to TSLP comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2 and HCDR3, said
  • the light chain variable region comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 12, and the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 13, SEQ ID NO: 18 or SEQ ID As shown in NO:19, the amino acid sequence of HCDR3 is shown in SEQ ID NO:14; the amino acid sequence of LCDR1 is shown in SEQ ID NO:15, and the amino acid sequence of LCDR2 is shown in SEQ ID NO:16 or SEQ ID NO: 20, the LCDR3 amino acid sequence is shown in SEQ ID NO: 17.
  • the amino acid sequences of HCDR and LCDR are defined according to Kabat.
  • the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, and wherein LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 15, Shown in SEQ ID NO:16 and SEQ ID NO:17.
  • the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 14, and wherein LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 15, Shown in SEQ ID NO:16 and SEQ ID NO:17.
  • the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 12, SEQ ID NO: 19 and SEQ ID NO: 14, and wherein LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 15, Shown in SEQ ID NO:16 and SEQ ID NO:17.
  • the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, and wherein LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO: 15, Shown in SEQ ID NO:20 and SEQ ID NO:17.
  • amino acid sequence of the heavy chain variable region of the TSLP antibody is shown in SEQ ID NO: 1, 3, 4, 5, 9 or 10.
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2, 6, 7, 8 or 11.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 2.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:11.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 9
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 7.
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 7.
  • the antibody comprises a heavy chain constant region and a light chain constant region.
  • the antibody comprises a heavy chain constant region selected from IgG1 subtype, IgG2 subtype, or IgG4 subtype, and the antibody comprises a light chain constant region selected from kappa subtype or lambda subtype.
  • the antibody comprises an IgG1 subtype heavy chain constant region and the antibody comprises a light chain constant region selected from a kappa subtype.
  • the IgG1 subtype constant region sequence is shown in SEQ ID NO: 23 or its mutant SEQ ID NO: 22, and the light chain constant region sequence is shown in SEQ ID NO: 21.
  • the antibody comprises a whole antibody that binds a TSLP antigen.
  • the antibody is an antigen-binding fragment of TSLP.
  • the antibodies are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, and scFv fragments.
  • the antibody is capable of binding human TSLP (e.g., SEQ ID NO: 28, 29, 34, or 35) and cynomolgus monkey TSLP (e.g., SEQ ID NO: 26 or 27).
  • human TSLP e.g., SEQ ID NO: 28, 29, 34, or 35
  • cynomolgus monkey TSLP e.g., SEQ ID NO: 26 or 27.
  • the antibody inhibits binding of human TSLP to a human TSLP receptor.
  • the antibody inhibits human TSLP and human TSLP receptor hTSLPR/IL7Ra (for example: a single-chain human TSLPR.IL7Ra fusion polypeptide of the amino acid sequence shown in SEQ ID NO: 32, 33 or SEQ ID NO: 30 and hTSLPR/IL7Ra knob/hole dimer formed by fusion of two chains of SEQ ID NO:31).
  • human TSLP and human TSLP receptor hTSLPR/IL7Ra for example: a single-chain human TSLPR.IL7Ra fusion polypeptide of the amino acid sequence shown in SEQ ID NO: 32, 33 or SEQ ID NO: 30 and hTSLPR/IL7Ra knob/hole dimer formed by fusion of two chains of SEQ ID NO:31.
  • nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of the first aspect.
  • a pharmaceutical composition which contains:
  • the pharmaceutical composition further contains other drugs for treating cancer (or tumor), autoimmune disease or inflammatory disease.
  • the TSLP-mediated disease is cancer (tumor), autoimmune disease or inflammatory disease.
  • the TSLP-mediated disease or condition comprises breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B-cell lymphoma, myeloma, asthma, idiopathic pulmonary Fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Netherton syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilia Cellular gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, psoriasis, chronic nephritis, chronic obstructive pulmonary disease, Systemic sclerosis, multiple sclerosis, keloids, ulcerative colitis, nasal polyposis, chronic
  • Figure 1 ELISA detection of chimeric antibody QP378379 binding to human TSLP protein.
  • Figure 2 ELISA identification of chimeric antibody QP378379 blocking human TSLP/TSLPR/IL7Ra protein binding.
  • Figure 3 ELISA identification of chimeric antibody QP378379 binding to cynomolgus monkey TSLP protein.
  • Figure 4 ELISA identification of humanized TSLP antibody binding to human TSLP protein.
  • Figure 5 ELISA identification of humanized anti-TSLP antibodies blocking the binding of human TSLP to TSLPR/IL7Ra.
  • Figure 6 ELISA identification of binding of humanized anti-TSLP antibody to cynomolgus monkey TSLP protein.
  • Figure 7 Reporter gene assay identified anti-TSLP antibody inhibits STAT5 signaling pathway assay.
  • Figure 8 ELISA identification of TSLP antibody binding to human TSLP protein.
  • Figure 9 ELISA identification of anti-TSLP antibodies blocking the binding of human TSLP to TSLPR/IL7Ra.
  • Figure 10 ELISA identification of binding of anti-TSLP antibodies to cynomolgus monkey TSLP protein.
  • Figure 11 Reporter gene assay identified anti-TSLP antibody inhibits STAT5 signaling pathway assay.
  • the "antibody” of the present invention includes not only intact antibodies, but also fragments, polypeptide sequences, and derivatives and analogs thereof that have antigen-binding activity.
  • the antigen-binding fragment refers to one or more portions of a full-length antibody that retain the ability to bind to an antigen (eg, HER2) and compete with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding portions.
  • an antigen eg, HER2
  • antigen-binding portions include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, Single-chain antibodies (for example, scFv), chimeric antibodies, which comprise at least a portion of an antibody sufficient to confer a polypeptide-specific antigen-binding ability.
  • an antigen-binding portion of an antibody e.g., the antibody fragment described above
  • a given antibody e.g., monoclonal antibody 2E12
  • the term "Fd fragment” means an antibody fragment consisting of VH and CH1 domains
  • the term “Fv fragment” means an antibody fragment consisting of VL and VH domains of a single arm of an antibody
  • dAb fragment means an antibody fragment consisting of Antibody fragments composed of VH domains (Ward et al., Nature 341:544-546 (1989))
  • the term "Fab fragment” means an antibody fragment composed of VL, VH, CL and CH1 domains
  • the term “F(ab' )2 fragment” means an antibody fragment comprising two Fab fragments connected by a disulfide bridge
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • variable domains of both the light chain (VL) and heavy chain (VH) portions are responsible for antigen recognition and specificity.
  • constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc.
  • the farther a constant region domain is from the antigen-binding site or amino terminus of an antibody the higher its number.
  • the CH3 and CL domains actually comprise the carboxyl termini of the heavy and light chains, respectively.
  • Chimeric antibody is an antibody molecule (or antigen-binding fragment thereof) wherein (a) the constant region or portion thereof has been altered, replaced or replaced such that the The antigen binding site (variable region) is linked to a different or altered type, effector function and/or species of constant region, or to an entirely different molecule (e.g., enzyme, toxin, hormone, growth factors, drugs, etc.); or (b) said variable region or part thereof is altered, replaced or replaced with a variable region having a different or altered antigen specificity.
  • mouse antibodies can be modified by substituting constant regions from human immunoglobulins for their constant regions. Due to replacement with human constant regions, the chimeric antibody can retain its specificity for recognizing an antigen while having reduced antigenicity in humans compared to the original mouse antibody.
  • Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the hypervariable regions of the receptor have been Substitution of residues in hypervariable regions (donor antibodies) with desired specificity, affinity, and capacity.
  • donor antibodies framework region residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance.
  • a humanized antibody will comprise substantially all of at least one, typically two, variable domains in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all Or substantially all of those FRs are human immunoglobulin lo sequences.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region.
  • Fc immunoglobulin constant region
  • Antibody affinity maturation in vitro is to simulate the in vivo maturation process of antibodies in vitro, that is, to use a certain antibody as a template for transformation, construct its mutant library, and screen the mutant library to obtain antibodies with greatly improved biological activity.
  • TSLP of the present invention includes variants, isoforms, homologs, orthologs and paralogs of TSLP.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies and humanized antibodies.
  • the TSLP-mediated disease of the present invention is not limited, as long as it is a disease related to TSLP.
  • the therapeutic response induced by the molecules of the present disclosure can be achieved by binding to human TSLP, and then inhibiting the binding of TSLP to its receptor, or killing overexpressed TSLP. cells.
  • autoimmune disease refers to diseases caused by the body's immune response to self-antigens resulting in damage to its own tissues, including but not limited to scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg -Strauss syndrome, Wegener's granulomatosis and pulmonary hemorrhage-nephritic syndrome, etc.
  • inflammatory disease refers to a general term for diseases in which inflammation is the main destroying factor. Inflammation, the biological response of tissues to noxious stimuli, is a lesion accompanied by tissue degeneration, circulatory disturbance, and fluid leakage.
  • inflammatory diseases include acute and chronic diseases including but not limited to asthma, hypersensitivity pneumonitis, atopic dermatitis, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis , chronic nephritis, Sjogren's syndrome, and multiple sclerosis.
  • the cancer (or tumor) mediated by TSLP of the present invention includes breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B-cell lymphoma, and myeloma.
  • Example 1 Cloning, Expression and Purification of TSLP and TSLP Receptor Recombinant Protein and Control Antibody Tezepelumab
  • Cloning design Design human TSLP, human TSLP (R127A, R130A), cynomolgus monkey TSLP, cynomolgus monkey TSLP (R127A, R130A), human TSLPR, human IL7Ra and other recombinant proteins, respectively, with His, flag, FC and other tags , for purification and identification. See Table 1 for the corresponding numbers of recombinant proteins and sequence numbers:
  • Transient expression the day before 293E cell transfection, the cells to be transfected were centrifuged to change the medium, and the cell density was adjusted to about 0.5 ⁇ 10 6 /ml. Prepare the plasmid and transfection reagent PEI. The amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1. The plasmid and PEI were mixed and allowed to stand for 15 minutes. The plasmid and PEI mixture was slowly added to 293E cells, placed in 8% CO2, 120 rpm, and cultured in a shaker at 37°C. On the fifth day of transfection, the cell supernatant was collected by centrifuging at 4700 rpm for 20 min in a horizontal centrifuge.
  • Purification of Protein A Use the equilibrium solution to pass through the column, at least 3CV, and the actual volume is 20ml. Ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, and the flow rate is 1ml/min; pass the supernatant of the culture solution after centrifugation through the column, and load 40ml of the sample , the flow rate is 0.33ml/min; use the balance liquid to pass through the column, at least 3CV, and the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the balance liquid, the flow rate is 0.33ml/min; use the eluent to pass through the column, and the UV280 rises
  • the elution peak (PAC-EP) was collected when the UV280 dropped to 15mAU, and the collection was stopped when the flow rate was 1ml/min. After the samples were collected, the PAC-EP was adjusted to neutrality with pH adjusting solution.
  • Example 2 Construction of BaF3-TSLPR-IL7Ra stably transfected cell line stably expressing TSLPR and IL7Ra
  • BaF3-TSLPR-IL7Ra that simultaneously expresses TSLPR and IL7Ra Stably transformed cell lines.
  • BaF3 cells (the original B cell line of mice) were purchased from Peking Union Medical College Hospital, and the plasmids pDNA3.1-TSLPR-IL-7Ra (QD1246) and PGL4.52 (promega) were co-transfected into BaF3 cells by electroporation.
  • Example 3 Hybridoma fusion screening and anti-human TSLP monoclonal antibody acquisition
  • mice Anti-human TSLP monoclonal antibody was generated by immunizing mice. The experiment used Balb/c white mice, female, 6 weeks old. Breeding environment: SPF grade. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12/12 hour light/dark cycle adjustment, a temperature of 20-25° C., and a humidity of 40-60%.
  • Cell fusion select mice with high antibody titer in serum for splenocyte fusion. Seventy-two hours before fusion, the selected mice were immunized with an intraperitoneal injection. Spleen lymphocytes were fused with myeloma Sp2/0 cells using optimized PEG-mediated fusion steps to obtain hybridoma cells. The fused hybridoma cells were resuspended with HAT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI), and distributed in 96-well cell culture plates (1 ⁇ 10 5 /150ul/ Wells), cultured at 37°C, 5% CO 2 .
  • HAT complete medium IMDM medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • the medium is HT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 250ul/well, 37°C , 5% CO 2 culture.
  • ELISA was used to detect the anti-TSLP antibody in the supernatant of the positive wells of the primary screening, which combined with the cynomolgus monkey TSLP recombinant protein QP313, and the clones that could block the combination of TSLP/TSLPR/IL7Ra were the anti-TSLP antibody positive clone wells.
  • the positive clones were expanded and transferred to 24/6-well plates in time, and the cell culture supernatants combined with human TSLP and cynomolgus monkey TSLP were detected again by ELISA, and the clone wells that could block the binding of TSLP/TSLPR/IL7Ra were obtained as The anti-TSLP antibody positive clone wells obtained the positive clone mab177A12.
  • the screening ELISA results are shown in Table 2.
  • the positive clone mab177A12 was subjected to 2-3 rounds of limiting dilution to single-cell clones, and the single-cell strains were cryopreserved.
  • Hybridoma monoclonal antibody sequencing to obtain antibody sequence extract mRNA from hybridoma-positive monoclonal cell lines, reverse-transcribe mRNA into cDNA, use cDNA as a template for PCR amplification, select PCR-positive clones for sequencing, and obtain the severity of monoclonal antibodies through sequence analysis chain variable region sequence.
  • SEQ ID NO: 1mab177A12 VH corresponds to HCDR1, HCDR2 and HCDR3 sequences are SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 2mab177A12 VL corresponds to LCDR1, LCDR2 and LCDR3 sequences are SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17; the amino acid sequences of HCDR and LCDR are defined in Table 3 according to the Kabat rules.
  • Cloning construction Using the sequence of the mouse monoclonal antibody obtained by sequencing the monoclonal antibody in Example 3 as a template, primers were designed to amplify the antibody VH/VK gene fragments by PCR.
  • the expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragment) was constructed and digested, and some special restriction endonucleases, such as BsmBI, were designed and constructed with different characteristics of recognition sequence and restriction site Expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragment). The vector was digested with BsmBI, and the gel was cut and recovered for later use.
  • the antibody VH/VK gene fragments amplified by PCR and the expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragments) recovered by digestion with BsmBI were added to DH5H competent cells at a ratio of 3:1. Ice bath at °C for 30 minutes, heat shock at 42°C for 90s, add 5 times the volume of LB medium, incubate at 37°C for 45min, coat LB-Amp plate, culture at 37°C overnight, pick single clones and send them for sequencing to obtain the target clones.
  • the light and heavy chain variable region sequences and protein expression numbers of each clone are shown in Table 4.
  • All antibody light chain variable regions in this table are fused with the kappa light chain constant region CL to form a chimeric antibody full-length light chain, and the antibody heavy chain variable region
  • the full-length heavy chain of the chimeric antibody is formed by fusing the constant region of human IgG1.
  • the sequences of CL and constant region of human IgG1 are as follows:
  • Transient expression adjust the cell density of HEK293E to 1 ⁇ 10 6 /ml, prepare the plasmid and transfection reagent PEI, the amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1.
  • the plasmid and PEI were mixed and allowed to stand for 15 minutes.
  • the plasmid and PEI mixture was slowly added to HEK293E cells, placed in 8% CO 2 , 120 rpm, and cultured in a shaker at 37°C. On the fifth day of transfection, the cell supernatant was collected by centrifuging at 4700 rpm for 20 min in a horizontal centrifuge.
  • Protein purification Protein A purification: Connect a purification column filled with 1ml Protein A filler to the SCG protein purification system; pass through the column with equilibrium solution 1 ⁇ PBS PH7.4, at least 3CV, and the actual volume is 20ml, to ensure that the pH of the solution flowing out of the final instrument and The conductance is consistent with the balance solution, and the flow rate is 1ml/min; after the cell culture solution is centrifuged in a refrigerated centrifuge at 4°C, the supernatant is passed through the column, the sample volume is 40ml, and the flow rate is 0.33ml/min; after the sample is loaded, use the balance solution 1 ⁇ PBS PH7.4 through the column, at least 3CV, the actual volume is 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, the flow rate is 0.33ml/min; use the eluent 50mM/L citric acid pH3.5 to pass through the column, UV280 Start to collect the
  • ELISA identification of chimeric antibody binding to human TSLP protein Coating: protein QP1328 (FC fusion of human TSLP) was diluted to 1 ⁇ g/ml with PBS, added to the ELISA plate according to the amount of 60 ⁇ l/well, and incubated at 4°C for 18-24h. Blocking: Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation Incubate different concentrations of human TSLP chimeric antibodies at room temperature for 1 hour, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST. Incubation of enzyme-labeled antibody: Enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 1:5000 times with blocking solution first, 60 ⁇ l/well, incubated at room temperature for 1 hour; TMB color development: use Biotek automatic plate washer to incubate Wash the ELISA plate, after washing 5 times with PBST, add the substrate chromogenic solution TMB according to the amount of 100 ⁇ l/well, and develop the color at room temperature in the dark for 10 minutes; Termination: After the color development is completed, quickly add the stop solution 1M H2SO4 according to the amount of 100 ⁇ L/well to terminate reaction.
  • HRP enzyme-labeled antibody
  • TMB color development use Biotek automatic plate washer to incubate Wash the ELISA plate, after
  • Reading On a microplate reader, measure the OD value at a wavelength of 450nm. The results were analyzed using Graphpad Prism software analysis for data entry and analysis. The results are shown in Figure 1, the chimeric antibody QP378379 binds to human TSLP recombinant protein.
  • ELISA identification of chimeric antibody blocking human TSLP/TSLPR/IL7Ra protein binding Coating: protein QP1171 (FC fusion protein of human TSLPR/IL7Ra) was diluted to 2 ⁇ g/ml with PBS, and added to the ELISA plate according to the amount of 60 ⁇ l/well, Incubate at 4°C for 18-24h. Blocking: Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation mix different concentrations of human TSLP chimeric antibody and 0.02ug/ml Biotin-QP1005 (biotinylated human TSLP FC fusion protein) in a 1:1 ratio, add to the blocked ELISA plate, 60 ⁇ l/well , at room temperature for 1 h, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST. Incubate the enzyme-labeled streptavidin (HRP) and dilute it 5000 times with blocking solution first, 60 ⁇ l/well, incubate at room temperature for 1 hour; TMB color development: use the Biotek automatic plate washer to wash the incubated ELISA plate, and wash it with PBST.
  • HRP enzyme-labeled streptavidin
  • ELISA identification of chimeric antibody binding to cynomolgus monkey TSLP protein coating: protein QP313 (FC fusion of cynomolgus TSLP) was diluted to 1 ⁇ g/ml with PBS, added to the ELISA plate according to the amount of 60 ⁇ l/well, and incubated at 4°C for 18- 24h.
  • Blocking Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation Incubate different concentrations of human TSLP chimeric antibodies at room temperature for 1 hour, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST. Incubation of enzyme-labeled antibody: Enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 1:5000 times with blocking solution first, 60 ⁇ l/well, incubated at room temperature for 1h; TMB color development: use Biotek automatic plate washer to incubate Wash the ELISA plate, after washing 5 times with PBST, add the substrate chromogenic solution TMB according to the amount of 100 ⁇ l/well, and develop the color at room temperature in the dark for 10 minutes; Termination: After the color development is completed, quickly add the stop solution 1M H2SO4 according to the amount of 100 ⁇ L/well to terminate reaction.
  • HRP enzyme-labeled antibody
  • TMB color development use Biotek automatic plate washer to incubate Wash the ELISA plate, after washing
  • Reading On a microplate reader, measure the OD value at a wavelength of 450nm. The results were analyzed using Graphpad Prism software analysis for data entry and analysis. The results are shown in Figure 3, the chimeric antibody QP378379 binds to cynomolgus monkey TSLP protein.
  • the heavy and light chain variable region germline genes with high homology to QP378379 were selected as templates, and the CDRs of the mouse antibody were transplanted into In the corresponding human template, the sequence of the variable region is formed in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues for back mutation combination. The amino acid residues are identified and annotated by the Kabat numbering system.
  • Design primers for PCR to build the VH/VK gene fragments of each humanized antibody, and then perform homologous recombination with the expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragments) to construct the antibody full-length expression vector VH- CH1-FC-pQD/VK-CL-pQD.
  • pQD signal peptide and constant region gene
  • the expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragment) was constructed and digested, and some special restriction endonucleases, such as BsmBI, were designed and constructed with different characteristics of recognition sequence and restriction site Expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragment). The vector was digested with BsmBI, and the gel was cut and recovered for later use. Recombinant construction of expression vector VH-CH1-FC-pQD/VK-CL-pQD.
  • VH/VK contains gene fragments required for recombination and expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) Fragments) were added to DH5H competent cells at a ratio of 3:1, ice-bathed at 0°C for 30min, heat-shocked at 42°C for 90s, added 5 times the volume of LB medium, incubated at 37°C for 45min, coated on LB-Amp plates, and incubated overnight at 37°C , pick single clones and send them for sequencing to obtain the clones of each purpose.
  • pQD with signal peptide and constant region gene (CH1-FC/CL) Fragments
  • Table 5 The clone numbers and proteins of the humanized construction of the QP378379 antibody.
  • the cell density was adjusted to 1 ⁇ 10 6 /ml, and the plasmid and transfection reagent PEI were prepared.
  • the amount of plasmid to be transfected was 100ug/100ml cells, and the mass ratio of PEI to plasmid was 2:1.
  • the plasmid and PEI were mixed and allowed to stand for 15 minutes.
  • the plasmid and PEI mixture was slowly added to HEK293E cells, placed in 8% CO 2 , 120 rpm, and cultured in a shaker at 37°C. On the fifth day of transfection, the cell supernatant was collected by centrifuging at 4700 rpm for 20 min in a horizontal centrifuge.
  • the purification column filled with 1ml ProteinA filler to the SCG protein purification system; use the balance solution 1 ⁇ PBS PH7.4 to pass through the column, at least 3CV, and the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the balance solution, and the flow rate 1ml/min; after the cell culture solution was centrifuged in a refrigerated centrifuge at 4°C, the supernatant was passed through the column, the loading volume was 40ml, and the flow rate was 0.33ml/min; 3CV, the actual volume is 20ml, to ensure that the pH and conductance of the solution flowing out of the final instrument are consistent with the equilibrium solution, the flow rate is 0.33ml/min; the eluent is passed through the column with 50mM/L citric acid pH3.5, and the eluent is collected when the UV280 rises to 15mAU.
  • Peak off (PAC-EP), stop collecting when UV280 drops to 15mAU, flow rate 1ml/min. After the sample collection is completed, adjust the PAC-EP to neutral with pH8.0 2M/L Tris-HCl adjustment solution. The ultrafiltration tube is centrifuged to replace the buffer with PBS. SDS-PAGE and HPLC-SEC identify the protein purity, the purity is greater than 95 % protein spare.
  • ELISA identification of humanized TSLP antibody binding to human TSLP protein Coating: protein QP1328 (FC fusion of human TSLP) was diluted to 1 ⁇ g/ml with PBS, added to the ELISA plate according to the amount of 60 ⁇ l/well, and incubated at 4°C for 18-24h. Blocking: Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation Incubate different concentrations of humanized anti-TSLP antibodies at room temperature for 1 hour, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST.
  • Incubation of enzyme-labeled antibody Enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 5000 times with blocking solution first, 60 ⁇ l/well, incubated at room temperature for 1 hour; TMB color development: use Biotek automatic plate washer to wash the incubated ELISA plate After washing with PBST for 5 times, add the substrate chromogenic solution TMB according to the dosage of 100 ⁇ l/well, and develop the color at room temperature in the dark for 10 minutes; termination: after the completion of the color development, quickly add the stop solution 1M H2SO4 according to the dosage of 100 ⁇ L/well to terminate the reaction.
  • Reading On a microplate reader, measure the OD value at a wavelength of 450nm. The results were analyzed using Graphpad Prism software analysis for data entry and analysis. The results are shown in Figure 4, the different variants of the humanized anti-TSLP antibody can bind to the human TSLP recombinant protein.
  • protein QP1171 FC fusion protein of human TSLPR/IL7Ra
  • PBS PBS
  • Blocking Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation mix different concentrations of human TSLP chimeric antibody and 0.02ug/ml Biotin-QP1006 (biotinylated human TSLP FC fusion protein) in a 1:1 ratio, add to the blocked ELISA plate, 60 ⁇ l/well , at room temperature for 1 h, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST.
  • Biotin-QP1006 biotinylated human TSLP FC fusion protein
  • Protein QP313 FC fusion of cynomolgus monkey TSLP
  • PBS protein QP313
  • Blocking Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation Incubate different concentrations of human TSLP chimeric antibodies at room temperature for 1 hour, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST.
  • Enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 1:5000 times with blocking solution first, 60 ⁇ l/well, incubated at room temperature for 1h; TMB color development: use Biotek automatic plate washer to incubate Wash the ELISA plate, after washing 5 times with PBST, add the substrate chromogenic solution TMB according to the amount of 100 ⁇ l/well, and develop the color at room temperature in the dark for 10 minutes; Termination: After the color development is completed, quickly add the stop solution 1M H2SO4 according to the amount of 100 ⁇ L/well to terminate reaction. Reading: On a microplate reader, measure the OD value at a wavelength of 450nm. The results were analyzed using Graphpad Prism software analysis for data entry and analysis. The results are shown in Figure 6, all variants of the humanized anti-TSLP antibody bind to the cynomolgus monkey TSLP protein.
  • Humanized QP420422 was constructed into phagemid vectors in scFv mode (VH-(GGGGS)3-VL) respectively, and used as wild-type sequences (that is, obtained by screening relative to affinity maturation) mutant sequence).
  • Overlap PCR over-lap PCR was used to splice VH, (GGGGS)3 linker, and VL, and the NcoI and NotI restriction sites were used to ligate into the phagemid vector.
  • Construction of phage display library use the constructed wild-type scFv as a template, design a mutation primer for each CDR, and use degenerate primers to introduce mutations in all CDR regions to construct a mutation library.
  • the PCR fragment was digested with NcoI and NotI, connected to the phagemid vector, and finally electrotransformed into Escherichia coli TG1. Degenerate primers for each CDR create an independent library.
  • ELISA detection After comparing and analyzing the sequenced clones and removing redundant sequences, the non-redundant sequences were converted into full-length IG (hIgG1, ⁇ ) for mammalian cell 293E expression. The full-length IG after affinity purification was tested by ELISA.
  • the light chain CDR2 mutation with QD422 as the parent is clone QD3625 as shown in Table 7:
  • ELISA identification antibody binding to human TSLP protein Coating: protein QP1328 (FC fusion of human TSLP) was diluted to 1 ⁇ g/ml with PBS, added to the ELISA plate according to the amount of 60 ⁇ l/well, and incubated at 4°C for 18-24h. Blocking: Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour. Incubation of antibodies: Incubate anti-TSLP antibodies at different concentrations for 1 hour at room temperature, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST.
  • Enzyme-labeled antibody Goat Anti-Fab needs to be diluted 5000 times with blocking solution first, 60 ⁇ l/well, incubated at room temperature for 1 hour; TMB color development: use Biotek automatic plate washer to wash the incubated ELISA plate After washing with PBST for 5 times, add the substrate chromogenic solution TMB according to the dosage of 100 ⁇ l/well, and develop the color at room temperature in the dark for 10 minutes; termination: after the completion of the color development, quickly add the stop solution 1M H2SO4 according to the dosage of 100 ⁇ L/well to terminate the reaction. Reading: On a microplate reader, measure the OD value at a wavelength of 450nm. The results were analyzed using Graphpad Prism software analysis for data entry and analysis.
  • protein QP1171 FC fusion protein of human TSLPR/IL7Ra
  • PBS PBS
  • Blocking Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation mix different concentrations of human TSLP chimeric antibody and 0.02ug/ml Biotin-QP1006 (biotinylated human TSLP FC fusion protein) in a 1:1 ratio, add to the blocked ELISA plate, 60 ⁇ l/well , at room temperature for 1 h, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST.
  • Biotin-QP1006 biotinylated human TSLP FC fusion protein
  • Protein QP313 FC fusion of cynomolgus monkey TSLP
  • PBS protein QP313
  • Blocking Use the Biotek automatic plate washer to wash the incubated ELISA plate and wash it twice with PBS. After washing, add 200 ⁇ l/well of 3% BSA blocking solution and block at room temperature for 1 hour.
  • Antibody incubation Incubate different concentrations of human TSLP chimeric antibodies at room temperature for 1 hour, use a Biotek automatic plate washer to wash the incubated ELISA plate, and wash 3 times with PBST.
  • Enzyme-labeled antibody Goat Anti-Fab needs to be diluted 1:5000 times with blocking solution first, 60 ⁇ l/well, incubated at room temperature for 1h; TMB color development: use Biotek automatic plate washer to incubate Wash the ELISA plate, after washing 5 times with PBST, add the substrate chromogenic solution TMB according to the amount of 100 ⁇ l/well, and develop the color at room temperature in the dark for 10 minutes; Termination: After the color development is completed, quickly add the stop solution 1M H2SO4 according to the amount of 100 ⁇ L/well to terminate reaction. Reading: On a microplate reader, measure the OD value at a wavelength of 450nm. The results were analyzed using Graphpad Prism software analysis for data entry and analysis. The results are shown in Figure 10, all variants of the anti-TSLP antibody bind to the cynomolgus monkey TSLP protein.
  • Anti-TSLP antibodies QP36110422, QP36160422, QP04203625 have better binding affinity to human TSLP protein than the control antibody AMG157.
  • Anti-TSLP antibodies QP04203625, QP36110422, and QP36160422 can all inhibit the downstream STAT5 signaling pathway caused by TSLP and its receptor binding, and the inhibitory IC50 value is better than that of the control antibody QP119120 (AMG157).

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Abstract

提供了人TSLP抗体及其医药用途。还提供了包含所述抗体CDR区鼠源抗体、嵌合抗体、人源化抗体、包含人TSLP抗体的药物组合物，以及所述抗体在制备用于治疗TSLP介导的疾病的药物中的用途。

Description

抗胸腺基质淋巴细胞生成素(TSLP)抗体及其用途 技术领域
本发明属于生物医药领域，涉及一种抗TSLP的抗体，本发明还涉及这种TSLP抗体的用途。
背景技术
人胸腺基质淋巴细胞生成素(thymine stromal lymphopoietin，TSLP)是一种IL-7样，由三对链内二硫键连接形成的四螺旋束组成短链的I型细胞因子，是IL-2细胞因子家族的一员，编码基因位于5q22.1染色体。其通过与异二聚体受体结合进行信号传导，该异二聚体受体由IL-7Rα亚基及与常见γ受体样链具有同源性的独特组分TSLPR组成，属于造血因子受体家族成员，只有受体复合物TSLPRα/IL-7Rα共同作用时才可具备高亲和力。TSLP由胸腺、肺、皮肤、肠及扁桃体中的上皮细胞以及气道平滑肌细胞、肺纤维母细胞及基质细胞表达。这些细胞响应于促炎性刺激产生TSLP，且TSLP通过其对于多种先天性免疫细胞的活性而驱动过敏性炎性反应，所述先天性免疫细胞包括树突状细胞、单核细胞及肥大细胞。
TSLP与其特异性受体TSLPR及IL-7Rα结合，形成三元复合物，启动信号传导。比如，通过Janus激酶1(JAK1)、JAK2磷酸化，激活信号转导和转录因子(STAT)1、STAT3、STAT5，启动促炎信号，促进DC成熟和活化，诱导功能性Ⅱ型辅助性T细胞(Th2)、调节性T细胞(Treg)和滤泡辅助T细胞(Tfh)表达，调节皮肤、肺和肠道黏膜屏障的炎症过程。TSLPR缺陷小鼠Th2免疫较低，但Th1免疫正常甚或增强，提示TSLPR数量是影响过敏症发展的重要因素。
TSLP在抗原提呈细胞和造血细胞成熟中至关重要。TSLP分布于多种免疫细胞(包括DC、Ⅱ型固有淋巴细胞(ILC2)、T细胞、B细胞、自然杀伤T细胞(NKT)、Treg细胞、嗜酸粒细胞、中性粒细胞、肥大细胞和巨噬细胞)和非免疫细胞(血小板和感觉神经元)。TSLP通过形成TSLP-TSLPR-IL-7Rα复合体而作用于多种细胞系，特别是髓样DC。TSLP可激活人外周血CD11c+DC，上调主要组织相容性复合物(MHC)Ⅱ类、OX40配体(OX40L/CD134L，CD252)、CD54、CD80、CD83和CD86以及激活标记物DC-LAMP的表达，促进幼稚型CD4+T细胞(Th0)分化为Th2细胞。
尤其，TSLP不能刺激髓系DC产生Th1极化细胞因子IL-12、促炎细胞因子TNF-α、IL-1β和IL-6，这是TSLP构建Th2免疫微环境的关键特征。TSLP-DC通过激活Th2效应记忆细胞并阻碍FOXP3+Treg产生，增强Th2免疫应答。TSLP与CD4+T细胞、CD8+T细胞和Treg细胞交互作用，进一步促进Th2细胞增殖和活化。TSLP尚可激活肥大细胞、固有淋巴细胞、上皮细胞、巨噬细胞等，协同IL-25、IL-33等上皮细胞趋化因子，共同促进Th2细胞因子(IL-4、IL-5和IL-13)生成。此外，TSLP可促进嗜酸粒细胞 激活和趋化。TSLP以浓度依赖方式显著延缓嗜酸粒细胞凋亡，通过上调CD18和细胞间黏附因子1(ICAM1)表达、下调L-选择素，诱导IL-6和趋化因子CXCL8、CXCL1生成，导致嗜酸粒细胞炎症。
TSLP能通过对髓样和淋巴样细胞进行作用，从而平衡体内天然免疫和适应性免疫，在启动及促进由Th2细胞介导的过敏性炎症中扮演着重要的角色。
TSLP在一些自身免疫性疾病及炎性疾病/病症中过表达，例如特应性皮炎、食物过敏、过敏性鼻炎、内瑟顿综合征、哮喘，指示该细胞因子在这些变应性炎性疾病的发病机制中的重要作用。在所述动物模型中，TSLP在皮肤或肺中的转基因过表达以及TSLP负调节子的基因靶效应的去除会导致与人特应性皮炎或哮喘非常类似的变应性炎性疾病。
特应性皮炎是一种慢性炎症性皮肤病。皮肤黏膜屏障缺损是过敏进程的导火线，而TSLP则可作为过敏进程发生发展的"警报"。首先，AD皮损为过敏原入侵提供了靶位点，上皮细胞内多种模式识别受体被触发，引起核因子κB(NF-κB)激活，产生活性氧(ROS)。通过产生损伤相关分子模式(DAMP)或促炎细胞因子(如IL-1α、高迁移率族蛋白B1(HMGB1)、尿酸、三磷酸腺苷或S100蛋白)，促进TSLP、粒细胞巨噬细胞集落刺激因子(GM-CSF)、IL-25、IL-33等炎症因子释放。TSLP促进未成熟DC上调OX40L和Notch配体的表达，下调IL-12水平，分泌Th2细胞趋化因子(CCL17和CCL22等)，继而刺激固有淋巴细胞增殖并产生Th2细胞因子。高水平TSLP还可直接刺激瞬时受体电位激肽1(TRPA1)阳性皮肤感觉神经元细胞，触发强劲瘙痒且加重皮损。于是，共同构建了促进过敏进程的Th2优势免疫微环境。
食物过敏，TSLP有利于朗格罕细胞(LC)的迁移、成熟和激活，促进DC极化，引起原位Th2免疫。同时，TSLP极可能作为过敏进程的启动因子，启动全身Th2免疫应答。皮内注射TSLP可诱发小鼠食物过敏，包括全身嗜碱粒细胞扩增、血清特异性IgE水平增高和肥大细胞肠内蓄积，促进消化道炎症反应。相反，降低TSLP水平或去除嗜碱粒细胞，则肠道食物过敏的易感性下降。这提示TSLP-嗜碱粒细胞轴可能是食物过敏潜在的治疗靶点。另外，TSLP介导食物过敏的潜在靶细胞还有DC、NKT细胞和Treg细胞。TSLP刺激DC成熟和激活，促进CD4+Th0细胞向Th2细胞增殖和分化，并且在外周血和食管中分泌大量Th2细胞因子。TSLP与NKT细胞TSLPR结合，可显著诱导NKT细胞募集食管上皮并产生大量IL-13，介导牛乳过敏。同时，TSLP在Foxp3+Treg细胞分化中发挥重要作用。而且，当TSLP水平波动会导致肠道免疫功能失衡，揭示TSLP对于维持肠道内环境稳定至关重要。
过敏性鼻炎是以上呼吸道Th2免疫为主的变应性疾病，其中上皮细胞源性组胺介导的炎症发挥重要作用，固有淋巴细胞ILC2产生的上皮细胞源性细胞因子(如IL-33、IL-25和TSLP)是启动和驱动过敏性免疫应答的关键。研究结果显示，致敏小鼠的鼻黏膜中组胺受体4(H4R)的表达增加，H4R作为一种潜在的DC激活和T细胞极化的调节剂，可诱导鼻上皮细胞产生TSLP，激活DC上OX40L信号，促进Th2免疫应答。IL-4、IL-5和IL-13等Th2细胞因子可进一步活化B细胞、嗜酸粒细胞和肥大细胞，构成全身 Th2免疫优势，激发过敏进程。与之相反，通过阻断H4R降低TSLP水平，则能减轻过敏性炎症。
哮喘是一种以气道炎症为特征的慢性疾病，临床特征表现为反复发作的喘息、气促、胸闷和咳嗽等症状。在全球范围内，大约有3亿人患有哮喘。近二十年来全球哮喘患病率大约每年以1％的速度递增。现有的治疗药物包括支气管扩张剂、糖皮质激素、两者的联合制剂(舒利迭、信必可)、白三烯调节剂、长效胆碱能受体拮抗剂(噻托溴胺)、IgE抗体等，都不能控制所有哮喘患者病情。
大量研究证明约2/3的严重哮喘表现为Th2类细胞因子过表达，TSLP是引起Th2类细胞因子过表达的一个重要因子。TSLP-TSLPR作用主要是通过JAK-STAT信号通路完成的。研究认为TSLP的上调，会与DC细胞上的TSLPR结合引起JAK活化，招募转录因子STAT5，引起下游信号转导，最终导致DC细胞的活化。DC细胞活化表现出共刺激分子的表达上调(如CD80、CD40、CD86)和趋化因子的分泌(TARC/CCL17、MDC/CCL22和I-309/CCL1)，从而提供给Th0向Th2细胞分化的有利微环境，引导Th2细胞为主的炎症反应，且伴随因子(IL-4、IL-13、IL-5)释放。TSLP转基因鼠易受特异抗原诱导而发生哮喘，而TSLP受体敲除的小鼠症状则明显减轻。从哮喘和炎症的发生机制分析，抗细胞因子(IL-4、IL-13、IL-5)药物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。TSLP与IL4、IL5等靶点明显不同，TSLP在炎症级联反应的早期上游活动，可能适用于广泛的重症不受控哮喘患者。
研究人员对于靶向TSLP的药物进行了大量的探索和研究。在螨尘诱导的小鼠哮喘模型中抗TSLP单克隆抗体可有效阻断TSLP/TSLPR作用，逆转气道炎症，预防组织结构改变，降低气道高反应性(AHR)以及TGF-β1水平。在血清蛋白诱导小鼠哮喘模型中，抗TSLP单克隆抗体有效地降低了Th2类因子(IL-4、IL-5等)的表达。抗TSLP单克隆抗体的安全性在猴体内也得到充分证实。而且，临床在研的抗TSLP单克隆抗体在早期临床中显示出良好的客观反应率，有效缓解受试患者的病症。
另外，研究人员发现在许多不同类型的肿瘤中，TSLP水平升高。肿瘤募集免疫细胞到肿瘤，通过表达IL-1a因子诱导肿瘤浸润性骨髓细胞高表达TSLP。而TSLP能进一步诱导肿瘤细胞表达抗凋亡蛋白BCL-2，从而使肿瘤细胞免于死亡。因此，TSLP对于肿瘤的生存至关重要。与TSLP暴露的肿瘤相比，乳腺癌细胞在不含TSLP的肿瘤中具有更高的死亡率。进一步研究表明，阻断乳腺癌模型中的TSLP可以显著抑制乳腺肿瘤的生长并阻止其肺转移。另外，阻断TSLP不仅可以预防乳腺癌肿瘤的生长，还包括许多TSLP升高的其他肿瘤，如胰腺癌，宫颈癌和多发性骨髓瘤。
因此，鉴于结合TSLP抗体具有广泛的适用性，基于临床需求，探索和研发抗TSLP的抗体具有重要的生物学和医学意义。
发明的公开
在本发明的第一方面，提供了一种结合TSLP的抗体，其包含重链可变区和轻链可变区，所述重链可 变区包含互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含互补决定区LCDR1、LCDR2和LCDR3，其中所述HCDR1氨基酸序列如SEQ ID NO:12所示，所述HCDR2氨基酸序列如SEQ ID NO:13、SEQ ID NO:18或SEQ ID NO:19所示，所述HCDR3氨基酸序列如SEQ ID NO:14所示；所述LCDR1氨基酸序列如SEQ ID NO:15所示，所述LCDR2氨基酸序列如SEQ ID NO:16或SEQ ID NO:20所示，所述LCDR3氨基酸序列如SEQ ID NO:17所示。其中HCDR和LCDR氨基酸序列根据Kabat定义。
在一些实施方案中，所述HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示，且其中LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示。
在一些实施方案中，所述HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:18和SEQ ID NO:14所示，且其中LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示。
在一些实施方案中，所述HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:19和SEQ ID NO:14所示，且其中LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示。
在一些实施方案中，所述HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示，且其中LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:20和SEQ ID NO:17所示。
在一些实施方案中，所述TSLP抗体的重链可变区的氨基酸序列如SEQ ID NO:1、3、4、5、9或者10所示。
在一些实施方案中，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2、6、7、8或者11所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，所述抗体的轻链可 变区的氨基酸序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:6所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:8所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:11所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:9所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
在一些实施方案中，所述抗体包含重链恒定区和轻链恒定区。
在一些实施方案中，所述抗体包含选自IgG1亚型、IgG2亚型或IgG4亚型的重链恒定区，且所述抗体包含选自κ亚型或者λ亚型的轻链恒定区。
在一些实施方案中，所述抗体包含IgG1亚型重链恒定区，且所述抗体包含选自κ亚型的轻链恒定区。
在一些实施方案中，所述IgG1亚型恒定区序列如SEQ ID NO:23或其突变体SEQ ID NO:22所示，且所述轻链恒定区序列如SEQ ID NO:21所示。
在一些实施方案中，所述抗体包含结合TSLP抗原的全抗体。
在一些实施方案中，所述抗体为TSLP的抗原结合片段。
在一些实施方案中，所述抗体为Fab片段、Fab’片段、F(ab’)2片段、Fv片段和scFv片段。
在一些实施方案中，所述抗体能够结合人TSLP(例如：SEQ ID NO:28、29、34或者35)和食蟹猴TSLP(例如SEQ ID NO:26或者27)。
在一些实施方案中，所述抗体抑制人TSLP与人TSLP受体结合。
在一些实施方案中，所述抗体抑制人TSLP与人TSLP受体hTSLPR/IL7Ra(例如：SEQ ID NO:32、33所示氨基酸序列的单链人TSLPR.IL7Ra融合多肽或者SEQ ID NO:30和SEQ ID NO:31两条链融合而成的hTSLPR/IL7Ra knob/hole二聚体)结合。
在本发明的第二方面，提供了核酸分子，其编码第一方面所述的抗体或其抗原结合片段。
在本发明的第三方面，提供了一种药物组合物，所述药物组合物含有：
(a)本发明第一方面所述的TSLP抗体；和
(b)可药用的赋形剂、稀释剂或药学上可接受的载体。
在一些实施方案中，所述的药物组合物中还含有治疗癌症(或肿瘤)、自身免疫性疾病或炎性疾病的其他药物。
在本发明的第四方面，提供了第一方面所述的抗体或者第三方面所述的药物组合物在制备用于预防或治疗TSLP介导的疾病的药物中的用途。
在一些实施方案中，所述TSLP介导的疾病为癌症(肿瘤)、自身免疫疾病或炎性疾病。
在一些实施方案中，所述TSLP介导的疾病或病症包含乳腺癌、胰腺癌、胃癌、宫颈癌、结直肠癌、肺癌、黑色素瘤、B细胞淋巴瘤、骨髓瘤、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、系统性红斑狼疮、类风湿性关节炎、克罗恩病、银屑病、慢性肾炎、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。
附图的简要说明
图1：ELISA检测嵌合抗体QP378379结合人TSLP蛋白。
图2：ELISA鉴定嵌合抗体QP378379阻断人TSLP/TSLPR/IL7Ra蛋白结合。
图3：ELISA鉴定嵌合抗体QP378379结合食蟹猴TSLP蛋白。
图4：ELISA鉴定人源化TSLP抗体结合人TSLP蛋白。
图5：ELISA鉴定人源化抗TSLP抗体阻断人TSLP与TSLPR/IL7Ra的结合。
图6：ELISA鉴定人源化抗TSLP抗体结合食蟹猴TSLP蛋白。
图7：报告基因实验鉴定抗TSLP抗体抑制STAT5信号通路实验。
图8：ELISA鉴定TSLP抗体结合人TSLP蛋白。
图9：ELISA鉴定抗TSLP抗体阻断人TSLP与TSLPR/IL7Ra的结合。
图10：ELISA鉴定抗TSLP抗体结合食蟹猴TSLP蛋白。
图11：报告基因实验鉴定抗TSLP抗体抑制STAT5信号通路实验。
实现本发明的最佳方式
一、术语：
为了更容易理解本发明，描述实施例之前，先对本发明某些技术和科学术语做说明。
除显而易见在本发明申请文件中的它处另有明确定义，本发明使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.Boil.Chem.,243,p3558(1968)中所述。
本发明所述“抗体”，不仅包括完整的抗体，还包括具有抗原结合活性的片段、多肽序列、及其衍生物和类似物。
所述抗原结合片段是指全长抗体的一个或多个部分，所述部分保持结合抗原(例如，HER2)的能力，与完整抗体竞争对抗原的特异性结合。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合部分。在一些情况下，抗原结合部分包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如单克隆抗体2E12)获得抗体的抗原结合部分(例如，上述抗体片段)，并且以与对于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合部分。术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature341:544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
如本发明所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
将轻链和重链两者分成结构同源性区和功能同源性区。所述术语“恒定”和“可变”是在功能上使用。在这点上，应当理解轻链(VL)和重链(VH)部分的可变结构域均决定抗原识别和特异性。相反地，轻链(CL)和重链(CH1、CH2或CH3)的恒定结构域赋予重要生物学特性例如分泌、经胎盘移动性(transplacentalmobility)、Fc受体结合、补体结合等。按照惯例，恒定区结构域离抗体的抗原结合位点或者 氨基末端越远，它的编号越大。N-末端是可变区并且在C-末端是恒定区；CH3结构域和CL结构域实际上分别包含重链和轻链的羧基末端。
嵌合抗体：所述术语“嵌合抗体”(或其抗原结合片段)是抗体分子(或其抗原结合片段)，其中(a)所述恒定区或其部分被改变、置换或更换，使得所述抗原结合位点(可变区)与不同或改变的类型、效应子功能和/或种类的恒定区连接，或者与赋予嵌合抗体新特性的完全不同的分子(例如酶、毒素、激素、生长因素、药物等)连接；或(b)所述可变区或其部分被改变、置换或更换为具有不同或改变的抗原特异性的可变区。例如，可以通过用来自人免疫球蛋白的恒定区替代其恒定区来修饰小鼠抗体。由于被人恒定区置换，所述嵌合抗体可以保留其识别抗原的特异性，同时与原始小鼠抗体相比在人体中具有降低的抗原性。
人源化：所述术语非人(例如，鼠)抗体的“人源化”形式是含有源自非人免疫球蛋白的最小序列的嵌合抗体。在大多数情况下，人源化抗体是人类免疫球蛋白(受体抗体)，其中来自受体高变区的残基被来自非人类物种(如小鼠、大鼠、兔或非人类灵长类动物)的具有所希望特异性、亲和力和容量的高变区(供体抗体)的残基置换。在一些情况下，人免疫球蛋白的框架区(FR)残基由相应非人类残基置换。此外，人源化抗体可以包含在受者抗体或供体抗体中未发现的残基。进行这些修饰以进一步改善抗体性能。通常，人源化抗体将包含基本上所有如下项：至少一个，典型地两个可变结构域，其中所有或基本上所有高变环对应于非人类免疫球蛋白的那些高变环，且所有或基本上所有FR是人类免疫球蛋白lo序列的那些FR。人源化抗体任选地还包含免疫球蛋白恒定区(Fc)，典型地人免疫球蛋白恒定区的至少一部分。有关进一步的细节，参见Jones等人,1986,Nature[自然]321:522-525；Riechmann等人,1988,Nature[自然]332:323-329；和Presta,1992,Curr.Op.Struct.Biol.[结构生物学的研究现状]2:593-596。另参见以下评论文章和其中引用的参考文献：Vaswani和Hamilton,1998,Ann.Allergy,Asthma&Immunol.[过敏、哮喘和免疫学年鉴]1:105-115；Harris,1995,Biochem.Soc.Transactions[生化学会会刊]23:1035-1038；Hurle和Gross,1994,Curr.Op.Biotech.[当前生物技术观点]5:428-433。
抗体体外亲和力成熟，是在体外模拟抗体的体内成熟过程，即以某种抗体为改造模板，通过构建其突变体库，对突变体库进行筛选，获得生物活性大大改善的抗体。
本发明的“TSLP”包括TSLP的变体、同种型、同系物、直系同源物和旁系同源物。
本发明的抗体包括鼠源抗体、嵌合抗体和人源化抗体。
本发明TSLP介导的疾病没有限制，只要它是与TSLP相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类TSLP，然后阻遏TSLP与其受体结合，或杀伤过表达TSLP的细胞。
如本发明所用术语“自身免疫病”是指自机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病，包括但不限于硬皮病、系统性红斑狼疮、类风湿性关节炎、Churg-Strauss综合征、韦格纳肉芽肿(Wegener's granulomatosis)和肺出血-肾炎综合征等。
如本发明所用术语“炎性疾病”是指炎症作为其主要破坏因子的疾病的统称。炎症为组织对有害刺激 的生物反应，其是一种病变，并且伴随有组织退化、循环紊乱和液体渗出。炎性疾病的实例包括急性和慢性疾病，包括但不限于哮喘、过敏性肺炎、特应性皮炎、鼻炎、克罗恩病、强直性脊柱炎、风湿热、纤维肌痛、银屑病关节炎、慢性肾炎、斯耶格伦氏综合征以及多发性硬化等。
本发明TSLP介导的癌症(或肿瘤)包含乳腺癌、胰腺癌、胃癌、宫颈癌、结直肠癌、肺癌、黑色素瘤、B细胞淋巴瘤、骨髓瘤。
二、具体实施例
本实验中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件。如分子克隆，实验室手册，冷泉港实验室，当代分子生物学方法，细胞生物学等等。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：TSLP及TSLP受体重组蛋白及对照抗体Tezepelumab的克隆及表达纯化
克隆设计：分别设计人TSLP、人TSLP(R127A,R130A)、食蟹猴TSLP、食蟹猴TSLP(R127A,R130A)、人TSLPR、人IL7Ra等重组蛋白，带His，flag，FC等标签不等，用于纯化及鉴定。重组蛋白编号及序列号对应详见表1：
表1：克隆设计构建TSLP及TSLP受体重组蛋白
Figure PCTCN2022095806-appb-000001
根据公开信息(WHO Drug Information Vol.30,No.1,2016)查询到安进公司抗TSLP抗体Tezepelumab(AMG157)的全长抗体序列(如下所示)，分别构建全长轻重链表达质粒QD119,QD120，瞬转表达生产蛋白，蛋白编号为QP119120,用于后面实验对照：
QD119/pQD-Tezepelumab-Vλ/SEQ ID NO:24:
Figure PCTCN2022095806-appb-000002
QD120/pQDH-Tezepelumab-H(IgG2)/SEQ ID NO:25:
Figure PCTCN2022095806-appb-000003
瞬转表达：293E细胞转染前一天待转染细胞离心换液，调整细胞密度约为0.5×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入293E细胞中，放入8％CO2，120rpm，37℃的摇床中培养,转染第五天，水平离心机4700rpm离心20min收集细胞上清。
蛋白纯化：
Protein A纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml,流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致,流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性.
实施例2：构建稳定表达TSLPR及IL7Ra的BaF3-TSLPR-IL7Ra稳转细胞株
为了开展TSLP-STAT5荧光素酶报告基因实验，用于筛选能阻断TSLP与TSLPR/IL7Ra结合并引起下游STAT5信号通路激活的抗体，本实验室构建了同时表达TSLPR及IL7Ra的BaF3-TSLPR-IL7Ra稳转细胞株。BaF3细胞(小鼠原B细胞株)购买于北京协和医院，通过电转染方式，将质粒PCDNA3.1-TSLPR-IL-7Ra(QD1246)及PGL4.52(promega)共转染入BaF3细胞，转染后48小时后，将所有细胞收集离心后加入新鲜培养基重悬，并添加G418 1.2mg/ml进行筛选，hygmycin 1mg/mL；每2-3天离心换液，总共筛选2-3周，使细胞活力达95％以上。种单克隆板，挑选单克隆，通过FACS检测稳定表达TSLPR及IL7Ra的BaF3-TSLPR-IL7Ra单细胞株。
实施例3：杂交瘤融合筛选及抗人TSLP单克隆抗体获得
小鼠免疫：抗人TSLP单克隆抗体通过免疫小鼠产生。实验用Balb/c白小鼠，雌性，6周龄。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫Balb/c小鼠，首次用弗式完全佐剂(CFA)免疫重组蛋白QP1005(human TSLP-his)50ug/只小鼠两周后，此后用重组蛋白QP1005(human TSLP-his)加弗式不完全佐剂(IFA)或者重组蛋白QP312(cyno TSLP-his)加铝盐Alum+CpG ODN 1826交替免疫，25ug/只小鼠。
细胞融合：选择血清中抗体滴度高的小鼠进行脾细胞融合。融合前72小时，腹腔注射冲刺免疫所选小鼠。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤Sp2/0细胞进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的IMDM培养基)重悬，分装于96孔细胞培养板中(1×10 5个/150ul/孔)，37℃、5％CO 2培养。融合后的第5天加入20％FBS的IMDM培养基(含2×HAT和1×OPI)，50ul/孔，37℃，5％CO 2培养。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，250ul/孔，37℃、5％CO 2培养。
杂交瘤细胞筛选：根据细胞生长密度，融合后第10-14天，进行ELISA检测筛选杂交瘤上清中抗TSLP抗体。取杂交瘤融合孔上清通过ELISA进行96孔板整板初筛，检测上清中抗TSLP抗体能结合人TSLP重组蛋白QP1328，即为初筛阳性孔。检测上清中抗TSLP抗体能阻断TSLP/TSLPR/IL7Ra的结合，即为初筛阳性孔。再取初筛阳性孔ELISA检测上清中抗TSLP抗体结合食蟹猴TSLP重组蛋白QP313，同时能阻断TSLP/TSLPR/IL7Ra的结合的克隆，即为抗TSLP抗体阳性克隆孔。将阳性克隆扩培并及时转移至24/6孔板中，通过ELISA再次检测细胞培养上清结合人TSLP及食蟹猴TSLP，同时能阻断TSLP/TSLPR/IL7Ra的结合的克隆孔，即为抗TSLP抗体阳性克隆孔，得到阳性克隆mab177A12。筛选ELISA结果见表2所示。将阳性克隆mab177A12进行2-3轮有限稀释至单细胞克隆，冻存单细胞株。
表2：结合和阻断ELISA筛选杂交瘤阳性克隆
Figure PCTCN2022095806-appb-000004
杂交瘤单抗测序获得抗体序列：取杂交瘤阳性单克隆细胞株提取mRNA，mRNA反转录成cDNA，以cDNA为模板PCR扩增，挑选PCR阳性克隆送测序，通过序列分析得到单克隆抗体轻重链可变区序列。
SEQ ID NO:1mab177A12 VH
Figure PCTCN2022095806-appb-000005
Figure PCTCN2022095806-appb-000006
SEQ ID NO:2mab177A12 VL
Figure PCTCN2022095806-appb-000007
SEQ ID NO:1mab177A12 VH对应HCDR1、HCDR2和HCDR3序列分别为SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14；SEQ ID NO:2mab177A12 VL对应LCDR1、LCDR2和LCDR3序列分别为SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17；HCDR和LCDR氨基酸序列根据Kabat规则定义具体见表3。
表3：mab177A12轻重链可变区CDR序列及编号
Figure PCTCN2022095806-appb-000008
实施例3：抗人TSLP单克隆嵌合抗体构建
克隆构建：以实施例3单抗测序获得鼠单抗序列为模板，分别设计引物PCR扩增抗体VH/VK基因片段。表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切,利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)。BsmBI酶切载体，切胶回收备用。PCR扩增的抗体VH/VK基因片段分别与BsmBI酶切回收表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)按3:1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。各克隆轻重链可变区序列及蛋白表达编号如表4所示，此表中所有抗体轻链可变区融合kappa轻链恒定区CL组成嵌合抗体全长轻链，抗体重链可变区融合人IgG1恒定区组成嵌合抗体全长重链，CL及人IgG1恒定区序列如下所示：
CL SEQ ID NO:21
Figure PCTCN2022095806-appb-000009
Human IgG-Fc SEQ ID NO:23
Figure PCTCN2022095806-appb-000010
表4：嵌合抗体构建克隆编号及蛋白
Figure PCTCN2022095806-appb-000011
瞬转表达：HEK293E调整细胞密度为1×10 6/ml，准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入HEK293E细胞中，放入8％CO 2，120rpm，37℃的摇床中培养,转染第五天，水平离心机4700rpm离心20min收集细胞上清。
蛋白纯化：Protein A纯化：在SCG蛋白纯化仪系统连接装填1ml ProteinA填料的纯化柱；用平衡液1×PBS PH7.4过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；细胞培养液经4℃冷冻离心机离心后，将上清过柱，上样体积40ml,流速0.33ml/min；上样结束后用平衡液1×PBS PH7.4过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致,流速0.33ml/min；用洗脱液50mM/L柠檬酸PH3.5过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH8.0 2M/L Tris-HCl调节液将PAC-EP调至中性.超滤管离心将buffer置换成PBS.SDS-PAGE及HPLC-SEC鉴定蛋白纯度，纯度大于95％蛋白备用。
ELISA鉴定嵌合抗体结合人TSLP蛋白:包被：用PBS将蛋白QP1328(人TSLP的FC融合)稀释为1μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：孵育不同浓度人TSLP嵌合抗体，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标抗体：酶标抗体Goat Anti-Fab(HRP)需先用封闭液以1：5000倍进行稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H2SO4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图1所示,嵌合抗体QP378379结合人TSLP重组蛋白。
ELISA鉴定嵌合抗体阻断人TSLP/TSLPR/IL7Ra蛋白结合:包被：用PBS将蛋白QP1171(人TSLPR/IL7Ra的FC融合蛋白)稀释为2μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：将不同浓度人TSLP嵌合抗体和0.02ug/ml的 Biotin-QP1005(生物素化的人TSLP的FC融合蛋白)按照1：1比例混合，加入封闭好的ELISA板中，60μl/孔，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标链霉亲和素Streptavidin(HRP)先用封闭液进行5000倍稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H 2SO 4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图2所示,嵌合抗体QP378379能阻断人TSLP与TSLPR/IL7Ra的结合。
ELISA鉴定嵌合抗体结合食蟹猴TSLP蛋白:包被：用PBS将蛋白QP313(食蟹猴TSLP的FC融合)稀释为1μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：孵育不同浓度人TSLP嵌合抗体，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标抗体：酶标抗体Goat Anti-Fab(HRP)需先用封闭液以1：5000倍进行稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H2SO4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图3所示,嵌合抗体QP378379结合食蟹猴TSLP蛋白。
实施例4：抗人TSLP单克隆抗体人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP378379同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。
1.抗TSLP抗体人源化分子克隆
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pQD/VK-CL-pQD。
利用在线软件DNAWorks(v3.2.4)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH/VK+30bp CH1/CL-3’.按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切,利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)。BsmBI酶切载体，切胶回收备用。重组构建表达载体VH-CH1-FC-pQD/VK-CL-pQD.VH/VK含重组所需基因片 段与BsmBI酶切回收表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)按3:1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
各克隆人源化设计轻重链可变区序列及蛋白表达编号如表5所示，此表中所有抗体轻链可变区融合kappa轻链恒定区CL组成全长轻链，抗体重链可变区融合人IgG1恒定区组成全长重链：
表5：QP378379抗体人源化构建克隆编号及蛋白。
Figure PCTCN2022095806-appb-000012
2.人源化抗TSLP抗体蛋白表达
HEK293E调整细胞密度为1×10 6/ml，准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入HEK293E细胞中，放入8％CO 2，120rpm，37℃的摇床中培养,转染第五天，水平离心机4700rpm离心20min收集细胞上清。
3.蛋白纯化，Protein A纯化
在SCG蛋白纯化仪系统连接装填1ml ProteinA填料的纯化柱；用平衡液1×PBS PH7.4过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；细胞培养液经4℃冷冻离心机离心后，将上清过柱，上样体积40ml,流速0.33ml/min；上样结束后用平衡液1×PBS PH7.4过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致,流速0.33ml/min；用洗脱液50mM/L柠檬酸PH3.5过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH8.0 2M/L Tris-HCl调节液将PAC-EP调至中性.超滤管离心将buffer置换成PBS.SDS-PAGE及HPLC-SEC鉴定蛋白纯度，纯度大于95％蛋白备用。
4.人源化TSLP抗体活性鉴定(Binding-ELISA)
ELISA鉴定人源化TSLP抗体结合人TSLP蛋白:包被：用PBS将蛋白QP1328(人TSLP的FC融合)稀释为1μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：孵育不同浓度人源化抗TSLP抗体，常温1h，使用Biotek自动洗板机对孵育 完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标抗体：酶标抗体Goat Anti-Fab(HRP)需先用封闭液5000倍进行稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H2SO4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图4所示,人源化抗TSLP抗体不同变体均能结合人TSLP重组蛋白。
5.人源化TSLP抗体活性鉴定(Blocking-ELISA)
实验步骤：用PBS将蛋白QP1171(人TSLPR/IL7Ra的FC融合蛋白)稀释为2μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：将不同浓度人TSLP嵌合抗体和0.02ug/ml的Biotin-QP1006(生物素化的人TSLP的FC融合蛋白)按照1：1比例混合，加入封闭好的ELISA板中，60μl/孔，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标链霉亲和素Streptavidin(HRP)先用封闭液进行5000倍稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H 2SO 4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图5所示,人源化抗TSLP抗体不同变体均能阻断人TSLP与TSLPR/IL7Ra的结合.
6.ELISA鉴定嵌合抗体结合食蟹猴TSLP蛋白
包被：用PBS将蛋白QP313(食蟹猴TSLP的FC融合)稀释为1μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：孵育不同浓度人TSLP嵌合抗体，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标抗体：酶标抗体Goat Anti-Fab(HRP)需先用封闭液以1：5000倍进行稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H2SO4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图6所示,人源化抗TSLP抗体各变体均结合食蟹猴TSLP蛋白.
7.Biacore检测抗TSLP抗体与人TSLP重组蛋白结合亲和力和动力学实验
通过Biacore S200测定抗TSLP抗体与人TSLP的亲和力，实验过程如下：
实验步骤：用Protein A芯片亲和捕获一定量的待测抗体，然后于芯片表面流经人TSLP蛋白 (QP1005)，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
实验结果如表6所示，人源化的抗TSLP抗体QP419423,QP420421,QP420422,QP420423及人源化前的嵌合抗体QP378379均结合人TSLP蛋白。
表6：抗TSLP抗体与人TSLP结合亲合力结果
Figure PCTCN2022095806-appb-000013
8.抗TSLP抗体抑制STAT5信号通路报告基因实验
实验步骤：取稳转的BaF3-TSLPR-IL-7Ra细胞株,用PBS清洗2次细胞，然后铺4E4/well至不透明反应板上,即2E6/ml,20ul/孔，培养基为不加IL-3的基础培养基，加入不同浓度的抗TSLP抗体(抗体中浓度为100ug/mL,4倍梯度稀释)，培养箱孵育20min后；加入终浓度2ng/mL的TSLP重组蛋白(QP1005)20ul/孔；培养箱培养6h后，通过One-Glo Luciferase Assay System(promega,E6120)试剂盒检测，加入60ul/孔试剂，仪器Bio-TECK读数，记录结果。
结果如图7所示，人源化的抗TSLP抗体QP419423,QP420421,QP420422,QP420423及人源化前的嵌合抗体QP378379均能抑制TSLP及其受体结合引起的下游STAT5信号通路。
实施例5：人源化抗TSLP抗体QP420422亲和力成熟
1.亲和力成熟抗体构建
构建人源化噬菌粒载体：人源化后的QP420422分别以scFv模式(VH-(GGGGS)3-VL)构建到噬菌粒载体中，作为野生型序列(即相对于亲和力成熟筛选得到的突变序列)。利用重叠PCR(over-lap PCR)拼接VH、(GGGGS)3接头、VL，采用NcoI和NotI酶切位点连接入噬菌粒载体。
构建噬菌体展示文库：利用构建好的野生型scFv为模板，每个CDR设计一条突变引物，采用简并引物，在所有CDR区引入突变构建突变文库。PCR片段经过NcoI和NotI酶切，连接到噬菌粒体载体中，最后电转化大肠杆菌TG1。每个CDR的简并引物建立一个独立的文库。
文库淘筛：文库经过phage包装出淘筛用的噬菌体颗粒后，利用生物素化的QP1005(人TSLP重组蛋白)抗原和链霉亲和素磁珠进行液相法淘筛，第一轮筛选浓度5nM,且每一轮筛选相对于上一轮都降低抗原浓度10倍。三轮淘筛之后，挑取250克隆进行噬菌体ELISA检测结合活性，阳性克隆进行测序。
ELISA检测：经过对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长IG(hIgG1,κ)进行哺乳动物细胞293E表达。亲和纯化之后的全长IG进行ELISA检测。
最终得到3个突变抗体，分别是以QD420(SEQ ID NO:5)为母本的重链CDR2突变如表7所示的克隆QD3611(SEQ ID NO:9),QD3616(SEQ ID NO:10)，以QD422(SEQ ID NO:7)为母本的轻链CDR2突变如表8所示的克隆QD3625(SEQ ID NO:11)。
表7：TSLP抗体亲和力成熟分子重链CDR2突变
Figure PCTCN2022095806-appb-000014
以QD422为母本的轻链CDR2突变如表7所示的克隆QD3625：
表8：TSLP抗体亲和力成熟分子轻链CDR2突变
Figure PCTCN2022095806-appb-000015
2.TSLP抗体活性鉴定(Binding-ELISA)
ELISA鉴定抗体结合人TSLP蛋白:包被：用PBS将蛋白QP1328(人TSLP的FC融合)稀释为1μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：孵育不同浓度抗TSLP抗体，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标抗体：酶标抗体Goat Anti-Fab(HRP)需先用封闭液5000倍进行稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H2SO4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。
结果如图8所示,抗TSLP抗体不同变体均能结合人TSLP重组蛋白,且优于对照抗体QP119120(AMG157)。
3.人源化TSLP抗体活性鉴定(Blocking-ELISA)
实验步骤：用PBS将蛋白QP1171(人TSLPR/IL7Ra的FC融合蛋白)稀释为2μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：将不同浓度人TSLP嵌合抗体和0.02ug/ml的Biotin-QP1006(生物素化的人TSLP的FC融合蛋白)按照1：1比例混合，加入封闭好的ELISA板中，60μl/孔，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标链霉亲和素Streptavidin(HRP)先用封闭液进行5000倍稀释，60μl/ 孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H 2SO 4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图9所示,抗TSLP抗体不同变体均能阻断人TSLP与TSLPR/IL7Ra的结合.
4.ELISA鉴定抗体结合食蟹猴TSLP蛋白:
包被：用PBS将蛋白QP313(食蟹猴TSLP的FC融合)稀释为1μg/ml,按照60μl/孔用量加入ELISA板中，4℃孵育18-24h。封闭：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBS清洗2次，清洗结束后，按照200μl/孔加入3％BSA封闭液，常温封闭1h。孵育抗体：孵育不同浓度人TSLP嵌合抗体，常温1h，使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，用PBST清洗3次。孵育酶标抗体：酶标抗体Goat Anti-Fab(HRP)需先用封闭液以1：5000倍进行稀释，60μl/孔，常温孵育1h；TMB显色：使用Biotek自动洗板机对孵育完成的ELISA板进行清洗，PBST洗5次后，按照100μl/孔用量加底物显色液TMB，常温避光显色10min；终止：显色完成后，按100μL/孔用量快速加入终止液1M H2SO4终止反应。读数：在酶标仪上，测定波长450nm的OD值。结果分析,使用Graphpad Prism软件分析进行数据录入与分析。结果如图10所示,抗TSLP抗体各变体均结合食蟹猴TSLP蛋白.
5.Biacore检测抗TSLP抗体与人TSLP重组蛋白结合亲和力和动力学实验
通过Biacore 8K测定抗TSLP抗体与人TSLP的亲和力，实验过程如下：
实验步骤：用Protein A芯片亲和捕获一定量的待测抗体，然后于芯片表面流经人TSLP蛋白(QP1005)，利用Biacore实时检测反应信号，从而获得结合和解离曲线。
实验结果如表9所示，抗TSLP抗体QP36110422、QP36160422、QP04203625与人TSLP蛋白结合亲和力优于对照抗体AMG157。
表9：抗TSLP抗体与人TSLP结合亲合力结果
Figure PCTCN2022095806-appb-000016
6.抗TSLP抗体抑制STAT5信号通路报告基因实验
实验步骤：取稳转的BaF3-TSLPR-IL-7Ra细胞株,用PBS清洗2次细胞，然后铺4E4/well至不透明反应板上,即2E6/ml,20ul/孔，培养基为不加IL-3的基础培养基，加入不同浓度的抗TSLP抗体(抗体中浓度为100ug/mL,4倍梯度稀释)，培养箱孵育20min后；加入终浓度2ng/mL的TSLP重组蛋白(QP1005)20ul/孔；培养箱培养6h后，通过One-Glo Luciferase Assay System(promega,E6120)试剂盒检测，加入60ul/孔试剂，仪器Bio-TECK读数，记录结果。
结果如图11所示，抗TSLP抗体QP04203625、QP36110422、QP36160422均能抑制TSLP及其受体结合引起的下游STAT5信号通路,且抑制IC50值优于对照抗体QP119120(AMG157)。

Claims (11)

  1. 一种抗TSLP抗体，其包含重链可变区和轻链可变区，所述重链可变区包含互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含互补决定区LCDR1、LCDR2和LCDR3，其中：
    HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:18和SEQ ID NO:14所示，LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示；或
    HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:19和SEQ ID NO:14所示，LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示；或
    HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示，LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:20和SEQ ID NO:17所示；或
    HCDR1、HCDR2和HCDR3分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示，LCDR1、LCDR2和LCDR3分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示。
  2. 如权利要求1所述的抗体，其中所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1、3、4、5、9或者10所示。
  3. 如权利要求1所述的抗体，其中所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2、6、7、8或者11所示。
  4. 如权利要求1所述的抗体，其中
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:2所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:5所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:11所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:9所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示；或
    所述抗体的重链可变区的氨基酸序列如SEQ ID NO:10所示，所述抗体的轻链可变区的氨基酸序列如SEQ ID NO:7所示。
  5. 如权利要求1所述的抗体，其中所述抗体包含抗TSLP抗原的全抗体、Fab片段、Fab’片段、F(ab’)2片段、Fv片段和sc-Fv。
  6. 如权利要求1所述的抗体，其中所述抗体包含选自IgG1亚型、IgG2亚型、IgG3亚型或IgG4亚型的 重链恒定区及其常规变体，且所述抗体包含选自κ亚型或者λ亚型的轻链恒定区及其常规变体。
  7. 一种分离的多核苷酸，其中所述多核苷酸编码如权利要求1-6任一项所述的TSLP抗体。
  8. 一种药物组合物，其含有如权利要求1-6任一项所述的TSLP抗体和可药用的赋形剂、稀释剂或载体。
  9. 如权利要求1-6任一项所述的抗体或权利要求8所述的药物组合物在制备用于预防或治疗TSLP介导的疾病的药物中的用途。
  10. 如权利要求9所述的抗体，其中所述TSLP介导的疾病包含癌症(肿瘤)、自身免疫性疾病或炎性疾病。
  11. 如权利要求9所述的抗体，其中所述TSLP介导的疾病包含乳腺癌、胰腺癌、胃癌、宫颈癌、结直肠癌、肺癌、黑色素瘤、B细胞淋巴瘤、骨髓瘤、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、系统性红斑狼疮、类风湿性关节炎、克罗恩病、银屑病、慢性肾炎、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。
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