Antibodies specific for human tslp and uses thereof

Abstract

The present application provides an antibody molecule binding to human thymic stromal lymphopoietin (TSLP) and antigen-binding fragments thereof, and use of the antibody molecule or antigen-binding fragments thereof for preventing or treating TSLP high expression related purposes. The antibody molecule and antigen-binding fragments thereof of the present application can bind to human TSLP with high affinity, block the interaction between TSLP and its receptor, and significantly inhibit the secretion of chemokines such as CCL17, OPG, and dendritic cells, and can be used for treating diseases and disorders related to TSLP signaling and / or TSLP cell expression, such as inflammatory diseases or tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular, the present application relates to antibody molecules and antigen-binding fragments thereof which specifically bind to thymic stromal lymphopoietin (TSLP), and the use of the antibody molecules and antigen-binding fragments thereof. BACKGROUND

[0002] Thymic stromal lymphopoietin (TSLP) is a cytokine produced by epithelial cells in response to proinflammatory stimuli, which was first isolated from the culture supernatant of thymic stromal cells by Friend in 1994. Human TSLP (hTSLP) has two isoforms, isoform I and isoform II. Isoform I is a 159-amino acid protein molecule, and isoform II is a 60-amino acid protein molecule.

[0003] The TSLP receptor (TSLPR) is a dimer composed of the IL-7R-alpha chain and the TSLPR-gamma chain. The functional TSLPR complex is composed of TSLPR and IL-7Rα. In human dendritic cells and monocytes, TSLP can only induce intracellular Stat3 and Stat5 phosphorylation when TSLPR and IL-7Rα are expressed simultaneously.

[0004] Studies have found that TSLP acts on dendritic cells (DC) and mast cells, driving the body's sensitized inflammatory response. TSLP can support the growth, differentiation of B lymphocytes, and T cell proliferation, especially playing an important regulatory role in the activation, differentiation, maturation and migration of dendritic cells. In vitro studies have shown that TSLP can activate CD11c dendritic cells, and activated DCs can express Th2 aggregation chemokines TARC and MDC. In addition, DCs activated by TSLP can also initiate the transformation of CD4 T cell precursors to Th2, and these Th2 cells can produce allergic factors IL-4, IL-5, IL-13 and tumor necrosis factor alpha, while reducing the production of IL-10 and IFN-γ. hTSLP-induced DC cells create a Th2-type microenvironment. hTSLP induces DC cells to produce CCL17, CCL22 and other chemotactic factors of chemotactic CD4+ Th2-type cells, and can also induce DC cells to express OX40L, thereby inducing T cells to express IL-4, IL-5 and IL-13. TSLP produced by epithelial cells can stimulate peripheral blood CD11c+ DCs to significantly up-regulate the expression of CD40 and CD80. TSLP can activate Stat5 molecules and other molecules in effector cells.

[0005] TSLP plays an important role in the development of allergic asthma. Clinical studies have found that the expression level of TSLP in the bronchoalveolar lavage fluid of asthma patients is increased, and is positively correlated with the severity of the disease, confirming that TSLP is closely related to human asthma. At the same time, preclinical data also show that TSLP plays an important role in the development of asthma, and targeting TSLP can inhibit multiple asthma-related biological pathways, including IL-4, IL-5 and IL-13; can inhibit the release of downstream T2 cytokines and various non-T2 cell-driven inflammatory responses. TSLP plays an important role in the early upstream activity of the inflammatory cascade, making it a clear target for the treatment of asthma. SUMMARY

[0006] The technical problem to be solved by the present application is to obtain a new antibody with high affinity and high functional activity to human TSLP through hybridoma screening and humanization technology.

[0007] To solve the above technical problems, the purpose of the present application is to provide an antibody molecule or antigen binding fragment thereof against human TSLP, which has higher affinity and cell activity to human TSLP than existing anti-TSLP antibodies.

[0008] The present application provides the following technical solutions:

[0009] In one aspect, the present application provides an antibody molecule or antigen binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) comprise a combination of heavy chain CDRs and light chain CDRs selected from the following:

[0010] (1) H-CDR1 (GYTFTNY), H-CDR2 (NPGSGG), H-CDR3 (ESEVGEGFAY) shown in SEQ ID NO: 7, 8, 9 in turn; and L-CDR1 (RASQDISNYLN), L-CDR2 (YTSTLHS), L-CDR3 (QQFDTLPYP) shown in SEQ ID NO: 10, 11, 12 in turn;

[0011] (2) H-CDR1 (NYFID), H-CDR2 (VINPGSGGTNFNEKFKG), H-CDR3 (ESEVGEGFAY) shown in SEQ ID NO: 13, 14, 9 in turn; and L-CDR1 (RASQDISNYLN), L-CDR2 (YTSTLHS), L-CDR3 (QQFDTLPYP) shown in SEQ ID NO: 10, 11, 12 in turn;

[0012] (3) H-CDRs 1 (GYTFTNYFID), 2 (VINPGSGGTN), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 15, 16, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQFDTLPYP), shown in SEQ ID NOs: 10, 11, 12, respectively;

[0013] (4) H-CDRs 1 (GYTFTNYFID), 2 (VINPGSGGTNFNEKFKG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 15, 14, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQFDTLPYP), shown in SEQ ID NOs: 10, 11, 12, respectively;

[0014] (5) H-CDRs 1 (GYTFTNY), 2 (DAFSGG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 7, 17, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0015] (6) H-CDRs 1 (NYFID), 2 (VIDAFSGGSNFNEKFKG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 13, 19, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0016] (7) H-CDRs 1 (GYTFTNYFID), 2 (VIDAFSGGSN), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 15, 20, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0017] (8) H-CDRs 1 (GYTFTNYFID), 2 (VIDAFSGGSNFNEKFKG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 15, 19, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0018] (9) H-CDRs 1 (GYAFTNY), 2 (NPGSGG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 23, 8, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0019] (10) H-CDRs 1 (NYFID), 2 (VINPGSGGTNFNEKFKG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 13, 14, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0020] (11) H-CDRs 1 (GYAFTNYFID), 2 (VINPGSGGTN), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 24, 16, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively;

[0021] (12) H-CDRs 1 (GYAFTNYFID), 2 (VINPGSGGTNFNEKFKG), 3 (ESEVGEGFAY), shown in SEQ ID NOs: 24, 14, 9, respectively; and L-CDRs 1 (RASQDISNYLN), 2 (YTSTLHS), 3 (QQGNTLPYT), shown in SEQ ID NOs: 10, 11, 18, respectively.

[0022] According to the domain composition of the variable region of heavy chain and the variable region of light chain of antibody molecule known in the art, the variable region of light chain or the variable region of heavy chain of the antibody molecule or the antigen-binding fragment thereof provided in the present application comprises the above domain components in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR is a framework region.

[0023] Preferably, in the antibody molecule or the antigen-binding fragment thereof provided in the present application, the variable region of heavy chain comprises a sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 3, 5 or 21 or an amino acid sequence having at least 75% identity to the amino acid sequence; and, the variable region of light chain comprises a sequence selected from the group consisting of the amino acid sequence shown in SEQ ID NO: 4, 6 or 22 or an amino acid sequence having at least 75% identity to the amino acid sequence.

[0024] According to the specific embodiments of the present application, in the antibody molecule or the antigen-binding fragment thereof provided in the present application, the variable region of heavy chain and the variable region of light chain can be selected from the following combinations:

[0025] (1) the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 3; and, the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 4;

[0026] (2) the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 5; and, the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 6;

[0027] (3) the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 21; and, the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to the amino acid sequence shown in SEQ ID NO: 22.

[0028] Wherein, the amino acid sequence difference caused by the "at least 75% identity" can exist in any framework region in the variable region of heavy chain or the variable region of light chain, or in any domain or sequence outside the variable region of heavy chain and the variable region of light chain in the antibody molecule or the antigen-binding fragment thereof of the present application. The difference can be caused by amino acid deletion, addition or substitution at any position, wherein the substitution can be conservative substitution or non-conservative substitution.

[0029] Preferably, the antibody molecule provided by the present application can be a murine antibody, a chimeric antibody or a fully or partially humanized antibody; the antigen binding fragment can be a half antibody or a scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2or Fv fragment of the antibody molecule.

[0030] Further preferably, the antibody molecule is a monoclonal antibody or a single chain antibody.

[0031] And preferably, the antibody molecule or the antigen binding fragment thereof further comprises a constant region, preferably a murine or human heavy chain constant region (CH) and / or a light chain constant region (CL); further preferably, the antibody molecule or the antigen binding fragment thereof comprises a heavy chain and a light chain, for example two heavy chains and light chains.

[0032] More preferably, the antibody molecule or the antigen binding fragment thereof comprises a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a kappa or lambda type light chain constant region.

[0033] According to the specific embodiments of the present application, the antibody molecule is a monoclonal antibody, preferably a murine or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG2 type and the light chain constant region is of kappa type.

[0034] For example, the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 75% identity to the amino acid sequence; the light chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity to the amino acid sequence.

[0035] In the context of the present application, "at least 75% identity" is any percentage number of identity between 75% and 100%, for example 75%, 80%, 85%, 90%, even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0036] The antibody molecule or the antigen binding fragment thereof provided by the present application is an antibody molecule or the antigen binding fragment thereof against thymic stromal lymphopoietin (TSLP); preferably, the TSLP is a mammalian TSLP, for example a human TSLP.

[0037] Experiments prove that the antibody molecule or antigen-binding fragment thereof of the present application can bind to TSLP with high affinity; can block the binding of TSLP to TSLPR; can inhibit BAF / 3-TSLPR cell reporter gene expression; can inhibit dendritic cells from secreting CCL17 and OPG. Moreover, the antibody molecule or antigen-binding fragment thereof of the present application does not recognize IL7.

[0038] In another aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding a light chain variable region, a heavy chain variable region, a heavy chain or a light chain comprised in the antibody molecule or antigen-binding fragment thereof of the present application.

[0039] The nucleic acid molecule of the present application can be cloned into a vector, which can be an expression vector, or a transformation or transfection vector. Therefore, in yet another aspect, the present application also provides a vector comprising the nucleic acid molecule of the present application. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome and a bacteriophage vector, etc.

[0040] The vector or nucleic acid molecule of the present application can be used to transform or transfect a host cell for the purpose of preservation or antibody expression, etc. Therefore, in still another aspect, the present application provides a host cell comprising the nucleic acid molecule and / or vector of the present application, or the host cell is transformed or transfected by the nucleic acid molecule and / or vector of the present application. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

[0041] The antibody molecule provided by the present application can be obtained by any method known in the art. For example, the heavy chain variable region and / or light chain variable region of the antibody molecule, or the heavy chain and / or light chain of the antibody can be obtained from the nucleic acid molecule provided by the present application, and then assembled into the antibody molecule with optional other domains of the antibody molecule; or the host cell provided by the present application is cultured under conditions allowing the heavy chain variable region and / or light chain variable region of the antibody molecule, or the heavy chain and / or light chain of the antibody molecule to express to assemble into the antibody molecule. Optionally, the method further comprises a step of recovering the produced antibody molecule.

[0042] The antibody molecule or antigen-binding fragment thereof, nucleic acid molecule, vector and / or host cell provided by the present application can be comprised in a composition, more particularly in a pharmaceutical composition, e.g. a pharmaceutical preparation, for various purposes according to actual needs. Therefore, in yet another aspect, the present application also provides a composition comprising the antibody molecule or antigen-binding fragment thereof, nucleic acid molecule, vector and / or host cell of the present application. The composition can be a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, adjuvant or excipient.

[0043] In a further aspect, the present application also provides the use of the antibody molecule or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application for the manufacture of a medicament for the prevention or treatment of an inflammatory disease or a tumor. Preferably, the inflammatory disease is selected from asthma, allergic dermatitis, chronic obstructive pulmonary disease (COPD) and allergic rhinitis; preferably, the asthma comprises Th2-type and non-Th2-type asthma; the tumor is selected from Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma and lung cancer.

[0044] In another aspect, the present application also provides a method for the prevention or treatment of an inflammatory disease or a tumor, comprising administering to a subject in need thereof the antibody molecule or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application. Preferably, the inflammatory disease is selected from asthma, allergic dermatitis, chronic obstructive pulmonary disease (COPD) and allergic rhinitis; preferably, the asthma comprises Th2-type and non-Th2-type asthma; the tumor is selected from Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma and lung cancer.

[0045] wherein the subject is a mammal, more preferably a human.

[0046] In a further aspect, the present application also provides the use of the antibody molecule or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application for the manufacture of a medicament for the prevention or treatment of an inflammatory disease or a tumor. Preferably, the inflammatory disease is selected from asthma, allergic dermatitis, chronic obstructive pulmonary disease (COPD) and allergic rhinitis; preferably, the asthma comprises Th2-type and non-Th2-type asthma; the tumor is selected from Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma and lung cancer.

[0047] Accordingly, the present application also provides a method for the diagnosis of an inflammatory disease or a tumor, comprising contacting the antibody molecule or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application with a sample from a subject. Preferably, the inflammatory disease is selected from asthma, chronic obstructive pulmonary disease (COPD), allergic dermatitis and allergic rhinitis; preferably, the asthma comprises Th2-type and non-Th2-type asthma; the tumor is selected from Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma and lung cancer.

[0048] wherein the subject is a mammal, more preferably a human.

[0049] Accordingly, in a further aspect, the present application provides a kit comprising the antibody molecule or antigen binding fragment thereof, nucleic acid molecule, vector, host cell and / or composition of the present application. The kit can be used for therapeutic or diagnostic purposes. BRIEF DESCRIPTION OF DRAWINGS

[0050] Embodiments of the present application will be described in detail below with reference to the attached drawings, wherein:

[0051] Figure 1 TSLP antibody titers in sera of 10 mice are shown.

[0052] Figure 2 TSLPR blocking ability of hybridoma monoclonal cell culture supernatants is shown, wherein Figure 2 A to Figure 2 D are results of the 1st to 4th batches of monoclonal cells, respectively.

[0053] Figure 3 Detection results of the inhibitory effect of hybridoma monoclonal cell culture supernatants on BAF / 3-TSLPR cell reporter gene expression are shown.

[0054] Figure 4 Detection results of the inhibitory effect of chimeric antibodies on BAF / 3-TSLPR cell reporter gene expression are shown.

[0055] Figure 5 Detection results of the blocking effect of antibodies on the binding of TSLP to TSLPR are shown.

[0056] Figure 6 Detection results of the binding kinetics of humanized antibodies to TSLP are shown, wherein Figure 6 A: hzD01Lm12; Figure 6 B: hzD01Hm5Lm4.

[0057] Figure 7 Detection results of the inhibitory effect of humanized antibodies on BAF / 3-TSLPR cell reporter gene expression are shown.

[0058] Figure 8 Detection results of the inhibitory effect of humanized antibodies on dendritic cell secretion of CCL17 and OPG are shown, wherein Figure 8 A: CCL17; Figure 8 B: OPG.

[0059] Figure 9 Detection results of the species cross-binding of humanized antibodies to human TSLP, mouse TSLP and monkey TSLP proteins, and human IL7 protein are shown, wherein Figure 9 A: hzD01Hm5Lm4; Figure 9 B: hzD01m12; Figure 9 C: AMG157; BLK: blank. DETAILED DESCRIPTION

[0060] The present application will be described below with reference to specific examples. Those skilled in the art will understand that the examples are only used to illustrate the present application, and do not limit the scope of the present application in any way.

[0061] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are all commercially available products unless otherwise specified. Among them:

[0062] Human TSLP: R&D Systems, catalog # CK16.

[0063] Mouse TSLP: U-CyTech, catalog # 51005-M08H.

[0064] Rhesus TSLP: R&D Systems, catalog # CR62.

[0065] Human TSLPR: U-CyTech, catalog # 29749-H08H.

[0066] Human IL7: R&D Systems, catalog # C086.

[0067] Human TSLPR (CRLF2) CDS: NM_022148.3.

[0068] Human IL7R CDS: NM_002185.4.

[0069] The following known antibodies were used as control antibodies:

[0070] AMG157: heavy chain shown in SEQ ID NO: 1, light chain shown in SEQ ID NO: 2. The fully synthetic AMG157 antibody light chain and heavy chain genes were cloned into eukaryotic expression vectors, respectively, to obtain AMG157 light and heavy chain expression plasmids, which were transformed into E. coli for amplification, and a large amount of plasmids containing AMG157 antibody light and heavy chains were obtained. After mixing with polyethyleneimine (PEI), they were co-transfected into HEK293 cells. After 5-6 days of cell transfection, the culture supernatant was obtained, and the expression supernatant was purified by ProA affinity chromatography column to obtain AMG157 antibody recombinant protein.

[0071] Heavy chain constant region of IgG2 subclass: SEQ ID NO: 25; light chain constant region of kappa subclass: SEQ ID NO: 26

[0072] Example 1 Screening of murine monoclonal antibodies

[0073] 1.1 Preparation of monoclonal cells

[0074] Ten Balb / c mice were immunized with human TSLP protein according to the routine method described in the literature (Lonberg, N., et al., nature 368 (1994) 856-859; Fishwild, D. M., et al., Nat. Biotechnol. 14 (1996) 845-851 and WO 98 / 24884), and then the anti-TSLP titers in the sera of the immunized mice were determined by antigen-specific ELISA.

[0075] A TSLP solution (in pH 9.6, 0.1M NaHCO3) with a concentration of 1 ug / ml was coated in a 96-well plate, 100ul per well, and incubated at 4°C overnight. Then the coating solution was washed away, and blocking solution was added to each well, and incubated at room temperature for 2 hours. The mouse serum was pre-diluted 400-fold in PBSA (PBS containing 1% BSA), and serially diluted 10 dilution gradients at 1:2 to 204800-fold. After the blocking solution was washed away, the diluted serum was added to the plate wells and incubated at room temperature for 1 hour. After the 96-well plate was washed with PBST (0.05% Tween20-PBS), 100ul / well of 1 / 30000 diluted secondary antibody (HRP-labeled goat anti-mouse IgG (Fc gamma)) was added, incubated at room temperature for 45 minutes, washed with PBST three times, added with TMB 20ul / well equilibrated at room temperature, incubated at room temperature for 10 minutes, and the absorbance was measured at 450nm. The serum TSLP antibody titers of the 10 mice are shown in Table 1. Figure 1 .

[0076] The mice with serum titers against TSLP greater than 1:100000 were given an additional intraperitoneal injection of TSLP four days before fusion to boost the immune response. The spleen cells obtained from the mice were fused with myeloma cells P3X63Ag8.653 using the electrofusion method.

[0077] The fused hybridoma cells were seeded in a 384-well plate, and after 14 days of culture, the enzyme-labeled plate was coated with human TSLP protein (1ug / ml, pH9.6, 0.1M NaHCO3), and HRP-goat anti-mouse IgG (Fc gamma) was added, and the A490 value was read on an enzyme-labeled instrument to detect the antibodies in the culture supernatant of the hybridoma clones. A positive result was determined when the A450 value of the test well was more than 3 times the A490 value of the negative control well.

[0078] A total of 4 batches of immunization, fusion and screening were performed. After detection and identification, a plurality of antibody-secreting positive hybridoma clones were obtained. The hybridoma cell clones secreting anti-human TSLP antibodies obtained through the combined experiment screening were single-cellized by the limited dilution method, and each hybridoma cell clone obtained after two rounds of subcloning only secreted one antibody.

[0079] 1.2 Detection of monoclonal cell supernatant

[0080] Four batches of monoclonal cells were expanded and the supernatant was taken for TSLPR blocking ability detection.

[0081] ELISA plate was coated with 1 ug / ml human TSLPR protein in NaHCO3 buffer (pH 9.6, 0.1 M) at 4°C overnight, blocked with 4% skim milk-PBS at 37°C for 2 hours, washed with PBST (0.05% Tween20-PBS) for three times, and then added with TSLP-biotin (Acro, cat#TSP-H82E0, 0.5 ug / ml) solution (in PBSA (PBS containing 1% BSA)) with negative clone supernatant as control. Equal volume of monoclonal cell culture supernatant was added and mixed at room temperature for 1 hour. After washing with PBST (0.05% Tween20-PBS) for three times, Streptavidin-HRP diluted at 1:40000 was added and incubated at 37°C for 1 hour. After washing with PBST (0.05% Tween20-PBS) again, TMB color developing solution was added, and the reaction was carried out in the dark for 10 minutes. After adding 2M H2SO4 to terminate the reaction, the A450 value was read by a microplate reader.

[0082] Blocking inhibition rate (%) = ((negative control well-monoclonal supernatant well) / negative control well)*100%. After detection and identification, a plurality of hybridoma monoclonals with blocking ability were obtained, as shown in Table 2A to Figure 2 Table 2B to Figure 2 D.

[0083] In addition, the inhibitory effect of the supernatant on the expression of the reporter gene was detected.

[0084] Human TSLPR and human IL7R molecules were overexpressed on BAF / 3 cells, and STAT5-luciferase reporter gene was stably transfected. 1E6 stably transfected cells were added to each well of a white bottom transparent plate for 4 hours of starvation. Medium was used as negative control (N.C.), and 100ul / well of negative control and clone supernatant were incubated with the same volume of human TSLP protein solution (200ng / ml, PBSA (PBS containing 1% BSA)) in U-bottom plate for 30 minutes. 50ul of TSLP-test sample mixed solution was added to 50ul / well of cells, and incubated at 37°C in a 5% CO2 cell incubator for 16 hours. 100ul of Bio-Glo Luciferase Assay Reagent was added and shaken for 5 minutes, and the chemiluminescence result was read. See Figure 3 .

[0085] According to the blocking experiment and the report gene expression inhibition experiment results, the antibody clone with strong ability was selected. After the hybridoma cells were cultured, the total RNA of the cells was extracted according to the RNAfast200 kit (Shanghai Feijie Biotechnology Co., Ltd.) instruction steps; the total RNA of the hybridoma cells was reversely transcribed into cDNA by using 5x PrimeScript RT Master Mix (Takara); the IgVL (Kappa) and Vh sequence of the antibody light chain variable region were amplified by using the degenerate primer (Anke Krebber. 1997) and Extaq PCR reagent (Takara); the PCR amplification product was purified by using the PCR clean-up Gel extraction kit (Macherey-Nagel company); the amplification PCR product was connected to the T vector according to the pClone007 Simple Vector Kit kit (Qingke Biotechnology Co., Ltd.) instruction book and transformed into the E. coli competent cells, and after the strain amplification and plasmid extraction, the DNA sequencing was performed to obtain the single clone antibody variable region sequence.

[0086] Clone A20

[0087] >A20-VH

[0088] QIQLVQSGPELKKPGETVKISCKASGYSFATYGVSWLKQAPGKGLKWMGWMNTKSGVPTYADDFKGRFAFSWETSASTAYLLITNLKNEDTASYFCARNGQSGYDDYFDYWGQGTTLTVSS

[0089] >A20-VL

[0090] DIQLTQTTSSLSASLGDRVTISCSASQVISNYLNWYQQKPDGTLKLLIYYTSSLQSGVPSRFSGSGSGTDYSLTISNLESEDIATYYCQQYRKLPLTFGAGTKLELK

[0091] Clone B01

[0092] >B01-VH

[0093] EVHLQQSGAELVRPGASVKLSCIVSGFNIKDDFLHWVKQRPEQGLEWVGWIDPENGDTEYASKFQGKATVTADTSSNTAYLHLSSLTSEDTAVYYCVHQDYDEAFAYWGQGTLVTVSA

[0094] >B01-VL

[0095] DVVMTQTPLTLSITIGQPASISCKSSQSLLDSDEKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK

[0096] Clone C13

[0097] >C13-VH

[0098] EVQLQQSGPELVKPGTSMKISCKASGFSFTDYTMNWVKQSHGKSLEWIGLINPYNRGPTYNPKFKGKATLAVDKSSTTAYMELHSLTSEDSAVYYCAIEGGYRYDGAWFAYWGQGTLVTVSA

[0099] >C13-VL

[0100] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLELK

[0101] Clone D01

[0102] >D01-VH (SEQ ID NO: 21)

[0103] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYFIDWVKQRPGQGLEWIGVINPGSGGTNFNEKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARESEVGEGFAYWGQGTLVTVSA

[0104] >D01-VL (SEQ ID NO: 22)

[0105] DIQMTQTSSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIK

[0106] Clone D02

[0107] >D02-VH

[0108] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGLHWVRLSPGKGLEWLGVMWSGGGTDYNAAFISRLTISKDNSKSQVFFKMKSLQAADTAIYYCARRNSENEGYALDYWGQGTSVTVSS

[0109] >D02-VL

[0110] QIVLTQSPVIMSASLGEEITLTCSASSSVTYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSTYMNTFGGGTKLEIK

[0111] Clone D04

[0112] >D04-VH

[0113] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGLHWVRLSPGKGLEWLGVMWSGGGTDYNAALISRLTISKDNSKSQVFFKMKSLQAADTAIYYCARRSSENEGYALDYWGQGTSVTVSS

[0114] >D04-VL

[0115] QIVLTQSPVIMSASLGEEITLTCSASSSVTYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSTYMNTFGGGTKLEIK

[0116] Example 2 Kinetic study of the affinity of anti-human TSLP murine antibodies in vitro.

[0117] The binding kinetic parameters of murine antibodies to human TSLP protein were analyzed using a Fortebio (BLITZ pro 1.1.0.28) instrument.

[0118] AMC bio-probe was soaked in kinetic buffer (PBS, containing 0.01% BSA, 0.002% Tween20, pH 7.4) for 10 minutes before the assay; then the probe was placed in kinetic buffer (PBS, containing 0.01% BSA, 0.002% Tween20, pH 7.4) containing 100 nM of mouse antibody for 1000 seconds to capture the mouse antibody; further, the probe was combined with a solution containing 100 nM of antigen (PBS, containing 0.01% BSA, 0.002% Tween20, pH 7.4) for a binding time of 600 seconds; then the probe was transferred to kinetic buffer (PBS, containing 0.01% BSA, 0.002% Tween20, pH 7.4) for dissociation for a time of 600 seconds. After the experiment, the blank control response value was deducted, and the software was used for 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding, and the results are shown in Table 1.

[0119] Table 1. Results of mouse anti-antigen binding kinetics detection

[0120] Response KD (M) kon (1 / Ms) kdis (1 / s) IgG D01 0.5081 2.83E-09 7.96E+04 2.25E-04 IgG D02 0.5132 3.28E-09 7.33E+04 2.41E-04 IgG D03 0.4585 1.03E-08 4.50E+04 4.65E-04 IgG D04 0.5195 3.63E-09 7.35E+04 2.67E-04 IgG D05 0.4726 1.09E-08 3.59E+04 3.93E-04

[0121] Example 3 Preparation of anti-human TSLP chimeric antibody

[0122] The molecule D01 with high affinity, high blocking ability and cell activity was selected, and the sequences of the heavy and light chain variable regions of the antibody and the CDR domains thereof are shown in Table 2.

[0123] Table 2. Sequences of mouse antibodies

[0124]

[0125]

[0126] The sequence of the heavy chain variable region of the mouse anti-human TSLP monoclonal antibody was spliced with the sequence of the heavy chain constant region of the published human monoclonal antibody IgG2 subclass, and was constructed into a mammalian cell expression vector; the sequence of the light chain variable region of the mouse anti-human TSLP monoclonal antibody D01 was spliced with the sequence of the light chain constant region of the published human monoclonal antibody κ subclass, and was constructed into a mammalian cell expression vector. The constructed heavy chain vector and light chain vector of the anti-human TSLP chimeric antibody were paired and mixed, polyethyleneimine (PEI) was used to transfect HEK293 cells, and the cell supernatant was collected about 7 days later, and the anti-human TSLP chimeric antibody protein xiD01 was obtained by using Protein A purification.

[0127] The chimeric antibodies were analyzed for binding kinetics to human TSLP protein using the Fortebio instrument according to the assay described in Example 2. The results are shown in Table 3.

[0128] Table 3. Results of binding kinetics assay of chimeric antibodies to antigen

[0129] Response KD (M) kon (1 / Ms) kdis (1 / s) xiD01 0.3993 9.85E-10 3.61E+05 3.56E-04

[0130] Cellular activity of the chimeric antibodies, i.e. the inhibition of reporter gene expression, was assayed.

[0131] As above, the ability of the antibodies to inhibit the activity of the reporter gene was investigated using a stable BAF / 3 cell line overexpressing human TSLPR, human IL7R and STAT5-luciferase. The BAF / 3 cells were overexpressed with human TSLPR and human IL7R molecules and stably transfected with STAT5-luciferase reporter gene. The stable cells 1E6 were added to white bottom transwell plates at 50ul per well and starved for 4 hours. The antibodies were diluted 1:2 in a series of 8 dilutions to 8 gradients at 1mg / ml in PBSA (PBS with 1% BSA) at 100ul / well in U-bottom plates and 200ng / ml human TSLP in PBSA (PBS with 1% BSA) solution, incubated together for 30 minutes. The 50ul TSLP-antibody mixed solution was added to 50ul / well cells and incubated at 37°C in a 5% CO2 cell incubator for 16 hours. 100ul Bio-Glo Luciferase Assay Reagent was added and shaken for 5 minutes and the chemiluminescence was assayed. The results are shown in Figure 4 .

[0132] Example 4 Humanization and affinity maturation of anti-human TSLP murine antibodies

[0133] The amino acid sequence regions of the six antigenic complementarity determining regions (CDRs) of the heavy and light chains of the murine antibodies and the framework regions supporting the conserved three-dimensional conformation of the antibodies were determined by combining the antibody encoding schemes of Kabat, Chothia, Abm and CCG. Subsequently, by searching known human antibody sequences, the human antibody heavy chain variable region sequence most similar to the murine antibody was selected, and the antibody framework region sequence thereof was selected as a template. The murine antibody heavy chain CDRs were combined with the human antibody framework region, and finally the humanized antibody heavy chain variable region sequence was generated. In the same way, the humanized antibody light chain variable region sequence was generated.

[0134] The binding activity of the antibody molecule in which the CDR of the mouse-derived antibody is directly grafted into the human framework region is reduced, and then the yeast display technology is used for affinity maturation modification of the molecule. The CDR region amino acids of the heavy chain variable region and the light chain variable region of the template molecule are randomly mutated to build a library, from which candidate antibody molecules with higher affinity are screened. And the individual amino acids in the framework region are back-mutated from human-derived amino acids to mouse-derived amino acids. Determine the back-mutation site, one is to compare the designed humanized antibody sequence and the original mouse-derived antibody sequence to check which amino acids are different; two is to check whether these amino acids are important for supporting antibody structure or important for binding to antigen, and at the same time it is necessary to check whether there are some potential post-translational modification sites, such as N (asparagine) glycosylation site, N deamidation site, D (aspartic acid) isomerization site, etc. The humanized sequence is as follows.

[0135] Heavy chain variable region

[0136] >hzD01 H (SEQ ID NO: 3)

[0137] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIDWVRQAPGQGLEWIGVINPGSGGTNFNEKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARESEVGEGFAYWGQGTLVTVSS

[0138] >hzD01 Hm5 (SEQ ID NO: 5)

[0139] EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIDWVRQAPGQGLEWIGVIDAFSGGSNFNEKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARESEVGEGFAYWGQGTLVTVSS

[0140] >hzD01 Hm6 EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIDWVRQAPGQGLEWIGVIDSFSGGINFNEKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCARESEVGEGFAYWGQGTLVTVSS

[0141] Light chain variable region

[0142] >hzD01 L

[0143] DIQMTQSPSSLSASVGDRVTISCRASQDISNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQFDTLPYPFGQGTKLEIK

[0144] >hzD01 Lm4 (SEQ ID NO: 6)

[0145] DIQMTQSPSSLSASVGDRVTISCRASQDISNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQFDTLPYPFGQGTKLEIK

[0146] >hzD01 Lm12 (SEQ ID NO: 4)

[0147] DIQMTQSPSSLSASVGDRVTISCRASQDISNYLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQFDTLPYPFGQGTKLEIK

[0148] The engineered humanized antibody variable region heavy chain gene is constructed into a mammalian cell expression vector containing the heavy chain constant region gene of the human monoclonal antibody IgG2 subclass; the light chain gene is constructed into a mammalian cell expression vector containing the light chain constant region gene of the human monoclonal antibody kappa subclass. The constructed heavy chain vector and light chain vector of the anti-human TSLP humanized antibody are mixed and paired, transfected into HEK293 cells using polyethyleneimine (PEI), and the cell supernatant is collected after about 7 days. The anti-human TSLP humanized antibody protein is obtained by using Protein A purification.

[0149] Example 5 In vitro blocking experiment of humanized antibody

[0150] APC is coupled to human TSLP protein using a Dojindo kit (Dojindo #LK21). In addition, TSLPR overexpression cells are constructed using human TSLPR CDS and CHO cells, and stable transfection cells are obtained by antibiotic selection for 4 weeks.

[0151] The antibody to be tested was diluted in a gradient of 3 times (PBSA (PBS containing 1% BSA)) from a starting concentration of 100 ug / mL, a total of 11 concentration points, 100 ul of antibody at each concentration point was added to a 96-well plate, and 100 ul of APC-labeled TSLP antigen (1.6 ug / mL) (in PBSA (PBS containing 1% BSA)) was co-incubated for 1 hour. 100 ul of TSLPR-overexpressing CHO cells (1E5 / well) were added and incubated for 1 hour, the cells were washed once with PBS containing 0.5% BSA, and the mean fluorescence value of the cell population was analyzed by flow cytometry. The results are shown in Figure 5 .

[0152] The detection results show that, in terms of blocking the binding of TSLP to TSLPR, the two humanized antibody molecules achieve a blocking effect that is basically equivalent to that of the control antibody AMG157. The sequences of the heavy and light chain variable regions and the CDR domains of the antibodies are shown in Table 4.

[0153] Table 4. Sequences of humanized antibodies

[0154]

[0155]

[0156] Example 6 In vitro binding experiments of anti-human TSLP humanized antibodies

[0157] In vitro affinity kinetics of anti-human TSLP humanized antibodies were studied according to the procedure of Example 2. The results are shown in Table 5 and Figure 6 A and Figure 6 B. Figure 6

[0158] Table 5. Results of binding kinetics detection of humanized antibodies and antigens

[0159] Response KD (M) kon (1 / Ms) kdis (1 / s) AMG157 0.3786 1.66E-10 6.66E+05 1.10E-04 hzD01 Hm5 Lm4 0.3685 6.37E-11 6.25E+05 3.98E-05 hzD01 Lm12 0.4262 9.83E-11 6.19E+05 6.08E-05

[0160] Example 7 In vitro cell-based assays of anti-human TSLP humanized antibodies

[0161] 7.1 Inhibition of BAF / 3-TSLPR cell reporter gene expression

[0162] According to the detection method described in Example 3 for chimeric antibodies, humanized TSLP antibodies were diluted in a gradient, and their ability to inhibit luciferase in BAF / 3-TSLPR cells was evaluated. The results are shown in Figure 7 .

[0163] 7.2 Inhibition of dendritic cell secretion of CCL17 and OPG

[0164] ​Human peripheral blood lymphocytes were used to sort dendritic cells according to the reagent instructions of the company (catalog # 130-090-506). 100ul (5E4 cells / well) was plated in a 96-well plate, and the cell spread was observed after 24 hours. The human TSLP protein was prepared in PBSA (1% BSA in PBS) to detect the concentration of 40ng / ml, and the antibody concentration was 4000ng / ml, which was diluted by 1:2 gradient (in PBSA (1% BSA in PBS)), and the antibody and TSLP solution were mixed in equal volume and incubated for 30 minutes. 100ul of the mixture was added to the cell culture well, and the culture was continued for 48 hours. The concentrations of the two cytokines in the supernatant were detected by CCL17 and OPG ELISA kit, and the results are shown in Figure 8 Figure 8 A and Figure 8 B.

[0165] Example 8 Species cross-binding of anti-human TSLP humanized antibody and cross-binding with structural similar molecule IL7

[0166] Human TSLP, mouse TSLP and monkey TSLP proteins, and human IL7 protein (1ug / ml, pH9.6, 0.1M NaHCO3) were coated in 96-well plates respectively at 4℃ overnight; blocked with 4% skim milk-PBS at 37℃ for 2 hours; washed with PBST (0.05% Tween20-PBS) for three times, and then anti-human TSLP humanized antibody was added and incubated at 37℃ for 1 hour. After washing with PBST for three times, HRP-goat anti-human IgG (Jackson #109-035-003) was added at a dilution of 1:10000 and incubated at 37℃ for 1 hour. After washing with PBST for three times, TMB color developing solution was added, and color development was carried out in the dark for 10min. 2M H2SO4 was added to terminate the reaction. The A490 value was read by an enzyme-labeled instrument.

[0167] The results show that the antibody molecules hzD01Hm5Lm4 and hzD01Lm12 of the application can recognize human TSLP and monkey TSLP molecules, and have no non-specific recognition with IL7. See Figure 9 9A, 9B and Figure 9 C in

[0168] The above description of the specific embodiments of the application does not limit the application, and those skilled in the art can make various changes or modifications to the application without departing from the spirit of the application, and all such changes or modifications shall fall within the scope of the appended claims of the application. SEQUENCE LISTING <110> Maiwei (Shanghai) Biotechnology Co., Ltd. Shanghai Puming Biotechnology Co., Ltd. Beijing Konosinc Technology Co., Ltd. ​<120> Antibodies specific for human TSLP and uses thereof <130> LC20110015 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 448 <212> PRT <213> artificial sequence <400> 1 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp 100 105 110 Gly Gin Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys 210 215 220 Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Phe Arg Val Val 290 295 300 Ser Val Leu Thr Val Val His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Thr Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 2 <211> 214 <212> PRT <213> artificial sequence <400> 2 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 3 <211> 119 <212> PRT <213> Artificial sequence <400> 3 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val lie Asn Pro Gly Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 107 <212> PRT <213> artificial sequence <400> 4 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Ser Cys Arg Ala Ser Gin Asp lie Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Phe Asp Thr Leu Pro Tyr 85 90 95 Pro Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 119 <212> PRT <213> artificial sequence <400> 5 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Phe Ile Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asp Ala Phe Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 6 <211> 107 <212> PRT <213> artificial sequence <400> 6 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gin Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 7 <212> PRT <213> artificial sequence <400> 7 Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> 8 <211> 6 <212> PRT <213> artificial sequence <400> 8 Asn Pro Gly Ser Gly Gly 1 5 <210> 9 <211> 10 <212> PRT <213> artificial sequence <400> 9 Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> artificial sequence <400> 10 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> artificial sequence <400> 11 Tyr Thr Ser Thr Leu His Ser 1 5 <210> 12 <211> 9 <212> PRT <213> artificial sequence <400> 12 Gln Gln Phe Asp Thr Leu Pro Tyr Pro 1 5 <210> 13 <211> 5 <212> PRT <213> artificial sequence <400> 13 Asn Tyr Phe Ile Asp 1 5 <210> 14 <211> 17 <212> PRT <213> artificial sequence <400> 14 Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 15 <211> 10 <212> PRT <213> artificial sequence <400> 15 Gly Tyr Thr Phe Thr Asn Tyr Phe Ile Asp 1 5 10 <210> 16 <211> 10 <212> PRT <213> artificial sequence <400> 16 Val Ile Asn Pro Gly Ser Gly Gly Thr Asn 1 5 10 <210> 17 <211> 6 <212> PRT <213> artificial sequence <400> 17 Asp Ala Phe Ser Gly Gly 1 5 <210> 18 <211> 9 <212> PRT <213> artificial sequence <400> 18 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 19 <211> 17 <212> PRT <213> artificial sequence <400> 19 Val Ile Asp Ala Phe Ser Gly Gly Ser Asn Phe Asn Glu Lys Phe Lys 1 5 10 15 Gly <210> 20 <211> 10 <212> PRT <213> artificial sequence <400> 20 Val Ile Asp Ala Phe Ser Gly Gly Ser Asn 1 5 10 <210> 21 <211> 119 <212> PRT <213> artificial sequence <400> 21 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30 Phe Ile Asp Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He 35 40 45 Gly Val He Asn Pro Gly Ser Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Ser Glu Val Gly Glu Gly Phe Ala Tyr Trp Gly Gin Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115 <210> 22 <211> 107 <212> PRT <213> artificial sequence <400> 22 Asp He Gin Met Thr Gin Thr Ser Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr He Ser Cys Arg Ala Ser Gin Asp He Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gin 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 7 <212> PRT <213> artificial sequence <400> 23 Gly Tyr Ala Phe Thr Asn Tyr 1 5 <210> 24 <211> 10 <212> PRT <213> artificial sequence <400> 24 Gly Tyr Ala Phe Thr Asn Tyr Phe Ile Asp 1 5 10 <210> 25 <211> 326 <212> PRT <213> artificial sequence <400> 25 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gin Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gin Pro Arg Glu 210 215 220 Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 26 <211> 107 <212> PRT <213> artificial sequence <400> 26 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105

Claims

1. An antibody molecule or antigen-binding fragment thereof against thymic stromal lymphopoietin (TSLP), said antibody molecule or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) comprise a combination of heavy chain CDRs and light chain CDRs selected from: (1) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NO:7,8,9 in sequence; and L-CDR1, L-CDR2, L-CDR3 shown in SEQ ID NO:10,11,12 in sequence; (2) H-CDR1, H-CDR2, and H-CDR3 are shown in sequence in SEQ ID NO: 13, 14, and 9; And, respectively shown as L-CDR1, L-CDR2, L-CDR3 in SEQ ID NO:10, 11, 12; (3) H-CDR1, H-CDR2, and H-CDR3 shown in SEQ ID NO:15, 16, and 9 respectively; and L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO:10, 11, and 12 respectively; and (4) H-CDR1, H-CDR2, and H-CDR3 are shown in sequence in SEQ ID NO: 15, 14, and 9; And, respectively, L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO:10, 11, and 12.

2. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region respectively contain the amino acid sequence shown in SEQ ID NO:3 and the amino acid sequence shown in SEQ ID NO:

4.

3. The antibody molecule or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region and the light chain variable region respectively contain amino acid sequences that have at least 75% identity with the amino acid sequence shown in SEQ ID NO:3 and amino acid sequences that have at least 75% identity with the amino acid sequence shown in SEQ ID NO:

4.

4. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule is a murine antibody, a chimeric antibody, or a fully humanized antibody; the antigen-binding fragment is a fragment of the antibody molecule such as scFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

5. The antibody molecule or its antigen-binding fragment according to claim 4, characterized in that, The chimeric antibody is a partially humanized antibody.

6. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule is a monoclonal antibody or a single-chain antibody.

7. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule or its antigen-binding fragment also includes a constant region.

8. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule or its antigen-binding fragment also includes a mouse or human heavy chain constant region (CH) and / or a light chain constant region (CL).

9. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule or its antigen-binding fragment comprises a heavy chain and a light chain.

10. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule or its antigen-binding fragment contains a heavy chain constant region of IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

11. The antibody molecule or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The antibody molecule is a monoclonal antibody.

12. The antibody molecule or its antigen-binding fragment according to claim 11, characterized in that, The antibody molecule is a humanized monoclonal antibody.

13. The antibody molecule or its antigen-binding fragment according to claim 11, characterized in that, The heavy chain constant region of the monoclonal antibody is of type IgG1 or IgG2, and the light chain constant region is of type kappa.

14. The antibody molecule or its antigen-binding fragment according to claim 13, characterized in that, The heavy chain constant region contains the amino acid sequence shown in SEQ ID NO:25, and the light chain constant region contains the amino acid sequence shown in SEQ ID NO:

26.

15. A nucleic acid molecule comprising a nucleotide sequence encoding an antibody molecule or an antigen-binding fragment thereof as described in any one of claims 1 to 14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the nucleic acid molecule of claim 15 and / or the vector of claim 16, or the host cell being transformed or transfected by the nucleic acid molecule of claim 15 and / or the vector of claim 16.

18. A composition comprising an antibody molecule or an antigen-binding fragment thereof as described in any one of claims 1 to 14, a nucleic acid molecule as described in claim 15, a vector as described in claim 16, or a host cell as described in claim 17.

19. The composition according to claim 18, characterized in that, The composition is a pharmaceutical composition, which optionally further comprises a pharmaceutically acceptable carrier, excipient or excipient.

20. Use of the antibody molecule or antigen-binding fragment thereof of any one of claims 1 to 14, the nucleic acid molecule of claim 15, the vector of claim 16, the host cell of claim 17, or the composition of claim 18 or 19 in the preparation of a medicament for the prevention or treatment of inflammatory diseases or tumors that highly express TSLP; in, The inflammatory diseases are selected from asthma, allergic dermatitis, chronic obstructive pulmonary disease, and allergic rhinitis; the asthma includes Th2 type and non-Th2 type asthma; the tumors are selected from Hodgkin's lymphoma, breast cancer, pancreatic cancer, melanoma, and lung cancer.

21. A kit comprising an antibody molecule or antigen-binding fragment thereof as described in any one of claims 1 to 14, a nucleic acid molecule as described in claim 15, a vector as described in claim 16, a host cell as described in claim 17, or a composition as described in claim 18 or 19.

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